Mechanisms of contractile dysfunction in the senescent rat diaphragm

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Mechanisms of contractile dysfunction in the senescent rat diaphragm
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ix, 77 leaves : ill., photos ; 29 cm.
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English
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Criswell, David S
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Aging -- Physiological aspects   ( lcsh )
Rats -- Physiology   ( lcsh )
Muscle contraction   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1994.
Bibliography:
Includes bibliographical references (leaves 70-76).
Statement of Responsibility:
by David S. Criswell.
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Typescript.
General Note:
Vita.

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University of Florida
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oclc - 33003017
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lcc - LD1780 1994 .C933
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Full Text










MECHANISMS OF CONTRACTILE DYSFUNCTION
IN THE SENESCENT RAT DIAPHRAGM




















By
DAVID S. CRISWELL


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1994














ACKNOWLEDGMENTS


I would like to acknowledge those who have been instrumental in this work;

particularly my mentor, Dr. Scott Powers and my other committee members: Dr. Stephen

Dodd, Dr. Daniel Martin, Dr. Michael Pollock, and Dr. Paul Davenport. They have all

given me invaluable guidance and instruction. Further, Drs. Powers and Dodd allowed me

to have free run of their laboratories and equipment. These experiments could not have

been completed without the technical assistance of my fellow students: Robert Herb, Jeff

DeLott, Jane Eason, and Scott Stetson.

I am especially grateful to Dr. Powers for his constant support, example,

motivation, and friendship throughout our association.

This work was made possible by funding from the American Lung Association-

Florida Affiliate.














TABLE OF CONTENTS


ACKNOWLEDGMENTS.....................................................................ii

LIST OF TABLES...........................................................................v

LIST OF FIGURES .......................................................................vi

ABSTRACT .................................................. ............................. viii

INTRODUCTION. ............................................. ............................

REVIEW OF RELATED LITERATURE ..................................................5..

Introduction. .............................................................................5..
Effects of Aging on Skeletal Muscle .................................................5. 5
Morphological Changes.................................................... ...... 5
Contractile Changes. ............................................ ..............6
Biochemical Changes........................................ ............... 8
Effects of Aging on the Diaphragm............................... ........... .. 11
Possible Mechanisms of the Force Deficits in Aging Skeletal Muscle........... 12
Potential Extrinsic Sources of Age-Related Muscle Dysfunction. .......... 12
Potential Intrinsic Mechanisms of Muscle Force-Generating Deficits...... 14
Summary. ........................................................................... 17

MATERIALS AND METHODS ................................................. ....... 18

Animals ............................................................................. 18
Experimental Design. .................................................. ... ............ 18
Experimental Protocol. ................................................................. 19
Experimental Procedures. ......................................... .......... ... 20
Diaphragm Strip in vitro Contractile Measurements ........................ 20
Biochemical Measurements. .................. ....... ............. ............22
Histochemistry. ................................................................. 24
Cross-Sectional Area and Specific Force Corrections. ...................... 27
Diaphragmatic Single Fiber Analysis ........................................ 28
Statistical Analyses. .................................................................... 30

RESULTS ........................................................................ ...... 31

Morphometric Characteristics. ...................................................... 31
Contractile Properties of the Costal Diaphragm. .............. ................... 31
Muscle Composition Measurements. .................................... .......... 32
Biochemical Measurements .................................................... 32
Histochemical Measurements. ......................... .......................... 33
Mathematical Correction of Diaphragmatic Specific Force ..................... 34
Myofibrillar Protein Correction. ............................................. 34








Muscle Water and Connective Tissue Corrections. .......................... 34
Diaphragmatic Single Fiber Specific Force. .................. ..................... 35
Myosin Heavy Chain Isoforms and Myofibrillar ATPase Activity. .............. 36

DISCUSSION............................................. ............................. 58

Overview of Principle Findings ...................................................... 58
Age-Related Changes in Diaphragmatic Force Production ....................... 58
Age-Related Changes in Diaphragm Composition. ............... ............... 59
Diaphragm Cross-Sectional Area and Specific Force Corrections. ............. 62
Diaphragmatic Skinned Single Fiber Measurements. ............................. 63
Age-Related Changes in Shortening Velocity of the Diaphragm. ................. 65
Summary and Conclusions. .......................................................... 68

REFERENCES.................................... ..................................... 70

BIOGRAPHICAL SKETCH............................................................... 77













LIST OF TABLES


Table page

1. Morphometric characteristics of adult (9 month old)
and senescent (26 month old) F-344 rats........................................38

2. In vitro contractile properties of costal diaphragm strips
from adult (9 month old) and senescent (26 month old) F-344 rats ............39

3. Protein composition of costal diaphragm and plantaris
from adult (9 month old) and senescent (26 month old) F-344 rats ............42

4. Histochemical quantification of connective tissue cross-
sectional area in muscle from adult (9 month old)
and senescent (26 month old) F-344 rats .........................................44

5. Histochemically determined succinate dehydrogenase (SDH)
activity and cross-sectional areas of single fibers from costal diaphragm
of adult (9 month old) and senescent (26 month old) F-344 rats ................45

6. Costal diaphragm Po normalized to muscle cross-sectional
area (CSA) and to CSA corrected for non-contractile material
in adult (9 month old) and senescent (26 month old) F-344 rats ...............46

7. Cross-sectional area and calcium-activated force
of costal diaphragm skinned single fibers from adult
(9 month old) and senescent (26 month old) F-344 rats ........................50

8. Maximum calcium-activated specific force (mN mm-2) of costal
diaphragm skinned single fibers classified by MHC phenotype from
adult (9 month old) and senescent (26 month old) F-344 rats....................51

9. Myosin heavy chain (MHC) isoform composition (percent
of total MHC pool) of costal diaphragm and plantaris
from adult (9 month old) and senescent (26 month old) F-344 rats ...........55














LIST OF FIGURES


Figure page

1. Force-velocity relationships for costal diaphragm in vitro strips
from adult and senescent F-344 rats. ...........................................40

2. In vitro power curves as a function of specific force for combined
adult and combined senescent diaphragm strips. ................................41

3. Photographs of picrosirius red/acid fuchsin collagen staining
in representative histochemical sections (magnification=400x) from
a) adult and b) senescent costal diaphragms. .......................... ....... 43

4. Specific isometric force of costal diaphragm in vitro strips
expressed both as N cm-2 of muscle cross-sectional area (CSA)
and as N cm-2 of myofibrillar CSA. Values are presented for the
senescent group (SEM) as a percentage of the adult values.
P-value represents statistical comparison of senescent and adult values. ......47

5. Specific isometric force of costal diaphragm in vitro strips
expressed both as N cm-2 of muscle cross-sectional area (CSA)
and as N cm-2 of connective tissue (C.T.)-free CSA, dry CSA,
and dry, C.T.-free CSA. Values are presented for the
senescent group (SEM) as a percentage of the adult values. ..................48

6. Correlational analysis of the relationship between costal
diaphragmatic specific force and a) relative dry mass of the costal
diaphragm (mg/g) and b) relative connective tissue content (mg/g)
of the costal diaphragm. ............................................................49

7. Specific isometric force of costal diaphragm in vitro strips
expressed as N cm-2 of muscle cross-sectional area (CSA)
compared to maximum calcium-activated specific force of skinned
single fibers expressed as mN mm-2. Values are presented for
the senescent group (SEM) as a percentage of the adult values.
P-value represents statistical comparison of senescent and adult values. ......52

8. Photograph of sodium dodecyl sulfate-polyacrylamide gel
electrophoresis of isolated single fibers from the costal diaphragm.
Lane 1: Diaphragm fiber bundle expressing all four MHC isoforms.
Lane 2: Single fiber expressing type IIdx MHC.
Lane 3: Single fiber expressing type I MHC. ....................................53

9. Comparison of costal diaphragmatic myofibrillar ATPase activity
from adult and senescent animals. Values are means SEM. ..................54









10. Photograph of a typical sodium dodecyl sulfate-polyacrylamide
gel electrophoresis of bundles of fibers from the costal diaphragm
illustrating the region of the gel containing the myosin heavy chains.
Lane 1 contains a senescent sample; lane 2 contains an adult sample...........56

11. Correlational analysis of the relationship between costal
diaphragmatic Vmax and a) myofibrillar ATPase activity of the costal
diaphragm (nmol min-1 mg-1) and b) relative type ib MHC content
of the costal diaphragm ........................................................... 57













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



MECHANISMS OF CONTRACTILE DYSFUNCTION
IN THE SENESCENT RAT DIAPHRAGM

By

David S. Criswell

August 1994

Chairman: Scott K. Powers
Major Department: Exercise and Sport Sciences

The purpose of these experiments was to test the hypothesis that age-related

alterations in muscle composition cause the known age-related diaphragmatic specific force

deficit. Intrinsic properties of the myofibrillar proteins and excitation-contraction (E-C)

coupling in the rat costal diaphragm were assessed via an isolated skinned single fiber

preparation. Secondly, the hypothesis that the age-related increase in diaphragmatic

shortening velocity (Vmax) is caused by alterations in the senescent diaphragm myosin

heavy chain (MHC) phenotype was examined. Isometric twitch and tetanic contractile

properties as well as isotonic force-velocity relationships were measured in vitro on costal

diaphragm strips from adult (9 month old; n=12) and senescent (26 month old; n=13)

specific pathogen free-barrier protected Fischer 344 rats. Costal diaphragm myofibrillar

protein concentration, calcium-activated myosin ATPase activity, MHC composition,

relative water content, connective tissue (C.T.) concentration, skinned single fiber maximal

specific force (specific Fo), succinate dehydrogenase activity, and histochemical relative

connective tissue cross-sectional area (CSA) were also measured. Diaphragmatic maximal

tetanic force (Po) normalized to strip CSA was 16.4% lower in the senescent diaphragms








(21.030.4 N.cm-2) compared to the adult (25.160.5 N.cm-2) (P<0.001). There was a

trend for myofibrillar protein concentration to be lower (12.5%) in the senescent

diaphragms compared to the adult (P=0.09), while diaphragm water content was

significantly higher in the senescent group (P<0.01). Histochemical analysis of C.T. CSA

revealed a 19.3% increase in the relative contribution of C.T. to the total CSA in the

senescent diaphragms (P=0.01). Normalizing diaphragmatic Po to dry mass, C.T.-free

CSA eliminated the senescent specific force deficit (P>0.05). In agreement, normalizing Po

to myofibrillar protein CSA resulted in no age group differences in specific force (P>0.05).

Specific Fo of costal diaphragm fibers did not differ between age groups (P>0.05).

Diaphragmatic Vmax was 17.5% higher in the senescent diaphragms compared to the adult

(P<0.001). This change was accompanied by a three-fold increase in the relative proportion

of type IIb MHC (P<0.001). Correlational analysis indicated that -30% of the variance in

Vmax could be predicted by changes in type IIb MHC composition (r=0.56; P<0.05).

These data support the hypothesis that the decline in in vitro maximal specific force

observed in the senescent costal diaphragm can be explained by age-related alterations in

the composition of the diaphragm muscle and the resulting reduction in myofibrillar protein

concentration.














INTRODUCTION


The diaphragm is the primary muscle of inspiration and is essential for maintenance

of normal ventilation in mammals. It is well established that humans exhibit an age-related

decline in maximal inspiratory pressure (1, 2) suggesting that the diaphragm may be subject

to aging effects similar to other skeletal muscles. Further, a recently published report (3) as

well as data from our laboratory indicate that the senescent rodent displays a reduction (as

compared to young animals) in the maximal diaphragmatic force generating capacity

normalized to the cross-sectional area of the muscle (i.e. specific force). This age-related

diaphragmatic dysfunction may reduce the ability of the aging individual to perform

coughing maneuvers and/or to maintain normal ventilation during an acute challenge such

as exercise or an asthma attack.

These experiments were designed to define the functional and phenotypic changes

associated with aging in the diaphragm of male Fischer 344 rats, and to determine the

intrinsic cellular mechanisms) responsible for the observed age-related decline in the

maximal isometric specific force of the diaphragm. Since the age-related diaphragmatic

specific tension deficit is observed in an in vitro preparation where the muscle is stimulated

to contract via an electrical field, the underlying mechanism must be intrinsic to the muscle.

In theory, one the following intrinsic mechanisms must be altered by the aging process,

thereby, resulting in a decrease in the maximal isometric specific tension of diaphragm

muscle: 1) myofibrillar protein concentration (i.e. the number of force-generating units in

parallel per unit area); 2) intrinsic properties of the contractile proteins (i.e. the amount of

force produced per force-generating unit); 3) excitation-contraction (E-C) coupling (e.g. the








amount of free calcium in the myoplasm during maximal activation); or 4) some

combination of these.

The amount of force generated by a muscle is dependent on the number of half-

sarcomeres in parallel (4). Therefore, to accommodate differences in the number of parallel

cross bridges in parallel due to the size of the muscle, force generation is typically

expressed relative to the cross-sectional area of the muscle (specific force). However, this

procedure is limited by the assumption that the relative contribution of myofibrillar protein

to the total muscle cross-sectional area remains constant. In fact, it has recently been

established that hindlimb unweighting and denervation which result in a reduction in

specific force also result in a reduction in myofibrillar protein concentration and that this

accounts for much of the specific force deficit (5). This suggests the possibility that the

observed age-related diaphragmatic specific force deficit may also be explained by a

reduction in myofibrillar protein concentration. Alternatively, hypertrophied muscle caused

by synergist ablation results in a reduction in specific force without a change in myofibrillar

protein concentration (5); therefore, other factors such as E-C coupling and/or intrinsic

properties of the contractile proteins can also mediate a specific force deficit under certain

conditions.

The potential contribution of altered myofibrillar protein concentration to the age-

related specific force changes observed in in vitro diaphragm strips from adult (9 mon. old)

and senescent (26 mon. old) Fischer 344 rats is examined in the present study. This is

accomplished directly by analyzing the myofibrillar protein concentration of samples from

adult and senescent diaphragms. A reduction in the myofibrillar protein concentration

necessitates an increase in non-contractile volume. Therefore, the present experiments also

sought to determine which specific muscle compartments) (i.e. myofibrillar protein,

connective tissue and/or water) were altered by the aging process. After determining the

concentration of each component, the CSA of the separate compartments were calculated by

modifying the formula used to calculate average total muscle CSA (see Materials and








Methods). This allows assessment of the relative importance of changes in each of the

major muscle components in mediating the age-related diaphragmatic specific force deficit.

The potential contribution of age-related alterations in excitation-contraction

coupling to the decline in specific force in the aging diaphragm is assessed by examining

the maximum specific force characteristics of individual skinned single fibers isolated from

the diaphragms of adult and senescent Fischer 344 rats. Since contraction and relaxation of

skinned single fibers is accomplished by directly changing the [Ca++] in the medium

bathing the myofibrils, this technique by-passes the excitation-contraction coupling

mechanism and therefore provides an assessment of its role in the age-related specific force

deficit.

In addition to the specific force deficit observed in the senescent diaphragm, we
have also found a significant increase in maximal velocity of shortening (Vax) of

diaphragm strips from old animals as compared to young adults (unpublished

observations). Therefore, the present experiments were also designed to further document

this paradoxical change and determine if it is mediated by changes in the relative

proportions of the myosin heavy chain (MHC) isoforms and/or biochemically determined

myosin ATPase activity.

The following specific hypotheses were tested:

1) Diaphragmatic myofibrillar protein concentration is less in senescent (26 mon.

old) specific pathogen free-barrier protected (SPF-BP) Fischer 344 rats compared

to adult (9 mon. old) SPF-BP Fischer 344 rats.

2) The observed deficit in maximal specific force generation in senescent rat

diaphragms is explained by the reduction in myofibrillar protein concentration.

3) The increase in maximal isotonic shortening velocity in senescent diaphragm

strips will be accompanied by, and significantly correlated with an age-related

increase in type II MHC expression and myofibrillar ATPase activity.





4

4) Maximal force generation per CSA in isolated skinned single fibers from

senescent rat diaphragms does not differ from the specific force generation of fibers

from adult diaphragms.














REVIEW OF RELATED LITERATURE


Introduction


A decline in locomotor skeletal muscle strength and speed of contraction with

advancing age has been commonly observed in both humans and animals. Indeed, an

overall loss of muscle mass is evident in senescence with the aging effects being most

prominent in muscles containing higher proportions of fast-twitch fibers. This age-related

atrophy and loss of muscle performance has the potential to severely reduce the functional

capacity of an individual.

It is the purpose of this review to discuss the known effects of aging on skeletal

muscle with emphasis on the diaphragm. Further, this review will examine several possible

mechanisms underlying the age-related muscle dysfunction observed in humans and

animals with special attention placed on the force deficits reported in aging skeletal muscle.



Effects of Aging on Locomotor Skeletal Muscle


Morphological Changes


Among the most consistent findings in skeletal muscle with advancing age is a

general muscle atrophy (6, 7, 8, 9). Theoretically, this atrophy could be the result of a

reduction in fiber number, a decrease in the cross-sectional area (CSA) of some or all fibers

in the muscle, or a combination of these. Lexell et al. (8) studied whole vastus lateralis

muscles autopsied from formerly healthy men ranging in age from 15 to 83 years who had








suffered sudden accidental death. By sectioning across the middle of the entire muscle, they

determined that muscle atrophy begins at early adulthood and progresses throughout life.

The atrophy was associated primarily with a loss of fiber number with all histochemically

defined fiber types being equally susceptible. There was also a significant age-related

reduction in CSA of the type II fibers which contributed to a lesser extent to the overall

atrophy. In contrast to this human study, Daw et al. (7) used the nitric acid digestion

technique to precisely quantify the fiber numbers in the soleus and extensor digitorum

longus (EDL) muscles of young and old rats. These researchers concluded that a loss of

fiber number could explain only approximately 25% of the age-related muscle atrophy in

the rat. Presumably the remaining 75% of the overall observed atrophy must have been the

result of decreases in the CSA of fibers. Compared to a loss of fiber number, there are

more studies implicating the reduction in CSA of type II fibers in the age-related muscle

atrophy (10, 11); however, this may be due, in part, to the difficulty in making

measurements of total fiber number. Collectively, it seems possible that both mechanisms

operate to some extent, the importance of which may vary between different species.


Contractile Changes


Of primary importance in the study of aging in skeletal muscle are the changes in

muscle contractile properties. Numerous investigators have reported age-related changes in

the force generating properties of muscle in both humans (6, 11) and animals (9, 12, 13,

14). In a large cross-sectional study of 33 young and old men, Klitgaard et al. (6) found

large age-related changes in both functional and biochemical properties of the elbow flexion

and knee extension muscle groups. Functionally, the old subjects exhibited lower values

for maximal isometric force, speed of movement, and specific tension (force per cross-

sectional area). Similarly, Larsson et al. (11) studied isometric and dynamic strength

changes in the quadriceps of 114 male subjects ranging in age from 11 to 70 years. Both








measures of muscular strength did not differ between adult age groups below age 50 years;

however, there was a progressive decline in strength after age 50. Further, the loss of

strength correlated closely with an observed selective atrophy of type II fibers.

Longitudinal studies of the effects of aging on human skeletal muscle are rare; however,

Aniansson et al. (15) reported strength changes in 23 elderly men over a seven year period.

After the seven years, isokinetic strength measures in the vastus lateralis muscle were

decreased 10-22% while the CSA of type IIa and IIb fibers were reduced 14-25%.

Unfortunately, reports of age-related contractile changes in in vitro preparations of

rodent locomotor muscles are less conclusive. In keeping with the consistent finding of

overall muscle atrophy with aging, total tetanic force generation is reduced in senescent

muscles (12, 13, 14). Most studies also demonstrate a prolongation of contraction time

(12, 14), time to peak isometric tension (13), and one-half relaxation time (12, 13, 14).

Another important measure of contractile function is the tension generated by the muscle

per unit of CSA (i.e. specific tension). An age-related reduction in specific tension would

indicate that the force reduction in senescent muscles is due to changes in the contractile

apparatus itself. Klitgaard et al. (13) examined male Wistar rats of different age groups (9,

24, and 29 months) and reported a decrease in specific tension with aging in the soleus

(predominantly slow twitch muscle) and the plantaris (a mixed fiber type muscle). Brooks

and Faulkner (14) reported a reduction in specific tension in the EDL of senescent mice;

however, no difference in specific tension was found in the soleus between adult and

senescent mice. Fitts et al. (12) found no differences in specific tension of the soleus

muscle in Long Evans rats of three age groups (9, 18, and 28 months). Finally, in

contradiction to the majority of the literature, Eddinger et al. (9) reported an increase in the

specific tension of both fiber bundles and isolated skinned fibers from the soleus of

senescent Fischer 344 rats. The explanation for these divergent findings is unclear.

Reports of the maximum rate of unloaded shortening (Vmax) of aging locomotor

muscles in vitro are also equivocal with most studies showing either a decrease in Vmax








with aging (12) or no change in Vmax (14). Again, the report of Eddinger et al. (9)

contradicts the literature by demonstrating an increase in Vmax of fiber bundles from the

senescent rat soleus.

In spite of the variable results presented above, overall there appears to be a

consensus that skeletal muscle becomes slower and weaker with advancing age and that

these changes are due, at least in part, to intrinsic changes in the contractile properties of the

muscle.



Biochemical Changes



Metabolic enzymes. In an effort to better understand the mechanisms underlying the

observed age-related changes in skeletal muscle morphology and function, many

investigators have examined the effects of aging at the protein level using analytical

biochemistry and more recently molecular biology techniques. The activity of metabolic

enzymes was one of the first biochemical measures to be reported with aging. In early

studies of human muscle biopsies, no age-related differences in the activities of oxidative

enzymes were observed (16, 17). More recently, studies that have controlled for the

activity level of the subjects have shown age-related decreases in muscle oxidative enzyme

activities (18) and in vitro maximal rate of oxygen consumption measured in muscle biopsy

samples (19). Animal studies generally indicate that aging of skeletal muscle results in

decreases in the activities of key metabolic enzymes of glycolysis, the Kreb's cycle, and 13-

oxidation (10, 20, 21, 22).

Histochemical fiber types. Another variable reported to change with advancing age is

the distribution of ATPase histochemically determined fiber types (23). As mentioned

earlier, Lexell et al. (8) concluded that the loss of muscle fibers with age affected all fiber

types equally; nevertheless, this issue remains equivocal. Larsson et al. (11) reported an

increase in the percent by number of type I fibers in the gastrocnemius of aging humans








while data from Klitgaard et al. (6) failed to show any changes in fiber type distributions in

biopsies from senescent human vastus lateralis or biceps brachii. It should be emphasized

that most human studies of muscle fibers (e.g. Larsson et al. (11) and Klitgaard et al. (6))

rely on a single needle biopsy and therefore sample only a very small portion of the muscle.

Lexell et al. (8), however, studied cross-sections of the entire muscle and, therefore, would

seem to provide more reliable data concerning the aging effects on fiber type distribution.

Studies of the aging rat indicate a pattern of fiber type shifts from fast (i.e. type Ha

and IIb) to slow (type I). The most consistent finding seems to be an increase in the relative

proportion (both by number and by CSA) of type I fibers in predominantly slow-twitch

muscles like the soleus (9, 20, 24) and the sartorius (25). Aging affects on fiber types in

predominantly fast-twitch muscles are less clear. In the rectus femorus, Kovanen and

Suominen (20) reported an age-related decrease in the number of type IIb fibers and a

concomitant increase in the number of type IIa fibers. Larsson et al (25) and Eddinger et al.

(24) failed to show any age-related change in the fiber type distribution of the EDL.

However, in a subsequent report, Eddinger et al. (9) reported an increase in the percentage

(both by number and by CSA) of type Ilb fibers in the EDL muscles of senescent barrier

raised Fischer 344 rats.

Myosin isoform expression. Histochemically determined fiber types are based on the

pH stability/lability of the various isoforms of the myosin heavy chains whereon lies the

myofibrillar ATPase activity. Employing the techniques of immunohistochemistry and

electrophoretic separation of myosin heavy chains (MHC) from individual fibers, it has

been established that some proportion of muscle fibers express more than one MHC and/or

myosin light chain (MLC) isoform (26, 27). This leads to the conclusion that ATPase

histochemistry which classifies fibers into distinct groups and subgroups may overlook

subtle changes in the phenotypic expression of fibers in response to aging or any other

stimulus. This is evident in the report of Klitgaard et al. (6) in which no age-related change

in the distribution of fiber types was observed, but the relative proportion of MHC I was








significantly elevated in comparison to the young group. This overall increase in the

percentage of MHC I in the muscle from aging subjects could be due to the selective

atrophy of type I fibers as reviewed above; however, in a companion paper by the same

authors (27) they report an increase in the incidence of MHC coexpression in the muscle

fibers of elderly subjects. This seems to indicate an age-related change at the level of gene

expression in favor of MHC I as well as possibly the selective loss of CSA from fibers

containing primarily MHC Ha or lib.

Recent evidence from our laboratory (28) suggests a fiber type specific effect of aging

on skeletal muscle with the general aging trend being to reduce the proportion of the faster

MHC isoform(s) present in the muscle and to increase the proportion of the slower MHC

isoform(s). Specifically, we found a significantly lower percentage of MHC IIb isoform

and a concomitantly higher percentage of MHC's Ia, IIdx, and I in the mixed

gastrocnemius muscle of 24-month-old specific pathogen free-barrier protected (SPF-BP)

Fischer 344 rats compared to 4-month-old rats. Further, the plantaris also showed an age-

related decrease in the percentage of MHC IIb and an increase in the percentage of MHC

Idx. Finally, the soleus demonstrated a complete disappearance of the MHC Ha band in

the old animals and a concomitant increase in the percentage of MHC I (28). These data are

in agreement with recently published reports of increases in the relative proportion of MHC

Idx with concomitant decreases in MHC IIb proportions in aging locomotor muscle from

non-barrier protected rats (29, 30).

Myofibrillar ATPase activity. Quantitative myosin ATPase activity is known to

correlate closely to the speed of shortening of the intact muscle as well as to the MHC

profile of the muscle (4). The evidence indicates agreement between this measure and

measures of Vmax and MHC profile. In other words, ATPase activity decreases with age

(31, 32, 33) which corresponds to a shift in MHC from fast to slow and a decrease in

Vmax. It should be noted that work by Florini and Ewton (34) using Fischer 344 rats raised

in a specific pathogen-free barrier protected colony has argued that the changes in








biochemical and functional properties observed in aging muscle may be secondary to

exposure to some unknown environmental pathogen and therefore, not directly the result of

aging. Specifically, Florini and Ewton (34) reported no difference between the myofibrillar

ATPase activity of skeletal muscle in young and old barrier protected rats.



Effects of Aging on the Diaphragm


Although limited data exists, the rodent diaphragm may respond to aging in a manner

similar to that of other skeletal muscles. Zhang and Kelsen (3) reported a general contractile

dysfunction in the diaphragms of aging golden hamsters with reductions in specific tension

and Vmax and a prolongation of the time to peak tension and one-half relaxation time.

Gosselin et al. (35) reported an age-related shift in MHC isoforms from fast to slow in both

the crural and costal regions of the rat diaphragm. It should be noted that these authors did

not separate the various isoforms of fast MHC (IIa, IIdx, and IIb). In fact, a more recent

report from Gosselin et al. (36) indicated a significant increase in the relative proportion of

MHC IIb and concomitant decreases in the proportions of MHC Ia and IIdx in the

diaphragm of senescent (24 mon. old) SPF-BP Fischer 344 rats compared to 6 mon. old

adults. Recent work in our laboratory employing the in vitro diaphragm strip preparation

has confirmed an age-related increase in diaphragmatic type IIb MHC proportion and has

shown significant increases (-25%) in the Vmax of 24 mon. old SPF-BP Fischer 344 rats

as compared to 4 mon. old rats. However, maximal tetanic isometric specific tension was

found to be reduced (-11%) in the 24 mon. old diaphragms (unpublished observations).

This age-related increase in Vmax appears to be peculiar to the diaphragm. Since the

diaphragm must maintain its work load throughout life, perhaps an age-associated

reduction in specific tension could not be met with a reduction in usage of the muscle, as

would be possible in locomotor muscles. The MHC shift towards type IIb and the resulting

right shift of the force/velocity relationship may serve to increase the power output of the








diaphragm at any given load. The possible mechanisms) for this suggested adaptation

remains to be elucidated.

Interestingly, there appears to be no age-related decline in the activities of metabolic

enzymes in the rat diaphragm (35, 37); nor do we find any evidence of fiber atrophy in any

fiber type in the senescent rat diaphragm (38, 39). Presumably this is due to the chronic

activity of the diaphragm and may indicate that the work of breathing does not change

significantly with aging in the rat.



Possible Mechanisms of the Force Deficits in Aging Skeletal Muscle


The remainder of this review will focus on the age-related force deficits reported in

locomotor and diaphragm muscle as reviewed above.


Potential Extrinsic Sources of Age-Related Muscle Dysfunction


The observed changes in skeletal muscle occurring with advancing age must have

some underlying mechanismss. These changes may originate within the muscle itself, or

they may be secondary to changes occurring in other organ systems which in turn affect the

muscle tissue. This section will briefly examine age-related changes in the nervous and

endocrine systems and how they may relate to altered muscle function.

Altered neural function. Many of the animal studies reporting a decline in muscle

force production with aging have employed maximal direct or field stimulation of the

muscles, thereby, eliminating direct effects of the nervous system. For this reason, this

review will not deal in depth with age-related changes in the nervous system. Nevertheless,

it is well documented that chronic changes in the neural activity patterns of neurons

innervating skeletal muscles have profound effects on the phenotype and functional

characteristics of the muscle (40). For example, Larsson (41) has suggested that some of








the age-related changes in muscle result from a gradual loss of large alpha-motoneurons

which innervate type II fibers and a subsequent reinnervation of some of these fibers with

smaller motoneurons. Indeed, this could explain the transition of fiber types and MHC

profile from fast to slow that is commonly observed in locomotor muscle with aging as

well as the overall loss of fiber number, however, assuming that there are no differences in

the intrinsic force generating capabilities of the various MHC isoforms (4, 42), this would

not explain the age-related reductions in specific force.

Altered endocrine function. Several hormones have direct or indirect actions on

skeletal muscle. One of the most important of these is thyroid hormone which plays an

important role in the normal development and differentiation of muscle. Nwoye et al. (43)

studied hypo- and hyperthyroidism induced in male rats and reported that thyroid hormone

exerted its effects on skeletal muscle even in the absence of normal neural innervation,

thereby demonstrating a direct action of thyroid hormone on muscle. Further, Izumo et al.

(44) documented changes in the specific mRNA's associated with the various isoforms of

MHC with hypo- and hyperthyroidism. These changes were found to be tissue specific

with hyperthyroidism resulting in an increase in the mRNA of the MHC isoform associated

with the greatest ATPase activity. For example, in the soleus which contains MHC I

(-90%) and MHC IIa (-10%), hyperthyroidism resulted in an increase in the mRNA for

MHC Ia and a decrease in the mRNA for MHC I. Conversely, in the EDL which contains

MHC Ha (-10%) and MHC IIb (-90%), hyperthyroidism resulted in an increase in the

mRNA for MHC IIb and a decrease in the mRNA for MHC IIa. Hypothyroidism was

found to exert the opposite effects. These findings seem to indicate that thyroid hormone

exerts its effects on skeletal muscle by directly altering the phenotype of individual muscle

fibers.

With regard to force development, both hyperthyroidism (45) and hypothyroidism

(46, 47, 48) have been shown to cause locomotor muscle atrophy and therefore a reduction

in the total force production of skeletal muscles as compared to euthyroid controls.








However, when the force is normalized to CSA of the muscle, no differences were found.

In contrast to this, data from our laboratory indicates a reduction in the maximal specific

tension of diaphragm strips from young adult hypothyroid rats (unpublished observations).

Although it has been established that the aging rat exhibits a progressive decline in mean

serum thyroid hormone (49, 50), this would not appear to explain the maximal specific

tension deficits observed in senescent locomotor muscle. In the diaphragm, however, it

appears that an age-related hypothyroidism could contribute to the reduction in specific

tension, but would seem to contradict our observed increase in MHC IIb expression.

In addition to thyroid hormone, other hormones and growth factors are known to

exert effects on skeletal muscle. Logically, age-related changes in any of these factors could

play a role in the aging of muscle. The insulin-like growth factors, specifically IGF-I, have

been shown to be closely linked to skeletal muscle growth and differentiation (51).

Addition of IGF-I to the culture medium of primary myofibers produced fibers with

significantly larger diameters, higher myosin synthesis rates, a longer myosin half-life,

more myonuclei per fiber length, and 187% increase in the accumulation of myosin (52).

Sonntag et al. (53) have demonstrated that serum IGF-I is reduced in the senescent rat,

thereby suggesting a possible relationship between IGF-I (or growth hormone) and aging

muscle.



Potential Intrinsic Mechanisms of Muscle Force-Generating Deficits



Experimental models in which a skeletal muscle is removed from an animal and

electrically stimulated (in vitro) eliminate the acute influences of factors extrinsic to the

muscle. Therefore, the age-related force deficit in skeletal muscle studied in vitro must be

due to changes intrinsic to the muscle itself. It should be noted that any potential intrinsic

changes may be the results of chronic age-related changes in extrinsic factors as reviewed

in the previous section. The following paragraphs will address several intrinsic








mechanisms which may be responsible for the observed in vitro force deficit in senescent

skeletal muscle.

Reduction in myofibrillar protein concentration. The force production of a muscle is

directly related to the number of contractile units in parallel (4). Therefore, to compare the

intrinsic force generating capacity of two muscles, the total force produced is typically

expressed relative to the CSA of the muscle (specific tension). However, the CSA of a

muscle includes not only the contractile proteins but also fluid and non-contractile material.

The extracellular space occupies 8-25% of the CSA of skeletal muscle from young adult

rats (54). With this in mind, the comparison of maximal specific tension between two

muscles would only be valid if both muscles exhibited similar ratios of contractile/non-

contractile tissue.

One possible intrinsic mechanism for the reduction in skeletal muscle specific tension

with aging is a reduction in this ratio (i.e. a decrease in the concentration of myofibrillar

protein accompanied by increases in the concentration of non-contractile tissue). It is well

established that skeletal muscle connective tissue increases in senescence over young adult

muscles. Biochemical determination of collagen concentration in the soleus and EDL

muscles of 24-25 month old rats indicates large increases in collagen accumulation (55,

56). Further, quantitative histochemical determination of endomysium connective tissue has

shown 50-75% increases in the connective tissue content per unit area in senescent rat

soleus and EDL muscles (56, 57). This relative increase in connective tissue could explain

much of the specific force deficit associated with aging. The relative water content of aging

muscle could also change the ratio of contractile/non-contractile material. It has recently

been reported that hindlimb unweighting in rats, which is known to cause a reduction in

specific force generation of the soleus, also causes an increase in the interstitial fluid

volume of this muscle (58). This increase in fluid content along with a reduction in the total

content of myofibrillar protein appears to explain the specific force deficit observed in the

unweighted soleus muscles (5, 58). Although no age-related alterations in muscle water








content have been reported in locomotor muscles (14), no data are available for the aging

diaphragm. Therefore, the relative increase in connective tissue and a potential increase in

water content both remain as possible explanations for the diaphragmatic specific force

deficit associated with aging.

Finally, the ratio of contractile/non-contractile tissue could also be altered by a

reduction in the expression of the myofibrillar proteins. Jaiswal and Kanungo (59) reported

that transcription rate and mRNA levels of the skeletal alpha actin gene are reduced in 30

mon. old Wistar rats as compared to 6 mon. old rats. These researchers found no age

differences in the transcription rate or mRNA for the MHC genes. Although limited data

exists, the studies reviewed here suggest that aging in skeletal muscle is associated with a

down-regulation of some of the myofibrillar protein genes accompanied by no change or

possibly an up-regulation of some of the non-contractile proteins.

Intrinsic properties of the myofibrillar proteins. Another potential mechanism for the

age-related force deficit in muscle is the possibility of intrinsic aging changes in the skeletal

muscle proteins. Srivastava and Kanungo (33) reported an age-related decline in the

availability of titratable thiol groups in myosin isolated from rat skeletal muscle. Since the

thiol groups associated with the MHC molecule are essential for normal myosin ATPase

activity, these researchers concluded that age-related changes in the conformational shape

of the MHC molecule may explain the decline in myofibrillar ATPase activity and in vitro

shortening velocity in senescent skeletal muscle (33). If aging does indeed alter the intrinsic

properties of the myofibrillar proteins, this could also contribute to the reduction in specific

tension independent of changes in the concentration of myofibrillar protein.

Excitation-contraction coupling mechanism. Stimulation of a muscle via a nerve or

artificially via electrical current results in muscular contraction by stimulating the

sarcoplasmic reticulum (SR) to release free calcium into the myoplasm. This represents a

third independent point in the contraction mechanism where aging could affect maximal

specific force production. Theoretically, aging could affect multiple sites in the SR and/or








myoplasm that could result in a reduction in the free calcium level during contraction.

Recent studies have reported that SR vesicles isolated from the skeletal muscle of old rats

exhibit a lower protein content and a reduced maximal calcium storage capacity when

compared to vesicles from young rats (60, 61).



Summary


In conclusion, the effects of aging on skeletal muscle may originate extrinsically (e.g.

from age-related changes in the neural or endocrine systems) or intrinsically (e.g. aging

effects on the muscle genome). In either case, however, the intrinsic properties of the

muscle are affected such that maximal force generation per CSA is reduced. This could

occur by 1) decreasing the content of myofibrillar protein in the aging muscle, 2) increasing

the content of non-contractile tissue in the muscle, 3) altering the structure and therefore the

function of the myofibrillar proteins in aging muscle, 4) altering the excitation-contraction

coupling mechanism in aging muscle, or 5) a combination of some or all of these factors.














MATERIALS AND METHODS


Animals


Adult (9 mon. old) and senescent (26 mon. old) specific-pathogen-free, barrier-

protected (SPF-BP) male Fischer 344 rats were obtained from the National Institute of

Aging (NIA). An animal model was chosen because the invasive nature of the proposed

experiments precludes the use of human subjects. The Fischer 344 rat is widely accepted

and recommended as a standard model of aging (62). Further, the physiological function

and fiber type distribution of the rat diaphragm resembles the human diaphragm. SPF-BP

rats were chosen, as suggested by Florini and Ewton (34), to ensure that the measured

effects were due to aging rather than to undocumented diseases.



Experimental Design


To test the hypotheses listed earlier (Introduction), healthy young adult and senescent

rats were examined for diaphragmatic in vitro contractile properties, myofibrillar protein

content, muscle morphometry, muscle water content, muscle connective tissue

concentration, and single skinned fiber maximal specific tension. Twenty-five male SPF-

BP Fischer 344 rats were individually housed and fed rat chow and water ad libitum while

being maintained on a 12/12 hour light/dark photoperiod for -14 days prior to beginning

the experiments. During this 14 day period, animals were handled daily to reduce contact

stress. The animals were then assigned to one of two experimental groups based on age.

Group 1) adults (9 months old; n = 12)

Group 2) senescent adults (26 months old; n = 13)








This project was approved by the University of Florida Institutional Animal Care and

Use Committee (IACUC) and followed the guidelines for animal use established by the

American Physiological Society.



Experimental Protocol


Animals were euthanized by intraperitoneal injection of sodium pentobarbital

(90mg/kg) and the entire diaphragm quickly removed and placed in a dissecting dish. A

small strip of the costal diaphragm (-0.3 x -2.0 cm) was carefully cut leaving a portion of

the central tendon and the rib attachment on the ends. This strip was utilized for in vitro

measurements of contractile function; specifically, peak isometric twitch tension, one-half

relaxation time, maximal rate of twitch tension development, peak isometric tetanic tension,

and isotonic force-velocity relationship. All contractile measurements were conducted with

the muscle at Lo.

The remaining diaphragm was carefully trimmed of fat and connective tissue

(including the central tendon), blotted and weighed on a Mettler analytical balance. The

costal portion of the diaphragm was then divided into five pieces: piece #1 was frozen in

liquid nitrogen and stored at -700 C for subsequent measurement of myofibrillar protein

concentration, Ca+-activated myosin ATPase activity, and myosin heavy chain profile;

piece #2 was weighed and frozen in liquid nitrogen for subsequent measurement of muscle

water content; piece #3 was frozen in liquid nitrogen for subsequent measurement of

connective tissue concentration; piece #4 was placed on aluminum foil at resting excised

length and frozen in liquid nitrogen, for subsequent histochemical sectioning (ATPase fiber

typing, quantitative histochemical succinate dehydrogenase activity, and connective tissue);

piece #5 was stored in relaxing solution at 50 C for subsequent single fiber measurements.

In order to provide a locomotor muscle comparison to the effects of aging on the

diaphragm, the right and left plantaris muscles were also removed, weighed, frozen in








liquid nitrogen and stored at -70 C for measurement of myofibrillar protein concentration,

myosin heavy chain profile, Ca+-activated myosin ATPase activity, muscle water, and

connective tissue content.



Experimental Procedures


Diaphragm Strip in vitro Contractile Measurements



Muscle preparation. After reaching a surgical plane of anesthesia, the entire

diaphragm was removed and placed in a dissecting chamber containing a Krebs-Hensleit

solution equilibrated with a 95% 02 / 5% CO2 gas. A muscle strip was dissected out from

the ventral costal region and suspended vertically between two light weight plexiglas

clamps in a jacketed tissue bath containing Krebs-Hensleit and 12p.M d-tubocurarine to

produce complete neuromuscular blockade. One clamp was fixed while the other was

connected to a transducer (Cambridge Technology, model 300B). The Cambridge

transducer is a combined force and position transducer and is capable of monitoring both

isometric and isotonic contractions. The force on the lever can be electronically controlled

from 0 to 80 g while the length change of the muscle is monitored, or the length of the

muscle can be held constant while force is monitored. The jacketed tissue bath was aerated

with gas (95% 02 / 5% CO2), pH was maintained at 7.4, and the osmolality of the bath

was 290 mOsmol. Temperature in the organ bath was maintained at -24 0.50C since

higher temperatures result in a deterioration of muscle function (63). After 15 min

equilibration in the bath, the muscle strip was field stimulated along its entire length with

platinum wire electrodes using a modified Grass Instruments S48 stimulator. A

supramaximal stimulation voltage was used equal to -150% of the minimum voltage

necessary for maximal activation of the muscle (-140 volts). We have demonstrated in pilot








experiments that this method of stimulation results in maximal force generation when

compared to direct muscle stimulation using stainless steel electrodes.

After the 15 minute equilibration period, the muscle strip was adjusted to its optimum

contractile length (Lo) at which maximal tetanic tension is obtained; this was accomplished

by systematically adjusting the length of the muscle using a micrometer while evoking

tetanic contractions (140 volts, 330 msec train duration, 50 Hz, and 2 msec pulse width).

After finding Lo, a series of isometric twitch contractions (-6-8, separated by 2 min rest

intervals), a series of isometric tetanic contractions (-4-6, separated by 3 min rest

intervals), and a series of isotonic tetanic contractions (12-15, separated by 2 min rest

intervals) were performed in random order. Following completion of the protocol, Lo was

measured using calipers with the strip still suspended between the two plexiglas clamps.

Isometric twitch contractions. Peak isometric twitch tension was determined from a

series of single pulses (140 volts, 2 msec duration). The Cambridge transducer output was

amplified and differentiated by operational amplifiers and underwent A/D conversion for

analysis using a computer based data acquisition system (GW Instruments-Series II). In

addition, half relaxation time (i.e. the time required for force to fall from maximum to half-

maximum; (1/2 RT)), and maximal rate of tension development were determined by

computer analysis of the force transduced output.

Peak isometric tetanic tension. A series of isometric tetanic contractions was produced

using a supramaximal (140 V) stimulus train of 50 Hz, 2 msec pulse width, and 1000 msec

duration (modified Grass Instruments S48 stimulator). Force was monitored by the

computerized ergometer previously described. Each tetanic contraction was separated by a

three minute recovery period.

Force-velocity measurements. The relationship between force and muscle shortening

velocity was assessed by measurement of shortening velocity (Cambridge transducer model

300B) at -12 isotonic loads (100 msec trains of 2 msec pulses at 50Hz) over the range of
-2-90% of maximal isometric tension (Po). The force-velocity data was fit to the Hill








equation (64) using least-squares techniques, and maximal velocity of unloaded shortening

(Vmax) was determined by solving for velocity when force equals zero.


Biochemical Measurements


Myofibrillar protein concentration. Myofibrillar protein was isolated using a

modification of the myofibril extraction technique described by Solaro et al. (65);

myofibrillar protein concentration was then determined using the biuret technique of

Watters (66). Briefly, the diaphragm and plantaris portions designated for myofibrillar

protein analysis were thawed and placed in a petri dish on ice. The muscle was then

carefully cleaned of fat and tendon and scissor minced. Precisely 100 mg of muscle was

then homogenized using a glass on glass homogenizer in 4 ml of sucrose buffer (250 mM

sucrose, 100 mM KCI, 5 mM EDTA, 20 mM Tris, pH = 6.8) and centrifuged for 15 min.

at 2500 x g. The supernatant was discarded and the pellet suspended in a KCI buffer

containing Triton X-100 to eliminate membrane ATPase components (175 mM KC1, 0.5%

Triton X-100, 20 mM Tris, pH = 6.8). This was centrifuged for 10 min at 2500 x g and

the supernatant discarded. The pellet was again suspended in the KC1 buffer containing

Triton X-100 and centrifuged at 2500 x g. This process was repeated in a KC1 buffer (175

mM KCI, 20 mM Tris, pH = 7.0) yielding a myofibrillar protein pellet of sufficient purity

for quantitative assessment of myofibrillar concentration (46).

This technique does not separate the insoluble connective tissue proteins from the

myofibrillar proteins. Since connective tissue concentration changes with aging, this would

not represent a constant error. Therefore, a more accurate myofibrillar protein concentration

was obtained by subtracting the connective tissue concentration (mg/g) as determined by

the method of Segal et al. (67) from the myofibrillar protein concentration (including

connective tissue) (mg/g) as determined by the method described above. The coefficient of

variation for this technique is -7% in our laboratory.








Calcium-activated mvosin ATPase activity. Immediately after measurement of

myofibrillar protein concentration, myosin ATPase activity was determined on the samples

using the technique described by Caiozzo et al. (46). Briefly, myofibrillar protein is

incubated in the presence of a maximally activating level of Ca++ (pCa = 4.0), and ATP.

The myosin ATPase enzyme hydrolyzes ATP to form ADP + Pi. Following cessation of

the reaction by addition of trichloroacetic acid, the concentration of Pi is determined via the

Fiske-Subbarow reaction (Sigma Chemical, St. Louis, MO).

Myosin heavy chain composition. The remaining myofibrillar protein (after biuret and

ATPase assays) was used for separation of myosin heavy chain (MHC) isoforms using

sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The myofibrillar protein

was diluted in glycerol to a concentration of -1 mg/ml (the samples can be stored in this

form at -200 C for up to two months). These glycerol samples were further diluted to a

concentration of -0.25 mg/ml in sample buffer containing 62.5 mM Tris (pH=6.8), 1.0%

SDS, 0.01% bromophenol blue, 15.0% glycerol, and 5.0% B-mercaptoethanol. The

protein was then denatured by incubation at -950 C for 3 min.

One to three gig of protein was loaded onto 22 cm vertical gels (Biorad Protean IIxi)

composed of 8% acrylamide and 40% glycerol. The samples were electrophoresed for 20

hours at -50 C using a constant voltage required to attain an initial current of 12 milliamps

per gel (68). Following electrophoresis, the gels were stained with Coomassie Blue R-250

for 30 min and destined in 30% methanol, 7% acetic acid for two hours. The relative

proportions of the myosin isoform bands (percent of total myosin pool) were analyzed

using a computer-based image analysis system (Targa M8 image capture board, Truevision;

Java video analysis software, Jandel Scientific) integrated with a high resolution video

camera (Video World, Inc.). The region of the gel containing the MHC bands was digitized

for each lane and the area and intensity of staining determined in duplicate for each band. In

our laboratory, the coefficient of variation for repeated measurement of relative MHC

composition of a given sample is < 3%.








Total muscle water content. Determination of total water content in a portion of the

costal diaphragm was made using a freeze drying technique incorporating a vacuum pump

with a negative pressure of- ImmHg. The frozen samples were placed in the vacuum

chamber and dried for 24 hours before measuring the dry mass. The dry mass of the

samples was unchanged after an additional 24 hours in the vacuum chamber, thereby

confirming complete drying. Relative muscle water content was calculated from the

difference between the wet weight of the diaphragm section (taken when the diaphragm

was removed from the animal) and the dry weight of the same section.

Connective tissue concentration. Muscle samples were analyzed for total protein

concentration and connective tissue concentration using the techniques described by Segal

et al. (67). Briefly, muscle sections were homogenized in 0.9% NaCl and allowed to stand

18 hours in 0.05 M NaOH to solubilize nonconnective tissue. Total muscle protein

concentration was measured from a sample of the intact digest. The remaining NaOH

digest was centrifuged for 15 min at 4000 x g to sediment collagenous protein. The protein

concentration of the supernatant was measured and the connective tissue protein

concentration calculated as the difference between protein concentration of the intact digest

and that of the digest supernatant. This technique has been shown to be a sensitive

measure of muscle connective tissue (54, 69).


Histochemistry


Succinate dehydrogenase. A portion of the costal diaphragm was blotted dry and

carefully placed flat on a square of aluminum foil. The muscle was then frozen by dipping

the foil directly into liquid nitrogen. Care was taken to insure that all samples were frozen at

the same unstressed excised length to avoid length-mediated variations in fiber cross-

sectional area. Frozen sections (10 p[m thickness) of the diaphragms were cut using a

cryostat (Reichert-Jung) at 200 C. The activity of succinate dehydrogenase (SDH) was








quantitatively determined in individual fibers of the diaphragm cross-sections using the

technique of Blanco et al. (70) and analyzed using a light microscope (Nikon-Alphaphot)

interfaced with a computerized image analysis system. Briefly the rationale and techniques

for this procedure are as follows. Since SDH is a mitochondrial membrane-bound enzyme,

it will not diffuse out of histochemically prepared cross-sections. This makes SDH a good

candidate for quantitative histochemical analysis. The principle of the Blanco et al. (70)

assay involves the progressive reduction of nitroblue tetrazolium (NBT) by electrons

released from the oxidation of succinate (the SDH reaction) to form a colored compound

(NBT-dfz) which serves as a reaction indicator.

To measure muscle fiber SDH activity, two to three cryosections were placed on a

cover slip and stored at -200 C until assay. Within 5 min. of removal from the -200 C

environment, the muscle sections were introduced into a Columbia jar containing the

reaction medium (5 mM EDTA, 0.75 mM sodium azide, 1.0 mM methoxyphenazine

methosulphate, 1.5 mM nitroblue tetrazolium, 80 mM succinate, 100 mM phosphate

buffer, pH=7.6). The reaction was stopped at exactly 3 min. by multiple rinses in distilled

water. The cover slips were then mounted and the sections digitized using a computerized

image analysis system (described earlier under "Myosin Heavy Chain Composition")

interfaced with a light microscope. The rate at which the reaction indicator was deposited

within a fiber (i.e. SDH activity) is calculated using the Beer-Lambert equation:

[NBT-dfz] / min. = A O.D. / k x L

Where A O.D. = the optical density change per minute measured at 570 nm (optical density

is defined as log [1/% transmission]), k = the molar extinction coefficient for NBT-dfz

(26,478 O.D. units / mole cm), and L = the length of the light path through the tissue

(10m thickness). The system was calibrated for optical density measurements and cross-

sectional area before and after analysis of each tissue section using neutral density filters

and a stage micrometer, respectively. Approximately 100 fibers from each muscle section

were analyzed. After an image of the muscle section is digitized, a cursor controller is used








to outline individual cells. The O.D. of the cell is determined by the average O.D. of all

pixels contained within the outlined cell. The A O.D. is determined by subtracting the O.D.

of the tissue section incubated without substrate from the measured O.D. of each cell and

dividing by the three minute reaction time. A photograph of a serial section stained for

myofibrillar ATPase was used to identify the fiber type of each cell analyzed for SDH

activity. We have previously reported this technique to be valid and reliable as compared to

a biochemical assay of SDH activity in muscle homogenate (71). Approximately 100 fibers

(30-40 of each fiber type) were sampled from each of ten adult and ten senescent costal

diaphragms.

ATPase fiber typing. Serial sections of the same diaphragms were used for

histochemical ATPase fiber typing (23) based on the acid lability/stability of the various

MHC ATPase isoforms. The relative proportions of the fiber types (I, Ha, and IIb) and

their cross-sectional areas were determined using computerized planimetry calibrated with a

stage micrometer.

Connective tissue histochemistry. The technique of Constantine (72) was used to

stain serial sections for connective tissue. This procedure combines Picro Sirius Red and

Acid Fuchsin to selectively stain both large and small collagen fibers. Further, this

technique has been used to report age-related quantitative changes in connective tissue

content of locomotor muscle cross-sections (57). Briefly, this dye stains connective tissue

bright red while leaving other tissues unstained. When the sections are visualized through a

green filter (570 nm) the connective tissue appears black and the remaining tissue white.

The stained sections were dehydrated, mounted with permount, and analyzed using the

computer based image analysis system by which the video signal of the section was

converted to a graphic image with two levels of gray (i.e. black and white). Computerized

planimetry was then used to quantitatively assess the number of black pixels (i.e. stained

for connective tissue) within a given calibrated area. In order to calculate the relative

proportion of the total CSA taken up by connective tissue for each sample, the entire








diaphragm section was analyzed (5-7 digitized images) excluding damaged or folded

regions if any.


Cross-Sectional Area and Specific Force Corrections


After the length of the muscle strip (Lo) and its mass were carefully determined, the

muscle strip cross-sectional area (CSA) was calculated using the formula:

CSA (cm2) = muscle mass (g) / [muscle length (cm) x muscle density (g/cm3)]

Assuming muscle density = 1.056 g/cm3 (73).

Myofibrillar protein CSA was calculated for the diaphragm strips using the same

formula with the exception that myofibrillar content (g) (i.e. myofibrillar protein

concentration (g/g) x strip mass (g)) was substituted for strip mass. This calculation

assumes that purified myofibrils have the same density as whole muscle. Although this

may not be the case, the difference would be small and should produce only a small and

constant effect on the CSA values obtained for both experimental groups. Likewise,

connective tissue CSA and dry mass CSA were also calculated using this formula

substituting strip connective tissue content (g) and dry mass content (g) for total strip mass,

respectively.

The maximal isometric force generated by each of the diaphragm strips was then
normalized to muscle CSA (specific force (Po)), myofibrillar CSA, connective tissue-free

CSA (strip CSA connective tissue CSA), dry mass CSA, and dry mass, connective

tissue-free CSA (dry mass CSA connective tissue CSA).

The dry mass, connective tissue-free CSA of the strips was also determined

independently by an alternate method employing the histochemical connective tissue

staining procedure. The contribution of connective tissue to the total dehydrated CSA of the

histochemical sections (i.e. cm2 of connective tissue / cm2 dehydrated CSA) was

determined as described above. The connective tissue CSA (cm2) of each strip was then








calculated by multiplying this relative connective tissue CSA (cm2/cm2) by the dry mass

CSA (cm2). Maximal tetanic force was then normalized to the histochemically determined

dry mass, connective-free CSA (dry mass CSA histochemical connective tissue CSA).

The total CSA of the costal diaphragm was calculated for each animal from the total

costal mass and the measured Lo of the in vitro strips. The total potential force production

(N) of each costal diaphragm was then estimated by multiplying the specific Po (N cm-2)

by the total CSA (cm2). (Note: This procedure assumes that all fibers in the costal

diaphragm have the same Lo. This introduces some error into the calculation. However, if

the geometry of the costal diaphragm does not differ between adult and senescent animals,

this would be a small, constant error.)


Diaphragmatic Single Fiber Analysis


Fiber preparation. A bundle of muscle fibers cut from the costal region of the

diaphragm was stored in a relaxing solution containing 7.0 mM EGTA, 1.0 mM free

Mg++, 4.38 mM ATP, 14.5 mM creatine phosphate, 20 mM imidazole, and sufficient KC1

to adjust ionic strength to 180 mM. This solution chemically removes the sarcolemma from

the fibers; fiber bundles can be stored up to 30 days in this solution (74). Activating

solutions of varying calcium concentrations were made by addition of CaC12 to relaxing

solution in the appropriate amounts to yield free calcium concentrations of pCa=4.0 and

pCa=3.0 (75).

Single fiber mechanical measurements. Five to ten single fibers were dissected from

each diaphragm sample for measurement of calcium-activated maximal isometric force

production. Individual fibers were attached on one end to a fixed clamp and on the other to

a sensitive isometric force transducer (Cambridge Technology model 400A) and suspended

horizontally above a plexiglas block containing multiple wells (-400.l/well). A solution

containing no calcium (relaxing solution) and a solution containing a maximally activating








concentration of calcium (pCa = 4.0 to pCa = 3.0) were pipetted into the separate wells

producing a meniscus which protrudes above the top surface of the plexiglas block. The

block was then manually moved such that the fiber was positioned within the meniscus of

the desired solution. At this point, the length of the fiber was set by employing an eyepiece

micrometer to adjust the fiber to 120% of its resting length (resting length was defined as

the point where the slack has just been removed). In approximately 50% of the fibers, the

sarcomere length was also determined by directing the beam of a helium-neon laser

(Spectra-Physics) to the fiber and observing the primary diffraction pattern. The distance

between the primary diffraction lines is inversely proportional to the sarcomere length and

was calibrated by first directing the laser through a grid of known dimensions on a

microscope slide. Sarcomere length was set at 2.5 p.m and maintained at this length during

the force measurements. The force developed during maximal calcium activated

contractions did not differ in the same fiber between the two methods of setting fiber

length. Fiber diameter was measured at multiple sites along the length of the fiber with a

calibrated microscope eyepiece micrometer. Fiber diameter was used to calculate fiber

cross-sectional area, assuming the fiber is a cylinder.

Movement of the fiber from well-to-well followed the sequence: relaxing solution,

calcium solution, relaxing solution; this sequence was repeated four to six times (separated

by 2 min rest intervals) with the fiber being exposed to activating solutions of pCa = 4.0

and pCa = 3.0, to insure maximal activation. The force developed did not differ between

the two calcium concentrations. If the same force could not be achieved ( 5%) at least

three times, the fiber was discarded. Force generation by the fiber was measured and

monitored by the force transducer interfaced to a computer and utilizing the same data

acquisition system described above for diaphragm strip measurements.

Mvosin heavy chain characterization. Following the contractile measurements, the

fibers were dissolved in 10 ptl of sample buffer and incubated at 950 C for 3 minutes to








denature the proteins. The entire volume (10 ptl) was then electrophoresed as described

previously for characterization of MHC profile (68).



Statistical Analyses


Comparisons between experimental groups (adult vs. senescent adult) for each

dependent variable with the exceptions of histochemical SDH data, isolated skinned single

fiber data, and MHC isoform distribution data were made by unpaired Student t-tests.

Histochemical single fiber SDH activities and CSA were analyzed by a 2 x 3 (age x fiber

type) factorial analysis of variance (ANOVA). Isolated skinned single fiber Fo, CSA, and

specific Fo were analyzed by unpaired t-tests for type I and type II fibers with the

Bonferroni procedure applied to correct for multiple comparisons. Myosin heavy chain

isoform distribution was analyzed by a 2 x 4 (age x MHC phenotype) factorial ANOVA

with special contrasts applied to determine where differences occurred. The relationships

between biochemical measures (e.g. myofibrillar protein concentration) and contractile

properties (e.g. maximal tetanic specific tension) were determined by Pearson product

moment correlation. Significance was established at P < 0.05.













RESULTS


Morphometric Characteristics


Mean (SEM) body mass and morphometric characteristics of the diaphragm and

plantaris muscles for both experimental groups are presented in table 1. Body mass did not

differ between groups. Likewise, costal diaphragm mass did not differ between groups,

therefore the ratio of costal diaphragm mass to total body mass did not differ between

groups. Further, the dimensions of the costal diaphragm in vitro strips (length and CSA)

taken at Lo were not different between groups. There was however, a strong trend for

crural diaphragm mass to be higher in the senescent animals as compared to the adults

(P-0.06). Conversely, plantaris muscle mass tended to be lower in the senescent group as

compared to the adult (P=0.07), causing a tendency for the plantaris mass / body mass ratio

to be reduced in the senescent animals (P=0.06).



Contractile Properties of the Costal Diaphragm


Table 2 reports the in vitro contractile properties (meansSEM) of costal diaphragm

strips taken from adult and senescent animals. Maximal tetanic force expressed per cm2 of

strip CSA was 16.4% lower in the senescent diaphragms as compared to the adults.

Assuming a constant fiber length at Lo, the total CSA of each diaphragm was calculated

based on each costal diaphragm mass and the measured Lo of the in vitro strip. Using this

total costal CSA (cm2) and the measured specific force (N cm-2), the potential for total

maximal tetanic force generation (N) for each costal diaphragm was estimated. Since








diaphragmatic mass and strip Lo did not differ between groups and the specific force was

reduced in the senescent group, the estimated total potential force generation by the costal

diaphragm was significantly lower in the senescent animals compared to the adults.

In vitro twitch characteristics (1/2 relaxation time and rate of tension development) did

not differ between the two experimental groups. Analysis of the isotonic force-velocity

relationship fitted with the Hill equation (64) indicated a 17.5% increase in Vmax of the

senescent diaphragm strips as compared to the adult strips (P<0.001). The a/Po ratio

differed statistically between adult and senescent groups (P=0.02) indicating greater

curvature in the force-velocity relationship of the senescent diaphragm strips compared to

the adult strips. The Hill equation was fitted to the force-velocity data from each in vitro

strip individually to calculate Vmax and the a/Po ratio. However, for illustrative purposes,

figure 1 presents the Hill equation fitted to the combined data for each age group. Finally,

maximal power of the diaphragm strips, calculated from the isotonic force-velocity data,

did not differ between age groups. Figure 2 displays the combined data illustrating the

relationship between diaphragmatic power and specific force for each age group.



Muscle Composition Measurements


Biochemical Measurements


Myofibrillar protein concentration, connective tissue protein concentration, and dry

mass per unit wet mass results (meansSEM) are presented in table 3 for the costal

diaphragm and plantaris muscles of adult and senescent rats. Costal diaphragm myofibrillar

protein concentration tended to be lower (P-0.09) in the senescent group and connective

tissue concentration tended to be higher (P=0.06) compared to the adult group. Further,

relative dry mass content (mg dry mass/g wet mass) was significantly lower in the

senescent diaphragms compared to the adults. The total diaphragmatic contents of








myofibrillar protein, connective tissue, and water were calculated by multiplying the

appropriate concentration by the total costal weight for each animal. Mean (SEM) values

(adult vs. senescent) were as follows: total myofibrillar protein: 75.14.2 vs. 70.23.3 mg

(P=0.37), total connective tissue: 20.23.4 vs. 29.63.1 mg (P=0.05), and total water:

53426 vs. 58914 mg (P=0.07).

For the plantaris muscle, myofibrillar protein concentration tended to be lower in the

senescent muscles compared to the adult (P--0.11). Further, connective tissue protein

concentration was significantly higher in the senescent muscles when compared to the adult

plantaris muscles (P=0.02). Finally, plantaris dry mass concentration did not differ

between groups.



Histochemical Measurements



Histochemical determination of connective tissue cross-sectional area. Table 4

presents the relative contribution of connective tissue to the total cross-sectional area of

dehydrated histochemical sections of the costal diaphragm and plantaris muscles from adult

and senescent rats. Figure 3 illustrates representative cross-sections of adult and senescent

costal diaphragms stained for collagen. The relative proportion of connective tissue CSA to

total CSA was 19.3% higher (P-0.01) in the senescent diaphragms and 51.1% higher

(P<0.001) in the senescent plantaris muscles compared to the adult diaphragms and

plantaris muscles, respectively.

Succinate dehydrogenase activity. Histochemically determined succinate

dehydrogenase (SDH) activity and cross-sectional areas of costal diaphragm single fibers

classified by fiber type are presented in table 5. The CSA of the type I, type Ha, and type

IIb fibers in the senescent costal diaphragms did not differ from the CSA of fibers with the

corresponding phenotype in adult costal diaphragms. Also, the SDH activity of individual

costal diaphragm fibers did not differ between groups in any of the three fiber types.








Likewise, the average SDH activity for the entire costal diaphragm cross-section did not

differ between age groups (adult mean(SEM) = 4.82+0.12 vs. senescent mean(SEM) =

4.670.11 mmol min-1 liter1). In contrast, the average SDH activity for the plantaris

muscle was significantly lower (P=0.02) for the senescent group compared to the adult

(adult mean(SEM) = 2.030.15 vs. senescent mean(SEM) = 1.41+0.18 mmol min-1 *

liter1).


Mathematical Correction of Diaphragmatic Specific Force


Table 6 presents the maximum tetanic force (Po) produced by the in vitro diaphragm

strips normalized to muscle CSA, connective tissue (C.T.)-free CSA, dry mass CSA, and

dry mass, C.T.-free CSA.


Myofibrillar Protein Cross-Sectional Area Correction


Mean (SEM) cross-sectional areas of the in vitro diaphragm strips did not differ

between the experimental groups (adult = 0.0215+0.001 cm2 vs. senescent =

0.0201+0.001 cm2). However, myofibrillar protein CSA was significantly lower

(P=0.003) in the senescent diaphragm strips (0.00181+0.00006 cm2) compared to the

adult strips (0.00215+0.00009 cm2). When Po for each diaphragm strip was normalized to

its myofibrillar protein CSA, the resulting specific force did not differ between age groups

(table 6, figure 4).


Muscle Water and Connective Tissue Corrections


Mean (SEM) C.T.-free CSA was not different between groups (adult =

0.0208+0.001 cm2 vs. senescent = 0.0193+0.001 cm2), while calculation of dry mass








CSA of the strips resulted in significantly lower values (P-0.03) for the senescent group

(0.00485+0.0003 cm2) compared to the adult (0.00598+0.0004 cm2). Calculation of dry

mass, C.T.-free CSA using the biochemical measurement of C.T. concentration resulted in

even greater differences (P-0.007) between adult and senescent values (adult =

0.005360.0003 cm2 vs. senescent = 0.00407+0.0003 cm2). Also, calculation of dry

mass, C.T.-free CSA using the histochemical measurement of C.T. CSA (adult =

0.00525+0.0003 cm2 vs. senescent = 0.00435+0.0002 cm2) was in close agreement with

the biochemical method.

The age-related deficit in mean specific force observed when Po was normalized to

muscle CSA (-16.4%) was lower when Po was normalized to C.T.-free CSA (-15.5%).

However, this was not significantly different. When Po was normalized to dry mass CSA,

the senescent mean specific force deficit was significantly reduced (-6.4%). Finally,
normalization of Po to dry mass, C.T.-free CSA of the strip completely eliminated the

senescent specific force deficit (table 6, figure 5).

Figure 6 illustrates the relationships between dry mass of the costal diaphragm and

specific force (P=0.005) and between connective tissue concentration and specific force

(P-0.03). The coefficients of determination for the relationships between dry mass and

connective tissue concentration and specific force are r2-0.31 and r2=0.18, respectively.



Diaphragmatic Single Fiber Specific Force


Table 7 presents mean (SEM) cross-sectional areas, maximal calcium-activated force

(Fo), and maximal specific force of isolated diaphragmatic skinned single fibers. Because

of the low sample size, all fibers exhibiting type II MHC phenotype (type Ia, type IIdx,

and type Ib) were analyzed together. Specific forces of individual fibers classified by

MHC phenotype for each animal are presented in table 8. In type I fibers, CSA, Fo, and

specific Fo did not differ between adult and senescent groups. In type II fibers, there were








trends for both CSA and Fo to be lower in the senescent fibers compared to the adult fibers;

however, specific Fo did not differ between groups. Figure 7 compares the specific force

of senescent type I and type II skinned single fibers to the specific force of senescent in

vitro diaphragm strips (both expressed as a percentage of the corresponding adult mean

value). The age-related specific force deficit present in the diaphragm strips is not present in

the calcium-activated single fiber preparation (figure 7). Figure 8 illustrates the

identification of MHC phenotype of single diaphragmatic fibers using polyacrylamide gel

electrophoresis (PAGE).


Myosin Heavy Chain Isoforms and Myofibrillar ATPase Activity


Figure 9 illustrates mean (SEM) calcium-activated myofibrillar ATPase activity for

the adult and senescent costal diaphragms. Senescent diaphragms tended to exhibit higher

ATPase activities as compared to the adults, however, this was not significant (P=0.20).

The relative myosin heavy chain isoform composition (meanSEM) of the costal

diaphragm and plantaris is presented in table 9. Paradoxically, the relative proportions of

both the type I MHC (P<0.001) and the type IIb MHC (P<0.001) were found to be higher

in the senescent diaphragms compared to the adult. Concomitantly, the proportion of type

IIdx MHC was significantly lower (P<0.001) in the senescent diaphragms when contrasted

to the adults. In the plantaris, there was a trend for the percentage of type IIb MHC to be

lower (P=0.12) in the senescent muscles, while the proportion of type I MHC was

significantly higher (P<0.001) in the senescent plantaris compared to the adult muscles.

Figure 10 shows a PAGE separation of the MHC isoforms illustrating the notable relative

increase in the type IUb MHC band in a senescent diaphragm sample as compared to a

typical adult diaphragm sample.

Finally, figure 11 illustrates the relationships between myofibrillar ATPase activity of
the costal diaphragm and Vmax (P=0.07) and between percent type IIb MHC in the costal





37

diaphragm and Vmax (P=0.005). The coefficients of determination for the relationships

between myofibrillar ATPase activity and percent type IIb MHC and Vmax are r2=0.14 and

r2-0.29, respectively.














Table 1. Morphometric characteristics of adult (9 month old)
and senescent (26 month old) F-344 rats.


Body mass (g)

Costal Diaphragm mass (mg)

Crural Diaphragm mass (mg)

Total Diaphragm mass (mg)

Costal mass/body mass ratio (mg/g)

Costal in vitro strip length at Lo (mm)

Right Plantaris mass (mg)

Plantaris mass/body mass (mg/g)


Adult Senescent


Adult
(n=12)

389.3 + 8.6

738 + 29

334 18

1072 46

1.89 0.06

24.8 0.4

298 + 15

0.77 0.04


Senescent
(n=13)

392.2 + 9.1

778 + 19

376 12

1154 + 27

1.98 0.04

24.6 0.4

270 5

0.69 0.02


P-value


NS

NS

NS (P=0.06)

NS (P=0.13)

NS

NS

NS (P=0.07)

NS (P=0.06)


Values are means SEM.
NS = non-significant (P>0.05).













Table 2. In vitro contractile properties of costal diaphragm strips
from adult (9 month old) and senescent (26 month old) F-344 rats.


Adult Senescent P-value
(n=12) (n=13)

Maximal tetanic Po (N cm-2) 25.16 0.46 21.03 0.38 P<0.001

Total potential force (N)* 7.09 0.3 6.29 0.2 P=0.03

1/2 RT (msec) 39.03 1.1 40.31 1.8 NS

+dP/dt (N *msec-1 cm-2) 0.23 0.01 0.20 0.01 NS

Vmax (lengths sec-1) 5.50 0.11 6.46 0.15 P<0.001

a/Po 0.25 0.01 0.20 0.02 P=0.02

Maximal power (mWatts cm-2) 254.61 9.43 236.50 10.41 NS


Values are means SEM.
NS = non-significant (P>0.05).
Po = Isometric specific force.
* Total force calculated from total costal diaphragm mass.
1/2 RT = Relaxation half-time following a twitch contraction.
+dP/dt = Maximal rate of specific force development during a twitch contraction.
Vmax = Maximal shortening velocity calculated from the Hill equation.
a/Po = Curvature of the force-velocity relationship, where a = a constant derived from the
Hill equation.







































- Adult

-- -- Senescent


o *
o
o


Specific Force (N cm2)






Figure 1. Force-velocity relationships for costal diaphragm in vitro strips from adult and
senescent F-344 rats.



























* Adult
0 Senescent


*
*


p I I I I


Specific Force


-2
(N cm )


Figure 2. In vitro power curves as a function of specific force for combined adult and
combined senescent diaphragm strips.


400-




300-


200


100-




0
0













Table 3. Protein composition of costal diaphragm and plantaris
from adult (9 month old) and senescent (26 month old) F-344 rats.


Adult Senescent P-value
(n=12) (n=13)

Costal Diaphragm

Dry mass 278.5 9.6 240.1 10.2 P=0.003
(mg/g wet mass)
Myofibrillar protein 102.9 5.8 90.0 4.5 NS (P=0.09)
(mg/g wet mass)
Connective Tissue protein 27.3 + 4.2 37.9 3.7 NS (P=0.06)
(mg/g wet mass)
Plantaris

Dry mass 256.8 + 8.2 259.7 + 3.3 NS
(mg/g wet mass)

Myofibrillar protein 106.6 6.1 89.1 + 7.3 NS (P=0.11)
(mg/g wet mass)

Connective Tissue protein 45.6 + 3.7 66.3 4.4 P=0.02
(mg/g wet mass)


Values are means SEM.
NS = non-significant (P>0.05).
* Does not include connective tissue protein (see Materials and Methods).








A.























B.







--lob















Figure 3. Photographs of picrosirius red/acid fuchsin collagen staining in representative
histochemical sections (magnification=400x) from a) adult and b) senescent costal
diaphragms.














Table 4. Histochemical quantification of connective tissue cross-sectional area in muscle
from adult (9 month old) and senescent (26 month old) F-344 rats.


Adult Senescent P-value
(n=12) (n=12)

Costal Diaphragm 12.13 0.58 14.47 0.63 P=0.01

Plantaris 11.05 0.38 16.70 1.04 P<0.001


2 2
Units are mm connective tissue per 100 mm2 of tissue.
Values are means SEM.













Table 5. Histochemically determined succinate dehydrogenase (SDH) activity
and cross-sectional areas of single fibers from costal diaphragm
of adult (9 month old) and senescent (26 month old) F-344 rats.


Adult Senescent P-value
(n=10) (n=10)

Type I fibers

SDH activity 6.07 + 0.60 5.91 0.40 NS

CSA (Lnm2) 1349 163 1359 + 72 NS

Type IIa fibers

SDH activity 6.16 0.59 5.62 0.52 NS

CSA (gLm2) 1424 159 1330 + 48 NS

Type IIb fibers

SDH activity 2.36 0.33 2.24 + 0.32 NS

CSA (jpm2) 3461 202 3290 186 NS


Units of SDH activity are mmol min-1 liter1 of tissue.
Values are means SEM.
NS = non-significant (P>0.05).














Table 6. Costal diaphragm Po normalized to muscle cross-sectional area (CSA)
and to CSA corrected for non-contractile material in adult (9 month old)
and senescent (26 month old) F-344 rats.


2
Specific Force (N cm-

Adult Senescent A (%)
(n=12) (n=13)

Normalized to muscle CSA 25.16 0.46 21.03 0.38 a 16.44

Normalized to C.T. free CSA 25.88 0.49 21.86 0.35 a 15.53

Normalized to dry CSA 91.37 3.05 85.52 1.99 b 6.40

Normalized to dry, C.T. free CSA 101.98 4.22 100.64 2.64 NS

Normalized to dry, C.T. free CSA 104.02 3.53 100.06 2.48 NS

Normalized to myofibrillar CSA 254.13 16.13 241.26 12.08 NS


Values are means SEM.
C.T. = connective tissue
* Biochemical determination of C.T. mass.
SHistochemical determination of C.T. CSA.
a P<0.001
b P=0.12
NS = non-significant (P>0.05).
























120
P<0.001
100


80


60

40


20


0
N cm2 muscle CSA N cm2 myofib. CSA








Figure 4. Specific isometric force of costal diaphragm in vitro strips expressed both as
N cm-2 of muscle cross-sectional area (CSA) and as N cm-2 of myofibrillar CSA.
Values are presented for the senescent group (SEM) as a percentage of the adult values.
P-value represents statistical comparison of senescent and adult values.





























A = -15.5%


P / muscle P / C.T.-free
CSA CSA


P Dry P / Dry, C.T.-free
CSA CSA


Figure 5. Specific isometric force of costal diaphragm in vitro strips expressed both as
N cm-2 of muscle cross-sectional area (CSA) and as N cm-2 of connective tissue (C.T.)-
free CSA, dry CSA, and dry, C.T.-free CSA. Values are presented for the senescent group
(SEM) as a percentage of the adult values.


A = -16.4%


A = -6.4%
















**


r = 0.56
SEE = 2.12


p I 1,I ~ I I II I a p I


20 25


30 35


Dry mass (mg g-1)


* *


*.*


*


r = 0.42
SEE = 2.36




0 10 20 30 40 50 60 70
Connective tissue (mg g-')


Figure 6. Correlational analysis of the relationship between costal diaphragmatic specific
force and a) relative dry mass of the costal diaphragm (mg/g) and b) relative connective
tissue content (mg/g) of the costal diaphragm.


10 L
1;


5


20













Table 7. Cross-sectional area and calcium-activated force in costal diaphragm skinned
single fibers from adult (9 month old) and senescent (26 month old) F-344 rats.


Adult Senescent P-value

Type I MHC n = 19 n=12

CSA (im2) 1200 110 1311 133 NS

Fo (mN) 0.073 0.007 0.077 + 0.009 NS

Specific Fo (mN mm-2) 62.5 6.7 60.9 6.4 NS


Type II MHC n = 14 n=9

CSA (gim2) 2481 + 273 1750 235 NS (P=0.08)

Fo (mN) 0.182 0.034 0.107 0.016 NS (P=0.11)

Specific Fo (mN mm-2) 70.0 9.3 65.5 9.3 NS


Values are means SEM.
n = the number of individual fibers across all animals.
Fo = single fiber maximal calcium-activated tetanic force.
NS = non-significant (P>0.05).













Table 8. Maximum calcium-activated specific force (mN mm-2) of costal diaphragm
skinned single fibers classified by MHC phenotype from adult (9 month old)
and senescent (26 month old) F-344 rats.


MHC Phenotype
Type I Type Ha Type IIdx Type IIb

Adult

Animal #1 76.07 (3) 41.37 (1) 144.73 (1) 94.43 (1)
Animal #2 80.85 (3) 109.04 (1)
Animal #3 69.44 (4) 67.56 (1)
Animal #4 51.88 (5) 136.22 (1)
Animal #5 50.16 (1) 36.32 (1) 85.85 (1)
Animal #6 43.21 (3) 55.26 (1) 50.20 (1)
Animal #7 63.16 (1)
Animal #8 46.28 (2) 56.25 (1)

Mean SEM: 61.9 6.3 44.3 5.7 76.3 + 13.3 99.0 16.7

Senescent

Animal #1 62.53 (3)
Animal #2 57.77 (2)
Animal #3 59.14 (1)
Animal #4 67.29 (2) 76.64 (2)
Animal #5 39.91 (1) 88.46 (2)
Animal #6 35.91 (1) 88.96 (1)
Animal #7 97.02 (2) 40.79 (1) 42.30 (2)
Animal #8 44.69 (1)

Mean SEM: 57.4 5.7 58.7 13.4 82.6 12.4


The values for each animal represent the mean of (n) fibers expressing that MHC
phenotype sampled from the costal diaphragm.























120
10 P<0.001
100


80


60


40


20


In vitro strip P Type I Type II

(N cm'2) Single Fiber F (mN mm"2)






Figure 7. Specific isometric force of costal diaphragm in vitro strips expressed as N cm-2
of muscle cross-sectional area (CSA) compared to maximum calcium-activated specific
force of skinned single fibers expressed as mN mm-2. Values are presented for the
senescent group (SEM) as a percentage of the adult values. P-value represents statistical
comparison of senescent and adult values.








































Lane 1 Lane 2 Lane 3










Figure 8. Photograph of sodium dodecyl Sulale-polyvacrylamide gel electrophoresis of
isolated single fibers from the costal diaphrIagnm.
Lane 1: Diaphragm fiber bundle expressing all four Vl MHC isofonns.
Lane 2: Single fiber expressing type lIdx MII1C.
Lane 3: Single fiber expressing type 1 Mi IC.
Figure 8.Poonaho oi* o*cl.,l~lcplarlniegleetohrsso
isolated~~~~~~~ sigefbr rmtl otldaham
Lanef, 1: Diaprag fie bnl epesigal b MCiofn
Lane ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 2: Sigefbrepesigtp lxM


























600


_2 500
0

400
o

S 300


200


100


0
Adult Senescent







Figure 9. Comparison of costal diaphragmatic myofibrillar ATPase activity from adult and
senescent animals. Values are means SEM.













Table 9. Myosin heavy chain (MHC) isoform composition (percent of total MHC pool)
of costal diaphragm and plantaris from adult (9 month old)
and senescent (26 month old) F-344 rats.


Adult Senescent P-value
(n=12) (n=13)

Costal Diaphragm

Type I MHC 20.4 0.5 24.7 0.7 P<0.001

Type Ha MHC 18.4 1.0 18.2 0.9 NS

Type IIdx MHC 54.9 1.3 38.3 1.6 P<0.001

Type IIb MHC 6.3 + 1.1 18.8 1.3 P<0.001

Plantaris

Type I MHC 1.5 0.2 3.8 + 0.5 P<0.001

Type Ha MHC 12.3 0.8 11.2 + 0.6 NS

Type IIdx MHC 64.0 2.5 67.3 + 1.5 NS

Type IIb MHC 22.2 2.4 17.7 + 1.4 NS (P=0.12)


Values are means SEM.
NS = non-significant (P>0.05).




































Lane I Lane 2








Figure 10. Photograph of a typical sodium dodecvl sulfate-polyacrvlamide gel
electrophoresis of bundles of fibers from the costll diaphragnm illustrating the region of the
gel containing the myosin heavy chains. Lane I contains a senescent sample: lane 2
contains an adult sample.


I ~ --~--- --
~I ---- -- ----


-- ---4L..
-- --




















* -4-


* **


r = 0.37
SEE = 0.65


I I


400


500


600


700


800


Myofibrillar ATPase Activity (nmol min-' mg-1)


*

,*


r = 0.54
SEE = 0.58


0 5 10 15 20

% Type IIb MHC


25 30


Figure 11. Correlational analysis of the relationship between costal diaphragmatic Vmax and
a) myofibrillar ATPase activity of the costal diaphragm (nmol min-1 mg-1) and b) relative
type lib MHC content of the costal diaphragm.


4
30


0


I* *
i I I I I


. I I I I I I


* 4141


3*
A'














DISCUSSION


Overview of Principle Findings


These experiments confirm the existence of a significant age-related reduction in

maximal in vitro tetanic specific force (Po) of the costal diaphragm. The data support the

hypothesis (#1) that the composition of the costal diaphragm is altered as the animal reaches

senescence with connective tissue and water content increasing and myofibrillar protein

concentration tending to decrease. Further, in support of hypothesis #2, it appears that

these increased concentrations of non-contractile material in the senescent diaphragm fully

account for the observed deficit in specific P0. This conclusion is corroborated by data that

fails to indicate any age-related deficit in calcium-activated maximal specific force of

isolated diaphragmatic skinned single fibers (supporting hypothesis #4). Finally,

hypothesis #3 is supported by data showing a large increase in senescent diaphragmatic

Vmax compared to adults which is accompanied by a dramatic increase in the relative

proportion of type IIb MHC in the costal diaphragm. However, these changes in type IIb

MHC proportion do not fully explain the age-related difference in Vmax. Paradoxically, the

percentage of type I MHC was also significantly increased in the senescent diaphragms.



Age-Related Changes in Diaphragmatic Force Production


Several recent studies have reported a reduction in diaphragmatic in vitro maximal

specific force of aging rodents (3, 36). This is confirmed in the present study wherein

specific P0 was found to be 16.4% lower in senescent diaphragms compared to adults. The








body mass of the senescent animals in the present study did not differ from the adult rats;

therefore, the effects of growth and maturation on the diaphragm were avoided. One

possible mechanism for the senescent specific Po deficit is an increase in non-contractile

mass (and CSA) with no change in contractile mass. This would effectively reduce the

contractile protein concentration by dilution and, therefore, result in a specific P0 reduction.

If this were the case, the total (absolute) force generating capacity of the diaphragm would

not be compromised. However, this is not true in the present study. The total mass of the

costal diaphragm and the costal mass / body mass ratio did not differ between groups;

therefore, the estimated total diaphragmatic force was significantly lower in the senescent

animals (table 2). This suggests that the observed specific P0 deficit in the diaphragm of

senescent rats compared to adults translates into a real reduction in the functional capability

of the diaphragm to generate inspiratory pressure.

Although no data is available concerning the workload on the senescent diaphragms,

activity of the oxidative enzyme, SDH, was unchanged in the senescent diaphragms as

compared to the adults whereas SDH activity in the plantaris was -31% lower in the

senescent animals. This is interpreted as evidence that the workload on the diaphragm is

similar between senescent and adults animals. Therefore, the age-related reduction in

maximal force generating capacity of the diaphragm would reduce the reserve

diaphragmatic force available to the senescent animals, which may be needed to maintain

adequate ventilation during times such as heavy exercise or periods of airway obstruction.

The physiological significance of this age-related reduction in diaphragmatic force

generation is unclear.



Age-Related Changes in Diaphragm Composition


If individual cross-bridges produce the same amount of force when activated, the

force produced by a muscle is a function of the number of parallel cross-bridges (4). This








forms the basis for the hypothesis that the aging process results in alterations in the

composition of the costal diaphragm such that the number of cross-bridges per unit area is

reduced (i.e. reduced myofibrillar protein concentration). The major components of skeletal

muscle include water, myofibrillar protein, and connective tissue. Conditions other than

aging which cause a reduction in skeletal muscle maximal specific Po, such as hindlimb

unweighting and denervation, have recently been shown to be associated with reductions in

the concentration of myofibrillar protein (5). On the other hand, compensatory
hypertrophy, which also causes a reduction in specific Po, is not associated with a

reduction in myofibrillar protein concentration (5). The present study reveals a strong trend

for myofibrillar protein concentration to be lower (-13%) in the senescent diaphragms

compared to the adult, but this did not reach statistical significance (P=0.09). Mean total

myofibrillar protein content (myofibrillar protein concentration (mg/g) x costal mass (g))

was -8% lower in the senescent diaphragms as compared to the adults, but this also was

not statistically significant. The technique used in the present study to assess myofibrillar

protein concentration is a modification of a procedure that has been widely used to

document changes in locomotor muscle myofibrillar protein concentration following

hindlimb unweighting and denervation (5). However, the magnitude of the changes in

myofibrillar protein concentration and specific force generation are much greater under

these conditions compared to the changes presently observed in the aging costal diaphragm

(5, 76, 77). In our hands, the coefficient of variation for this technique is -7%; hence, this

technique may lack the resolution necessary to statistically identify relatively small age-

related changes in diaphragmatic myofibrillar protein concentration. Nevertheless, the

failure to demonstrate any age-related change in total costal diaphragm mass in the presence

of the observed increases in diaphragmatic connective tissue concentration and water

content, strongly suggest that myofibrillar protein concentration is reduced in the senescent

diaphragms.








It is well established that connective tissue concentration increases with aging in

locomotor muscle (78) and diaphragm (79); this is confirmed in the present study. Also,

total diaphragmatic connective tissue content (mg) was significantly higher in the senescent

diaphragms as compared to the adult.

This is the first study to examine age-related changes in water content of the

diaphragm. The aging process reportedly does not alter the water content of locomotor

muscles in the rodent (14). This was confirmed in the plantaris muscle of senescent and

adult rats in the present study; however, the relative concentration and the total content of

water were found to be increased in the senescent diaphragms. The location of the

increased diaphragmatic water content (i.e. intracellular vs. extracellular) remains

unknown. A recent report has shown that hindlimb unweighting results in an increase in

locomotor muscle water content and that this occurs in the interstitial space (58). Therefore,

it is possible that aging could result in a similar increase in interstitial water content of the

diaphragm muscle. In fact, the observed age-related increase in the water content of the

diaphragm may be linked to the accumulation of connective tissue also observed in the

senescent diaphragms. One functional property of collagen fibers is that they resist changes

in tissue configuration and volume (80). Also, collagen fibers immobilize the

glycosaminoglycans (GAG) found in the interstitial space (80). Due to the highly

hydrophilic nature of GAGs, these molecules are important in establishing the osmotic

pressure of the extracellular space. Therefore, the quantity and type of GAGs present in a

tissue greatly influence the interstitial volume of the tissue (80). Given this relationship

between collagen content, GAGs, and tissue water content, it seems reasonable to speculate

that the increase in connective tissue in the aging diaphragm may result in the accumulation

of GAG molecules in the interstitial space of the muscle, thereby, increasing the osmotic

pressure and causing an age-related accumulation of water. Future experiments would be

necessary to determine the role, if any, that GAGs play in mediating the age-related

increase in water content in the costal diaphragm.








In summary, these observations suggest that two mechanisms interact to result in

alterations in the composition of the senescent diaphragm. First, an accumulation of water

in the diaphragm and second, an accumulation of connective tissue. Although the measured

values for myofibrillar protein concentration (mg/g) and content (mg) were not statistically

different between the senescent and adult diaphragms, the estimated total diaphragmatic

force generation potential, as discussed previously, suggests that total myofibrillar protein

content may, in fact, be reduced in the senescent diaphragms.


Diaphragm Cross-Sectional Area and Specific Force Corrections


Kandarian and coworkers (5, 58) have pointed out that the information gained from

examining alterations in specific Po of muscles is limited by the failure of this procedure to

distinguish between intrinsic changes in the contractile machinery and changes in the

concentration (or CSA) of the contractile proteins. Taylor and Kandarian (5) have recently

introduced a method of normalizing force production of a muscle to the myofibrillar protein

CSA rather than to total CSA. This procedure is effective in determining the force generated

per unit area of contractile protein. In the present data, the significant mean specific Po

deficit noted in the senescent diaphragm strips was eliminated when normalized to

myofibrillar protein CSA (figure 4). This implies that the age-related reduction in

diaphragmatic myofibrillar protein concentration accounts for the specific Po deficit

observed in senescent diaphragms compared to adults. Further, since in this in vitro

preparation, a given unit area of myofibrillar protein produced the same amount of force in

both adult and senescent diaphragms, it is inferred that any possible age-related alterations

in excitation-contraction coupling and/or the intrinsic properties of the contractile proteins

did not contribute to the specific Po deficit.

As noted above, a reduction in the relative contribution of myofibrillar protein to the

total CSA could occur by increasing the quantity of some non-contractile material.








Therefore, to determine which non-contractile components) of muscle were responsible

for the age-related reduction in diaphragm myofibrillar protein concentration, the

concentrations of connective tissue and water were determined in the costal diaphragm.

This allows the assessment of the role of connective tissue and water on the specific Po

production both separately and together. This analysis reveals that elimination of the

contribution of connective tissue to the strip CSA only slightly reduces the senescent

specific force deficit; whereas, elimination of the contribution of water to the strip CSA
reduces the senescent specific Po deficit by 61% (from -16.4% to -6.4%). Finally,

elimination of both connective tissue and water from the strip CSA results in no differences

between adult and senescent specific forces (figure 5). These data confirm that the
senescent specific Po deficit is a function of reduced myofibrillar protein concentration due

to increases in water and connective tissue concentration with water being the primary

factor.
Correlational analyses reveal significant relationships between specific Po and relative

dry mass of the diaphragms and connective tissue concentration (figure 6). The relatively
low coefficients of determination can be explained by the variation in specific Po and dry

mass/connective tissue concentration within the separate age groups. In other words, the

natural variation in specific Po within each age group cannot be explained or predicted by

variations in dry mass and/or connective tissue concentration, even though the specific Po

variation between age groups can be explained by these factors.


Diaphragmatic Skinned Single Fiber Measurements


The data presented in tables 7 and 8 represent the first comparison of contractile

characteristics of single diaphragm fibers from mature adult and senescent animals. A

recent study has compared the contractile characteristics of skinned single fibers isolated

from locomotor muscle of adult (6-9 months old) and senescent (27 months old) rats (81).








Similar to the present findings in the diaphragm, these investigators reported no age-related

changes in the specific Fo of fast- and slow-twitch fibers. The lack of age-related change in

the specific Fo lends further support to the conclusion that the intrinsic force generating

capacity of the sarcomeres of aging diaphragm muscle is not altered. This conclusion

agrees with data examining force production in locomotor muscles (82).
The mean fiber CSA, single fiber maximal calcium-activated tetanic force (Fo), and

specific Fo (mN mm-2) in the present study are in agreement with published values

presented for type I (slow) fibers from adult rat diaphragm (83). However, the mean
specific Fo for type II fibers in the present study is lower (-25%) than the mean specific Fo

for fast fibers presented by Eddinger and Moss (83). Further, the mean type I and type II

fiber specific Fo presented in the present study are 30-40% lower than specific Fo values

reported for skinned single fibers from adult locomotor muscles (84, 85). One possible
reason for this discrepancy in diaphragmatic type II fiber specific Fo is that Eddinger and

Moss (83) classified fibers based on single fiber Vmax and ATPase histochemistry.

Therefore, the fibers in this study labeled as "fast" may have been predominantly type IIb
fibers. Examination of individual single fiber Fo (table 8) from the present study indicates

that the specific Fo for the type IIb fibers tend to be higher than the mean specific Fo

reported for all type II fibers. Although the sample size was too small for statistical

comparison of each type II MHC phenotype separately, the proportion of each phenotype

sampled is similar for both adult and senescent groups; therefore, combining all type II

fibers for group comparison should yield a valid conclusion. A possible reason for the

lower specific Fo values in the present study, as compared to locomotor muscle fibers,

involves the method employed to determine the CSA of the single fibers. In the present
study, the diameter of each single fiber at Lo was measured with the fiber submerged at a

constant depth in relaxing solution. Subsequent comparison of fiber diameters obtained

using this method with diameters of the same fibers measured in air reveals a constant

-20% increase in the submerged fiber diameter compared to the fiber in air. Theoretical








calculations indicate that inflation of a fiber diameter by 20% would result in a consistent

31% reduction in the calculated specific Fo. This accounts for the discrepancy between the

present data and the locomotor muscle fiber specific Fo cited above (84, 85), since these

investigators did, in fact, measure fiber diameters in air.



Age-Related Changes in Shortening Velocity of the Diaphragm


The age-related increase in diaphragmatic Vax observed in the present data is a

unique finding and contradicts previous reports of unaltered (14) or decreased (12) Vmax in

locomotor skeletal muscle of senescent rodents. Despite the age-related reduction in

maximal diaphragmatic force generation, comparison of the in vitro diaphragmatic power

curves for adult and senescent animals (figure 2) revealed no obvious age-related

differences in diaphragmatic power over the range of isotonic specific forces surrounding

maximal power production. Further, maximal power did not differ between groups (table

2). This suggests that the senescent diaphragm may have effectively compensated for its

reduced force generating capacity by increasing its velocity of shortening. It is unknown

whether this alteration is a direct adaptation to the decreased force generating capacity of the

diaphragm or if it is the result of some other change in the aging diaphragm.

Since Vmax is thought to be primarily determined by the rate of ATP hydrolysis by

the myofibrillar ATPase enzyme (4), it was postulated that the underlying mechanism for

the aging increase in Vmax was an increase in the maximal myofibrillar ATPase activity in

the diaphragm. Also, that the relative proportions of the fast (type II) MHC isoforms would

be increased in the senescent diaphragms. The second postulate was supported by the data

showing a large increase in the relative proportion of type IIb MHC in the senescent

diaphragms as compared to the adults. Interestingly, the proportion of type I MHC was

also increased in the senescent diaphragms (table 9). This paradoxical increase in the MHC

isoforms possessing both the fastest (type IIb) and the slowest (type I) ATPase activities








may explain why myofibrillar ATPase activity did not differ statistically between the age

groups. It appears that the aging diaphragm may be subject to two separate and opposing

mechanisms. One mechanism, common to all aging skeletal muscle, causes a shift in MHC

expression from fast-to-slow (29, 30). A second mechanism, unique to the diaphragm,

causes a shift in MHC expression from slow-to-fast (36). The first mechanism may be the

result of an age related hypothyroidism (49), but the second mechanism remains a mystery.

One possibility is that there is a change in the neural output to the diaphragm as the animal

ages which differs from the aging changes in neural output to locomotor muscles. Support

for this notion can be found in a study of the neuromuscular junctions of young and old

mice (86). These investigators reported that mean resting membrane potentials and

miniature end plate potential (m.e.p.p.) amplitudes were higher in the old diaphragms as

compared to the young. Further, they found that the m.e.p.p. frequency did not differ

between young and old diaphragms whereas m.e.p.p. frequency was reduced in the old

locomotor muscles examined. The significance of these findings are unclear but provide a

direction for future research.

There are three primary factors that determine the velocity of shortening of skeletal

muscle when expressed in units of muscle lengths per second: 1) length of the sarcomeres,

2) cross-bridge cycling rate, and 3) cross-bridge shortening distance per molecule of ATP

hydrolyzed. Comparative physiologists have observed that, across species, speed of

shortening is inversely correlated with sarcomere length (87, 88). This is presumably due

to an increased number of shortening units in series. Nevertheless, no evidence exists to

suggest that sarcomere length changes with aging. The theory proposed by A. F. Huxley

(89) describing thick and thin filament interactions remains the most widely accepted model

of muscular contraction (shortening and force generation). In this model, sarcomere

shortening is the result of three steps: the attachment of the cross-bridge to actin, the
"working stroke" of the cross-bridge, and the detachment of the cross-bridge. Shortening

velocity is limited by the rate constant for dissociation of the myosin head from the actin








active site which, in turn, is closely associated with the hydrolysis of ATP. As noted

previously, myofibrillar ATPase activity is closely related to Vmax in skeletal muscle;

however, recent reports suggest that the cross-bridge shortening stroke may be subject to

alterations independent of ATPase activity. For example, Maruyama et al. (90) found that

Vmax is reduced in isolated single fibers after incubation of the fibers in ethylene glycol.

Based on this data and the relationship between shortening velocity and ATP concentration

in the ethylene glycol treated fibers, these authors concluded that this treatment reduced

both actomyosin ATPase activity and the shortening distance per mole of hydrolyzed ATP.

Hofmann et al. (91) reported that extraction of C-protein from isolated rabbit muscle

skinned fibers resulted in an increase in Vmax at submaximal levels of calcium activation.

Since C-protein is known to be involved in the structure of the thick filament (91), its

removal may alter the mechanics of the cross-bridge stroke. Finally, Lowey et al. (92)

studied the in vitro movement of actin filaments across stationary S1 myosin fragments

(which contain the ATPase enzyme and the light chain binding regions) and found that

complete extraction of the myosin light chains resulted in a dramatic reduction in the

velocity of actin movement without altering the ATPase activity of the myosin fragments.

These data indicate that under certain conditions, changes in muscle Vmax may be

attributable to factors other than changes in myofibrillar ATPase activity.

In the present study, correlational analyses of the relationships between Vmax and

both myofibrillar ATPase activity and percentage of type Ib MHC (table 10) suggest that

other mechanismss, in addition to the increase in type IIb MHC proportion, must be

involved in mediating the dramatic increase in Vmax in the aging diaphragm. It is possible

that some unknown age-related change in the rat diaphragm causes an increase in the

sarcomere shortening distance per mole of ATP hydrolyzed. This remains an interesting

area for future research.








Summary and Conclusions


It has been noted that the maximal in vitro specific Po of a muscle is determined by

the following factors: the maximal force generated by the individual force generating units

(i.e. cross-bridges), the proportion of myofibrils per unit of muscle CSA, and the degree of

sarcomere activation via excitation-contraction coupling. The present study was designed to

systematically examine these factors to determine the mechanism responsible for the age-
related deficit in specific Po observed in the rat diaphragm. This was accomplished by

examining the components of the diaphragm muscle (i.e. myofibrillar protein, water, and

connective tissue) in adult (9 month old) and senescent (26 month old) male Fischer 344

rats, and normalizing diaphragmatic in vitro force production to strip myofibrillar protein

CSA, connective tissue-free CSA, dry mass CSA, and dry, connective tissue-free CSA.

Further, the maximal calcium-activated specific force (specific Fo) in diaphragmatic skinned

single fibers was also assessed. Theoretically, if an age-related change in the force

generating capacity of individual cross-bridges was primarily responsible for the aging

specific Po deficit, then diaphragm strip Po normalized to myofibrillar protein CSA and

single fiber specific Fo would also demonstrate an age-related deficit. If aging changes in

the excitation-contraction coupling mechanism were primarily responsible for the aging

specific Po deficit, then diaphragm strip Po normalized to myofibrillar protein CSA would

exhibit an age-related deficit but single fiber specific Fo would not. Finally, if an age-

related alteration in diaphragmatic myofibrillar protein concentration was primarily

responsible for the aging specific Po deficit, then neither diaphragm strip Po normalized to

myofibrillar protein CSA nor single fiber specific Fo would exhibit the age-related force

deficit.

The results of this study show that normalization of diaphragmatic in vitro force to

myofibrillar protein CSA eliminates the difference observed in specific Po between adult

and senescent diaphragms. Also, that specific Fo of diaphragmatic skinned single fibers








does not differ between adult and senescent animals. Therefore, an age-related reduction in

the proportion of myofibrils per unit area of diaphragm muscle is identified as the primary

factor mediating the senescent specific Po deficit. Further, the data indicate that the aging-

induced reduction in relative myofibrillar protein CSA primarily results from an age-related

increase in diaphragmatic water content, with an increase in connective tissue concentration

and possibly a decrease in total myofibrillar protein content also contributing. The age-

related increase in diaphragmatic connective tissue concentration occurs in the extracellular

compartment as can be seen in the histochemical connective tissue data presented in the

present study (figure 3). However, the location (i.e. intracellular vs. extracellular) as well

as the underlying mechanisms) of the aging-induced increase in diaphragmatic water

content remains unknown. This provides an intriguing avenue for future research in the

area of aging muscle.

Another finding of this study is that diaphragmatic Vax is increased by 17.5% in the

senescent animals as compared to the adults. This is apparently due in part to an increase in

the proportion of type IIb MHC in the aging diaphragm. This could occur by an

upregulation of the type IIb MHC gene and/or a decrease in the degradation rate of this

protein relative to the other myosin isoforms. The age-related change in Vmax may be a

neurally mediated adaptation to the decreasing force generating capacity of the diaphragm as

the animal ages. This is very speculative and provides an interesting area for future

research.














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BIOGRAPHICAL SKETCH


David S. Criswell graduated from the University of Mississippi in 1988 with a

Bachelor of Science degree in biology. An interest in the scientific basis of sport and

exercise led him to pursue graduate studies in the field of exercise physiology. In 1990 he

earned a Master of Science in Exercise and Sport Sciences at the University of Florida.

David continued his graduate work at the University of Florida, under the direction of Dr.

Scott K. Powers, focusing on the adaptive responses of skeletal muscle to various stimuli

including exercise training, pharmacological treatments, and aging. Upon receiving a Ph.D.

in Health and Human Performance, David has accepted a postdoctoral training fellowship

at the University of Texas Medical School in Houston, under the direction of Dr. Frank

Booth.







I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.


-cott K. Powers, Chair
Professor of Exercise and Sport Sciences
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosop


Stepih L.Ma
Associate Professor of Exercise and Sport
Sciences
I certify that I have read this study and that in my opinion it conforms o c ptable
standards of scholarly presentation and is fully adeate, in scope and qui a/a
dissertation for the degree of Doctor of Philosoe Y i /


chael L. Pollock
Professor of Exercise and Sport Sciences
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.


A. Ddniel Martin
Associate Professor of Physical Therapy
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosop.


iaul W. Davenport //
Associate Professor of Veterinary Medicine

This dissertation was submitted to the Graduate Faculty of the College of Health and
Human Performance and to the Graduate School and was accepted as partial fu fillment of
the requirements for the degree of Doctor of Philosoph

August 1994f H h a H
De n, Colle e of Health and Human Performance


Dean, Graduate School
















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UNIVERSITY OF FLORIDA
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