Respiratory Muscle Overload Training and Diaphragm Remodeling

Permanent Link: http://ufdc.ufl.edu/UFE0041908/00001

Material Information

Title: Respiratory Muscle Overload Training and Diaphragm Remodeling
Physical Description: 1 online resource (144 p.)
Language: english
Creator: Smith, Barbara
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010


Subjects / Keywords: diaphragm, hypertrophy, muscles, regeneration, respiratory, strength, training
Rehabilitation Science -- Dissertations, Academic -- UF
Genre: Rehabilitation Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation


Abstract: RESPIRATORY MUSCLE OVERLOAD TRAINING AND DIAPHRAGM REMODELING It is well-known that the diaphragm undergoes rapid atrophy and contractile dysfunction with inactivity, but the effect of overload training on the respiratory muscle pump is less understood. We investigated whether overload training produced by intermittent tracheal occlusion facilitated muscle fiber hypertrophy and regeneration, or whether the loads induced damage. Sixteen animals underwent placement of a tracheal cuff and were randomly assigned to receive either ten sessions of brief occlusions (n=8, OCCL) or observation (n=8, SHAM). After the intervention, the costal diaphragm, third parasternal intercostal, and soleus muscles were examined for fiber morphology, myosin heavy chain isoform composition and cross-sectional area, and presence of embryonic myosin. In the OCCL animals, type IIx/b fibers were 26% larger in the medial costal diaphragm (p < .01) and 18% larger in the intercostals (p < .01). A modest yet significant increase in embryonic myosin occurred in the intercostals of OCCL animals (p < .05). These results indicate that tracheal occlusion may facilitate rapid, preferential type II fiber hypertrophy in the respiratory muscle pump. Additional study is suggested to determine whether the training offers a performance benefit.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Barbara Smith.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Martin, Anatole D.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041908:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041908/00001

Material Information

Title: Respiratory Muscle Overload Training and Diaphragm Remodeling
Physical Description: 1 online resource (144 p.)
Language: english
Creator: Smith, Barbara
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010


Subjects / Keywords: diaphragm, hypertrophy, muscles, regeneration, respiratory, strength, training
Rehabilitation Science -- Dissertations, Academic -- UF
Genre: Rehabilitation Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation


Abstract: RESPIRATORY MUSCLE OVERLOAD TRAINING AND DIAPHRAGM REMODELING It is well-known that the diaphragm undergoes rapid atrophy and contractile dysfunction with inactivity, but the effect of overload training on the respiratory muscle pump is less understood. We investigated whether overload training produced by intermittent tracheal occlusion facilitated muscle fiber hypertrophy and regeneration, or whether the loads induced damage. Sixteen animals underwent placement of a tracheal cuff and were randomly assigned to receive either ten sessions of brief occlusions (n=8, OCCL) or observation (n=8, SHAM). After the intervention, the costal diaphragm, third parasternal intercostal, and soleus muscles were examined for fiber morphology, myosin heavy chain isoform composition and cross-sectional area, and presence of embryonic myosin. In the OCCL animals, type IIx/b fibers were 26% larger in the medial costal diaphragm (p < .01) and 18% larger in the intercostals (p < .01). A modest yet significant increase in embryonic myosin occurred in the intercostals of OCCL animals (p < .05). These results indicate that tracheal occlusion may facilitate rapid, preferential type II fiber hypertrophy in the respiratory muscle pump. Additional study is suggested to determine whether the training offers a performance benefit.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Barbara Smith.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Martin, Anatole D.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041908:00001

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2 2010 Barbara Kellerman Smith


3 To Gram


4 ACKNOWLEDGMENTS This journey could not have begun without the assistance of my wonderful family, friends, and mentors. I thank my husband Brian and family for their immeasurable love, support and guidance. I sincerely appreciate the ongoing support of my doctoral committe e, consisting of Drs. Paul Davenport, Orit S hec h tman, Krista Vandenborne and chaired by Dr. Danny Martin, and value their patience and wisdom. Dr. Davenport personif ies the enthusiasm of a scientist moved by the joy of discovery and continually motivates m e Dr. S hechtma my studies have been invaluable to me, and I appreciate her thoughtful questions and of this pr oject as well as her vast wisdom and assistance with future planning. I cannot imagine a finer model of the clinician scientist than Dr. Martin, and will always be grateful to him for his many years of support, patience and compassion. I am especially than kful to the patients who inspired my research pursuits and provide continued motivation to further the body of knowledge in ca rdiopulmonary rehabilitation. Doctoral studies were supported by a research assistantship (January July 2007, R01HD42705) and trai ning fellowship (August 2007 August 2010 T32 HD043730 ) from the National Institutes of Health. The author gratefully acknowledges the Foundation for Physical Therapy for their Promotion of Doctoral Studies (PODS I and II) awards, co co sponsored by the Car diovascular and Pulmonary Section, APTA and endowed in honor of Scot C. Irwin, DPT, CCS, an educator, advocate, and true pioneer in cardiopulmonary physi cal therapy. M entorship training and laboratory supplies were provided through the Group Advantaged Tr aining of Research program, co sponsored through University of Florida and Howard Hughes Medical Institute. Psalm 150:6.


5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 SPECIFIC AIMS ................................ ................................ ................................ ..... 13 2 REVIEW OF LITERATURE ................................ ................................ .................... 16 The Respir atory Pump Generates the Work of Breathing ................................ ....... 16 The Diaphragm Muscle ................................ ................................ .................... 16 Form and Function of the Costal Diaphragm Regions ................................ ...... 18 Accessory Muscles of Inspiration ................................ ................................ ..... 20 The Effects of Mechanical Ventilation on the Diaphragm ................................ ........ 22 Mechanisms of Ventilator In duced Diaphragmatic Dysfunction ........................ 23 Evidence for Diaphragm D ysfunction in Ventilated Humans ............................ 26 Inspiratory Weakness Reinforces Prolonged M echanical V entilation ............... 27 Inspiratory Muscle Training Effects on the Ventilatory Pump ................................ .. 29 Evidence that Inspiratory Muscle Strength Training ( IMST ) Improves Inspiratory M uscle S trength ................................ ................................ ........... 29 IMST and Diaphragm Fiber Remodeling ................................ .......................... 32 Mechanisms of myofiber h ypertrophy ................................ ........................ 34 Overlo ad training and myofiber damage ................................ .................... 36 Summary and Significance ................................ ................................ ..................... 39 3 MATERI ALS AND METHODS ................................ ................................ ................ 44 Research Design and Data Analysis ................................ ................................ ...... 44 Justification for an Animal M odel ................................ ................................ ...... 44 Experimental Design ................................ ................................ ........................ 45 Rationale ................................ ................................ ................................ .......... 45 Power Analysis ................................ ................................ ................................ 46 Methods ................................ ................................ ................................ .................. 47 Surgical Procedures ................................ ................................ ......................... 47 Occlusion Protocol ................................ ................................ ........................... 48 Tissue Analysis ................................ ................................ ................................ 48 Fiber phenotype and cross sectional area ................................ ................. 49 Muscle fiber remodeling ................................ ................................ ............. 50 Myofiber regeneration embryonic myosin ................................ ................. 51


6 Protein i mmunoblotting ................................ ................................ .............. 52 Statistical Analysis ................................ ................................ ............................ 53 4 RESULTS ................................ ................................ ................................ ............... 58 AIM 1 Respiratory Muscle Fiber Hypertrophy ................................ ........................ 58 Effect of Tracheal Occlusion on Fiber Cross Sectional Area ............................ 58 Cross sectional area of the diaphragm ................................ ...................... 58 Cross sectional area of the third intercostal ................................ ............... 59 Cross sectional area of the soleus ................................ ............................. 59 Regional diaphragm cross sectional area ................................ .................. 59 Effect of Tracheal Occlusion on Fiber Phenotype ................................ ............ 60 Fiber phenotype of the medial diaphragm ................................ .................. 60 Fiber phenotype of the third intercostal ................................ ...................... 61 Fiber phenotype of the soleus ................................ ................................ .... 61 Regional costal diaphragm expression of fiber phenotype ......................... 62 AIM 2: Respiratory Muscle Damage and Regeneration ................................ .......... 63 Effect of Tracheal Occlusion on Respiratory Muscle Mor phology .................... 63 Morphological assessment of the medial costal diaphragm ....................... 65 Morphological assessment of the third parasternal intercostal .................. 65 Morphological assessment of the soleus ................................ ................... 66 Morphological asses sment of the regional diaphragm ............................... 66 Effect of Tracheal Occlusion on Muscle Fiber Regeneration ............................ 67 Embryonic myosin expression in the diaphragm ................................ ........ 67 Embryonic myosin expression in the third intercostal ................................ 67 Embryonic myosin expression in the Soleus ................................ .............. 68 5 DICUSSION ................................ ................................ ................................ ............ 84 Principal Findings ................................ ................................ ................................ ... 84 Training Elici ted Fast Fiber Hypertrophy ................................ ................................ 85 Regional Heterogeneity in the Costal Diaphragm ................................ ................... 88 Regional Heterogeneity in Cross Sectional Area ................................ ............. 88 Regional Differences in Fiber Phenotype ................................ ......................... 90 Sustained Damage was not Present in Overloaded Muscle ................................ ... 91 Limited Presence Embryonic Myosin after Training ................................ ................ 93 Study Limitations ................................ ................................ ................................ .... 96 Application of the Model ................................ ................................ .......................... 98 6 CLINICAL APPLICATION OF OCCLUSION TRAINING: Case Report of an Infant with Post Operative Weaning Failure ................................ .......................... 100 Case Report ................................ ................................ ................................ .......... 100 Diagnostic Background ................................ ................................ .................. 100 Clinical Presentation ................................ ................................ ....................... 101 Respirator y Muscle Testing ................................ ................................ ............ 102 Training Program ................................ ................................ ............................ 102


7 Data Analysis ................................ ................................ ................................ 103 Training Outcomes ................................ ................................ ......................... 104 Discussion ................................ ................................ ................................ ............ 104 Inspiratory Occlusions and Cardiac Function ................................ ................. 106 Considerations for Respiratory Mechanics ................................ ..................... 108 Maturation of the Ventilatory Pump ................................ ................................ 108 Mechanical Ventilation and Weaning of Infants ................................ .............. 109 Conclusions ................................ ................................ ................................ .......... 111 7 CONCLUSIONS ................................ ................................ ................................ ... 119 LIST OF REFERENCES ................................ ................................ ............................. 121 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 144


8 LIST OF TABLES Table page 3 1 Categories for classification of myofiber features determined by point counting. ................................ ................................ ................................ ............. 55 4 1 Histological cross sectional area assessment of the medial costal diaphragm, third pa rasternal intercostals, and soleus in sham occluded animals and animals treated with intermittent tracheal occlusion ................................ ........... 69 4 2 Histological remodeling of the medial costal diaphragm. ................................ .. 69 4 3 Histological assessment of the third parasternal intercostals ............................. 70 4 4 Histological assessment of the soleus mus cles ................................ .................. 70 4 5 Cross sectional area assessment of the dorsal, medial and ventral costal diaphragm in sham occluded animals and animals treated with intermittent tracheal occlusion. ................................ ................................ .............................. 71 4 6 Proportion of fiber phenotype expression in the respiratory muscles. ................ 71 4 7 Area fraction of fiber phenotype expression in the respiratory muscles. ............. 72 4 8 Regional diaphragm phenotype proportions ................................ ....................... 72 4 9 Regional diaphragm phenotype area fractions ................................ ................... 73 4 10 Quantitative assessment of fiber remodeling in the respiratory muscles. ........... 73 4 11 Area fraction of abnormal cells in the diaphragm. Intercostal, and soleus muscles. ................................ ................................ ................................ ............. 74 4 12 Quantitative assessment of fiber remodeling in the regions of the costal diaphragm ................................ ................................ ................................ .......... 74 4 13 Area fraction of abnormal cells in the regions of the costal diaphragm. .............. 75 4 14 Embryonic myosi n positive fibers in the respiratory muscles ............................. 75 6 1 Suggested clinical indications and contraindications for inspiratory muscle str ength training. ................................ ................................ ............................... 112 6 2 Vital signs during the course of occlusion training sessions. ............................ 113 6 3 Respiratory performance variabl es during inspiratory occlusion ....................... 113 6 4 Ventilator settings and pulmonary mechanics during the course of treatment. 115


9 LIST OF FIGURES Figure page 2 1 Regional architecture of the rodent diaphragm. ................................ .................. 40 2 2 Su mmary of mechanical ventilation effects on atrophy and contractile dysfunction in the diaphragm. ................................ ................................ ............. 41 2.3 Protein synthesis and myogenic regeneration each contribute to training induced muscle hypertrophy ................................ ................................ ............... 42 2 4 Myogenic regeneration after skeletal muscle overload ................................ ....... 43 3 1 Schematic representation of the experimental design. ................................ ....... 56 3 2 Experimental apparatus ................................ ................................ ...................... 57 4 1 Cross sectional area (CSA) of the medial costal diaphragm .............................. 76 4 2 Fiber CSA in the third parasternal intercostal muscles ................................ ....... 77 4 3 Diaphragm, intercostal, and soleus muscle immunoh istochemistry for myosin heavy chain isoform. ................................ ................................ ........................... 78 4 4 Hematoxylin and eosin stained images of the study muscle s ............................ 79 4 5 Categories of fiber remodeling in the diaphragms of the experimental sample.. ................................ ................................ ................................ .............. 80 4 6 Embryonic myosin positive cells ................................ ................................ ......... 81 4 7 Proportions of embryonic myosin positive fibers in the respiratory mu scles ....... 82 4.8 Verification of embryonic myosin in intercostal muscle ................................ ....... 83 6 1 Type I Truncus Arteriosus ................................ ................................ ................ 116 6 2 Inspiratory muscle testing and training device ................................ .................. 116 6 3 Ventilatory parameters and strength ................................ ................................ 117 6 4 Spontaneous ti dal volume and maximal inspiratory pressure ........................... 118


10 LIST OF ABBREVIATION S A A Area fraction C dyn Dynamic compliance COPD Chronic obstructive pulmonary disease CPAP Continuous positive airway pressure CSA Cross sectional area dP/dt Rate of pressure development FRC Functional residual capacity HR Heart rate IAA Interrupted aortic arch IGF 1 Insulin like growth factor 1 IMST Inspiratory muscle strength training IMV Intermittent mandatory ventilation MAP Mean arterial pressure maxRPD Maximal rate of pressure development MHC Myosin heavy chain MIP Maximal inspiratory pressure MRF Myogenic regulatory factor MV Mechanical ventilation OCCL Occlusion training PBS Phosphat e buffered saline PEEP i Intrinsic positive end expiratory pressure PVR Pulmonary vascular resistance R awdyn Dynamic airway resistance (inspiratory or expiratory) RIPA Radio immunoprecipitation assay


11 RR Respiratory rate SBT Spontaneous breathing trial SDS P AGE Sodium dodecyl sufate polyacrylamide gel electrophoresis SHAM Sham training SpO 2 Pulse oximetry saturation of oxygen TA Truncus arteriosus TTI Tension time index V E Minute ventilation VIDD Ventilator induced diaphragm dysfunction VSD Ventricular septal defect V T Tidal volume ZAP Zone of apposition


12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy RESPIRATORY MUSCLE OVERLOAD TRAINING AND DIAPHRAGM FIBER REMODELING By Barbara Kellerman Smith August 2010 Chair: Anatole D. Martin Major: Rehabilitation Science It is well known that the diaphragm undergoes rapid atrophy and contractile dysfunction with inactivity, but the effect of overload training on the respiratory muscle pump is less understood. We investigated whether overload training produced by intermittent tracheal occlusion facilitated muscle fiber hypertrophy and regeneration, or whether the loads induced damage. Twelve animals underwent placement of a tracheal cuff and were randomly assigned to receive either ten sessions of brief occlusions (n=6, OCCL) or observation (n=6, SHAM). After the intervention, the costal diaphragm, third parast ernal intercostal, and soleus muscles were examined for fiber morphology, myosin heavy chain isoform compositio n and cross sectional area and presence of embryonic myosin. In the OCCL animals, type IIx/b fibers were 27% larger in the medial costal diaphra gm (p<.05) and 22% larger in the intercostals (p<.01) A modest yet significant increase in embryonic myosin occurred in the intercostals of OCCL animals (p <.05). These results indicate that tracheal occlusion may facilitate rapid, preferential type II fib er hypertrophy in the respiratory muscle pump. Additional study is suggested to determine whether the training offers a performance benefit.


13 CHAPTER 1 SPECIFIC AIMS Mechanical ventilation (MV) is a medical therapy used to sustain alveolar ventilation and rest the inspiratory muscles of patients with respiratory failure Paradoxically, this life saving therapy can induce severe weakness and atrophy of the diaphragm, referred to as ventilator induced diaphragm dysfunction (VIDD). While the median duratio n of MV is approximately 4 days VIDD can make it progressively difficult to discontinue the use of MV, termed weaning. Nearly 20 percent of mechanically ventilated adults will experience difficulty with weaning (1) In addition, 5 to 10% of ventilated patient s develop chronic ventilator dependence (2) Because prolonged MV is associated with disproportionate healthcare costs and high mortality (3, 4) it is imperative to understand the mechanisms responsible for preventing or treating VIDD. Animal studies ha ve repeatedly de monstrated that MV elicits rapid diaphragm fiber dysfunction (5 9) Significant diaphragm atrophy occurs within six hours of controlled MV in small mammals, and contractile dysfunction increases in a dose time response (10, 11) The inactiv e human diaphragm can also atrophy within three days of controlled MV (12) L ess is known about respiratory responses to increased activity and rehabilitation Inspiratory muscle strength training (IMST) has shown promise to prevent or reverse the effects of VIDD. A recent randomized, controlled clinical trial from our laboratory demon strated that IMST increased maximal inspiratory pressure (MIP) and facilitated ventilator weaning in MV dependent adults (13) Inspiratory exercises may promote intercostal mu scle fiber hypertrophy in ad ults with chronic obstructive pulmonary disease (COPD), in conjunction with increased strength (14) It is unknown whether overload training can elicit significant diaphragm muscle fiber hypertrophy


14 Animal models show that chr onic respiratory loading increases slow fiber size and phenotype expression in the diaphragm, but the intense, prolonged loads also elicit significant muscle damage (15 17) However, the previous loading models did not resemble the intermittent, moderate loads used for rehabilitation. We employed a novel model of tracheal occlusion to transiently overload the respiratory pump. The overall objective of following experiments was to investigate wh ether tracheal occlusion elicit s positive respiratory muscle fiber remodeling, including hypertrophy, or whether the overload generates fiber damage The projec ts were designed to achieve the following specific aims. Specific Aim #1: To determine whether intermittent tracheal occlusion generates hypertrophy and phenotype remodeling of inspiratory muscle fiber s Sub aim 1a: To determine whether the fiber cross sectional area of the diaphragm and third intercostal muscles differs in an imals that receive tracheal occlusion Sub aim 1b: To determine whether the expression of myosin heavy chain isoforms differs in the diaphragm and intercostal muscles of animals that undergo tracheal occlusion Rationale: It has been suggested that the highly active respiratory muscles may be more susceptible to disuse atrophy (5, 18) Alternatively m echanical loading activates pro tein synthesis and regeneration, and upregulates diaphragm myosin heavy chain (MHC) mRNA expression within hours (19) In th e costal diaphragm, the baseline regional fiber composition is similar I t is not known whether occlusion training modifies the regional fiber size or phenotype of the diaphragm, due to varied biomechanics.


15 Hypothesis: The fiber cross sectional area will be significantly greater in the medial costal diaphragm and intercostal muscles of animals that undergo ten session s of tracheal occlusion, without evidence of a shift in MHC phenotype composition. Specific Aim #2: To determine whether transient tracheal occlusion induces muscle fiber damage and regeneration in the respiratory muscles Sub aim #2a: To determine whether cellular remodeling is more prevalent in the diaphragm and intercostal muscles of animals that receive tracheal occlusion Sub aim #2b: To determine whether diaphragm expression of embryonic myosin heavy chain is greater in the respiratory muscles of occlusion trained animals Rationale: Sustained overload produces diffuse injury to the diaphragm and intercostal muscles (20) However, i nte rmittent occlusion could induce less secondary inflammation than prior models that imposed a constant load (21) The reports in the literature differ regarding regional costal diaphragm damage. The medial region undergoes the greatest shortening with ventilation and therefore may be more susceptible to remodeling (22) Hypothesis: Respiratory muscle regeneration wi ll significantly increase in animals that undergo ten sessions of tracheal occlusion without a significant presence of damage in the respiratory muscles or costal diaphragm regions T he following experiments were designed to increase the current understa nding of respiratory strength training and muscle remodeling. Translational applications will be discussed in a case report of clinical inspiratory occlusion training. It is hoped that these findings will help advance our understanding of respiratory muscl e plasticity, in order to promote motor recovery of the ventilatory muscle pump and facilitate ventilator weaning.


16 CHAPTER 2 REVIEW OF LITERATURE The Respiratory Pump Generates the Work of Breathing The Diaphragm Muscle Ventilation is the passage of air from the atmosphere into the alveoli of the lungs, and represents the first step to supply oxygen to body tissues. The thin, elliptically shaped diaphragm is the primary muscle of the inspiratory pump. T h is muscular membrane spans the internal diameter of the ribcage and separates the thoracic and abdominal body compartments. The diaphragm muscle consists of costal and crural segments which are thought to assume divergent secondary functions due to differences in muscular architecture The bony origin s of t he costal diaphragm include the xiphoid process and the seventh through twelfth ribs and costal cartilages. The crural diaphragm arises from the transverse processes of the first three lumbar vertebra e, and this region contains a larger number of muscle sp indles (23) Crural fibers differ from costal fibers in both anatomy and function. The crural diaphragm can act as an accessory esophageal sphincter and exerts considerable influence on non ventilatory maneuvers such as coughing and emesis (24) Its non ventilatory physiological function s are distinct from the costal diaphragm, despite similar timing of motor unit activation during inhalation (24, 25) The larger costal segment accounts for approximately 80% of the weight of diaphragm. Its greater oxi dative capacity and mobile anatomical origins reflect the primary ventilatory function of the costal diaphragm (26) The diaphragm is capped by a centrally located tendon, which gives the muscle a dome shaped appearance and acts as the muscular insertion for both the costal and crural segments The fibers of the costal diaphragm fan out circumferentially around the


17 inner portion of the thoracic cage. The stiffness of the central tendon is much greater than that of muscle tissue (27) and thus muscular cont raction results in a flattening of the diaphragm muscle tissue similar to the movement of a piston. Because of the closed architecture of the thoracic cavity, fiber orientation can differ considerably at different reg ions of the costal diaphragm and influ ence the efficiency of contracting fibers At its bony attachment s, the crural diaphragm contractile forces yield no direct motion of the vertebral column. On the other hand, the costal diaphragm interacts with the thoracic cage at the zone of apposition, and the abdom inal contents act as a fulcrum. Coordinated d iaphragm contraction results in a decrease in intrathoracic pressure, descent of the abdominal contents accompanied by a rise in abdominal pressure, and expansion of the ribcage. The architecture o f the thoracic cage, fiber length, and fiber orientation differ between regions of the costal diaphragm, but regional myosin heavy chain (MHC) isoform proportions do not appear to vary (28) The diaphragm bears a mixed fiber composition, reflective of both its continuous ventilatory function and its intermittent roles in vocalization, expulsion, and protective reflexes. Sieck reported that the rodent diaphragm contains ~37% type I slow, oxidative fibers, 30% type IIa fast, oxidative fibers, 25% type IIx fas t, intermediate fibers, and approximately 8% type IIb fast, fatigable fibers (29) Although 86% of rat diaphragm fibers express a single MHC isoform, type IIx and IIb MHC is co expressed in approximately 12% of rat diaphragm myofibers (29) The MHC isoform composition of the diaphragm can be influenced by training. Aerobic conditioning results in a greater oxidative capacity and slow phenotype shift in the costal, but not the crural diaphragm (30) Although larger mammals including


18 humans do not express the type IIb MHC isoform, the human diaphragm metabolic, architectural and innervation properties are similar to those of the rodent. Innervation of the diaphragm is organized somatotopcially. In the rat, the ventral costal and crural regions are innervated by C4 nerve segments, medial regions by C5, and dorsal regions by C6 nerve roots (31) Diaphragm muscle contractions follow a (32) In skeletal muscles, small motor unit s with high resistance and a low rheobase are recruited first, and small ventilatory demands result in the contraction of primarily slow motor units (33) Greater efferent drive results in rate modulation of active motor units as well as additional recruit ment of larger motor units (34, 35) The neuromechanical efficiency of the diaphragm influences its rate modulation and motor unit recruitment, as well as the recruitment of accessory ventilatory muscles (36) Form and Function of the Costal Diaphragm Regi ons Although the diaphragm comprises less than 0.5% of body mass (37) it spans a large relative surface area, and its heterogeneous muscular attachments may yield regional differences in function. Scientists have delineated the diaphragm previously, based upon somatotopic innervations, anatomical origins, or functional properties. For the following experiments, the regions of the costal diaphragm have been differentiated according to the functional divisions described by Poole and colleagues (26) ( Fig ure 2 1 ). Important regional distinctions for our experiments included the zone of apposition, metabolism, mechanical advantage, and eccentric contractions. Zone of apposition : The portion of the diaphragm that is apposed to the thoracic cage extends from the bony origin of the diaphragm to the point where it begins to turn away from the chest wall. This zone of apposition (ZAP) couples the pressures exerted


19 through the ribcage and abdomen. Mechanical stress generated by the costal diaphragm varies regional ly, because a larger ZAP permits contracting fibers to generate more stress (38) In other animal models the length of the ZAP is greatest at the medial costal region of the diaphragm, and the ZAP is smallest in the ventral costal region (38) During contraction, fibers along the ZAP exert a cephalad force on the thoracic cage and facilitate expansion of the lower lung segments. Bioenergetic considerations : Although MHC isoform expression does not differ substantially between diaphragm regions, the v entral costal segment exhibits a lower oxidative enzyme capacity (39) The magnitude of blood flow in the costal diaphragm may also account for regional differences in oxidative capacity (40) Medial costal segments appear to receive the greatest blood flo w both at rest and with exercise, while dorsal segments receive the smallest flow rate (41) On the other hand, ventral sternal diaphragm sections exhibit lower rates of glycogen depletion during aerobic exercise (42) The findings could indicate that the ventral regions relied less upon glycogen as an energy substrate, or instead, the region was recruited to a lower degree with exercise. Mechanical advantage : In quadruped animals, the diaphragm generates ~40% of inspiratory pressure during quiet breathing (22) Inspiratory pressure generation can be influenced by the mechanical advantage ( ) of a given region as well as muscle tension (43, 44) The magnitude of depends upon mechanical strain and the change in lung volume M L ) There are reg ional differences for in the costal diaphragm that are largely dictated by mass. Both t he mass and the is greatest in the medial costal diaphragm and the lowest in dorsal regions (41) Regional differences in are preserved during passive shortening, quiet breathing and forceful ventilatory efforts


20 (22, 45) The ratios of passive to active muscle shortening are highly correlated and suggest that these regional variations serve to minimize the work of breathing (46) Eccentric loading and injury : Ins piration is typically associated with shortening contractions of the diaphragm, but lengthening loads may be imposed under certain conditions. During expulsive reflexes and vigorous exhalation, diaphragm contractions stiffen the thoracic cage as the muscle is lengthened (47) Medial costal regions of the diaphragm may specifically contract in an eccentric fashion during obstructed inspiration (48) Eccentric contractions of skeletal muscles have been associated with a high er incidence of muscle fiber damage (49) but eccentric training may also yield greater fiber hypertrophy and strength gains, compared to concentric contractions (50) Thus, respiratory occlusion could potentially result in eccentric loading, damage, and h ypertrophy. However, the diaphragm does not contract in isolation. Large respiratory efforts generate additional motor unit recruitment in accessory ventilatory muscles. Additionally, phasic activation of the abdominals can yield a pre inspiratory diaphrag matic stretch (51) Therefore, accessory ventilatory muscles may influence the degree of diaphragm stress and remodeling. Accessory Muscles of Inspiration T he diaphragm contracts the earliest among the muscles of inspiration (36) and its displacement ac counts for approximately 70% of the change in resting tidal volume (52) Although the diaphragm is the primary muscle of the inspiratory pump, synergistic ventilatory muscles contract in a predictable fashion during breathing. T he scalene and inspiratory i ntercostal muscles are also recruited during quiet inspiration in humans (36) The negative intrathoracic pressure generated by diaphragmatic descent is offset by stiffening and expansion of the ribcage by the intercostal muscles. This cooperation


21 prevents inefficient, paradoxical movements between the thorax and the abdomen. Without co contraction of the accessory intercostal muscles, isolated diaphragm contractions result in inward displacement of the upper thoracic cage (53) On the other hand, accessory muscle contraction in the presence of diaphragm paresis results in expansion of the upper ribs and narrowing of the lower ribcage (53, 54) Of the accessory inspiratory muscles, the human and canine intercostals are the best understood. The individual in tercostals are heterogeneous muscles, and the ventilatory function of these muscles is specified to both its mechanical advantage and its distribution of neural drive (34, 55) T he exte rnal intercostals originate at the costal tubercles and continue to the ventral costal cartilage. In each thoracic segment, f ibers run from the lower margin of the upper ribs anteriorally and inferiorly to the lower rib. At most thoracic interspaces, the e xternal intercostals primarily exert an inspiratory moment on the thora cic cage. In quadrupeds, t he inspiratory mechanical advantage of the external intercostals increases from rostral to caudal segments (44) In opposition to the external intercostals, the internal intercostal muscles originate ventrally at the sternocostal articulations and each segment run s posteriorally and inferiorly from the lower margin of the upper rib to the lower rib. In the upper thoracic segments, the internal intercostals are functionally divided into dorsal ( interosseus) and ventral, paras terna l regions. Based upon the kinematics of the rostral thoracic cage and the neural drive, parasternal intercostals act as inspiratory muscles, and they are recruited within the first 10% of the inspiratory time (56) The inspiratory mechanical advantage of t he parasternal intercostals is greatest at the second and third thoracic segments (57) After the first few costal segments, the mechanical advantage and drive


22 to the internal intercostal muscles transition rapidly from inspiratory to expiratory action (55, 56) (55) The timing and amplitude of motor unit r ecruitment in the accessory muscles match their mechanical advantage (34) C ontraction of the inspiratory muscle pump establishes a negative pressure gradient, which drives airflow into the lungs. The diaphragm contracts for ~30 40% of the life cycle of an organism in order to continually meet its ventilatory requirements (58) The ventilatory pump must be activated with sufficient timing, amplitude and coordination to meet ventilatory motor drive. With additional respiratory demands, the degree and intensity of accessory muscle recruitment increases to match the amplitude and timing of breathing (52) When ventilatory demand is unmet due to illness or injury, MV may be initiated to maintain the movement of air from the atmosphere to the alveoli. The Effects of Mechanical Ventilation on the Diaphragm Intensive care therapies, including mechanical ventilation ( MV ) can save many lives following a life threatening injury or illness. MV attenuates the mecha nical work of the inspiratory pump and results in decreased descending corticospinal drive to the diaphragm (59) Ventilatory support can be modified to regulate the number of assis ted breaths, level of pressure and/or tidal volume s upport or mode of con trol Progressively higher levels of MV support can diminish or cease diaphragmatic electromyography ( EMG ) critically ill patients (60) Paradoxically, MV can facilitate dysfunction of inactive dia phragm sarcomeres, termed ventilator induced diaphragm dysfunction (VIDD), a condition identified clinically by progressive inspiratory weakness. VIDD results in structural and functional changes to the diaphragm, manifested by atrophy and contractile dy sfunction. Numerous animal models have confirmed that


23 periods of controlled MV between six and 48 hours result in diaphragmatic atrophy (61 64) Atrophy of the inactive diaphragm occurs in conjunction with rapidly down regulated protein synthesis (5, 65) followed shortly by a heightened, sustained state of proteolysis (7, 65, 66) Initially, MV induced diaphragm inactivity affects all muscle fiber types, but sustained ventilation appears to preferentially atrophy fast fatigable fibers (62, 63, 67, 68) Controlled MV initiate s atrophy signaling cascades in the diaphragm up to eight times faster than in the limb muscles (5, 18) suggesting that this chronically active muscle may be more susceptible to disuse atrophy (8) In humans, proteolytic gene expre ssion significantly increases within the first several hours of MV (69) Mechanisms of Ventilator Induced Diaphragmatic Dysfunction The diaphragm is an active muscle characterized by a high duty cycle, strong oxidative capacity and large number of mitoch ondria. Therefore, the inactive, ventilated diaphragm is vulnera ble to damage from oxidative stress (Figure 2 2) (6, 9) Oxidative stress is thought to play a key role in disturbances of muscle ion channels, resulting in an influx of intracellular calcium and ineffective calcium sequestration by the sarcoplasmic reticulum (8) In addition, the antioxidant defenses of the ventilated diaphragm become less efficient, and mitochondria become particularly susceptible to damage (7, 70) Preliminary work from our collaborators indicates that 3 4 hours of controlled MV alters state 3 and 4 mitochondrial respiration in the human diaphragm by ~25% (unpublished pilot data). O xidation of phospholipids, nucleic acids, and protein in the diaphra gm may also activate apoptosis and proteolysis. Caspase signaling cas cades in receptor s, sarcopla smic reticulum and mitochondria trigger apoptotic activity of myonuclei or myofibers (11) T he rate of myonuclear apoptosis matches the rate of proteolysis, p reserving the diaphragmatic myonuclear domain in VIDD (11)


24 Severe disturbance results in apoptosis of skeletal myocytes Within several hours of controlled MV, gene expression of a number of apoptotic and proteolytic regulatory molecules becomes significa ntly upregulated (5) Proteolysis can be mediated by lysosomal, calcium regulated, and ubiquitin proteosomal pathways. The myofibrillar proteins account for over 50% of muscle proteins (8) and ubiquitin proteosomal pathways are primary mediators of atro phy in adult muscle fiber s (71, 72) U biquitin related proteolysis is activated by the forkhead box (Foxo) transcription factors and occurs through initiation (E1), elongation (E2), and ligation (E3) protein families Two atrophy specific E3 ligases atrog in 1 and muscle ring finger 1 (MuRF1) modul ate the rate and extent of myofibrillar protein decomposition. The Foxo transcription factors also initiate lysosomally mediated autophagy in specific cases of muscle atrophy (73) Although only a small portion of myofibrillar degradation occurs through autophagy, the lysosome may facilitate the breakdown of sarolemma, sarcoplasmic reticulum, and mitochondrial proteins (74) The calcium mediated proteolytic m olecule s calpain and caspase 3 are thought to initiate myofibrillar deconstruction (10) because the E3 ubiquitin ligases cannot degrade intact contractile elements Proteolytic drive is accompanied by down regulation of key hypertrophic t ranscription factors within regenerative and protein synthesis signaling cascades (63, 64) In addition to muscle fiber atrophy, the contractile function of the diaphragm is impaired by MV Controlled MV induces rapid and profound decrements in specific twitch and tetanic isometric force in multiple animal models in a time course that parallels the rate of diaphragm atrophy (10, 62, 67, 75 78) Significant reductions in


25 diaphragmatic force can be detected within 6 hours of controlled MV (10, 76, 78) The literature suggests that period s of 6 to 72 hours o f cont rolled ventilation impair the specific tension of the diaphragm by 18 to 60% (10, 64, 76, 77, 79) Impairments in isometric and isotonic specific force can be detected throughout the physiological and supra maximal ranges of the force frequency curve (10 64, 77) in proportion to MV duration (77, 78) While a number of factors can accelerate diaphragm contractile dysfunction, other influences can be ruled out. Diaphragm force impairments not appear to result from a shift in relative fiber type proporti ons within the first 72 hours of MV (64, 76, 77) In conjunction, MV does not alter phrenic n erve conduction latency or the duration of the compound muscle action potential (80) suggesting that nerve conduction and membrane inexcitability do not co ntribu te substantially to VIDD. A ddition ally repetitive passive shortening of the dia phragm by MV does not alter the length tension properties of the muscle (67, 68, 77) However, some neuromuscular blocking medications can exacerbate diaphragm force impairmen ts brought about by MV (81) Some scientist s attribute diaphragm contractile dysfunction to reduced quantities of fast MHC protein and mRNA (65, 79) but these findings have not been universally reported (78) On the other hand, ultrastructural damage of the diaphragm and intercostals can be visualized within 2 days of MV, accompanied by reduced mitochondrial numbers and blunted mitochondrial respiration (82) Ion channel disruption and ultrastructural damage of the diaphragm likely contribute to excitatio n contraction uncoupling (76, 78) In summary, diaphragm inactivity induced oxidative stress rapidly promotes mitochondrial


26 dysfunction, proteolysis, and excitation contraction uncoupling, resulting in atrophy and contractile dysfunction. Evidence for Diaphragm Dysfunction in Ventilated Humans C linical research illustrates that diaphragm dysfunction also occurs in ventilated humans. The first evidence of VIDD in humans consisted of a post mortem report of fiber atrophy in neonate s who required extend ed periods of ventilation (83) More r ecent ly, research by Levine (12) illustrated that passively ventilated adults ( age: 3516 years ) free from active pulmonary or infectious diseases, exhibited rapid and profound diaphragmatic atrophy. In this study, o rgan donors with terminal brain injuries experienced 57% atrophy of type I fibers and 53% atrophy in type II fibers within a median 34 hours of controlled MV, compared to the diaphragms of older adults who underwent thoracic surgeries (MV duration : 2.40.5 hours ). Although MHC gene expression did not appear to change, patients expressed markedly elevated mRNA levels of atrogin 1 and MuRF1 These compelling data indicate that clinically meaningful proteolysis occurs in humans after short periods of control led MV. Moreover, they indicate that VIDD can affect ventilated humans of any age. Additional factors in critical care clinical practice influence the developmen t of VIDD in humans N euromuscular blocking agents and corticosteroids medications may impair excitation contraction coupling and accelerate the formation of critical illness myopathy (84) Hyperglycemia, systemic inflammatory response syndrome, and multiple system organ failure each place patients at greater risk for ICU acquired weakness of the d iaphragm (85) Clinically meaningful decreases in inspiratory and limb muscle strength occur within one week of continuous MV, and inspiratory muscle weakness is


27 associated with delayed weaning (86) Scientists acknowledge that VIDD is a major clinical pr oblem that can delay or prevent weaning (1, 87 89) Inspiratory Weakness Reinforces Prolonged Mechanical V entilation To minimize the risk of VIDD, weaning efforts should begin as soon as possible (90, 91) W eaning readiness trials use short period s of unassisted breathing, termed spontaneous breathing trials, to test readiness for extubation (92, 93) Even after short periods of MV, the ventilatory characteristics of patients who successful ly wean from MV differ from those who fail (94 102 ) Patients who fail extubation often develop a rapid, shallow ventilatory pattern during a spontaneous breathing trial (102) However, many causes of early failed extubation s do not necessarily predict weaning failure in patients w ho require prolonged MV (1) After longer periods of MV, the reasons for weaning failure are more complex and cannot be explained by weaning predictors based upon respiratory mechanics (1) Additional risk factor s for prolonged weaning include cardiac insufficiency, use of myoto xic drugs, sepsis and advanced age, as well as inspiratory muscle dysfunction (87, 103) Of these factors, it is acknowledged that a principal reason for weaning failure in alert, difficult to wean patients is insufficient ventilatory capacity, in relation to required breathing loads (1, 101, 104 106) I nspiratory weakness has been identified as a crucial contributor to ventilator dependence (97, 101, 107) Specifically, patients who repeatedly fail to wean require large pressures to generate a tidal bre ath, in comparison to the peak pressure capacity of the inspiratory pump (101) At rest, passive respiratory mechanics are unable to reliably differentiate patients who fail a weaning trial from those who pass (108) However, patients with respiratory musc le weakness often exhibit a deterioration of pulmonary mechanics during periods of unassisted breathing Most notably, intrinsic


28 positive end expiratory pressure (PEEP i ) increases, which may occur due to active expiratory muscle contraction s and early term ination of exhalation (109, 110) At end expiration, lung volumes progressively expand, resulting in d ynam ic hyperinflation. Progressive hyperinflation is accompanied by increased airway resistance and decreased pulmonary compliance during failed breathing trials (100, 106) In the presence of dynamic hyperinflation, tidal breathing occurs over a less compliant region of the respiratory pressure volume curve As a result, the pressure load increases during a tidal inhalation (100) Excessive resistive ventilatory loads may occur due to airway obstruction by edema, bronchoconstriction, or mucus plugging, while pulmonary congestion, effusion, ascites, or skeletal injury can elevate elastic loading. Chronic co morbid diseases such as CO PD may exacerbate acute changes in resistive and elastic ventilatory loads. Patients with repeated weaning failure often experience tidal pressure load s (P di /P dimax ) t hat approximate 40% of maximal transdiaphragmatic pressure (101) W orkloads of this magni tude are unsustainable in healthy adults who undergo breathing or extremity loading tasks (101, 106, 111) Clinically, t he pressure demands of inspiration and the timing of breaths have been integrated into a tension time index, TTI (TTI = P di /P dimax T i / T tot where P di /P dimax = proportion of maximal transdiaphragmatic pressure required for tidal inhalation, and T i /T tot = duty cycle). Values of TTI that exceed 0.15 ha ve been identified as a major pathophysiological characteristic of repeated weaning failu re in infants, children and adults (101, 104, 106, 112) Moreover, TTI underscores the relationship between r espiratory muscle weakness and breathing pattern s during weaning failure.


29 In conjunction with altered mechanics and increased energetic requirement s of breathing, accessory muscle recruitment may increase in patients who fail a weaning trial. Some prior investigators have interpreted accessory muscle use as a sign of diaphragmatic fatigue (97, 113) Indeed, accessory muscle use and increased diaphrag matic TTI frequently occur with weaning failure (104, 109) However, the evoke d tension of the diaphragm does not deteriorate in patients who fail to wean, compared to successfully weaned patients, indicating that inspiratory fatigue does not predicate weaning failure (114) Rather, clinical signs of physiological or psychological distress, each associated with recruitment of ve ntilatory accessory muscles, likely preceded the onset of diaphragm fatigue (100, 109, 115) Notab ly, the relationship between weaning failure and inspiratory weakness implies that two primary stra tegies may facilitate weaning. Clinical w eaning success ma y be achieved by decreasing the loads which oppose breathing, or by increasing the strength of the ventilatory muscles. Inspiratory Muscle Training Effects on the Ventilatory Pump Evidence that Inspiratory Muscle Strength Training ( IMST ) Improves Inspirat ory M u scle S trength While d iaphragmatic weakness is a well recognized characteristic of prolonged MV (86, 104, 107) l ess is known about the role of inspiratory muscle strengthening to facilitate weaning. The first reports of concurrent respiratory muscle training and ventilator weaning were published nearly 30 years ago and consisted of isolated reports and case series (116 119) E arl y studies of inspiratory muscle training provided sustained breathing loads designed to improve ventilatory endurance. Pa tients received training with isocapnic hyperpnea (116, 117) or by alinear resisters that provided flow dependent breathing loads (118, 119) In these reports, training produced


30 significant maximal inspiratory pressure ( MIP ) gains that were associated with weaning (116 119) However, n ot all reports have shown positive effects of inspirat ory training Light, sustained resistive breathing can be associated with hypoventilation and oxygen desaturation in patients, without an improvement in breathing performa nce (120) MV assisted breaths at higher pressure settings (121) Although patients needed to briefly generate greater pressures to trigger the ventilator, the elastic loads of inspiration were abolished by MV. Therefore, it is not surprising that respiratory muscle efficiency did not improve and this method of training did not accelerate weaning. Weaning success necessitates the prevention or reversal of re spira tory weakness, particularly with extend ed MV (1, 105, 107, 109, 114, 122) Strengthening can be achieved with training overloads appropriate for the capacity of the patient. Reliable inspiratory muscle strength training ( IMST ) loads can be provided by weig hted plungers, occlusions, or commercially available threshold trainers (123, 124) Conventional t hreshold IMST devices use a spring loaded, one way valve, and subjects must generate a minimum amount of inspiratory pressure, in order to overcome the tensio n of the spring (125 127) Below the threshold pressure value the poppet valve of th e training device remains closed and patients do not receive any inspiratory tidal volume. During valve closure, small volume fluctuations occur due to gas compression a nd chest wall compliance, and the pressure effort of the inspiratory muscles is quasi isometric. Once the target pressure has been reached, the poppet valve opens and the diaphr agm contraction becomes isotonic in nature as airflow begins Threshold IMST trainers deliver flow independent lo ads under most airflow rate s (123) While respiratory


31 endurance exercises require light, sustained loaded breaths, IMST typically provide s moderate to high intensity loads for brief durations, akin to limb strength train ing (125, 128) The application of IMST to patient populations was based upon inspiratory performance benefits that were established in healthy human subjects. Studies in healthy adults consistently demonstrated that four to six weeks of moderate to high intensity IMST (50% to 100% of MIP, brief durations) yield an average 45% gain in MIP (69, 129 131) More intense training loads produced greater relative MIP improvements, and some of the highest relative pressure gains have occurred with training sets o f 1 repetition maximal inspirations (131) In addition to improved strength IMS T decr eased the inspiratory neural drive, in proportion to the MIP gain (69, 132) With improved MIP, subjects perceived standard inspiratory loads as smaller (129) and the detection of the smallest threshold pressure loads decreased (69) In conjunction, IMST appears to improve ventilatory performance After training, s ubjects could maintain tidal volume with a faster airflow and produced greater power while breathi ng against inspiratory loads (69, 129, 131) Th us, m oderate to high intensity IMST resulted in robust strength increases in that lower ed drive, reduc ed load perception and improved ventilatory performance Just as IMST improved strength in healthy adults, it can facilitate recovery from respiratory mu scle weakness, including VIDD. However, c ompared to their healthy counterpar ts, ventilated adults achieved a more modest strength benefit MIP improvement s between 16 and 140% (excluding the outlier, average g ain 27%) have been reported after 2 4 weeks of IMST (13, 127, 133 135) A lower MIP gain in patients


32 may be due to a lower training intensity (between 30% and 50% of MIP) Short, intense IMST sessions appeared to elicit as large a strengthening effect as m oderate, sustained training modes. Clinical training studies show that many difficult to wean patients can achieve liberation from MV when IMST accompanies the weaning efforts (125, 127) In clinical studies, the greatest rates of ventilator weaning o ccurred with higher training intensities. Moreover, trained patients exhibit ed improved MIP upon weaning (126, 127) Our laboratory recently completed a randomized, controlled trial of 69 ventilated adults and demonstrated a significant weaning benefit of IMST, compared to sham inspiratory training (128) After an aver age 44 continuous days of MV, 71 % of IMST subject s weaned from MV, compared to 47 % of sham trained subjects (p<.05) Other medical co morbidities, treatments or medications did not differ appreciably between the groups. The collective data impl y that IMST can improve ventilatory performance and enhance weaning from MV yet it is less understood how IMST remodels the inspiratory pump IMST and Diaphragm Fiber Remodeling Although rapid stre ngth and functional gains have been reported with IMST, fiber hypertrophy has not been traditionally considered to occur within the first 4 we eks of training (136) However, other physiological evidence indicates that strong overload training can elicit br isk muscle remodeling In the limb muscles, expression of myogenic growth factors significantly increase within hours of single resistance training bouts, and adaptations are reinforced with repeated training sessions (72, 137) The evidence in extremity m uscles suggests that IMST could facilitate rapid diaphragm remodeling D iaphragm fiber hypertrophy has been examined in animals, after sustained inspiratory load ing. These efforts were largely unsuccessful due to the use of


33 experimen tal designs that did n ot implement high tension, low repetition contractions, in concordance with accepted principles of strength training. Early loading studies used bands to reduce the internal tracheal diameter of animals, creating a resistive load that was sustained for da ys to weeks (15, 16, 138) The continuous res istive loads expanded the proportion of slow oxidative fibers and oxidative activity in the diaphragm and increase d diaphr agm mass and fatigue resistance (15, 17) Although a chronic inspiratory overload appears to increase oxidative capacity, muscle growth and some components of contractile function, it produc ed a high rate of mortality (16) This su ggests that the intervention elicit ed unsustainable physiological stresses (17, 139) Short yet physiologically challenging durations of inspiratory resistive breathing (2 continuous hours at 50% MIP) have been shown to increase slow MHC gene expression (19) However, this training dura tion has also been associated with plasma membrane damage and sarcomere disruptio n (140) T o minimize sarcomere damage alternative animal training studies utilized alinear resistive masks to provide low intensity inspiratory training (21, 139) E ight weeks of light respiratory muscle conditioning ind uced type I and IIa hypertrophy (13 9) and moderate tra ining increased type IIa and IIx/b cross sectional area (CSA). In spite of the modest fiber hypertrophy, inspiratory training did not improve evoked tension of the diaphragm. Alternatively the extent of the force adaptation may have be en limited by a low training intensity (TTI = 0.05) (21, 139) Collectively, the animal training studies indicate that diaphragm fiber hypertrophy is possible, yet t he se studie s cannot elucidate if or how the diaphragm remodels with strengthening, because they did not administer strength training. In contrast, t his investigation employed a novel model of intermittent


34 tracheal occlusion that applied a high intensity, brief strengthening stimulus to the inspiratory muscles. We postulated that the respiratory muscles would demonstrate robust histological plasticity, in the presence of this transient, intense pressure stimulus Mechanisms of myofiber h ypertrophy The primary histological outcome variable in this study wa s muscle fiber CSA, an estimate of fiber hypertrophy. Muscle hypertrophy ultimately results from a net increase in protein synthesis, in relation to protein degradation. Atrophy and hypertrophy function antagonistically to mediate muscle size, and these ev ents share a number of key regulatory signaling mechanisms. Resistance training can mediate muscle fiber hypertrophy through a network of interrelat ed signal transduction pathways, result ing in elevated protein synthesis or myogenic activation ( briefly sum marized in F igure 2 3 ). Muscle fiber size, MHC isoform expression and regeneration adaptations may each be modified by changes in activity. Growth of mature, terminally differentiated myofibers is driven by an increase in synthesis and reduction in proteol ysis (141) Myogenesis and protein synthesis can facilitate fiber hypertrophy independently (142, 143) yet activity mediated hypertrophy in intact animals likely r esults from coordina tion of these events Protein synthesis is largely mediat ed by the PI3K Akt mTOR signaling pathway, and can be initiated by receptor specific binding of insulin like growth factor I ( IGF 1 ) molecules (144, 145) and activation of P I3K Downstream of IGF 1 and PI3K, Akt molecules influence protein turnover activity and muscle g rowth through a linkage to the F oxo transcription factors. Akt phosphorylation prevents nuclear translocation of phosphorylated F oxo, an action that inhibits expression of proteolytic regulatory genes such as atrogin 1 and Murf1. The Akt Foxo interaction i s a primary regulatory point for maintenance of muscle protein balance Akt concurrently promotes downstream activity


35 of the mammalian target of rapamycin (mTOR) isoforms. In particular mTORC1 is a potent stimu lator of ribosomal translation and a crucial regulator of eukaryotic initiation and elongation factors (144) M echanical strain alone is capable of induc ing hypertrophy through Akt mTOR signaling, independent of a functional IGF 1 receptor (146) The Akt mTOR signaling proteins are integral modulators of protein synthesis but it is not fully understood how these signals dictate the rate of ribosomal translation or specify protein synthesis (147) While hypertrophy depends upon protein synthesis the extent of mature fiber growth can be lim ited by the myonuclear domain when muscle regenerative pathways are inhibited (148 150) Research in limb muscle suggests that the extent of muscle fiber hypertrophy is restricted to a myonuclear domain approximating 2000 m 2 without addition of myonucle i (151, 152) Regeneration can be promoted when mechanical strain deforms key cytoskeletal proteins and induces an inflammatory reaction. Pro regenerative paracrine and autocrine molecules include hepatocyte growth factor, nitric oxide, IL 6, fibroblast gro wth factor, and IGF 1. Systemic and local growth factors and circulating cytokines bind to cell receptors, resulting in mechanical chemical signal transduction (153, 154) P ro regenerative signal transduction promotes activation of satellite cells periphe rally located muscle fiber progenitors that can be anatomically and functionally differentiated from other myonuclei (155) The fate of activated satellite cells, termed myoblasts depends upon the environmental niche as well as the subsequent expression of influential myogenic regulatory transcriptional factors ( MRFs). IGF 1 facilitates anti apoptotic signaling through PI3K Akt, and promotes ERK signaling, resulting in progression of the


36 mitogenic cycle (156) During proliferation, activated satellite ce lls express the Pax7, MyoD and Myf5 MRFs. S ome proliferating myoblasts yield daughter cells responsible that renew the satellite cell pool Other daughter cells undergo withdrawal from the cell cycle and subsequent differentiation. The cyclin dependent ki nase inhibitor, p21 promotes cell cycle arrest and facilitates differentiation. D uring differentiation, Pax7 and MyoD downregulate, accompanied by the upr egulation of Mrf4 and myogenin. In addition anti apoptotic Akt/PI3K/mTOR transcription factors module the rate and extent of differentiation (157, 158) Differentiating myoblasts can fuse to existing myofibers to promote repair and facilitate fiber growth, become myonuclei, or fuse together t o create new, regenerating myotube s (Figure 2 4) (159, 160) A dditional myonuclei are thought to preserve the myonuclear domain to support hypertrophied muscle tissue (152, 161) With terminal differentiation, n ascent and repaired myofibers express developmental MHC isoform s and subsequently mature into adult fibers Together, signal transduction for myogenic activation and protein synthesis synergistically promote muscle growth in response to overload training. Overload training and m yofiber damage In addition to CSA our experiments analy zed diaphragm fiber damage and regeneration in an animal model of tracheal occlusion Respiratory loads can induce widespread diaphragm injury (162) M yofibrillar damage can be influenced by the mode (stretch vs. contraction), intensity and duration of the mechanical stimulus, as w ell as the contractile state of the muscle (shortening vs. lengthening). Diaphragm damage has been foun d with both acute and chronic loads. To study acute muscle damage in the diaphragm, experimental designs typically delivered a single sustained, injuriou s


37 overload in an animal model. Damage responses differ in region s of the diaphragm and between accessory muscles. The costal diaphragm appears more susceptible to acute injury than the crural diaphragm or accessory muscles of respiration (162 165) The ext ent and timing of diaphragm damage responses occurs in proportion to the duration and intensity of the respiratory load. One day after a single, injurious load, s ignificant proportions of injured myofibers and inflammatory cell infiltration become apparent with light microscopy and continue for three days (163, 166, 167) In contrast to severe sustained loads (~70% MIP), moderate loads (~45% MIP) do not induce significant diaphragm injury (163) With continuous load, damage is prolonged. Six days of continuous respiratory loading increased the pooled proportions of abnormal muscle fibers and connective tissue in the costal diaphragm (162) Substantial remodeled and inflamed diaphragm fibers persist after t hirty days of chronic respiratory loa ding (17) After a mechanical or chemical injury muscle fiber remodeling occurs in four related, overlapping phases: degeneration, inflammation, regeneration, and fibrosis (Reviewed in (168) ) Degeneration begins rapidly following mechanical overloads. E xcessive m echanical strain disrupts the myofibrillar scaffolding and sarcolemma proteins, and elevates calcium infl ux, oxidative stress and proteolytic signaling (20, 169 172) P hysiological strength training loads and damaging contractions trigger proport ionate levels of oxidative stress and proteolysis (166, 173) Mo derat e to severe calcium ion influx can impair excitation contraction uncoupling, leading to measureable decrements in evoked tension (174 176) D egeneration can be measured within five minute s of severe eccentric overload s in the limb muscles (177) and may continue for days. T he activation of proteolytic pathways and fiber degeneration corresponds to a


38 rapid inflammatory response M acrophages and lymphocytes migrate to injured tissue, and lev els of local and circulating pro inflammatory cytokines and growth factors increase (167) P ro inflammatory molecules mediate the immune response s of remodeling dictate the outcome of subsequent fiber adaptations. Pro regenerative molecules include macroph ages, interleukin 6, hepatocyte growth factor, and IGF 1. In human limb muscle, rege nerative activity peak s approximately two weeks after introducing mechanical loads (168, 178) Rodent tissue may remodel more rapidly; expression of proliferation genes increases within 12 hours and peaks within three days (179, 180) Next, e xpression of immature myosin heavy chains increases in differentiated myotubes In differentiated myotubes, g ene expression of embryonic myosin increases approximately three to seven days and peak s within seven to ten days of a training load or damage stimulus (172, 180) Regenerative markers may be detected earlier into training, before measureable hypertrophy can be detected (159) Alternatively, apoptotic dominance or elevated myostatin expression can inhibit regeneration and promote fibrosis (181) However, physiological strengthening loads typically inhibit myostatin gene expression, in the absence of muscul ar disease or myotoxic medications (182, 183) Force recovery accompan ies muscle fiber regenerative and remodeling but impaired evoked force may continue in the presence of fibrosis, due to decreased myofibrillar protein content (184) The phases of mus cle fiber remodeling can be characterized by specific histological adaptations Connective tissue and changes in muscle fiber shape or size indicate the remodeling state of the muscle. D egeneration may be characterized by disruption of ultrasctuctural feat ures or cellular incursion of plasma fibronectin, and


39 increased proportions of inflammatory cells within the interstitium characterize early remodeling (166, 167) Elevated ratios of inflamed or necrotic fibers occur during inflamm ation and can persist dur ing regeneration and fibrosis (166, 184) Proportions of centrally nucleated fibers and small fibers increase during myofiber regeneration (185) In contrast incomplete or inhibited regeneration can be visualized by the replacement of myofibrillar tissue with excessive connective tissue and elevated presence of fibroblasts (168, 186) While histo log ical assessment s of muscle remodeling cannot i d entify the unde rlying cause s of remodeling or whether they are beneficial or maladaptive, they provide valuable insight regarding the timing and degree of fiber remodeling. Summary and Significance This work is significant because the rate of ventilatory failure continues to increase, despite advance ments in critical care medicine (187) Patients who require prolonged MV experience significant disability and risk additional m edical co morbidities and death (3, 188, 189) Despite improved critical care practices, i ncidence of weaning failure has increased at a rate five times faster than the incidence of hospital admissions and c hronic ventilator dependence is projected to grow ~250% by 2020 (187) VIDD remains a substantial problem and reflects a need to explore o ptimal respiratory muscle rehabilitation practices The respiratory loading regime in this experimental design resembles brief airway occlusions utilized for clinical respiratory testing (190) and could also be adapted for train ing patients who cannot vol untarily participate in respiratory exercises. The experiment s contribute novel information regarding the timing and extent of inspiratory muscle training responses. R esults of this study may bear particula r significance to the rehabilitation of patient s w ith respiratory failure and diaphragm atrophy


40 Figure 2 1. Regional architecture of the rodent diaphragm. Figure printed with permission from Poole et al, MSSE, 1997; 29(6): 740.


41 Figure 2 2. Summary of mechanical ventilation effects on atrophy and contractile dysfunction in the diaphragm.


42 Figure 2.3. Protein synthesis and myogenic regeneration each contribute to training induced muscle hypertrophy. A number of molecules fr om the PI3K Akt family participate in cross talk to mediate atrophy and regeneration processes and thereby manage protein homeostasis.


43 Figure 2 4. Myogenic regeneration after skeletal muscle overload. Quiescent satelli te cells are activated by mechanical loading or injury, and myoblasts begin to proliferate. Some replicating myoblasts will not differentiate and will replenish the satellite cell pool. Other myoblasts differentiate and fuse to existing myofibers to repair damage or add myonuclei. Yet others fuse together, yielding new, immature myofibers.


44 CHAPTER 3 MATERIALS AND METHOD S Research Design and Data Analysis The prima ry objective of this investigation was to identify selected histological adaptation s to overload training in respiratory muscle using an animal model of tracheal occlusion. Phase one of the investigation examined fiber hypertrophy and myosin heavy chain ( MHC ) phenotype adaptations of the respiratory pump ( Experiment 1 ). Next the muscles were compared for histological differences in damage and regeneration ( Experiment 2 ). Justification for an Animal M odel Invasive procedures such a s surgery are needed to obtain human diaphragm specimens. Due to medically tenuous status of mechanical ventilation ( MV ) dependent patients, diaphragm biopsies would involve excessive risk when an alternative animal model of training can be applied Animal model s of MV have advanced the scientific understanding of ventilator induced diaphragm dysfunction ( VIDD ) (10, 64, 66, 67, 76 79, 170, 191) and the timeframe for atrophy appears to be translatable to ventilated humans (12) The S prague Dawley rat was c hosen due to physiological similarities to humans in training and disuse atrophy muscle remodeling (192) Sixteen juvenile male rats (12 16 weeks) were selected for the study. Animals were housed at the University of Florida Animal Care Services Center, in accordance with the criteria established by the Institutional Animal Care and Use Committee (IACUC). Tracheal occlusion experiments were approved by the University of Florida IACUC and met the stipulations of the Helsinki Declaration A 12:12 hour


45 reverse light: dark cycle and ad libitum diet of animal chow and water were provided to the animals throughout the experimental period. Experimental Design The study design is outlined in Figure 3 1 Animals were randomly assigned to one of two groups, and then underwent surgic al placement of a tracheal cuff Cuff placement was followed by a post surgical recovery period Upon recovery, the animals began ten sessions of the assigned experimental intervention. Every day, e ach animal in the sham treated group (SHAM n=8 ) was placed in a plethysmo graph and the tracheal cuff was connected to a pressure line. The cuff was never occluded for SHAM trained animals. Animals assigned to t he occlusion group (OCCL, n=8 ) were also placed in a plethys m ograph daily, and the tra cheal cuff was connected to a pressure line. OCC L animals received intermittent tracheal occlusions for a 10 minute daily s ession After a two week intervention, animals were euthanized The muscle tissue was preserved, a nd then analyzed by a masked i nvest igator for hypertrophy, damage, and regeneration. Rationale The costal diaphragm, third parasternal intercostal and soleus muscles were selected for analysis. We focused upon the medial costal diaphragm because its large appositional area results in a lar ge degree of active shortening during inspiration and therefore may be amenable to remodeling. In addition, the majority of the published injury, training and atrophy studies published findings from medial diaphragm segment s, and this data can be compare d to our training regime. W e also contrast ed the data from the medial costal diaphragm to ventral and dorsal regions to determine whether regional training differences existed. The third intercostal muscle was analyzed because the


46 parasternal region has a st rong inspiratory function that is activated early in a tidal breath (55) Although the fiber composition of the soleus is predominantly slow and oxidative, this muscle was selected because its duty cycle is among the greatest in the limb muscles of small q uadrupeds (58, 193) Power Analysis A power analysis was conducted to determine the sample size for the projected hypertrophy effect. Because the experimental model was novel, we applied existing atory training in Wistar rats (139) In this work, rats underwent 60 minutes of alinear resistive breathing through a snout mask, three times weekly for eight weeks. Two important components of the resistive training model differed from the experimental de sign employed by the current study: training occurred for eight weeks rather than two, and the protocol consisted of low level training (average tension time index: .02) rather than brief, intense occlusion loads. The power analysis for this work indicated that a group size of 5 animals was sufficient to yield statistical significance for hypertrophy of fast, glycolytic fibers, and 3 animals for significant differences in oxidative fiber hypertrophy (139) Despite the differences in the experimental models, we anticipated that the effect elicits high ventilatory drive yielding strong to maximal respiratory efforts (194) In addition, the rodent facemasks resulted in airwa y leaks, while occlusions were inescapable and confirmed by cuff pressures and plethysmography in the curr ent study. Therefore we hypothesiz ed that tracheal occlusion should provide a greater strength and hypertrophy stimulus, despite the shorter training duration. In a sample of four


47 determined groups of 13 animals were needed. However, the analysis pooled the values of type I, IIa, and IIx/b fibers, resulting in lar ge cross sectional area ( CSA ) variability D = 1.58 confirmed that a sample size of six animals per gr oup was sufficiently powered ( 1 ) Methods Surgical Procedures For the placement of the tracheal cuff, the animals were anesthetized using isofluorane gas ( 2 5% in O 2 ). During the procedure, the body temperature was maintained at 37 C with a heating blanket Anesthetic plane of sedation was confirmed by an absent wi thdrawal reflex to a noxious paw pinch. Animals breathed room air without MV support. With a ventral incision, the trachea was exposed A n inflatable vascular occluder was sutured around the trachea, and the actuating line was routed and externaliz ed dorsa lly, between the scapulae (Figure 5 2A) The e xternaliz ed line could be connected to an air filled syringe, in order to inflate or deflate the cuff bladder. Inflation of the cuff elicited total tracheal occlusion while deflation restored airway patency an d permitted unobstructed breathing The external pressure line was securely stitched in place, and the tracheal incision sutured. Animals received doses of buprenorphine (.01 .05 mg/kg body weight ( BW ) ) and carprofen (5mg/kg BW) for pain control and rehydr ated with .01 .02 mL/g BW of normal saline, prior to withdrawal of anesthesia. During a 4 day recovery period and for the subsequent experimental duration, animals were provided with routine pain medication (buprenorphine .01 .05 mg/kg BW every 12 24 hours and carprofen 5mg/kg BW every 24 hours) and were


48 maintained in standard housing in the University animal care facility. Animals were closely monitored for signs of respiratory distress, infection, or pain. Occlusion Protocol After the surgical recovery p eriod, animals underwent their assigned training intervention for ten consecutive days All sessions occurred in the morning, and lasted 15 minutes. Prior to the first session all animals were acclimatized to the study plethysmograph for 15 minute s (Figur e 5 2B) During this session, animals were observed and no experimental interventions occurred. For the experimental sessions the SHAM grou p returned to the study plethys m o graph for 15 minutes daily for observation. The SHAM animals received no interventi ons during monitored plethysmo graph sessions. The OCCL group received intermittent, total tracheal occlusions during experimental sessions. The cuff pressure to produce reliable, complete tracheal occlusion was determined from previous work The tracheal cuff was inflated for approximately 5 1 0 seconds in order to elicit 5 8 strong respiratory attempts, and then deflated for approximately 30 second s. The occlusion cycle was repeated for a total of 10 minutes. Oscilloscope tracings of the c uf f pressure and the plethysmograph were monitored during each session to confirm the onset and removal of occlusions. Tissue Analysis One day after the last session, animals were anesthetized with isofluorane gas ( 2 5% in O 2 ) Once animals reached a surgic al plane of anesthesia, evidenced by lack of corneal reflex or absent limb withdrawal to a paw pinch they were euthanized by decapitation. The diaphragm, third intercostal and soleus muscles were isolated and extracted. The left side of each muscle was fl ash frozen in liquid nitrogen for molecular


49 analysis. The right side d muscles equilibrated at 4 C for 3 5 minutes at resting length (195) and then were frozen in isopentane cooled in liquid nitrogen. Specimens were stored in a 70 C freezer until histol ogical analysis. Fiber p henotype and CSA To determine the phenotype properties of the muscle tissue, 10 m transverse serial sections were acquired using a cryostat microtome (Microm HM505, Walldorf, Germany). In order to obtain minimal fiber diameters the orientation of the sample was adjusted in 5 degree increments, as needed Cross sections were confirmed with a low power dissecting microscope (Omano OM2344, Wirtz, VA). The sections were air dried at 25 C for 30 minutes. Slides were rinsed in 1X phos phate buffered saline (PBS) solution and then permeabilized with 0.5% Triton X100 in 1X PBS. The samples were incubated with primary antibodies for laminin 1:200 (anti rabbit, IgG, Lab Vision ), type I myosin heavy chain 1:15 (anti mouse A4.840, IgM, Developmental Studies), and type IIa myosin heavy chain 1:50 (anti mouse SC 71, IgG, Developmental Studies ) for 60 minutes and then rinsed three times for five minutes in 1X PBS. Sections were incubated with secondary antibodies f or rhodamine 1:40 (goat anti rabbit, Invitrogen ), Alexa Fluor 350 1:333 (goat anti mouse, IgM, Invitrogen ) and Alexa Fluor 488 1:133 (goat anti mouse, IgG, Invitrogen ) Rockford, IL) and 1X PBS, for 60 minu tes in a darkened, humidified tray. Three final rinses for 5 minutes in 1X PBS were then performed. Cover slips were mounted with Vectashield fluorescent mounting medium (Vector Labs, Burlingame, CA) and secured with nitrocellulose lacquer. The A4.840 and SC 71 antibodies generat ed by Helen M. Blau were obtained from the Developmental Studies Hybridoma Bank developed under


50 the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. Samples were visualized using fluorescence microscopy at 10 0 x magnification and N21 GFP and A4 cube filters. Type I fibers il luminated blue under the A4 filter type IIa filters fl uoresced green using a GFP filter and type IIb/x fibers remained free of fluorescence. Cell bord ers were visualized with red fluorescence under the N21 filter. At least five randomly selected images were captured under each filter (Leica DM LB Solms Germany ). The images were encoded and black and white threshold images calculated for fiber CSA using Scion Image (NIH) software. The CSA of each mus cle sample was calculated from at least 300 fibers per muscle specimen. A masked investigator quantified fiber CSA and phenotype proportion s. MHC isoform proportions were determined using the encoded images and recorded in a spreadsheet The area fraction ( A A ) for each fiber phenotype was also calculated, to account for the proportion of the CSA consumed by a given MHC isoform The phenotype A A accounted for the number of fibers with a gi ven phenotype as well as the CSA of the fibers Phenotype A A = ( CSA of phenotype)/(total CSA of all phenotypes) 100% Muscle fiber remodeling To analyze the extent of inspiratory muscle fiber remodeling associated with each group 10 m transverse serial sections we re obtained from a cryostat cooled to 20 C ( Microm HM505, Walldorf, Germany ). Sections were stained with hematoxylin and eosin, and then the specimens were mounted with Permount medium (Fisher Scientific), cover slips applied and secured with a nitrocell ulose lacquer. S pecimens were visualized with brightfield microscopy (Leica DM LB Solms Germany) at 40 0 x


51 magnification. For each animal, t wenty images were acquired in all of the study muscles Dig ital images of the tissue were encoded, and then analyzed for qua litative evidence of remodeling. An analysis of m orphological remodeling was undertaken using a systematic point counting technique (162, 196) ( Table 3 1 ) Point counting was conducted using Adobe Photoshop CS3 software (Ad obe Corporation, San Jose CA). Next, a 9x7 point grid was generated by Photoshop and superimposed onto each digital image. Partial fibers within the images were excluded from analysis. At each point intercept of the grid, underlying tissue at the top right quadrant was classified i nto one of nine morphological categories and recorded onto a spreadsheet. A n (A A ) was calculat ed for normal and remodeled muscle fibers, inflammatory cells, and connective tissue. Area fraction (A A ) = ( counts in category)/(total count) 100% A ma sked investigator completed the morphological analysis. The investigator was trained by an externally trained scientist (Sunita Mathur, PhD, PT), and then inter rater reliability of fiber classification was examined using the standardized classification ca tegories and definitions, over identical images. The correlation between the investigator and Dr. Mathur was 0.95. Myofiber regeneration embryonic myosin Slides were air dried at 25 degrees Celsius for 30 minutes. Slides were quick rinsed in 1X phosphate buffered saline (PBS), fixed in 1:1 acetone methanol solution for five minutes at room temperature, then rinsed three times for five minutes in 1X PBS. Rockford, IL) for 60 minutes at 25 C and then rinsed three times for five minutes in 1X PBS. Sections were incubated in primary antibodies for laminin 1:200 (anti rabbit, IgG,


52 LabVision) and embryonic myosin heavy chain (eMHC) 1:20 (anti mouse F1.652, IgG, Developmental Stud ies) in 10% normal goat serum, and 1X PBS overnight at 4 C The F1.652 antibody developed by Helen M. Blau was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Depar tment of Biology, Iowa City, IA 52242. The next day, each section was rinsed five times using 30 mL of 1X PBS syringe flushed onto the sample. Secondary antibody incubation occurred with rhodamine 1:500 (goat anti rabbit, Invitrogen) and Alexa Fluor 488 1: 300 (goat anti mouse, Invitrogen) in 10% normal goat serum and 1X PBS, for two hours at 25 C in the dark. Sections were flush rinsed five times with a 30 mL syringe of 1X PBS in the dark. Specimens were mounted in Vectashield fluorescent mounting medium w ith DAPI (Vector Labs, Burlingame, CA) and cover slips secured with nitrocellulose lacquer. Samples were visualized us ing fluorescence microscopy at 2 0 0 x magnification and using N21, GFP and A4 fil ter cubes. At least five randomly selected images were ca ptured under each filter (Leica DM LB Solms Germany). The images were encoded and the proportion of eMHC positive fibers was calculated from at least 15 0 fibers per muscle specimen. Positive fibers were interpreted to be intensely fluorescing areas within the exact borders of the sarcolemma. Faintly fluoresced fibers or areas that fluoresced only a portion of a cell were not counted. For each animal, proportions of eMHC positive cells were calculated automatically using a spreadsheet Protein i mmunoblottin g We confirmed presence of eMHC in the animals, using a subset of eight animals. The following methods were used to extract protein. Frozen intercostal muscle was minced (~30 mg), placed in a radio immunoprecipitation assay (RIPA) buffer and


53 protease inhib itor (1:1000) cocktail, and then triturated using a DAKO mortar and pestle. Then, samples were vortexed and centrifuged at 16,000 G for 20 minutes at 4 C The supernatant was carefully removed and the remaining pellet discarded. We calculated the concentration of protein in each muscle supernatant against a known standard, using BioRad DC protein assay reagents (BioRad, Hercules, CA). Samples were tested i n triplicate and concentration read with a Bio Rad Microplate Manager plate reader, at a wavelength of 580 nm. Aliquots of the isolated protein were diluted to equal concentration, combined with LDS buffer and reducing agent, and then incubated at 70 degr ees Celsius for 10 minutes. One dimensional SDS PAGE was conducted with the Nu Page mini gel system and bis tris 4 12% mini gel (Invitrogen, Carlsbas, CA), for 40 minutes at 200V. Gels were transferred to a nitrocellulose membrane (35V for one hour), and t he membrane s were stained with Ponceau S to identify molecular mass markers. Membranes were blocked with 5% milk in TBS T for one hour, then incubated with primary antibody in a 4 C room overnight (F1.652, 1:50 in milk blocking buffer). After six rinses i n TBS T, a peroxidase conjugated secondary antibody was added (goat anti mouse, IgG, 1:2000 dilution, Rockland laboratories, Gilbertsville, PA) and incubated for one hour at room temperature. Six final 5 minute TBS T rinses were completed. We applied enhan ced chemiluminescence (ECL) reagents (luminal substrate and hydrogen peroxide) from Bio Rad, and then exposed the protein bands with x ray film. The intensity of the bands was quantified using Image J software (NIH). Statistical Analysis The animal demographic characteristics were assessed using an independent t test. All data were examined to determine whether they met assumptions for normality


54 and homogeneity of variance. To account for the vastly different baseline fiber compositions be tween the diaphragm, intercostal, and soleus muscles, immumohistochemical tests were analyzed with separate, repeated measures analysis of variance ( ANOVA ) with one within group factor (MHC isoform: type I, IIa, and IIx/b fibers), and one between group fac tor (training: OCCL or SHAM) Diaphragm regional analyses were compared using a 3 way ANOVA, with two within group factors (region: ventral, medial, dorsal diaphragm and MHC isoform: type I, IIa, and IIx/b fibers), and one between group factor (training: O CCL or SHAM). A 3 way mixed ANOVA (one within subjects factor: remodeling category, two between subjects factors: training group, muscle) compared the training effect on A A of muscle fiber remodeling. When needed, interactions were assessed using pair wise comparisons with a Bonferroni correction. Statistical testing for embryonic myosin was completed using the Mann Whitney U test. For all analyses, the level of significance was established at p<0.05.


55 Table 3 1. Categories for classification of myofib er features determined by point counting. # Category Definition 0 no count space, artifact, epimysial connective tissue, nerve, large blood vessels. no count 1 normal muscle, capillary polygonal fiber with acidophilic cytoplasm, plasma membrane and peripheral nuclei or capillary (small blood vessel with endothelium only). normal 2 internal nuclei fiber with > 1 internally located nuclei (sarcoplasm between nucleus and sarcolemma), 8 pixels of sarcoplasm between nucleus and sarcolemma. re modeled 3 small angulated fiber (a) small fiber ( < 1/3 the lesser fiber diameter of the degrees or (c) fiber with > 2 acute angles (< 90 o ) re modeled 4 Inflamed, necrotic fiber fiber with > 1 inflammatory cell or necrotic mass of inflammatory cells and muscle debris without plasma membrane. re modeled 5 abnormal cytoplasm, lipofuscin includes: (a) fiber with pale acidophilic peripheral cytoplasm and enlarged peripheral nuclei with or without visible nucleoli, or (b) fiber with pale acidophilic peripheral cytoplasm and deep acidophilic tral region, or (c) split or who rled fibers, or (d) vacuoles or (e) uneven cytoplasm stai ning unrelated to processing or (f) fiber with dull or light gray staining, or (g) cytoplasmic fragmentation or (h) lipofuscin (brown yellow pigmentation > area of a muscle nucleus). re modeled 6 inflammatory cell cell in the interstitium that has a roun d shaped nucleus consistent with a monocyte, macrophage, or lymphocyte. re modeled 7 collagen or fibroblast protein fibrils of endomysial or perimysial connective tissue or a cell located in the interstitium with spindle shaped nucleus that is consistent with a fibroblast. collagen 8 adipocyte empty space surrounded by cell membrane consistent with size and shape of adipocyte. adipose


56 Figure 3 1. Schematic representation of the experimental design.


57 A B Figure 3 2. Experimental apparatus. A. Illustration of the tracheal occluder. B. Experimental sessions were conducted in a plethysmograph, and oscilloscope tracings recorded pressure and volume fluctuations during occlusion. The arrow distinguishes the pre ssure line to the tracheal cuff.


58 CHAPTER 4 RESULTS Of the sixt een animals that underwent occluder placements complete datasets were obtained from twelve Two animals did not complete the experimental sessions due to clinical signs of infection in a sham trained animal (n=1, SHAM) or occluder in an occlusion trained animal failure (n=1, OCCL), and diaphragm tissue was unavailable in two others (one animal from each group). Therefore, analyses were conducted with n=12 diaphragms, and n=14 intercostal and s oleus muscles. Animals in the SHAM group weighed 252 (48) grams at the study onset and 299 (35) grams upon conclusion. Animals in the OCCL group weighed 253 (45) grams at the start of the study and 303 (16) grams on completion The a nimals were 14 week old littermates and t here were no group differences in the weight or complication rate. AIM 1 Respiratory Muscle Fiber Hypertrophy Effect of Tracheal Occlusion on Fiber Cross Sectional Area Cross sectional area of the diaphragm Tables 4 1 and 4 2 repo rt the cross sectional area ( CSA ) of the medial costal diaphragm. Two way, repeated measures analysis of variance ( ANOVA ) was used to examine the effect of training on CSA in the medial costal diaphragm. The data did not meet the assumption of sphericity, p=.005) and data were analyzed using the Greenfield Geisser and more conservative Lower Bound sphericity corrections. Using the more conservative Lower Bound correction, the interaction between fiber type and training group was signif icant for CSA ( F( 2, 20) = 9.066, p<.01 ) There were no significant main effects for training group. Post hoc tests indicated that


59 the CSA of type IIx/b fibers was significantly larger in the OCCL animals than in the SHAM group ( p<.05 ) The significant interaction is depicted in Figure 4 1. Cross sectional area of the third intercostal The CSA data for the intercostals are available on Tables 4 1 and 4 3 In the IC muscle, the sphericity assumption was met for repeated measures ANOVA. There was a significant interaction between traini ng group and fiber type (F(2, 24) = 6.310 p<.01). Post hoc pair wise contrast indicated that for the type IIx/b fibers, the CSA was significantly larger in th e OCCL group (Figure 4 2) In the OCCL group, t he CSA of type I fibers was 11.7% greater, type IIa was 17.9% larger, and type IIx/b was 18.6 % greater than the CSA in the SHAM group. CSA measurements in type I and type IIa fibers did not differ between the training groups. Type IIa fibers were larger th an type I (p<.05), and type IIx/b fibers were significantly larger than both type IIa and type I fibers, (p<.001). There was no group main effect. Cross sectional area of the soleus Tables 4 1 and 4 4 report the results of the repeated measures ANOVA for C SA in the soleus muscle. While the interaction between fiber type and training gr oup was not significant (F(1, 12) = .405 p>.05), there was a significant main effect for fiber type on CSA (F(1, 12) = 27 176 p<.001). The CSA of type I fibers was larger th an type IIa fibers. Group assignme nt did not influence CSA (F(1,12 ) = .002 p>.05). Regional diaphragm cross sectional area The three way ANOVA with repeated measures was employed to examine regional CSA in the animals, and results are reported in Tabl e 4 5 A Greenhouse Geiser corrections was utilized to correct violations of the sphericity assumption A region phenotype inter action occurred for CSA (F(1.922 19.225 ) = 4.209 p<.05). Post


60 hoc tests revealed CSA was larger in the type IIx/b fibers of the me dial diaphragm ( F (2,33) = 3.422, p<.05 ). Overall, there was a significant main effect of MHC isoform on CSA. Fibers identified as type 2x/b were significantly larger than type 1 and type 2a (p<.001), regardless of group assignment. The main effect for gro up was not significant. Effect of Tracheal Occlusion on Fiber Phenotype Fiber phenotype of the medial diaphragm Representative immunohistochemistry images of the diaphragm (Figure 4 3 A and B) illustrate the MHC isoform distributions in the OCCL and SHAM groups. The mixed phenotype composition of the rat medial diaphragm was consistent with other reports (29) The sphericity assumption was met for the repeated measures ANOVA procedure. The interaction between group assign ment and fiber phenotype composition was not significant in the medial costal diaphragm ( F( 2, 20 ) = 1.428, p>.05 ). In addition, t he MHC isoform proportions did not differ between the training groups The proportion of type IIx/b fibers in the diaphragm was lower than proportions of type I (p<.005) or IIa (p<.05) fibers. Tables 4 2 and 4 6 detail the medial diaphragm MHC isoform proportions. The phenotype A A represents the sampling area occupied by a given myosin heavy chain ( MHC ) isoform. Phenotype A A depe nds upon the number of cells expressing an isoform as well as the fiber CSA. Tables 4 2 and 4 7 provide the A A of MHC isoforms in the medial costal diaphragm. The diaphragm phenotype A A data did 025), and within subjects effects we re contrasted using a Greenhouse Geisser correction. There was no significant interaction between fiber type and training gr oup for phenotype area fraction ( F ( 1.282 ) = 2.907, p>.05 ) although a trend was identified, p=.1 0. Although the main


61 effect for training was not significant, a strong main effect for fiber type identified that type IIx/b fibers occupied a greater A A in the diaphragms of all animals, compared to type I or IIa fibers ( p<.001 ). Fiber phenotype of the th ird intercostal The third intercostal contains a majority of fast intermediate or fast glycolytic fibers, with the balance of the fibers equally divided between slow and fast oxidative fibers ( Figure 4 3, images C D) In the intercostals a Greenhouse Geiser sphericity The interaction between MHC isoform and training g roup did not reach significance ( F( 1.397 16.769 ) = 1.470 p>.05 ) There was a significant main effect for fiber type on proportion, indicating that type IIx/b fibe rs were more prevalent in the intercostal muscle samples than either type IIa or type I fibers ( F( 1.397, 16.769 ) = 246.681, p<.001). The proportions of type IIa and type I fibers did not differ from one another. T he main effect for training group was not significant. Intercostal phenotype proportions are listed on Tables 4 3 and 4 6. For fiber phenotype A A there was no significant interaction between training group and A A of the respective MHC isoforms (F( 2, 24 ) = 1.952, p>.05) and the training groups did not differ. Due to the larger CSA and high prevalence of type IIx/b fibers in the third intercostal muscle, the fiber type main effect for was strongly significant for A A Type IIx/b fi bers occupied a greater A A than type I and IIa fibers (F( 2,24 ) = 1 954.258 p<.001). Summaries of A A in the intercostal tissue are available in Tables 4 3 and 4 7 Fiber phenotype of the soleus Immunohistochemistry illustrations of the soleus can be found on Figure 4 3 ( images E F ). As a postural hindlimb muscle with a relatively high duty cycle, the soleus was included as an activity control muscle. In the study sample, t he soleus contain ed an


62 extremely high p roportion of slow, oxidative fibers, consistent with the p ostural function of the muscle. The interaction between MHC isoform expression and training group was not significant (F( 1,12 ) = 0 335 p>.05). However, there was a significantly higher proportion of type I slow fibers than type IIa fibers in soleus specim ens ( F( 1,12 ) = 969.979 p<.00 1 ). No type IIx/b fibers were identified in any of the muscle specimens. The soleus phenotype proportions did not differ between the OCCL and SHAM groups ( F( 1,12 ) = 1.0, p>0.05). Fiber phenotype proportions in the soleus can be found on Table 4 4 and 4 6 The phenotype A A for MHC expression is summarized in Tables 4 4 and 4 7. In the SOL, no significant interaction between training group and fiber phenotype occurred (F( 1, 12 ) = 0.557, p>.05) and there were no A A differences bet ween the training groups. The A A occupied by type I fibers was significantly larger than the A A of type IIa fibers ( F( 1, 12 ) = 1958.441 p<.001 ) Regional costal diaphragm expression of f iber p henotype Table 4 8 depicts regional phenotype proportions in the costal diaphragm. The assumption of sphericity was met for every factor. There was an interaction between the training group, region of the diaphragm, and MHC isoform on phenotype proportion ( F( 4, 40 ) = 3. 535 p<.05 ) Analysis of the interaction revealed that the proportion of type IIx/b fibers in the ventral diaphragm was significantly reduced in the SHAM training group ( F( 2 2 0 ) = 5 .33 3 p <0.05 ). In all regions of the costal diaphragm, there were fewer type II x/b fibe rs, compared to type IIa or type I fibers ( F( 2,20 ) = 1 3.668, p<.001 ) A mixed MHC isoform composition was consistent throughout the costal diaphragm. Table 4 9 illustrates the A A of regional diaphragm MHC isoforms in the OCCL and SHAM trained anim als. The sphericity assumption for repeated measures was met. A


63 significant 3 way interaction was detected between the diaphragm region, fiber phenotype and training group for area fraction ( A A ), (F( 4 40 ) = 4.683, p<.01 ). Post hoc analyses revealed that the A A of type IIx/b fibers was lower in the ventral diaphragm only in the SHAM trained animals, compared to the medial region ( F( 4,20 ) = 4. 173 p<.05 ) Although a greater proportion of fibers in the diaphragm expressed oxidative MHC isoforms, the CSA of t ype IIx/b fibers was larger than the oxidative fibers. As a result, the A A of type IIx/b fibers was greater than that of type IIa or type I fibers, regardless of the region (F( 2,20 ) = 5 3. 335, p<.001 ). In summary, the results showed type IIx/b fiber hypert rophy of the medial costal diaphragm and intercostal muscles, without training interactions in the soleus. Regional analysis indicated a CSA difference between the medial costal diaphragm and other regions. Additionally, we identified an unanticipated redu ction in the expression of type IIx/b fibers of the SHAM trained ventral costal diaphragm. AIM 2: Respiratory Muscle Damage and Regeneration Effect of Tracheal Occlusion on Respiratory Muscle Morphology The categorical assessment of normal and remodeled mu scle in the medial diaphragm, intercostal, and soleus is detailed in Table 4 10, and representative hematoxylin and eosin ( H&E ) images of each muscle can be found at Figure 4 4 In every muscle group, the majority of fibers were classified as normal. Norma l fibers were characterized by polygonal shaped cells with multiple peripherally located myonuclei, tightly arranged fascicles, and limited quantities of endomysial connective tissue or inflammatory cells. Connective tissue consisted of endomysium and peri mysium tissue that stained pale pink, or fibroblasts contained within collagen.


64 The re were no significant group differences in the A A of normal and remodeled muscle tissue and connective tissue. The diaphragms of all animals contained a lower A A of normal tissue and greater A A of connective tissue, compared to the intercostals and soleus muscles (p<.001). A significantly smaller A A of abnormal fibers was identified in the soleus (p<.001). Many diaphragm images contained one or more abnormal features, sugg estive of remodeling tissue. Tissue remodeling consisted of connective tissue, internally nucleated fibers, small or angular fibers, inflamed fibers, or inflammatory cells. Inflammatory cells were identified as round basophilic cells with dark round or mul ti lobar nuclei ( Figure 4 5, black arrows). Inflammatory cells could be found in the interstitium and were occasionally identified in association with infiltration of connective tissue ( Figure 4 5, white arrows) or an inflamed or necrotic myofiber. Inflamed cells were distinguished by the presence of one or more inflammatory cells, a disrupted plasma membrane and muscle fiber fragments ( Figure 4 5 yellow arrows ). Internally nucleated fibers contained one or more myonuclei located at least eight pixe ls inside the plasma membrane ( Figure 4 5 green arrows). Small or angulated fibers were characterized by spindle shaped or spear like projections or a diameter less than 1/3 the diameter of the five largest fibers in a field ( Figure 4 5, blue arrows). For each muscle tested, the A A for internally nucleated fibers and inflammatory cells exceeded those of small, angular fibers and inflamed or necrotic fibers (p<.01, all contrasts). The most common diaphragm remodeling attribute was inflammatory cell infiltra tion (A A = 1.40.2 %), while intern ally nucleated fibers occurred most frequently in


65 the intercostal muscles (A A = 1.50.4 %). The diaphragms contained larger proportions of inflamed or necrotic tissue than the intercostal or soleus muscles. Morphological a ssessment of the medial costal diaphragm The majority of diaphragm tissue consisted of normal muscle fibers. There was significantly less connective tissue than normal muscle (.105 .013 versus .866 .012, p<.001), and the A A of remodeled tissue was sign ificantly smaller than the A A of connective tissue (.029 .005 versus .105 .013, p<.005). Of the remodeling attributes in the diaphragm, inflammatory cells occupied the largest A A (.014 .002), followed closely by internally nucleated fibers (.012 .0 02). Small, angular fibers and inflamed necrotic fibers were identified relatively less often (p<.05). Group assignment did not significantly affect diaphragm A A composition. Table s 4 10 and 4 11 detail the A A of remodeled cells in the medial diaphragm. M orphological assessment of the third parasternal intercostal The third parasternal intercostal muscle is an accessory inspiratory muscle that is recruited during quiet breathing. Morphology classification indicated that the majority of the intercostal musc le was comprised of normal muscle fibers (91.6 0.8%, pooled, F(2, 20) = 209.719 p<.001) Normal muscle occupied a significantly larger A A than connective tissue or remodeled fibers ( p<.001), while the A A of connective tissue exceeded the A A of remodeled muscle fibers (p<.001). Only minute quantities of remodeled tissue were identified. Internally nucleated cells occupied the largest A A of remodeled fibers (pooled proportion: .01 .001). By contrast, inflamed necrotic fibers occupied a signi ficantly lower proportion of the examined tissue (p<.001). Tables 4 10 and 4 11 list the remodeling features of the intercostal.


66 Morphological assessment of the soleus In the soleus muscle, the A A majority consisted of normal muscle fibers. A significant main effect for category revealed that the A A for normal muscle exceeded that of connective tissue, and the A A of connective tissue was larger than the A A of abnormal tissue (.916 .006 versus .071 .005 versus .013 .002, p<.001). There were no signifi cant group differences. A closer examination of soleus tissue adaptation illustrated that the most common remodeling feature was inflammatory cells (.01 .001). Inflammatory cells occupied a significantly greater A A in the soleus than inflamed fibers, the most infrequent remodeling category (p<.001). Table s 4 10 and 4 11 report the remodeling attributes of the soleus. Morphological a ssessment of the regional diaphragm Table 4 12 summarizes the A A of normal, remodeled, and connective tissue in the costa p=.009), and a Greenhouse Geiser correction was employed (corrected = .591). The interactions between group assignment, tissue classification, and diaphragm region were not significant. There w as a significant main effect of fiber classification for tissue A A ( F(1.182, 17.546) = 1619.122, p<.001 ): normal tissue exceeded the prevalence of connective tissue or remodeled cells. Group assignment had no effect on remodeling. In each region of the costal diaphragm, the majority of cells were classified as normal muscle fibers. Most diaphragm images contained at least one abnormal feature. Table 4 13 summ arizes the A A of each remodeling characteristic in the costal diaphragm regions. The most common remodeling category in any region was presence of inflammatory cells. Inflammatory cells occupied a significantly greater A A compared


67 to other remodeling categories (p<.005). However, no remodeling characteristic occupied an A A that exceeded 3%. Effect of Tracheal Occlusion on Muscle Fiber Regeneration Embryonic myosin expression in the diaphragm The Kolmogorov Smirnov test (D) was used with a Lillefors significance correction, in order to determine whether the data were normally distributed. The embryonic myosin ( eMHC ) proportions for the SHAM trained diaphragm (D ( 6 ) = .299, ns) did not significa ntly deviate from normality. On the other hand, the distribution in the OCCL group was significantly non normal ( D ( 6 ) = .326 p <.05). More over, Shapiro Wilk tests confirm ed the sample distributions were significantly non normal. Therefore, non parametri c data analyses were conducted, and the results reported as median/inter quartile range. A representative image of eMHC positive fibers in the diaphragm is depicted in Figure 4 6, image A. The Mann Whitney test indicated that proportions of eMHC positive fibers were consistent with control levels reported in the literature (197) and did not significantly differ between the groups (Z=.321, p>.05 ). Table 4 1 4 and Figure 4 7 list the proportions of eMHC positive fibers in the medial diaphragm. Embryonic myos in expression in the third intercostal Embryonic myosin expression in the third parasternal intercostal is depicted in Figure 4 6B and summarized in Table 4 14 and Figure 4 7. The CSA of eMHC positive fibers in the intercostal muscles was small (465 78 m 2 ). According to the Kolmogorov Smirnov and Shapiro Wilk tests, the assumption of normality was not met in the dataset. In the 3 rd intercostal muscle, a Mann Whitney U test indicated that eMHC positive fibers were more prevalent in the OCCL animals ( Z = 2. 128, p<0.05)


68 We examined the presence of eMHC protein in the intercostal tissue with Western Blot (Figure 4 8). Analysis revealed small, distinct bands at 200kDa that coincided with the limited expression of eMHC seen on immunohistological exam. The sign al intensity tended to be larger in the OCCL animals, but this difference was not significant (p=.23). Embr yonic myosin expression in the s oleus Figure 4 6C depicts an example of an eMHC positive fiber located in the soleus. Significant Kolmogorov Smirnov and Shapiro Wilk tests indicated that the dataset was non normally distributed. Mann Whitney U test showed no differences in the proportion of eMHC posit ive fibers in the soleus (z= 57 1 p>.05). Soleus expression of embryonic myosin approximated zero in this sample (Table 4 14 and Figure 4 7).


69 Table 4 1 Histological cross sectional area assessment of the medial costal diaphragm, third parasternal intercostals, and soleus in sham occluded animals and animals treated with intermittent tr acheal occlusion. T he type IIx/b fibers in the medial costal diaphragm and intercostals were larger in occluded animals. Type I f ibers Type IIa f ibers Type IIx/b f ibers Medial d iaphragm SHAM 1358 45 1456 83 3278 233 OCCL 1480 97 1599 119 4141 159* 3 rd parasternal i ntercostal # SHAM 1350 136 1513 127 3738 183 OCCL 1464 79 1757 122 4397 153 # Soleus SHAM 2406 1 16 1970 55 OCCL 25 05 1 65 1864 228 Values are mean SE. Significant interaction in diaphragm: increased type IIx/b CSA in OCCL animals, p<.0 1 # Significant interaction in intercostals: increased type IIx/b CSA in OCCL animals, p<.01 Table 4 2. Histological remodeling of the medial costal diaphragm. Phenotype proportion, cross sectional area and area fraction of the medial costal diaphragm in sham occluded animals and animals treated with intermittent tracheal occlusion. Type I f ibers Type IIa f ibers Type IIx/b f ibers Fiber count proportion SHAM 0.34 0.03 0.37 0.01 0.29 0.03 OCCL 0.40 0.03 0.36 0.03 0.24 0.01 Cross sectional area, m 2 SHAM 1358 45 1456 83 3278 233 OCCL 1480 97 1599 119 4141 159 Phenotype area fraction SHAM 0.22 0.02 0.27 0.01 0.51 0.03 OCCL 0.29 0.02 0.29 0.03 0.42 0.04 Values are mean SE. Significant phenotype main effect for proportion : decreased proportion of type IIx/b fibers, p<.005 Significant interaction for CSA: increased type IIx/b CSA in OCCL animals, p<.0 1 Significant phenotype main effect for A A : increased A A of type IIx/b fibers, p<.001


70 Table 4 3. Histological assessment of the third parasternal intercostals. Phenotype proportion, cross sectional area and area fraction of the third parasternal intercostals in sham occluded animals and animals treated with intermittent tracheal occlusion. Type I Fibers Type IIa Fibers Type IIx/b Fibers Fiber count proportion SHAM 0.12 0.02 0.21 0.01 0.67 0.02 OCCL 0.19 0.03 0.17 0.02 0.64 0.03 Cross sectional area, m 2 # SHAM 1350 136 1513 127 3738 183 OCCL 1464 79 1757 122 4397 153 Phenotype area fraction SHAM 0.05 0.01 0.11 0.01 0.84 0.02 OCCL 0.08 0.01 0.08 0.01 0.84 0.02 Values are mean SE. Significant phenotype main effect for fiber proportion: increased proportion of type IIx/b fibers, p<.001 # Significant interaction for CSA: increased type IIx/b CSA in OCCL animals, p<.01 Significant phenotype main effect for A A : increased A A of type IIx/ b fibers, p<.001 Table 4 4. Histological assessment of the soleus muscles. Phenotype proportion, cross sectional area and area fraction of the soleus muscle in sham occluded animals and animals treated with intermittent tracheal occlusion. Type I f ibe rs Type IIa f ibers Type IIx/b f ibers Fiber count proportion SHAM 0.92 0.02 0.08 0.02 OCCL 0.94 0.02 0.06 0.01 Cross sectional area, m 2 SHAM 2406 1 16 1970 55 OCCL 25 05 1 65 1864 228 Phenotype area fraction SHAM 0.93 0.02 0.07 0.02 OCCL 0.95 0.01 0.05 0.01 Values are mean SE. Significant phenotype main effect for fiber proportion: increased proportion of type I fibers, p<.00 1 Significant phenotype main effect for A A : increased A A of type I fibers, p<.001


71 Table 4 5. Cross sectional area assessment of the dorsal, medial and ventral costal diaphragm in sham occluded animals and animals treated with intermittent trac heal occlusion. Type I f ibers Type IIa fi bers Type IIx/b f ibe rs Dorsal d iaphragm (m 2 ) SHAM 1347 109 1429 130 3296 245 OCCL 1233 91 13 49 131 3103 190 Medial d iaphragm (m 2 ) SHAM 1358 45 1456 83 3278 233 OCCL 1480 97 1599 119 4141 159 Ventral d iaphragm (m 2 ) SHAM 134 4 1 24 1379 128 28 48 3 05 OCCL 1398 1 38 1391 101 32 49 294 Values are mean SE. Significant region phenotype interaction: p<0.05 versus the SHAM condition. Table 4 6 Proportion of fiber phenotype expression in the respiratory muscles. Type I f ibers Type IIa f ibers Type IIx/b f ibers Medial d iaphragm SHAM 0.34 0.03 0.37 0.01 0.29 0.03 OCCL 0.40 0.03 0.36 0.03 0.24 0.01 3 rd parasternal i ntercostal SHAM 0.12 0.03 0.21 0.01 0.67 0.02 OCCL 0.19 0.03 0.17 0.01 0.64 0.02 Soleus SHAM 0.92 0.02 0.08 0.02 OCCL 0.94 0.02 0.06 0.02 Values are mean SE. Significant main effect in diaphragm: decreased proportion of type IIx/b fibers, p<.005 Significant main effect in intercostal: increased proportion of type IIx/b fibers, p<.001 Significant main effect in soleus: increased proportion of type I fibers, p<.00 1


72 Table 4 7. Area fraction of fiber phenotype expression in the respiratory muscles. Type I f ibers Type IIa f ibers Type IIx/b f ibers Medial d iaphragm SHAM 0.22 0.02 0.27 0.01 0.51 0.03 OCCL 0.28 0.02 0.29 0.03 0.42 0.04 3 rd parasternal i ntercostal # SHAM 0.05 0.01 0.11 0.01 0.84 0.02 OCCL 0.08 0.01 0.08 0.01 0.84 0.02 Soleus SHAM 0.93 0.02 0.07 0.02 OCCL 0.95 0.02 0.05 0.02 Values are mean SE. Significant main effect for diaphragm: increased A A of type IIx/b fibers, p<.001 # Significant interaction for intercostal: increased A A of type IIx/b fibers, p<.001 Significant main effect for soleus: increased A A of type I fibers, p<.001 Table 4 8 Regional diaphragm phenotype proportions. The proportion of type IIx/b fibers was greater in the sham occluded ventral diaphragm, compare d to the other groups. Type I f ibers Type IIa f ibers Type IIx/b f ibers Dorsal d iaphragm SHAM 0.37 0.02 0.35 0.01 0.26 0.01 OCCL 0.37 0.03 0.37 0.03 0.29 0.02 Medial d iaphragm SHAM 0.34 0.03 0.37 0.01 0.29 0.03 OCCL 0.40 0.03 0.36 0.03 0.24 0.01 Ventral d iaphragm SHAM 0.40 0.03 0.37 0.03 0.23 0.01 OCCL 0.35 0.02 0.35 0.03 0.30 0.03 Values are mean SE. Significant 3 way interaction: p<0.05 versus the OCCL condition.


73 Table 4 9. Regional diaphragm phenotype area fractions Histological assessment of phenotype proportion of the dorsal, medial, and ventral costal diaphragm in sham occluded animals and animals treated with intermittent tracheal occlusion. Type I f ibers Type IIa f i bers Type IIx/b f ibers Dorsal Diaphragm SHAM 0.27 0.03 0.24 0.03 0.49 0.04 OCCL 0.25 0.03 0.28 0.03 0.46 0.03 Medial Diaphragm SHAM 0.22 0.02 0.27 0.02 0.51 0.04 OCCL 0.28 0.02 0.29 0.02 0.42 0.04 Ventral Diaphragm SHAM 0.32 0.02 0.29 0.03 0.39 0.04 # OCCL 0.25 0.03 0.25 0.02 0.50 0.03 Values are mean SE. # Significant 3 way interaction: p<0.01 versus the OCCL condition Table 4 10 Quantitative assessment of fiber remodeling in the respiratory muscles. Numbers represent area fractions of normal fibers, remodeled fibers or connective tissue in sham occluded animals and animals treated with intermittent tracheal occlusion. Normal f ibers Remodeled f ibers Connective f issue Medial diaphragm SHAM 0.867 0.051 0.028 0.008 0.103 0.060 OCCL 0.864 0.026 0.030 0.003 0.108 0.027 3 rd parasternal i ntercostal SHAM 0.914 0.004 0.018 0.002 0.068 0.005 OCCL 0.918 0.003 0.032 0.004 0.051 0.003 Soleus # SHAM 0.917 0.007 0.008 0.007 0.075 0.005 OCCL 0.914 0.010 0.018 0.007 0.068 0.008 p<.001 Significant main effect for category: differences between A A of normal, remodeled and connective tissue # p<.001: Significantly lower A A of remodeled fibers in soleus muscle p<.001: Significantly greater A A of connective tissue in diaphragm muscle


74 Table 4 11. Area fraction ( A A ) of abnormal cells in th e diaphragm, i ntercostal, and soleus muscles. Internal n uclei Small, angular f ibers Inflamed, necrotic f ibers Inflammatory c ells Medial d iaphragm* SHAM 0.01 4 0.00 3 0.005 0.002 0.006 0.0 02 0.01 3 0.003 OCCL 0.0 10 0.00 3 0. 007 0.0 02 0.00 5 0.00 2 0.01 5 0.00 3 Third i ntercostal SHAM 0.00 9 0.00 4 0. 001 0.0 00 0.00 0 0.00 0 0.01 2 0.00 4 OCCL 0.021 0.004 0.001 0.000 0.001 0.000 0.009 0.004 Soleus # SHAM 0.002 0.002 0.006 0.003 0.000 0.000 0.009 0.002 OCCL 0.004 0.002 0.003 0.003 0.000 0.000 0.010 0.002 Values are mean SE. Significant main effect for category in diaphragm: inflammatory cells versus internal nuclei, small/angular fibers, and inflamed/necrotic fibers p<.05 A A Internal nuclei of intercostal significantly greater than inflamed necrotic A A : p<.001 # A A Inflammatory cells of soleus significantly greater than inflamed necrotic A A : p<.001 Table 4 12. Quantitative assessment of fiber remodeling in the regions of the costal diaphragm. Numbers represent area fractions of normal fibers, remodeled fibers or connective tissue. Normal fi bers Remodeled f ibers Connective t issue Dors al d iaphragm SHAM 0.833 0.009 0.048 0.004 0.090 0.003 OCCL 0.827 0.019 0.050 0.008 0.095 0.007 Medial d iaphragm SHAM 0.853 0.022 0.040 0.009 0.107 0.030 OCCL 0.855 0.010 0.037 0.007 0.108 0.011 Ventral d iaphragm SHAM 0.833 0.017 0.049 0.008 0.092 0.0 15 OCCL 0.803 0.038 0.066 0.020 0.103 0.016 Values are mean SE. Significant main effect for category: normal>connective tissue>remodeled fibers (p<0.001 versus other categories)


75 Table 4 13. Area fraction of abnormal cells in the regions of the costal diaphragm. The prevalence of inflammatory cells was significantly greater than other remodeling characteristics. Internal n uclei Small, angular f ibers Inflamed, necrotic f ibers Inflammatory c ells Dors al d iaphragm SHAM 0.010 0.004 0.002 0.004 0.005 0.002 0.028 0.005 OCCL 0.012 0.004 0.007 0.003 0.006 0.002 0.028 0.004 Medial d iaphragm SHAM 0.013 0.004 0.005 0.002 0.006 0.013 0.014 0.003 OCCL 0.010 0.003 0.864 0.026 0.005 0.002 0.015 0.003 Ventral d iaphragm SHAM 0.012 0.005 0.002 0.002 0.008 0.012 0.028 0.004 OCCL 0.012 0.005 0.005 0.002 0.023 0.011 0.029 0.003 Values are mean SE. Significant main effect: p< 0.05 versus internal nuclei, small/angular fibers, and inflamed/necrotic fibers Table 4 14. Embryonic myosin positive fibers in the respiratory muscles. A significantly greater p ercentage of positive fibers was present in the intercostal muscles of the occluded animals. Embryonic myosin positive fibers Medial Diaphragm SHAM 0.42 (0 .36 0.60 )% OCCL 0.94 (0 .00 1.00 ) % 3 rd Parasternal Intercostal SHAM 0.00 ( 0.00 0 6 3)% OCCL 1.20 ( 0.68 3 37 )% Soleus SHAM 0.00 (0.00 1.51 )% OCCL 0.98 ( 0.00 1.31 )% Note: v alues are median interquartile range. p<0.05 versus the SHAM condition


76 Figure 4 1. Cross sectional area (CSA) of the medial costal diaphragm. The CSA of type IIx/b fibers was significantly greater in t he occluded group (ANOVA, *p<.01 ).


77 Figure 4 2. Fiber CSA in the third parasternal intercostal muscles. The CSA was significantly greater in the type II x/b fibers of the occluded animals (ANOVA, # p<.01). #


78 A B C D E F Figure 4 3 Diaphragm, intercostal, and soleus muscle immunohistochemistry for myosin heavy chain isoform A). Diaphragm from OCCL animal. B). Diaphragm from SHAM animal. C). Third intercostal from OCCL animal. D). Third intercostal from SHAM animal. E). Soleus from OCCL animal. F). Soleus from SHAM animal. Type I fibers fluoresce blue, type IIa illuminate gre en, and type IIx/b fibers remain free of fluorescence. Images were captured with 40X magnification and 10X objective. Scale bar represents 100 m


79 A B C Figure 4 4 Hematoxylin and eosin stained images A) Diaphragm B) 3 rd Parasternal Intercostals C) Soleus Images were captured with 40X magnification and 10X objective, scale bar represents 50 m


80 A B C D Figure 4 5 Categories of fiber remodeling in the diaphragms of the exp erimental sample. Color coded arrows highlight inflammatory cells (black), collagen/fibroblasts (white), inflamed or necrotic tissue (yellow), D) internally nucleated fibers (green) and small round or angular fibers (blue) Images were captured with 40X ma gnification and 10X objective, scale bar represents excursion 50 m


81 A B C E F Figure 4 6. Embryonic myosin positive cells. A). Diaphragm from OCCL animal. B). Diaphragm from SHAM animal. C). Third intercostal from OCCL animal. D). Third intercostal from SHAM animal. E). Soleus from OCCL animal. F). Soleus from SHAM animal. Positive cells fluoresce green. Images were captured with 20X magnification and 10X objective, scale bar represents 100 m.


82 F igure 4 7 Proportions of embryonic myosin positive fibers in the respiratory muscles. The expression of embryonic myosin was greater in the intercostals of t he occluded animals (Mann Whitney U *p<.05).


83 A B Figure 4 8. Verification of embryonic myosin in intercostal muscle. A). Western Blot images showed faint, distinct bands at 200 kDa, consistent with eMHC. B). The signal intensity of bands tended to be greater in the occluded animals, but the results were not statistically significant (p>.05). 200 kDa OCCL OCCL OCCL OCCL SHAM SHAM SHAM SHAM


84 CHAPTER 5 DICUSSION Principal Findings These experiments provide novel information regarding the effects of brief, intense overload training on respiratory muscle remodeling. In Aim 1, we hypothesized that brief occlusions would provide a sufficient stimulus to facilitate muscle hypertrophy wit hout significantly altering the fiber phenotype. The findings from this project support the postulate that occlusion training was associated with rapid, preferential hypertrophy of IIx/b respiratory muscle fibers. In the occlusion trained ( OCCL ) animals, t he type IIx/b fibers was 27% larger in the medial diaphragm, and 22% greater in the 3 rd intercostal. The data also indicate that the cross sectional area ( CSA ) of the medial costal diaphragm was greater in type IIx/b fibers, compared to the dorsal and vent ral regions. We did not identify a shift in myosin heavy chain ( MHC ) isoform composition after training. An additional unexpected finding revealed significant group region differences in the ventral diaphragm expression of type IIx/b MHC. The data sugges t that the nature of remodeling could vary based upon regional structure and function. In Aim 2, we tested the hypothesis that the intensity of tracheal occlusion would induce myogenic activity in the respiratory muscles, yet the brevity of the overload w ould minimize fiber damage. In agreement with our hypothesis, a limited degree of fiber remodeling was present in the respiratory muscles. However, the training program only induced modest regeneration of the third intercostal muscle. Detailed interpretati ons of the findings follow in subsequent sections of this chapter.


85 Training Elicited Fast Fiber Hypertrophy After ten sessions of tracheal occlusion, the CSA of type IIx/b fibers was increased in the medial diaphragm and third intercostals. Increased diap hragm fiber CSA has been reported in other models of muscle plasticity and could provide alternative justification for our findings. For example, an enlarged fiber CSA without force improvement occurs in models of passive stretching, injury or myopathy (19 8 200) It should be noted that we did not identify concurrent histological markers of injury and regeneration typical of these alternative models. Diaphragm fiber hypertrophy has also been reported following tetrodotoxin nerve blockade or denervation indu ced inactivity (201) It is possible that placement of the occluder cuff could have injured a nearby phrenic nerve. Denervation related hypertrophy occurs predominately in slow, oxidative fibers, with corresponding type IIx/b atrophy can be detected within two weeks of nerve injury (202) A denervation CSA remodeling pattern was not present the current study, and we did not visualize morphological evidence of denervated or regenerating fibers. Therefore, it is unlikely that these other factors influenced fi ber CSA. Notably, significant CSA differences were detected after only 10 training sessions, a timeframe that is considerably shorter than typical hypertrophy responses expected clinically. In adults with chronic obstructive pulmonary disease ( COPD ) signi ficant intercostal fiber hypertrophy has been found within five weeks (14) In young adults, four weeks of intense strength training resulted in fast fiber hypertrophy of the limb muscles (203) We are not aware of clinical reports of diaphragm fib er hyper trophy after training. In contrast, r odent tissue remodeling is thought to occur more rapidly than in humans. Significant increases in limb muscle CSA have been reported after as few as 14 days of functional overload training (204, 205) Group variation in fast fiber CSA after high


86 intensity limb strengthening was comparable to the respiratory CSA differences attained with ten days of tracheal occlusion. The r apid ity of respiratory remodeling could be influenced by the high baseline activity of respiratory motor units. Muscles with a high duty cycle respond rapidly to changes in activity (206) Furthermore, m uch more is known about respiratory responses to inactivity than with increased activity. With quiescence, diaphragm gene expression downregulates eight times more rapidly than limb muscle (5) Initially, slow and fast fiber atrophy equally, but prolonged inactivity preferentially atrophies fast fibers (61, 68) Altered diaphragm activity also affects neural coupling and signal transduction by trophic fac tors within days These mechanisms significantly alter diaphragm protein (206, 207) The OCCL animals exhibited hypertrophy only in type IIx/b fibers. There is other evidence of pr eferential fast fiber hypertrophy in intense strength training of respiratory and limb muscles, in agreement with our findings (21, 205) I n the respiratory muscles, a less intense, flow resistance training protocol elicits modest CSA enlargement in all ph enotypes (14, 139) The se disparate results likely arise from differences in training intensity and duration. The extent of hypertrophy can vary based upon motor unit recruitment, which in turn depends upon the intensity of the training load. At resting tidal volume, the diaphragm generates ~10% of its peak pressure, by predominately activating slow, oxidative fibers (33) Fast, fatigable fibers are not thought to be recruited during eupnea. Oxidative fibers generate lower specific tension but remain fatigue resistant. With increasing levels of inspiratory drive, diaphragm motor units contract at a higher


87 frequency, although individual motor unit recruitment appears programmed according to the inspiratory volume (208) Nevertheless, significant type IIx/b fiber recruitment requires intense loads. In order to promo te fast fiber hypertrophy of the respiratory pump, the training intensity must be sufficient to recruit these fibers. Additionally, t raining intensity should exceed 60% of peak force in order to significantly increase skeletal muscle protein synthesis (209) Occlusion maneuvers generate strong recruitment of the ventilatory pump. Sieck (33) estimated occluded inspiratory attempts recru ited only 50% of feline diaphragm motor units The most recent neurophysiological models identify that 40 50% of type IIx/b fibers are recruited during tracheal occlusion (210). Additionally, u p to 10 ventilatory attempts may be necessary to achieve maxima l transdiaphragmatic pressures during occlusion (211) Our studies imposed 5 8 seconds of tracheal occlusion and yielded 5 10 ventilatory attempts per occlusion. It is likely that strong to maximal efforts in the animals produced robust activation of the r espiratory pump and provided an atypical loading stimulus to preferentially remodel fast fibers It is known that t he greatest transdiaphragmatic pressures can be ac hieved with expulsive reflexes (33) Our ability to detect oxidative fiber hypertrophy coul d have been limited by statistical power. A power analysis was based upon published evidence of training induce d diaphragm hypertrophy in r odent s (139) In the referenced study, moderate respiratory endurance exercise promoted a small degree of hypertrophy in type IIx/b fibers, and five animals per group were needed to show training differences in type IIx/b fibers. However, only three animals per group were needed to achieve significance with


88 type IIa fibers and only one animal per group was needed to show differences in type I fibers. In contrast, a post hoc power analysis of our animals confirmed that 54 (effect size: .544) animals per group would be needed to detect type I hypertrophy, and 55 (effect size .540) animals per group needed for type IIa hyper trophy. Therefore, the preferential changes in fast fiber CSA were attributed to the training protocol, and this project appears adequately powered. Regional Heterogeneity in the Costal Diaphragm Regional Heterogeneity in CSA Our findings showed that type IIx b fibers in the medial costal diaphragm were larger than in the other regions. Additionally, training facilitated type IIx/b hypertrophy in the medial costal diaphragm, beyond baseline regional differences. A number of factors could have influenced reg ional fiber remodeling. Diaphragm contractions yield inspiratory pressures that can be affected by the neural drive to the muscle, its strength, and force length and force velocity properties of the muscle. Some of these feature s could differ regionally. R egional costal motor unit recruitment has been studied little but is not thought to vary substantially (212) It is known that t he number of sarcomeres in parallel dictates specific force, and varies due to fiber size as well as muscle size. Medial costal muscle size is thicker than other regions and thus may generate more force. In conjunction, the r egional architecture may influence force length and force velocity properties of the costal diaphragm. Muscle length and lung volume each influence the mechan ical effic iency of the inspiratory pump. As a result, d iaphragmatic pressure varies based upon the operating muscle length and regional shortening. The degree of diaphragm muscle shortening correlates well with zone of apposition ( ZAP ) shortening (213) an d relative ZAP


89 shortening correlates to the work of breathing (22) The ZAP is larger and the degree of appositional shortening is greater in the medial costal diaphragm, compared to adjacent regions braced by vertebral and sternal attachments. Functionall y, ZAP excursion is facilitated by t he ribcage muscles during inspiration. Accordingly, c ontraction of the accessory chest wall muscles optimizes length tension properties in the medial diaphragm (214, 215) This project did not measure or mani pulate muscl e length, yet regional shortening may favor a high work output in the medial region. Regional architecture may have also influenced force velocity attributes during v (216) and force velocity properties determine the work performed by a contracting muscle and describe its mechanical efficiency. The velocity of muscle shortening decreases as te nsion progressively increases. While t racheal occlusion generated strong diaphragm efforts that may have approxi mated optimal isometric force, e ven greater muscle tension can be generated by lengthening contractions. The mid costal diaphragm is reported to lengthen during occluded inspiratory efforts, while o ther regions shorten (48) The force velocity properties of the lengthening ventilatory pump can be quantified functionally by trans diaphragmatic pressure. Peak trans diaphragmatic pressures occur during expulsive reflexes (33, 217) We note that trachea l occlusions induced both inspiratory and expiratory efforts by animals. Furthermore, s trong expiratory efforts produce diaphragmatic lengthening (47, 48) Because mid costal fibers can lengthen with occluded inspiration or expiration, they may be more sus ceptible to remodeling. Our studies were not designed to measure force adaptations with training, but this is suggested for future studies.


90 Regional Differences in Fiber Phenotype In addition to significant CSA differences in the medial diaphragm, we found a reduced presence of type IIx/b fibers in the sham trained (SHAM) ventral diaphragm, compared to other regions and MHC isoforms. MHC isoform expression can be affected by activity and loading properties. Muscles recruited more frequently at lower forces typically display a greater prevalence of oxidative fibers. Attributes of oxidative fibers include greater capillarization, higher contents of oxidative enzymes, and greater glycogen depletion with exercise. Our results suggested a greater oxidative phenot ype in the untrained ventral diaphragm region, and showed relatively increased expression of fast fatigable fibers in the ventral region of occluded animals. Occasional reports in the literature suggest the ventral diaphragm may be more oxidative than oth er regions (218) oxidative activity and MHC composition are similar or lower to other regions (28, 201, 219, 220) Reid noted an exceptionally low oxidative capacity in type IIb fibers of the ventral ste rnal hamster diaphragm (39) and glycogen utilization is lowest in the ventral diaphragm, both at rest and with aerobic exercise (42) Additionally, the diaphragm oxygen consumption and blood flow are correlated (26) Sexton and Poole (221) noted that bloo d flow of the rodent diaphragm is lowest in the ventral costal region. In the dorsal, medial, and OCCL ventral regions of our sample, fiber type proportions and phenotype area fraction ( A A ) closely resemble reference values reported in the literature, but the SHAM ventral proportions are lower than most other reports. In addition, l ow intensity endurance inspiratory exercise does not significantly alter MHC proportions in the diaphragm (21, 139, 222) Thus, the altered ventral MHC composition in the SHAM t rained animals could be an incidental statistical artifact.


91 Greater metabolic differences appear to occur between the costal and crural diaphragm (39, 219, 223) than between costal diaphragm regions. Sustained Damage was not Present in Overloaded Muscle In the diaphragm third intercostal and soleus, normal fibers were by far more prevalent than connective tissue or remodeling fibers. The A A of connective tissue and abnormal cells found in the diaphragm and intercostal muscles was comparable to those found i n untrained or sham trained respiratory muscles (163) Additionally, there were no significant group differences in the prevalence of abnormal or connective tissue for any of the muscles. The results indicate that either the occlusion training did not elic it significant fiber disruption or that tissue repair had already occurred. We specifically examined differences in fiber shape and structure, degeneration and regeneration, fibrosis, and cellular reactions. We found a higher A A of connective tissue in th e diaphragm compared to the other muscles studied. However, we did not find the excessive quantities of collagen and fibroblasts associated clinically with respiratory diseases in adults (224) or infants (225) Connective tissue deposition may occur in pro portion to the severity of an injury, and can indicate incomplete or impaired regeneration (185) While a dipose deposition can arise in conjunction with ex cessive tissue fibrosis no ne were observed in any images. The most commonly observed abnormal cell type iden tified was inflammatory cells. While n eutrophils have a polymorphic nucleus and migrate to the site of injury almost immediately after severe respiratory loading (163) t heir presence may delay upregulation of muscle regenerative transcriptional f actors (181) On the other hand, macrophages can promote activation and proliferation of satellite cells (168) It should be noted that w e did not conduct immunohistochemical analysis to differentiate between


92 inflammatory cells. Necrotic and inflamed cells were seen infrequently, suggesting that mechanical loads were insufficient to result in extensive injury. A dditionally, a ngular fibers have been reported in instances of denervation and these cells may occur in small groups (185) Although surgical place ment of an occluder cuff could potentially elicit a phrenic nerve injury, low proportions of angular fibers in our diaphragm images suggest that the phrenic nerves did not sustain damage. Additionally, the occluder placement was proximal to the superficial location of the phrenic nerve. Centrally nucleated fibers are a histological indicator of muscle regeneration. Elevated proportions in the diaphragms of COPD or Duchenne muscular dystrophy models suggest ongoing damage and regeneration (20, 226) In this study, the A A of internally nucleated fibers fell within the 3% proportions contained in normal adult skeletal muscle (185) After eccentric contraction induced muscle injury, increased centrally nucleated fibers appear within 48 72 hours and peak by 7days (184) We did not observe other traits of regenerating fibers, such as granular, basophilic, or split features (185) Additional cytoplasmic changes can be described as h yaline, lipofuscin ic states or chronic loads (185) We found very few cytoplasmic changes in the respiratory muscles. The low presence of damage could be due to the assessment timeframe. Single injurious loads have been reported to increase calpain activity and promote ear ly accumulation of macrophages and neutrophils (227) While i njury can increase the prevalence of abnormal cells in the diaphragm, and to a lesser extent the parasternal intercostals (163) evidence of damage may be delayed (165) Significant morphological changes occur within one day of injury, and peak at three days. In the absence of


93 additional mechanical overloads, proportions of connective tissue and abnormal cells begin to decrease toward baseline after 4 days (165, 166) Models of widespread chemical or thermal injury reveal inflammation, necrotic cells and histological signs of regeneration up to 14 days after injury (184) Repeated respiratory loads can perpetuate histological signs of muscle damage. Two hours of daily resistive loading for four co nsecutive days damaged cell membranes and increased the prevalence of sarcomere disruption in the diaphragm (19, 140) While abnormal cell A A peaks on the third day after constant loading, proportions of connective tissue and abnormal fibers remain elevate d in subsequent days (162, 166) After 30 days of tracheal banding, the A A of abnormal fibers was five times higher than the unbanded animals, despite a larger diaphragm mass (17) While we did not find histological signs of damage in the respiratory muscles in the sample force measurements provide the most sen sitive gauge of muscle injury. It should be noted that evidence of ultrastructural disruption and inflammation is commonly presen t during resistance training in human and animal muscles and may accompany large and significant force gains. Therefor e we suggest that future studies examine the effects of occlusion on evoked respiratory muscle force. Limited Presence Embryonic Myosin after Training We found modest yet significant increases in intercostal expression of embryonic myosin, but in contrast to our hypothesis, we did not find elevated embryonic myosin in diaphragms of occluded animals. The immunohistological findings were su pported by the limited A A of intern ally nucleated fibers and minimal increases in embryonic myosin ( eMHC ) protein with immunoblotting The intercostal eMHC levels were statistically different between training groups, but absolute quantities did not reach e xpected values


94 that occur with injury (181, 228) In contrast, strength training in humans elicited similar proportions of eMHC positive fibers ( mean: 3%) to the levels found in the intercostal muscles of the OCCL animals (229) Embryonic MHC expression o ccurs in a similar timeframe to morphological features of damage and regeneration. Embryonic myosin is one of the earliest MHC isoforms expressed in development. During prenatal development, proliferating myoblasts exit the cell cycle and differentiate. Di fferentiation activates expression of eMHC. Developmental MHC forms predominate in prenatal and early postnatal periods, and then expression downregulates (230 232) After skeletal muscle fiber maturation in early infancy, adult MHC isoforms prevail throug hout the life span (233) Reemergence of eMHC can be observed periodically, following mechanical overload or other injury. Specifically, myoblasts that exit the cell cycle, differentiate and fuse together express eMHC, distinguishing nascent or regenerating fibers. Myf5 appears to initiate eMHC activation, while MyoD and calcineurin facilitate its expression (234) Widespread eMHC positive cells can also be found in DMD and SCI models of neuromuscular injury and regeneration (226, 235) Regeneration has bee n tracked in rodents following a single mechanical, chemical or thermal injury. As with other quantitative markers of damage or regeneration, the prevalence of eMHC varies with the size and severity of the injurious load (184) Gene expression of e MHC trai ls upregulat ion of proliferative transcrip tion factors by 2 3 days. S ignificant increases in expression occur after three days, peak by seven days and remain elevated at 14 days (180) Expression of immature MHC may be delayed or


95 inhibited by overabundant inflammation (181) The CSA of regenerating fibers is smaller, and many co express a slow MHC isoform (228) In the current study, fewer than1% of diaphragm fibers fluoresced for eMHC, regardless of group assignment. This rate approximates the expected no rmal values in rats (159, 160) The limited diaphragm eMHC expression could indicate that myogenesis was unnecessary because sig nificant damage did not occur. A l ack of damage could be due to an insufficient training intensity or duration. W hile w e observed vigorous respirat ory attempts by the animals, we did not measure occlusion generated pressures. On the other hand, recruitment of accessory respiratory muscles may have spared the diaphragm from excessive strain during OCCL. Larger mammals and hum ans frequently recruit accessory muscles and alter the ir breathing pattern during heavy respiratory loading. Each of these strategies may deter fatigue in the diaphragm (109, 114) A low diaphragm eMHC expression accompanied by increased expression in the loaded intercostals may indicate that animals adopted a different ventilatory recruitment strategy during occlusions, in order to minimize diaphragm fatigue or injury. Regeneration may have also been inhibited by stress related transcriptional activity in muscle. Our collaborators illustrated that occlusion acu tely elevated mRNA expression of stress response transcriptional pathways in the medial thalamus (236) T wo weeks of daily occlusion s elevated serum corticosterone levels (unpublished pilot data) and heighten ed stress related behavior in occlusion trained animals (237) The n euroendocrine responses to stress differ based upon the nature of the stressor. Therefore, psychological stressors engender different physiological adaptations than metabolic stres sors (238) Chronic stress ors increase basal levels of serum


96 corticosterone and diminish acu te respons iveness to stressors (239) The resultant increases in circulating glucocorticoids could attenuate muscle fiber regeneration When e xcess c irculating glu cocorticoids accompany psychological stress they reduce muscle protein synthesis and in crease gene expression of pathways leading to degeneration atrophy and apoptosis (240) In addition, GCs elevate myostatin levels in skeletal muscle (182, 241) While myostatin may not alter satellite cell proliferation or directly induce atrophy, it inhibits myoblast differentiation by blocking signaling molecules downstream of Akt (158, 242) In rodents, four weeks of daily restraint stress resulted in increases of li mb muscle myostatin, caspase 3, p53, and p38 MAPK, and facilitated sig nificant muscle fiber atrophy. Additionally, the authors attributed the altered apoptosis and protein metabolism signal ing to high levels of glu cocorticoids (240) Myostatin and glucocor ticoids cooperatively oppose protein synthesis and muscle regeneration (153, 243) Chronic stress specifically decreased MyoD and phosphorylated Akt protein expression (240) Conversely, IGF 1 antagonizes myostatin and glucocorticoid mediated catabolic act ivity in skeletal muscle, and supports h ypertrophy through PI3K Akt mTOR protein synthesis pathways or myogenic regeneration. Additional control of regeneration occurs through cyclin and MAPK signaling, mediated by MRF transcription factors (156) U ltimately, net muscle growth necessitates that protein synthesis exceeds proteolysis. Study Limitations Application of the study findings is limited by some aspects of the design. The magnitude and timing of protein synthesis after resistance exercise var ies between rodents and humans (147) and we cannot make direct translations. Also, the experiments were terminal, and therefore pre post changes in muscle CSA, phenotype,


97 and myogenic activity could not be directly measured. However, inclusion of an opera ted, sham trained animal group served to control for the effects of growth, surgery, and daily handling during the experiment. The refore, the similar group demographic characteristics suggest that occlusions did not alter animal growth. Replication of the experiment may be restricted because occlusion training loads were not quantified. The efficiency of respiratory muscle contractions varies by lung volume, and we were unable control lung volume during training. In addition, we could not determine with pr ecision the exact pressure load generated by the animals. Plethysmograph recordings allow investigators to non invasively track pressure and volume fluctuations, usually on sedated animals. Although animals were housed in a plethysmograph for daily trainin g sessions, pressure fluctuations with occlusion were obscured by movement artifact of animals. In future work, the in clusion of esophageal pressure transducers may provide addit ional information In addition to the design related limitations, the tissue analysis methods were limited in scope. Since h istological analyses utilize only a small p ortion of a muscle specimen, they therefore can be influenced by the sampling region of the mu scle. D iaphragm samples were taken midway between its costal attachment and the central tendon insertion, because in larger mammals, fiber size and connective tissue content differ at the attachments (196, 244) Additionally, samples were taken from the midbelly of the intercostal and soleus muscles (185) We suggest that hist ological findings be verified with quantification of protein synthesis and myogenesis biomarkers. Also, while remodeling was examined after ten days of training, acute adaptations to occlusion remain unknown.


98 Finally, the study did not analyze whether spec ific respiratory muscle force (force per unit area) or ventilatory function were altered by tracheal occlusion training. Although fiber size influences peak tension in skeletal muscle, the quasi isometric training does not replicate the timing or biomechan ical properties of tidal breathing. Therefore, we do not know whether in vivo pressure flow properties of the ventilatory muscles, and subsequently breathing pattern, differed in the OCCL animals Application of the Model The tracheal occlusion model prov ides important applications for the physiology of IMST. In contrast to many other animal models of loading, it demonstrates that rapid fast fiber hypertrophy of the respiratory muscles is possible, without eliciting substantial structural injury. Compared to previous loading models, ours most closely replicated principles of brief, intense overloads to elicit hypertrophy. Clinical inspiratory exercises have typically involved modest, sustained trainin g regimes. Modest resistive inspiratory training in adul ts with COPD facilitated slow and to a lesser extent fast fiber hypertrophy in the intercostal muscles (14) Training also resulted in significant gains in respiratory strength and endurance. While training improved function and fiber CSA, a more vigorou s training intensity may have generated preferential type IIx/b hypertrophy. On the other hand, respiratory loading to exhaustion can induce diaphragm damage in healthy adults and patients with COPD (245) In addition, maximal inspiratory pressure ( MIP ) o f patients was lower in the patients 24 hours after the injurious bout. Our findings reinforce that intense respiratory loads may facilitate preferential type II fiber hypertrophy, but brevity of the mechanical overload is essential to avoid widespread dam age and decrements in excitation contraction coupling.


99 The training model also suggests that poorly cooperative patients may benefit from occlusion training. Occlusion rapidly increases neural drive, and yields maximal efforts within approximately ten ins piratory attempts. Adults achieve MIP within 20 25 seconds and children reach maximal effort in ~15 seconds (190, 211) Strong to near maximal training intensities can be elicited by occlusion without active patient cooperation. Occlusion IMST could be app lied to equine or canine veterinary training as well as clinical pediatric or neurologically impaired patients. In the next chapter, we describe an infant recipient of occlusion IMST and recommend that future research examine training effects in these emer ging areas.

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100 CHAPTER 6 CLINICAL APPLICATION OF OCCLUSION TRAININ G: CASE REPORT OF AN INFANT WITH POST OPERATIVE WEANING FA ILURE Case Report Diagnostic Background This report describes an infant female born at 34 weeks gestation and diagnosed with DiGeorge syndrome and type I truncus arteriosus (TA) at birth. DiGeorge syndrome is a chromosomal disease characterized by gene deletion at the long arm of chromosome 22 ( del22q11.2). Associated signs and symptoms of del22q11.2 frequently include palate and pharyngeal defects, hypoparathyroidism, thymus insufficiency, learning disabilities, and congenital heart disease. Although DiGeorge syndrome does not alter primary lung physiology, respiratory complications are common, due to impaired immune function and bronchopharyngeal structural deficits (247) Approximately 40% of children with del22q11.2 have a concurrent congenital heart defect (248) The child in this case presented with TA, a developmental anomaly that occurs when the pulmonary artery and aorta do not mature from a single large arterial trunk. This single vascular outlet receives blood from the right and left ventricles, and deliver s blood to the systemic, coronary and pulmonary vascular systems. There are three types of TA, depending upon the anatomy of the pulmonary arteries. In type I TA, a small vessel emerges from the left side of the truncus and then rapidly divides into unders ized right and left pulmonary arteries (Figure 6 1). Approximately one third of patients born with TA carry del22q11.2 (248, 249) Other congenital heart defects associated with TA include patent ductus arteriosus, ventricular septal defect (VSD), patent f oramen ovale, and interrupted aortic arch. Corrective procedures consist of repair of the VSD and establishment of communication between the left ventricle and

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101 the aorta; construction of a valved conduit between the pulmonary arteries and right ventricle; and anastomosis of interruptions in the aorta. Early surgical repair reduces an otherwise high infant mortality from pulmonary hypertension and hypoxemia (248, 250) Clinical Presentation The infant underwent surgical repair of TA, VSD and interrupted aor tic arch, at 11 days of age. Her post operative course was complicated by feeding intolerance requiring gastric tube placement and Nissen fundoplication for gastro esophageal reflux disease, as well as respiratory insufficiency and delayed extubation follo wing each of the surgical interventions. She was originally discharged to home at 81 days of age. Three days later, she was readmitted with increased gastric tube discharge and respiratory distress, and she was intubated upon arrival. During the hospitali zation, the infant received treatment for presumed pneumonia and management of the gastric wound. However, weaning from mechanical ventilation ( MV ) was limited by pulmonary hypertension and left ventricular dysfunction. On hospital day #39, she underwent p ulmonary valve replacement with a 9 mm allograft, VSD re closure with a core matrix patch, right pulmonary artery patch, right ventricular tract augmentation with a core matrix patch, an d atrial catheter placement, followed by delayed sternal closure. Thro ughout the hospitalization, the infant required continuous MV support. Despite optimization of her post operative cardiac status, she failed three extubation trials. Each time, failure was attributed to increased work of breathing and hypercapnic respirato ry failure accompanied by hypoxemia. On hospital day #53, we were consulted to evaluate the patient for inspiratory muscle strength training (IMST). Her medical team felt that other pharmacological, surgical, and clinical therapies had been optimized, and her parent consented to

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102 assessment and treatment. At University of Florida, Institutional Review Board approval is not required for case studies of three or fewer patients, but her parent provided consent for a case report. Table 6 1 lists common clinical indications and contra indications for IMST. Although the patient could not actively participate and follow commands, we judged that IMST was indicated due to (1) a sustained decline from baseline respiratory function; (2) repeated hypercapnic respiratory failure, indicating ventilatory muscle dysfunction; (3) pre existing cardiac dysfunction; and (4) medical and nutritional stabilization. Respiratory Muscle Testing On the day of evaluation, the infant was 3.5 months of age, and weighed 3.5 kg. W e assess ed minute ventilation on baseline MV settings and estimated strength of the ventilatory muscles using maximal inspiratory pressure (MIP). MIP was tested with a 15 second inspiratory occlusion maneuver, the preferred strength testing mode for infants and youn g children (211, 251) The patient was tested in supine with approximately 40 degree s of head and trunk elevation. The patient was briefly disconnected from the ventilator, and then o cclusion was provided by way of a unidirectional valve attached to the en dotracheal tube, permitting exhalation. With each subsequent exhalation, the patient exhaled toward residual volume, but a standardized testing volume could not be imposed. The most negative pressure achieved in 15 seconds was recorded as the MIP, and the best of four trials was used for day to day comparison. Training Program The training options were limited for the patient, because all commercially available devices have ~30 mL of dead space and are intended for use in the adult population. Anatomic dea d space varies with size and posture. Neonates and small

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103 infants have an anatomic dead space estimated at 2.25 3.0 ml/kg of body mass (252) The combined dead space of our monitoring sensor and unidirectional valve was measured by volume displacement and was found to be approximately five mL. Insertion of a pressure transducer added another seven mL of dead space (Figure 6 2). ATS guidelines suggest that the dead space of respiratory testing equipment remain below 1.5 mL/kg body mass (251) To account for dead space volume, the occlusion maneuvers used to measure MIP were used to deliver IMST. Training sessions were conducted six days per week and consisted of four sets of 15 second occlusions (8 12 inspiratory attempts), with three minutes of rest betw een sets. In addition to IMST sessions, the infant also underwent daily breathing trials at progressively reduced pressure support and intermittent mandatory ventilation (IMV) levels. Data Analysis W e calculated ventilatory muscle performance during testin g and training sessions. Ventilatory parameters were determin ed using a neonatal respiratory monitor (CO 2 SMO Plus with Capnostat neonatal adaptor Philips Respironics, Murrysville, PA) connected to a laptop computer. An intrinsic pneumotachograph and press ure transducer captured airflow and pressure at a rate of 100 Hz, and airflow was integrated to obtain volume. During occluded breaths pressure performance variables were calculated for the most negative inspiratory effort. Rate of pressure development (d P/dt) was measured as the time to reach peak MIP. Maximal rate of pressure development (maxRPD) was the largest pressure gain in a 10 msec time interval. Additionally, t he most negative occlusion pressure was confirmed by a pressure transducer attached to a

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104 side port ( Sper Scientific, Scottsdale, AZ ) Data were integrated with AnalysisPlus software (Philips Respironics, Murrysville, PA). Training Outcomes The infant participated in 13 IMST sessions over 15 days. During IMST sessions, respiratory parameters and vital signs were monitored continuously (Table 6 2). The infant did not experience desaturation or hypercapnia during or after training, and transien t increases in systolic blood pressure and heart rate returned to baseline levels within approximately three minutes. MIP increased 14% from 55.4 to 63.3 cm H 2 O (Table 6 3). Time to reach MIP quickened by 27%. As a result of pressure and time improvement s, the infant was able to generate negative pressure more rapidly. With training, inspiratory dP/dt increased 43% from 92 cm H 2 O/s to 132 cm H 2 O/s. We detected small fluctuations in inspiratory flow and volume on waveform tracings, otracheal tube was not cuffed. Small quantities (~20 mL) of air leaked around the tube during IMST bouts. In addition to strength gains, IMST improved breathing function. During the training approp riate values ( Figure 6 4 ). B aseline r espiratory rate decreased by 34% (Figure 2 3), and rest ing spontaneous tidal vol umes increased by nearly 60% (Figure 2 4) After the 13th IMST session, the patient was extubated to a high flow nasal canula. She was disc harged to home using supplemental oxygen with a nasal canula two weeks after extubation. Discussion We describe an infant who required 68 days of MV and experienced repeated post operative weaning failures. MIP increased with training, accompanied by faste r pressu re timing of occluded breaths. It is known that t he rate of pressure generation can

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105 be influenced by a number of muscular factors, including the myosin heavy chain (MHC) isoform composition, cross sectional area (CSA) and tissue elasticity (253, 2 54) Neural drive can also affect dP/dt (255) A faster dP/dt resulted from a greater MIP as well as a faster pressure generation time. We are unable to determine the relative degrees of muscular and neural remodeling exclusively from non invasive measures of ventilatory performance, and suggest additional respiratory testing, including occlusion pressure, electromyography, and phrenic stimulation. Improved pressure and time performance appeared to translate into gains in breathing pattern. The breathing pa ttern became slower and deeper, and the infant weaned from MV after 13 days of training. A number of factors may affect the ability of infants and young children to wean from MV. Important influences include fluid status, medications, cardiopulmonary fun ction, and strength (256) During our 15 by less than 100 mL daily, and blood urea nitrogen (7 10 mg/dL) and creatinine (0.1 0.2 mg/dL) measurements remained at age expected values. Analgesics (fenta nyl, 2 4 mcg prn, daily average: 8 mcg) and inotropic (milrinone, 200mcg/mL, 0.3 mL/hr) medications were not altered during training, and she did not receive corticosteroids or neuromuscular blocking agents that could have influenced skeletal muscle contra ctility. Nutritiona l management was not modified. In addition, t he chest radiograph and laboratory values remained stable. Thus, many variables that can shape ventilatory nd benefit from IMST. morbid factors that can impair respiratory function. Despite her pre existing pulmonary

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106 hypertension, hypoxemia was controlled, as reflected by resting arterial b lood gases (pH: 7.44, PaCO 2 : 39 torr, PaO 2 : 80 torr, SaO 2 : 96%, HCO 3 : 26 mEq/L). Moreover, baseline dynamic resistance and compliance did not differ substantially from reference values for infants (257, 258) and did not fluctuate substantially over the cou rse of IMST ( Table 6 3 ). After cardiac surgery in infants and children pneumonia, delayed sternal closure and pulmonary hypertension ca n independently deter MV weaning (259) Although each of these risk factors transpired for the child, they were correcte d or change during IMST or influence changes in ventilation and strength. We conclude that the enhanced ventilatory performance was at least partially attributed to the IMST intervention. Inspiratory Occlusions and C ardiac F unction existing cardiac function was impaired, special attention was given to potential cardiovascular effects of IMST. Negative thoracic pressure can increase preload (right sided venous return) as well as afterload (left ventricular sy stolic pressure and myocardial workload). When ventilatory mode shifts from mechanically assisted to unassisted breathing, intrathoracic pressure decreases, facilitating venous return to the right ventricle. However, the relative right ventricular overload during inspiratory occlusion was tempered by flow limited venous return even with large negative pressure fluctuations (260) Inspiratory occlusion may also affect pulmonary vascular function or left ventricular work. Occlusion generates a large increas e in pulmonary blood flow and elevate s pulmonary pressure. However, hypoxemia and lung inflation elevate pulmonary vascular resistance (PVR) to a greater degree than intrathoracic pressure

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107 swings (257) Neither hypoxemia nor overinflation applied to the in fant. Clinical monitoring verified that SpO 2 was maintained. Even though small quantities of air leaked around the endotracheal tube, lung volumes decreased during the IMST sets. Together, the PVR and the diameter of the pulmonary arteries modulate pulmona ry shunt in congenital heart diseases. In this case, cardiothoracic surgery mitigated a large shunt and consequential left ventricular overload. While r epeated episodes of concurrent inspiratory occlusion and hypoxemia can diminish arterial pressure, stim ulate LV diastolic dysfunction and induce multifocal infarcts in an animal model (261) w e do not anticipate that the findings apply to our patient T he animal model produced a cumulative overload that was 45 times more intense than the clinical training p rotocol, and PaO 2 of the experimental animals dropped to ~25 mm Hg every three minutes. In contrast, IMST occlusion durations were short, SpO 2 remained well above 90% during and after occlusions, and we observed no acute or chronic changes in blood pressur e. Even though large fluctuations in negative pressure can transiently increase LV afterload and amplify myocardial oxygen consumption (262) nevertheless IMST did not induce a lasting shift in cardiopulmonary function. Echocardiograms were taken weekly during the intervention, including the day prior to initiating IMST, and within 24 hours of extubation. After extubation, LV fractional shortening decreased slightly from 36.2 to 34.9% (z score from 1.32 to 1.83), while the RV diastolic dimension was reduced slightly from 2.61 to 2.41 cm. LPA diameter increased from .49 to .51 cm (z score: .11 to .16), RPA diameter increased from .44 to.45 cm, (z score: 0.5 2 to 0.5) and the

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108 Pulmonary Artery Index (Nakata) decreased from 155 to 146. Despite pre existing impairments, it does not appear that IMST altered cardiac function appreciably. Considerations for Respiratory Mechanics We did not find a large difference in dynamic compliance during training. Absolute compliance values are low in infants (5 10 mL/cm H 2 O) compared to adults (120 180 mL/cm H 2 O). When compliance is referenced to body mass though, it fluctuates little across the lifespan (2 5 ml/kg/H 2 O). Infa nts have a highly compliant chest wall due to low mineralization of the thoracic cage. Consequently, the chest wall does not exert outward recoil pressure against the relatively stiff lung tissue, and the functional residual capacity ( FRC ) of infants is co mpar atively lower than adult volumes. Pediatric FRC varies by height, weight, and age, and is not significantly influenced by presence of congenital heart disease (263) Airway resistance of infants and toddlers (20 40 cm H 2 O/L/s) may be 10 20 times great er than in adults (1 2 cm H 2 O/L/s), due to a particularly low airway caliber within the respiratory zone (18 th generation and smaller) (257) Airway caliber varies with lung inflation. Above FRC, airway resistance decreases slightly, but small volume chang es below FRC correspond to large gains in resistance. At low lung volumes, airways close in dependent regions of the lung. The interaction between the compliant thoracic cage and relatively stiff, resistive lung tissue results an airway closing volume abov e FRC in infants (264) However, end expiratory volumes are greater than FRC in infants, due to the postinspiratory activity of the diaphragm and upper airway muscles (265) Maturation of the Ventilatory Pump As neonates grow, so does the diaphragm. Rapid diaphragm growth results in si gnificant increases of fiber CSA and muscle thickness (15, 266) During this period of

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109 rapid postnatal growth, adult MHC isoforms replace immature MHC. Initially the newborn and infant respiratory muscles contain a lower propo rtion of type I fibers than children and adults (15, 267) Fast isoforms replace developmental MHC and correlate with increases in specific force, maximum shortening, and actomyosin ATPase activity (268) Expression of immature MHC accounts for 60% of the variance in fatigue resistance postnatally (269) The infant respiratory muscle pump is capable of generating a relatively high diameter of the infant ribcage translates small q uantities of tension into relatively large pressures. However, the costovertebral angles approximate 90 degrees in newborns. This inefficient orientation minimizes the contributions of intercostal muscles to tidal volume changes, until costal angles become more acute with age (270) In conjunction, MIP has not been found to correlate significantly to infant size (271) Although infants are capable of generating high MIP values, this capacity is counteracted by high minute ventilation and ox ygen consumption requirements. Furthermore, i nspiratory load consumes a high proportion of capacity in infants, compared to adults (251) Although the patient generated a normal MIP, her ventilatory loads were multiplied due to high minute ventilation and metabolic rates, as well as the presence of pulmonary arterial stenosis and elevated myocardial work Mechanical Ventilation and Weaning of Infants MV alters the timing and mechanics of ventilation in infants. Inspiratory and expiratory volume mediated reflexes normally d ominate the control of breathing in early infancy, prior to the maturation of chemoreflexes (272) In non ventilated neonates there is greater variability in expiratory than inspiratory timing, but variability differences erase

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110 during MV (273) With MV, la rge inspiratory volumes prolong exhalation without altering the timing or amplitude of diaphragm activity during inhalation. The timing of spontaneous and mandatory ventilated breaths do not differ substantially (274) Although MV generates large tidal vol umes, it results in decreased diaphragm movement (275) Ventilator weaning of pediatric patients can involve reduction of the programmed mandatory ventilation rate or gradual reduction of inspiratory pressure support. As our owed and spontaneous tidal volume increased, the ventilator IMV settings were reduced progressively. Reductions in ventilator settings typically accompany extubation readiness tests o n minimal ventilatory support. However, w eaning readiness indices such as the rapid, shallow breathing index (RSBI = f/V T ) and Compliance, Resistance, Oxygenation, Pressure Index (CROP Index = C dyn MIP PaO 2 /PAO 2 f) have showed poor predictive value when applied to ventilat ed infants and children (256) Likewise, strength alone does not reliably predict weaning success (251) On the other hand, tension time index ( TTI ) is a highly significant predictor of weaning in infants and children (112) Successful pediatric MV weaning requires improvements in both respiratory streng th and ventilatory timing. After IMST, our patient demonstrated improved strength and ventilatory pattern However, few other data are available regarding the role of respiratory training to facilitate ventilator weaning in infants. A single published stud y of neonates demonstrated that flow resistive inspiratory exercises increased endurance and improved resting tidal volume (276) In the previous study, peak airway pressures did not differ after training, but airway

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111 occlusion tests lasted for only three b reaths while the infants were asleep. It is unlikely the investigators obtained maximal pressures by these brief occlusion tests. A difference in the training intensity could also explain the greater MIP outcome reported in our patient. Nevertheless, the r esults suggest that training may improve breathing pattern and performance, in general agreement with our results. Conclusions Brief inspiratory occlusion offers a training alternative for infants or patients who cannot follow directions for IMST with threshold or resistive devices. Our report is clinically meaningful, because developmental delay, hospital stay, cost, and mortalit y are significantly greater for infants who fail MV weaning after cardiac surgery (277 279) IMST may be feasible for stable infants with clinical signs of VIDD and warrants further study. Specific suggestions for further study include acute effects of ins piratory occlusion on central arterial pressures and myocardial function; effects of occlusion training on heart rate variability and circulating glucocorticoids; cardiac functional and histological properties of occlusion training in an animal model, and breathing pattern changes alter occlusion training.

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112 Table 6 1 Suggested clinical indications and contraindications for inspiratory muscle strength training. Clinical Indications for Inspiratory Muscle Strength Training Contraindications for Inspirat ory Muscle Strength Training Inspiratory strength below age and gender predicted normal values and/or ventilatory loads estimated to elicit fatigue (per tension time index ) Hemodynamic instability (systolic BP <90 mm Hg, or resting HR > 110 bpm) or requirement of continuous vasopressor medications Decline from pre morbid level of ventilatory function acute requirement for assisted ventilation Evidence of uncontrolled infection (temperature >36.0 C or > 38.5 C, white blood cell count > 19/mm 2 ) Failure to wean with routine clinical methods (i.e. reduced IMV, pressure support, lengthening spontaneous breathing trials) Acute pulmonary instability: untreated hemothorax or pneumothorax, unstable fractures Gas exchange maintained with minimal ventilatory support (ie. F I O 2 < .6, IMV <8, PS <15, PEEP <8) Presence of seizure activity, ventriculostomy, or evolving neurological injury Current or previously prescribed medication known to impair skeletal muscle excitation contraction coupling (such as corticosteroids, beta blockers, neuromuscular blockade, aminoglycoside antibiotics, immunosuppressants) Inability to follow commands

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113 Table 6 2. Vital signs during the course of occlusion training sessions Pre Post Training d ay Heart rate Respiratory rate Mean arterial pressure SpO 2 Heart rate Respiratory rate Mean arterial pressure SpO 2 1 156 70 62 96 166 77 61 98 2 167 51 73 94 182 76 80 96 3 166 51 71 100 182 57 82 96 4 176 49 83 90 180 60 80 99 5 6 170 61 59 97 175 60 64 98 7 150 43 64 96 170 55 64 97 8 160 46 51 96 175 58 57 98 9 165 54 59 93 180 57 59 97 10 187 50 61 94 190 53 64 96 11 164 62 52 97 174 67 70 99 12 13 165 57 56 98 169 51 65 94 14 147 47 64 98 162 49 65 96 15 148 46 55 96 162 68 72 95 Note: Vital signs were monitored continuously. Reported values were ta ken from the onset of daily training and within two minutes of completing the final exercise set. The patient did not receive train ing on days 5 and 12.

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114 Table 6 3 Respiratory performance variables during inspiratory occlusion. Parameters were obtained from the single ins piratory effort that yielded maximal inspiratory pressure Training d ay Maximal inspiratory pressure (cm H 2 O) Time to maximal inspiratory pressure ( s) dP/dt (cm H 2 O/s) Max RPD (cm H 2 O/s) Max RPD/MIP (%/s) 1 55.4 0.66 91.5 410 740 2 3 63.6 0.42 153. 1 615 967 4 5 6 63.3 0.52 125. 4 300 474 7 58.6 0.59 109. 7 250 427 8 66.3 0.6 122.8 335 505 9 64.6 0.53 125. 1 615 952 10 65.6 0.45 153.3 540 823 11 62.1 0.46 143.0 445 717 12 13 62.5 0.55 125.5 305 488 14 60.3 0.5 131 .0 295 489 15 63.3 0.48 132 0 575 908 Notes: MIP: maximal inspiratory pressure; dP/dt: inspiratory pressure development; Max RPD: maximal rate of pressure development; max RPD/MIP: maximal rate of pressure development normalized to strength

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115 Table 6 4 V entilator settings and pulmonary mechanics during the course of treatment. The baseline ventilation of the infant was assessed in an awake, restful state prior to starting inspiratory muscle strength training bouts. Training Day I MV rate Total rate Spontaneous inspired volume (ml) Pressure Support (cm H 2 O) PEEP (cm H 2 O) C dyn (mL/cm H 2 O) C dyn (mL/cm H 2 O/kg) R awi (cm/mL/s) R awe (cm/mL/s) 1 24 44 16 12 5 2 24 65 29 12 5 3 22 64 28 12 5 4 20 60 28 12 5 10. 2 2.9 13.9 14.8 5 18 64 29 12 5 6 18 48 29 12 5 7 16 51 26 12 5 8 20 46 38 12 5 4. 2 1.2 32.3 35.2 9 18 44 27 10 5 12.5 3.6 17.1 18.1 10 10 59 34 10 5 11. 7 3.4 11.7 12.2 11 10 47 28 10 5 12. 4 3.5 14.2 15.3 12 10 48 36 10 5 13 8 52 30 10 5 12. 1 3.5 16.6 17.1 14 8 40 25 10 5 15 8 42 32 10 5 8. 2 2.3 23.4 24.4 Notes: IMV: intermittent mandatory ventilation; PEEP: positive end expiratory pressure; C dyn : dynamic compliance; R awi : dynamic inspiratory airway resistance; R aw e : dynamic expiratory airway resistance

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116 Figure 6 1. Type I Truncus Arteriosus. The right and left ventricles communicate due to a septal defect, and blood exits a common vessel to enter the pulmonary, coronary, and systemic vascular systems. In type I truncus arteriosus, the pulmonary arteries emerge from the left side of the truncus and then divide into right and left branches. Used with permission from A.D.A.M. Images. Figure 6 2. Inspiratory muscle testing and training device. A one way valve prevented inhalation, while exhalation was un impeded.

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117 Figure 6 3 Ventilatory rate slowed during inspiratory muscle strength training Assisted and total ventilator rate. The IMV rate was progressively decreased as spontaneous ventilatory rate slowed.

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118 Figure 6 4. Spontaneous tidal volume a nd maximal inspiratory pressure A 14% increase in MIP was accompanied by a nearly 60% improvement in tidal volume. Lines are regression lines.

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119 CHAPTER 7 CONCLUSIONS This study provides the first evidence that respiratory muscle strength training may el icit muscle fiber hypertrophy in the respiratory muscles. Specifically daily, intermittent tracheal occlusion was associated with increased cross sectional area (CSA) in fast fatigable fibers of the medial costal region of the diaphragm and the third para sternal intercostals. A shift in myosin heavy chain isoform composition did not accompany CSA differences. Significant group differences were detected after just ten days of training. Muscle fiber hypertrophy was not associated with evidence of fiber injur y. While expression of embryonic myosin did not vary in the diaphragm, modest regeneration was present in the intercostal muscles of occluded animals. The results indicate that occlusion may be a feasible mode for strengthening patients without inducing mu scle damage. Clinically, our case patient experienced a modest increase in maximal inspiratory pressure after two weeks of training and functional gains that included a slower, deeper breathing pattern and ventilator weaning. These results are significant because respiratory muscle contractile dysfunction is thought to be a primary contributor to ventilator dependence. We recommend that future studies examine the effect of tracheal occlusion on the diaphragm gene expression of synthesis and myogenic transcr iptional regulators. In addition, it is essential to determine the effects of occlusion upon evoked force of the diaphragm and resultant breathing pattern. Moreover, translational models should examine human diaphragm remodeling following preoperative insp iratory muscle strength training A more complete understanding of respiratory muscle remodeling will

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120 help scientists to develop effective rehabilitation strategies to prevent or reverse ventilator induced diaphragm dysfunction and improve ventilator weani ng.

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144 BIOGRAPHICAL SKETCH Barbara Kellerman Smith received a Bachelor of Science Degree in molecular b iology from Grove City College, an d a Master of Physical Therapy d egree from University of Pittsburgh. She has practiced continuously as a physical therapist si nce 1994, almost exclusively in intensive care and acute cardiopulmonary practice. She studied respiratory sensation and exercise at University of Florida and received a post entry level Master of Health Science degree in p hysical t herapy in 1998. In 2007, she returned to the Doctor of Philosophy in rehabilitation s cience program at University of Florida, studying muscle strength and functional adaptations to inspiratory muscle strength training.