Fatigue in skeletal muscle

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Fatigue in skeletal muscle
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MacIntosh, Brian Robert, 1952-
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Muscles   ( mesh )
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Thesis:
Thesis (Ph.D.)--University of Florida, 1979.
Bibliography:
Bibliography: leaves 100-105.
Statement of Responsibility:
by Brian Robert MacIntosh.
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Typescript.
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Vita.

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University of Florida
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FATIGUE IN SKELETAL MUSCLE


BY


BRIAN ROBERT MACINTOSH
















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




UNIVERSITY OF FLORIDA


1979














ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to

Dr. W. N. Stainsby, Chairman of my Supervisory Committee,

for his valuable assistance and counsel over the past

four years. Acknowledgement is also due the other

members of my Committee: Dr. M. Fried, Dr. A. B. Otis,

Dr. P. Posner and Dr. C. W. Zauner. Each has unselfishly

contributed time and effort to provide me with the

guidance I needed to complete the requirements for this

degree.

Special thanks are expressed to Donna T. Dolbier, who

provided technical assistance and to Dr. L. Bruce Gladden

who collaborated with me on several research projects

during his Post-doctoral tenure with Dr. Stainsby.

Financial support for me during the pursuit of the

Ph.D degree has been provided by the following agencies

and departments:

NIH, grants to Drs. Stainsby, Otis and Cassin;
Department of Physiology (Teaching Assistantship);
College of Nursing (Teaching Assistantship).

The research reported in this dissertation has been

supported by The American Heart Association, Florida

Affiliate Grant # AG 7 and Sponsored Research Seed Grant

awarded to Dr. Stainsby.

I would like to thank Wendy Auerbach for doing an

excellent job of typing this manuscript.

ii















TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS . . ii

LIST OF FIGURES . . v

LIST OF TABLES . . vii

ABSTRACT . . viii

INTRODUCTION . . 1

EVENTS LEADING TO SKELETAL MUSCLE CONTRACTION 5

POSSIBLE FATIGUE MECHANISMS AND CURRENT THEORIES
OF FATIGUE . . 9

Central Nervous System . 9

Neuromuscular Junction Failure . 10

Attenuated Calcium Release . 11

Reduced Capacity of the Contractile Apparatus .. 13

GENERAL METHODS . . 16

02 UPTAKE AND DEVELOPED TENSION . 22

Introduction . . 22

Methods . . 23

Results . . 26

Discussion . . 34

EVALUATION OF CHANGES IN THE TWITCH CONTRACTION
ASSOCIATED WITH FATIGUE . 41

Introduction . . 41

Methods . . 42

Results . . 48

Discussion . . 59

iii








Page

EFFECTS OF RESPIRATORY ACIDOSIS ON THE TWITCH
CONTRACTION . . 68

Introduction . . 68

Methods . . 69

Results . . 70

Discussion . . 79

SUMMARY . . 89

02 Uptake and Developed Tension . 89

Time-Course of the Twitch Contraction in
Fatigue . . 90

Acidosis and the Twitch Contraction 91

PROPOSED HYPOTHESES FOR SKELETAL MUSCLE FATIGUE 92

Depletion of Calcium at Lateral Sacs .. 92

Compartmentalization of Calcium Within the
Lateral Sacs . 93

Attenuated Trigger for Release of Calcium 93

Reduced Binding Sensitivity for Calcium 94

CONCLUSIONS . . 95

APPENDIX . . 96

BIBLIOGRAPHY . . 100

BIOGRAPHICAL SKETCH .. . 106














LIST OF FIGURES


Page

1. Excitation-Contraction-Coupling . 7

2. Gastrocnemius-Plantaris Muscle Preparation 18

3. Sample Experiments; VO2 versus Developed
Tension . . 28

4. V02 versus Developed Tension; all Data,
Normalized . . 29

5. Twitch and Twin Contractions: Developed
Tension and dP/dt . 39

6. Twitch Characteristics from Fast Traces of
Developed Tension and dP/dt . 43

7. Procedure for Fatiguing Contractions with
Periodic Fast Traces . 46

8. Q, P02, PCO2 and pH During and Following
Fatiguing Contractions . 50

9. Developed Tension, Half Relaxation Time and
Contraction Time . 53

10. Peak Rate of Force Development and Peak Rate
of Relaxation . . 57

11. Tetanic Contraction Before and After Fatiguing
Contractions . . 58

12. Twitch Developed Tension and dP/dt Before,
During and After Ischemic Fatigue 60

13. Half Relaxation Time versus Developed Tension
for Dantrolene, Reduced Stimulation Voltage
and Ischemic Fatigue . 61

14. The Effects of Preceding Contractions on
Developed Tension . 63

15. Procedure for Ventilation and Sampling Pattern. 72








Page

16. Arterial and Venous [H+] . 76

17. PO2 During Different Ventilatory States 78

18. Developed Tension, dP/dt and -dP/dt During
Different Ventilatory States . 81

19. Contraction Time and Half Relaxation Time
During Different Ventilatory States 83















LIST OF TABLES

Page

I 02 UPTAKE AND DEVELOPED TENSION BEFORE AND
AFTER FATIGUE IN SERIES 1 . 30

II RELATIONSHIP BETWEEN 02 UPTAKE AND
DEVELOPED TENSION IN SERIES 2-5 . 32

III PHOSPHORYLCREATINE ANALYSIS DURING
CONTRACTIONS AND FOLLOWING THE RECOVERY
PERIOD . . 55

IV STATISTICS FOR DIFFERENCES BETWEEN
VENTILATION STATES FOR EACH GROUP 74

V STATISTICS FOR TWITCH CHARACTERISTICS
VERSUS BLOOD GASES . 84


vii














Abstract of Dissertation Presented to the Graduate
Council of the University of Florida
In Partial Fulfillment of the Requirements for the
Degree of Doctor of Philosophy


FATIGUE IN SKELETAL MUSCLE


By


Brian Robert MacIntosh


June 1979



Chairman: Wendell N. Stainsby, D.Sc.
Major Department: Physiology


The in situ dog gastrocnemius-plantaris muscle

preparation has been used to study fatigue. Skeletal

muscle fatigue (reduced force output for a given stimulus)

results from a thirty minute period of isometric

contractions at 2.5 to 20/sec. This fatigue is not a

result of failure of motor nerve propagation or transmitter

release. The ratio of oxygen uptake to developed tension

(total tension minus resting tension) is unaltered during

or following fatiguing contractions. The economy of force

production is unaltered by twin impulse stimulation,

relative ischemia or administration of moderate doses of

curare or succinylcholine.


viii








When developed tension is reduced due to repetitive

stimulation for thirty minutes at 2.5, 5 or 10/sec

contractions, the time to peak tension and half relaxation

times are unaltered. The peak rates of force development

and of relaxation are reduced proportionally to the

reduction in developed tension.

Following a forty minute period of recovery, the

twitch developed tension remains greatly attenuated, but

tetanic (200 msec of 100/sec stimulation) developed tension

is virtually the same as it was before the stimulation

period. Phosphorylcreatine is restored to resting levels

within forty minutes of recovery. Also, at this time,

blood flow and oxygen uptake have returned to pre-fatigue

values and venous PO2, PCO2 and pH are at resting levels.

The fatigue observed in these experiments appears to

be due to a reduction in the intensity of activation

obtained with a single impulse. Energy sources are

available and with maximal activation the contractile

mechanism is capable of the same force output it had

before the fatiguing contractions.

Further experiments were conducted to determine if

intracellular acidosis could have been the cause of the

reduced intensity of activation. A sixty to ninety

minute period of hypoventilation with an air mixture high

in 02 (arterial P02 was maintained at 75-100 mm Hg) resulted

in a reduction in arterial pH to 7.08. There was no

reduction in twitch developed tension associated with this

ix









acidosis. It is likely that intracellular pH fell as

much during the respiratory acidosis as it did during

fatiguing contractions at 10/sec. The fatigue observed

during contractions at 10/sec could not be a result of

intracellular acidosis.

It can be concluded from these experiments that

twitch fatigue is not a result of energy deficiency,

reduced capacity of the contractile elements, intra-

cellular acidosis (induced by reduced ventilation for

sixty to ninety minutes) or neuromuscular junction

failure. By the process of elimination it appears

that twitch fatigue results from a reduced activation

of the myofilaments during a twitch contraction. This

may be due to either a reduced sarcoplasmic Ca2+

concentration during contraction or a reduced response

of the myofilaments at a given Ca2+ concentration.














INTRODUCTION

The word fatigue has been used in the past with

several various definitions. Some authors (1, 52) equate

fatigue with exhaustion. Others (47, 50) use the word

fatigue to represent an inability to maintain a particular

work output. More recently (23, 30) skeletal muscle

fatigue has been defined as a reduced capacity of the

muscle to develop tension. Edwards et al. (23) and Fitts

and Holloszy (30) have observed that a twitch contraction

can still be attenuated when the force generating capacity

of the muscle is fully recovered.

A single impulse does not maximally activate the

contractile apparatus (17) and therefore does not permit

the full contractile response of which the muscle is

capable. The capacity to develop tension must be

evaluated under conditions of maximal activation. This

can be accomplished with a caffein or K+ induced

contracture or a maximal tetanic contraction.

If fatigue is defined as a reduced capacity of the

muscle to generate force, then what is it called if there

is an attenuated response to a single impulse? It appears

to be a separate phenomenon and probably occurs by a

separate mechanism (since a muscle recovers full capacity

to develop tension before twitch tension recovers to

pre-fatigue values). Edwards et al. point out (23) that

1








this "twitch fatigue" may be associated with perception

of increased effort necessary to maintain a given workload

or force output. For the purposes of this dissertation,

fatigue will be used as a generalization referring to a

reduced response of the muscle to a given stimulation.

Twitch fatigue will refer to an attenuated response to a

single impulse.

Wilson and Stainsby (66) have reported that twitch

developed tension of the in situ dog gastrocnemius-

plantaris muscle remains attenuated for hours following

a series of contractions at 10-14 per second. Fitts and

Holloszy (30) have reported that tetanic developed tension

recovers quickly following a period of repetitive

stimulation. This may also be the case for the in situ

dog gastrocnemius-plantaris muscle. If this is so, then

the fatigue observed by Wilson and Stainsby is only twitch

fatigue. This preparation, then, would provide a model

for studying twitch fatigue independent of tetanic fatigue.

Very little is known of the fatigability of the canine

gastrocnemius-plantaris muscle or of the mechanisms

responsible for that fatigue. The purpose of the present

study was to provide further information regarding the

fatiguing effects of repetitive stimulation on the

gastrocnemius-plantaris muscle.

To facilitate the reader's understanding of skeletal

muscle fatigue, a brief description of pertinent muscle

physiology and current theories of fatigue precedes the

sections describing the experiments which have been done.






3

The dog in situ gastrocnemius-plantaris muscle group

has been used throughout the series of studies reported

herein. To avoid repetition, the general procedures and

description of the preparation appear as a separate

chapter before any of the studies. The specific

procedures used in each study are described separately

in a Methods section for that study.

In the first study, the relationship between oxygen

uptake and developed tension has been determined for

skeletal muscle before, during and after fatiguing

contractions. These experiments were done to determine

whether or not the energy used by a muscle relative to the

amount of tension developed is altered by fatigue. There

are reports that indicate a change in either direction can

be expected (7, 21, 28).

Further studies were conducted to determine whether

or not changes occur in the time-course of the twitch as

a result of repetitive stimulation. Changes in the rate

of force development and in the time-course of a twitch

contraction have previously been interpreted as indications

of changes in the duration and intensity of activation of

the muscle (17, 36). These measurements may facilitate

an understanding of the mechanisms) responsible for the

fatigue.

A third series of experiments has been conducted to

study the effects of respiratory acidosis on the twitch

contraction. Acidosis has been claimed to be one of








the major causes of fatigue (29). If this is the case,

respiratory acidosis should reduce the developed tension

of a twitch contraction.

In the final chapter of this dissertation, a brief

discussion of the possible theories for the mechanism of

fatigue observed in these experiments is presented. It

is evident that further research will be necessary to

permit evaluation of these theories with respect to

fatigue of the canine gastrocnemius-plantaris muscle

group. However, considerable evidence has accumulated

as a result of my studies, which disputes several of the

current theories of fatigue.














EVENTS LEADING TO
SKELETAL MUSCLE CONTRACTION

Muscular contraction is the result of a sequence of

chemical and physical events beginning with activity in

the central nervous system (CNS)(or sensory input to the

CNS). Failure or impairment at any site in this process

will result in a reduced contractile response of the

muscle. Fatigue and twitch fatigue, then, are results

of such failure. The identification of the site(s) of

failure in fatigue would provide a better understanding

of the mechanisms) effecting the fatigue. Below, a

brief discussion of the normal sequence of events leading

to contraction is presented. CNS control of motor nerve

activity is complex and will not be described. For

simplicity, this discussion is based at the cellular level.

This sequence of events is described in a number of text-

books (45, 62) and is illustrated in Figure 1. Following

the presentation of events leading to contraction, each

step in the sequence is considered as a potential site

for a mechanism of fatigue.

The sequence of events occurring at the nerve

terminal may be susceptible to failure. The arrival of

an action potential at the nerve terminal triggers the

release of acetylcholine from the terminal bouton.

Synaptic vesicles fuse to the terminal membrane and

5








release their contents into the synaptic cleft.

Acetylcholine diffuses the short distance across the

cleft (500 A).

Binding of acetylcholine to specific receptors

causes a transient increase in permeability of the muscle

membrane to Na+ and K+. This results in depolarization

of the end plate. The resulting change in membrane

potential is called the end-plate potential. Destruction

of the acetylcholine is accomplished by acetylcholines-

terase which is located among the receptors on the post-

synaptic muscle membrane. Reconstitution of synaptic

vesicles is accomplished by reuptake of choline and

subsequent acetylation in the Golgi apparatus (enzyme:

choline-acetyl-transferase). Portions of the Golgi

complex, containing acetylcholine, are pinched off,

forming new synaptic vesicles.

A single action potential on a motor neuron usually

generates an end plate potential large enough to bring

the adjacent membrane area to threshold. Propagation of

an action potential over the membrane ensues. Transverse

tubules, located at regular intervals along the length of

the muscle fiber permit rapid communication with deep

portions of the muscle. Depolarization of transverse

tubules triggers release of calcium from the lateral sacs.

Lateral sacs are the terminal portions of the sarcoplasmic

reticulum lying adjacent to the transverse tubules. The

Ca2+ released from the lateral sacs raises the sarcoplasmic













NERVE


MUSCLE 2.


LONGITUDINAL
TUBULE


TRANSVERSE
TUBULE

LATERAL SAC


5 MYOFILAMENTS


FIGURE 1.


The sequence of events occurring in
excitation-contraction coupling are
listed below. The numbers refer to
numbered events shown in the diagram
above.
1. An action potential travels along a
motor nerve.
2. Acetylcholine which has been released
from the nerve terminal binds to
receptors on the muscle membrane,
causing depolarization an end-plate
potential.
3. When the end-plate potential reaches
a threshold value an action potential
is fired. This action potential is
propagated over the entire muscle
mIembrano, and causes depolarization
of the transverse tubules.
4. Depolarization of the transverse tubules
triggers release of Ca2+ from the lateral sacs.
5. Ca2+ which has been released, binds to troponin
which is associated with the thin myofilaments.
Contraction results.
6. Ca2+ is reaccumulated by an active transport
mechanism located in the longitudinal
tubules. Relaxation occurs.








free Ca2+ concentration. The subsequent binding of Ca2+

to troponin results in activation of the contractile

proteins in the muscle. The amount of Ca2+ released in

response to one action potential propagated over the

muscle membrane is not sufficient to saturate the troponin

molecules and therefore, incomplete activation occurs (17).

For complete activation and therefore maximal force

production, a period of nerve activity at a high frequency

is necessary. Relaxation occurs as Ca2+ is sequestered

(active transport) by the longitudinal sarcoplasmic

reticulum. Following reuptake, calcium is translocated

along the longitudinal reticulum to the lateral sacs,

completing the Ca2+ cycle (67). The mechanism of this

translocation is unclear.














POSSIBLE FATIGUE MECHANISMS AND
CURRENT THEORIES OF FATIGUE

Central Nervous System Fatigue

Events initiating muscular contraction originate

from sensory input or directly in the central nervous

system. Any study of fatigue during exercise of the

whole animal must consider the possibilities of central

inhibition resulting in reduced muscular performance.

There are conflicting reports concerning the potential

for a central component in muscular fatigue. For example,

Merton (44) found that maximal voluntary effort was not

different from the response of the muscle to maximal

tetanic stimulation of the motor nerve. He was studying

brief contractions of the adductor pollicis of humans.

Conversely, Asmussen and Mazin (1) have reported that

"diverting activity" (visual stimulation) permits greater

muscular performance than that which is accomplished when

the eyes are closed. Further experiments demonstrated

that immediate recovery from exhausting exercise (with

eyes closed) occurred if the eyes were subsequently

opened.

It is apparent from the work of Asmussen and Mazin

(1) that central effects can alter muscular performance.

It is important to keep in mind though, that under some








circumstances (i.e., brief maximal effort) fatigue appears

to be due entirely to peripheral mechanisms (44).

Neuromuscular Junction Failure

In the normal sequence of events preceding a muscular

contraction, an action potential is propagated over the

muscle membrane. The occurrence of a normal muscle action

potential is dependent on transmitter release and muscle

membrane properties. Repetitive stimulation may alter

these properties, and this could result in alterations in

the contractile response. Merton (44) found no change

in fatigue in the electromyogram resulting from maximal

stimulation despite an attenuation of force output.

Bergmans (3) studying human extensor digitorum brevis

observed no change in the surface electromyogram during

fatiguing contractions. Electromyography is not the

most sensitive technique for measuring the membrane

response, but any large alteration in muscle action

potential generation and propagation would probably

have been detected.

Using small muscle bundles, and measuring intracellular

potentials, Hanson (37) noted only minor changes in the

rat soleus muscle resting potential and action potential

following repetitive stimulation. The amplitude of the

action potential was reduced in fatigue, but was restored

within a few minutes of recovery. Grabowski (35) noted

a reduced amplitude of the muscle action potential of

fatigued frog muscle fibers. A reduced amplitude could








also be produced in a rested muscle by reducing extra-

cellular Na+ concentration. Under these conditions,

twitch height is not altered. It would appear from the

results of these experiments that following a period of

fatiguing contractions, the amount of neurotransmitter

released is sufficient to raise the end-plate potential

to threshold, and the muscle membrane is capable of

propagating a muscle action potential.

Attenuated Calcium Release

If a normal action potential is propagated over a

muscle membrane, but less calcium is released from the

lateral sacs, the contractile response will be attenuated.

The reduced amount of Ca2+ released would result in a

lower peak sarcoplasmic Ca2+ concentration and therefore

a reduced activation. Direct measurement of Ca2+ release

in a fatigued muscle has not been reported. Despite this,

several authors have concluded that the mechanism

responsible for the fatigue they observed was reduced

Ca2+ release (15, 19). This conclusion is based on

results from one of two techniques: either a) all other

possibilities are eliminated or b) inference is obtained

from analysis of changes in the time to peak tension and

the peak rate of force development for a twitch. In the

former, evaluation of the functional state of the

neuromuscular junction and of the force generating

capacity of the muscle has revealed that these processes

are unaltered in the fatigued muscle. This leads one to







believe that the muscle has a reduced amount of Ca2+

released. In the latter, it is assumed that relaxation

occurs simultaneously with Ca2+ reuptake (4). Under these

circumstances, a reduction in contraction time would

result from a reduction in duration of activation (more

rapid reaccumulation or shorter duration of release). A

reduction in peak rate of force development without a

concommitant reduction in contraction time indicates

reduced activation, and this is interpreted as a reduced

amount of Ca2+ released. Brust has made observations

similar to these (reduced rate of force development in

fatigue with no change in contraction time) on mouse

soleus muscles in vitro, (10) and concluded that fatigue

was due to reduced Ca2+ release.

Similar observations would be expected if there was

an increase in the Ca2+ concentration at which binding to

troponin and subsequent contractile activity occurs. It

has been observed by Fuchs et al. (32) that the affinity

of troponin for Ca2+ can be altered by pH. This

possibility must be considered when dealing with

inferences from measurements of contraction time and

rate of force development. Fitts and Holloszy (29) have

presented data indicating that reduced pH may be

associated with fatigue. They support the theory that

reduced activation (and reduced rate of force development)

is due to a reduced affinity of troponin for Ca2+.








Another situation may occur in the muscle for

which the rate of force development declines with developed

tension while contraction time remains unchanged. A

reduction in contractile capacity would give the same

results. This possibility must be given consideration.

Some authors have tested for, and found, changes in the

contractile capacity of the muscle under study (30, 44).

These are discussed below.

Reduced Capacity of the Contractile Apparatus

Fatigue may be the result of a reduced ability of the

contractile proteins to generate tension. This could be

a result of either: i) damage to myofilaments (i.e.,

misalignment or inactivation) or ii) restricted availability

of energy. In either case, the effect would be a reduced

force generation under conditions of maximal activation.

This capacity to develop tension has been traditionally

tested with either a K+ contracture or a caffein

contracture. Both of these procedures result in maximal

activation (Ca2+ concentration high enough to saturate

the contractile apparatus). Tetanic stimulation has also

been used to evaluate the capacity of a muscle to generate

tension.

Fitts and Holloszy (30) observed that tetanic force

was reduced in the rat soleus muscle following a series

of tetanic contractions. They noted that recovery of the

force generating capacity occurred relatively quickly

(within minutes). No insight into the mechanism








responsible for the fatigue observed by these authors is

provided. Since recovery occurred quickly, it is obvious

that permanent damage to the myofilaments was not a

mechanism of the fatigue.

Spande and Schottelius (56) studied fatigue in the

mouse soleus muscle in vitro. They found that the

magnitude of the reduction in developed tension was

inversely proportional to the phosphoryl-creatine (PC)

concentration. PC serves as an immediate source of high

energy phosphate (-P), for rephosphorylation of ADP and

may also be involved in a transport capacity for ~P from

mitochondria to myofilaments (40, 55). The experiments

by Spande and Schottelius (56) involved contractions with

periods of anoxia and/or glucose deprivation, and this

must be kept in mind when comparing their results with

those of other authors. Under these circumstances

reduced energy availability appears to be related to the

fatigue. Fitts and Holloszy (29) have measured PC

changes during and following fatiguing contractions in

rat muscle. They found no relationship between PC and

the amount of fatigue or recovery from fatigue.

The final common mediator of energy availability is

the level of ATP in the muscle. Edwards reported that

ATP and PC concentrations were reduced in isolated

mouse soleus muscles during prolonged tetani under

anaerobic conditions. This was also the case when

muscles were fatigued in the presence of cyanide and






15

iodoacetic acid. In the former case, lactate accumulated

but in the latter case there was no accumulation of

lactate. It was noted that prolongation of relaxation

was associated with a reduction in ATP and PC levels.

This provides an indirect method of evaluating energy

availability in the muscle. Relaxation would be expected

to be prolonged since it is dependent on reuptake of Ca2+.

Sequestering Ca2+ is an active transport process which

requires ATP (13). Reduced levels of ATP may also slow

the relaxation phase of individual cross-bridges. ATP

is required to permit dissociation of the actin and

myosin molecules (65). The extreme of this situation

occurs when rigor bonds form in the absence of ATP.

It can be concluded from the above discussion that

fatigue can be the result of any of several mechanisms.

The possibility exists that multiple mechanisms function

at once. For example, a reduced release of Ca2+ may be

accompanied by a limitation of energy availability. This

situation would complicate the elucidation of the

mechanisms) responsible for the fatigue.

The following chapters present the details of

experiments conducted in an effort to gain an under-

standing of fatigue in the gastrocnemius-plantaris

muscle group of the dog.














GENERAL METHODS

Mongrel dogs of either sex weighing 9-18 kg were

used in these studies. They were anesthetized with

intravenous sodium pentobarbitol, 30 mg/kg, with

additional 30 mg injections as needed. The animals

were intubated and maintained on a respirator throughout

the experiment. A Beckman LB-2 gas analyzer sampled gas

from the endotracheal tube continuously. Ventilation

was adjusted to maintain end-tidal CO2 at 4.5 .25 %.

Rectal temperature was monitored with a thermocouple,

and kept between 37.5 and 380C by appropriate adjustment

of a heating pad placed under the thorax of the supine

dog.

The left gastrocnemius-plantaris muscle was exposed

via an incision along the medial aspect of the left hind

limb. Muscles overlying the medial head of the

gastrocnemius-plantaris muscle group were tied twice

with butcher's cord and cut between the ties. These

muscles are: sartorius, gracilis, semitendinosis and

two heads of semimembranosis. All veins draining into

the popliteal vein were ligated except those branches

coming from the gastrocnemius-plantaris muscle (see

Figure 2). Any veins draining the muscle but not

entering the popliteal vein were ligated. These were

16





















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only minor vessels which occur along the anterior or

lateral surfaces of the muscle. The popliteal vein was

cannulated. A cannulating type electromagnetic flow

probe (Narco Biosystems) (3mm I.D.) was placed in the

outflow tubing. The venous effluent was returned to the

dog via another cannula in the external jugular vein.

Heparin, 2000 U/kg (12 mg/kg) was administered I.V.

initially and 1000 U/kg was given half way through the

experiment, prevented coagulation of blood in the tubing.

A thin cannula passed through the wall of the outflow

tubing and threaded within it to the muscle provided a

sampling port for venous blood. A thermocouple was placed

alongside this thin tube. The tip of the probe was

within 1 cm of the muscle. The blood temperature here

was assumed to be an average temperature of all parts of

the muscle. A heat lamp focused on the abdomen and hind

limbs was used to maintain muscle temperature near 370C,

while the muscle was at rest. During contractions, the

lamp was turned off and the muscle temperature was

permitted to rise. The contralateral femoral artery was

cannulated and a Statham pressure transducer was connected

to the cannula. Output of the transducer was recorded

on a Grass polygraph model #5.

The Achilles tendon was severed close to the

calcaneous and securely fixed in an aluminum clamp.

The clamp was hooked to a slide bar which was fastened

to the cantilever beam of an isometric lever. Force






20

was measured with a displacement transducer detecting the

displacement of the free end of the cantilever beam. The

transducer output was linear for forces up to 20 kg. A

displacement at the transducer of 0.1 mm gave a full

scale deflection on the recorder. Output of the displace-

ment transducer (tension) was amplified and recorded

directly. The amplified tension signal was also

differentiated with respect to time (Gould-Brush dif-

ferentiator). The differentiated and direct signals

were recorded on a Gould-Brush Model 2400 recorder.

Blood flow and muscle temperature were also recorded

continuously. The maximal rate of change of the

amplified force signal never exceeded 80 v/sec. The

differentiator was calibrated with ramp signals and was

found to be linear through 120 v/sec.

The sciatic nerve was dissected free from

surrounding tissue. All branches of the nerve not

innervating the gastrocnemius-plantaris muscle were

severed. The nerve trunk was double ligated about 4

cm proximal to the muscle and cut between the ties. A

tubular stimulating electrode was placed on the distal

stump of the nerve. The nerve was stimulated with a

Grass Model SD9 stimulator with square pulses 0.2 msec

in duration and of 2-4 volts. This voltage was double

that necessary to produce a maximal contraction.

Contractions were isometric. The lever-arm of the

myograph was bolted to a cast iron base which was








clamped to the table. Bone nails were placed in the

tibia and femur (one each). These nails were firmly

attached to the base of the myograph. A turnbuckle

strut, placed between the lever-arm and one of the

bone nails prevented flexing of the lever-arm. The

muscle length was set 1-2 mm shorter than the length at

which developed tension was greatest (optimal length).

Optimal length was determined by measuring the developed

tension (total tension minus resting tension) of

contractions at (0.2/sec) at various lengths.














02 UPTAKE AND DEVELOPED TENSION

Introduction

Oxygen uptake (VO2) of muscle can increase more than

40 times resting levels during repetitive stimulation (57).

At low frequencies of stimulation, VO2 is proportional

to the isometric developed tension (AT) (total tension

minus rest tension) (66). This relationship was

observed for contractions following a period of fatiguing

contractions at 10-14 per sec for 30 minutes (66). By

stimulating the motor nerve with twin impulses, AT can

be increased. It is not known whether the proportionality

between VO2 and AT persists for twin impulses stimulation

before or after fatiguing contractions. It is of interest

to determine whether or not the muscle is capable of

increasing its VO2 following fatigue, and if so, to see

if AT is still proportional to V02.

The purpose of this study is to investigate the

effect of fatigue and twin impulse stimulation on the

ratio between VO2 and isometric developed tension in

the in situ dog gastrocnemius muscle. Further experiments

have also been conducted to determine the V02:AT relation-

ship for muscle "fatigued" by curare infusion or ischemia

during repetitive stimulation.








Methods

The preparation described in the General Methods

section was used in these experiments. Five series of

experiments were completed to determine the relationship

between muscle V02 and AT.

Oxygen uptake by the muscle was calculated from the

venous outflow and the arteriovenous blood oxygen content

difference. Arterial samples were taken from the

contralateral femoral artery. Venous samples were taken

from the popliteal vein cannula via a thin catheter

threaded through the wall of the venous outflow tubing

to the end of the cannula close to the muscle group.

The blood samples, 0.8 ml each, were collected in glass

tuberculin syringes sealed with mercury-containing caps

and kept in ice until analyzed for 02 content with a

Lex 02 Con analyzer.

Series 1 and 2

Contractions began at the rate of 1/sec, and 02

uptake and developed tension were measured after a steady

level had been attained. Next, the muscle was fatigued

by stimulating it at a rate of 10-20 impulses / sec for

30-40 minutes. This reduced the developed tension in a

single twitch to about one-third to one-half of the pre-

fatigue level. The muscle was allowed to recover for 30-

40 minutes so that the resting VO2 approached the pre-

fatigue level. Three pairs of blood samples were taken

five minutes apart as the muscle continued to recover.








After this recovery period, the muscle was stimulated

at the same rate as before (1/sec) with twin impulses

(two impulses, 6.5 v in amplitude, 0.2 msec in duration

and separated by 10-20 msec), and 02 uptake and developed

tension were measured. The time between the twin impulses

was set by adjusting the delay between impulses until a

smooth contraction was obtained. Post fatigue stimulation

with twin impulses returned the developed tension

approximately to the level of single impulse stimulation

pre-fatigue. In the second series of experiments, both

single and twin impulse contractions were done before

and after the fatiguing contractions.

In each type of contraction, the muscle was allowed

to contract for at least four minutes before arterial and

venous samples were taken to ensure that developed tension

and blood flow had reached a steady level. After an

additional two to three minutes of contractions, a

second pair of arterial and venous samples was collected.

The 02 uptake rates calculated from the two pairs of

blood samples were averaged and the resting 02 uptake

rate was subtracted to give the net 02 uptake per minute.

This value was divided by the muscle weight and the

number of contractions per minute to give the 02 uptake

in microliters of 02 per gram of wet muscle per

contraction (Pl 02 g 9-1 C-1). Developed tension was

expressed as grams of developed tension per gram of wet

muscle (g-g-1).







Series 3

Oxygen uptake and developed tension were measured

during the fatigue process. In separate experiments,

muscles were stimulated at rates of 3, 4, 5 and 6 impulses

per second. After the first five minutes of contractions,

blood samples were collected periodically as the muscle

fatigued during contractions for two hours. The decrease

in developed tension ranged from 34 to 45% over the two

hour period. Sixty to 80% of this decrease occurred in

the first 30 minutes. Although blood flow and developed

tension were sometimes changing rapidly, there was almost

no change in the arteriovenous blood oxygen content

differences. This allowed application of the Fick

equation for 02 uptake calculation with confidence (64).

Series 4

Oxygen uptake and developed tension were measured in

muscles during different levels of reduced blood flow

produced by partially occluding the arterial inflow.

The muscles were stimulated to contract at one twitch

per second throughout these experiments.

Series 5

It is possible that a portion of the fatigue

observed in the experiments of Series 1-3 might be due

to presynaptic neural failure or neuromuscular junction

failure, particularly in the first series of experiments

in which the nerve-muscle preparation was stimulated at

rates of 10-20 impulses / sec for 30-40 minutes. To








investigate this possibility, two experiments were done,

in which neuromuscular transmission was completely

blocked by repeated injections of either curare or

succinylcholine. After the drug was given, the nerve

was stimulated at the rate of 20 impulses / sec for

30 minutes. Muscle contraction did not occur during this

30 minute period because of the presence of the blocking

drug. Developed tenion (at a stimulation rate of 1/sec)

was measured before the drug was injected and after the

effects of the drug had worn off. Therefore, any

difference in developed tension before and after the

period of high frequency stimulation with curare block

would be due to either nerve or neuromuscular junction

failure since the muscle did not contract during the 30

minutes of stimulation.

Neuromuscular fatigue was mimicked in a fresh muscle

by infusing curare into the animal at different rates to

block muscle contraction to varying degrees. 02 uptake

and developed tension were measured at the different

levels of neuromuscular blockade. The stimulation rate

was 1/sec.

Results

Resting 02 uptake for the gastrocnemius-plantaris

muscle averaged 7.7 Pl 02-g- 1min-1. This is somewhat

higher than average values previously reported (27, 57)

but well within the usual range. Mean arterial blood

pressure remained above 100 mmHg throughout all of the

experiments.








In the first series of experiments, analysis of

variance for repeated measures (8) on the ratios

between 02 uptake and developed tension observed before

and after fatigue revealed no significant difference

(p>.25). Table I shows the 02 uptake and developed

tension for each of the muscles both before and after

fatigue.

The results of the 02 uptake and developed tension

measurements in series 2-5 are summarized in Table II

and illustrated in Figures 3 and 4. Table II shows the

linear regression equations relating 02 uptake and

developed tension for each experiment. These equations

were calculated from data which included values from

the fatigued muscle as well as the fresh muscle. The

slopes of all but one (Experiment 8, p=.09) of the lines

are significantly different from zero (p<.05), despite

the small number of points used to determine each

regression equation. It is obvious from Figure 3 and

Table II that there was considerable variability between

animals. This has always been observed in this preparation

(27, 57, 66). However, despite differences in absolute

values between different animals, the same pattern of

response was observed in all cases. VO2 per contraction

and AT were directly related.

Results of four sample experiments from series 2-5

are shown in Figure 3. Figure 4 shows that all of the
























O .8

I2

N2
W Is

0.

O



0 100 200 300 400
TENSION (g.g-I)







FIGURE 3. Results of four sample experiments.
Numbers refer to individual experiments.
Thirteen is from Series 2 (circled
numbers = post fatigue). Sixteen is
from Series 3. Nineteen is from
Series 4. Twenty-two is from Series
5.






































TENSION (% of greatest tension)


FIGURE 4.


Data from Series 2-5 normalized to the same
scale. Developed tension in percent of the
greatest developed tension in each experiment.
02 uptake in percent of the 02 uptake at the
greatest developed tension. Numbers refer to
individual experiments. Seven to fourteen are
Series 2 (circled numbers = post fatigue). Fifteen
to eighteen are Series 3. Nineteen to twenty-one
are Series 4. Twenty-two to twenty-five are
Series 5. The asterisk denoWts (100%, 100%)
which is common to all of the experiments. The
line in this figure is the line of identity (X=Y).



















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33

data follow the same pattern when normalized to the same

scale. In this figure, developed tension is plotted as

the percent of the highest tension developed in each

individual experiment, and 02 uptake is plotted as the

percent of the 02 uptake at the highest developed tension.

Most importantly, Figures 3 and 4, and Tables I and II

show that the relationship between 02 uptake and developed

tension was unchanged by the various treatments.

In two experiments, muscle contraction was completely

blocked by repeated injections of curare or succinylcholine

while the nerve was stimulated 20 times per second for

30 minutes. Injection of the blocker was discontinued

after the stimulation period and the effects of the

blocker were mostly dissipated within 10 minutes.

Developed tension was still at least 90% of the control

value. The observed reduction in contraction strength

may have been due to incomplete recovery from the

neuromuscular block. This 10% reduction in developed

tension can be compared with the 50-70% reduction

observed in the other experiments in which muscle

contraction was not blocked. It appears that most if

not all of the reduced contractile response was due to

alterations beyond the neuromuscular junction.

As pointed out in the Methods, the fatigued muscles

in the first and second series of experiments were allowed

to recover for 30-50 minutes. After this time, the

resting 02 uptake approached the pre-fatigue level.








However, developed tension recovered very little during

this time and was still only one-third to one-half of

the pre-fatigue value.

Discussion

Isometric developed tension at constant muscle length

was varied in this study by four methods: 1) twin impulses

stimulation, 2) fatigue produced by 30 minutes of

contractions at 20/sec, 3) ischemia caused by partial

occlusion of arterial inflow to the muscle, and 4) partial

block of neuromuscular transmission with curare. Figures

3 and 4, and Tables I and II show that none of these

treatments changed the relationship between 02 uptake

and developed tension. Stimulating the fatigued muscle

with twin impulses restored developed tension to pre-

fatigue values. Fatigue did not increase the 02

requirement per unit of force developed, even when the

tension developed by the fatigued muscle was returned

to the pre-fatigue level by twin impulses stimulation.

The fatigued gastrocnemius-plantaris muscle is therefore

capable of increased developed tension and increased

V02. In addition, the 02 requirement per unit of force

developed was not altered during the development of

fatigue.

The dog gastrocnemius-plantaris muscle group has

certain advantages in studies of fatigue. Based on

histochemical staining properties, the dog gastrocnemius

contains only two motor unit types (43). These two








correspond to types FR and S (SR), (fast, fatigue

resistant and slow, fatigue resistant respectively),

described by Burke and colleagues (11) for hindlimb

muscles of the cat. Even though the dog gastrocnemius-

plantaris muscle group contains both FR and S units,

and the cat soleus muscle contains only type S units,

homogenates of cat soleus muscle have less than one-

third of the succinate oxidase activity of homogenates

of the dog gastrocnemius-plantaris muscle group (43).

From this, one might expect all of the dog gastrocnemius-

plantaris muscle units to be more resistant to fatigue

than any of the units of cat soleus muscles. However,

Burke and colleagues (11) have warned against extra-

polation of histochemical and biochemical properties

to physiological properties.

There are several possible causes of the fatigue

observed in these experiments. In Series 1-3, fatigue

could have resulted from a failure in excitation-

contraction coupling, substrate depletion, accumulation

of metabolites, or a combination of these factors.

Since there are both FR and S fiber types in the

gastrocnemius, the fatigue might have been predominantly

in one of the fiber types.

It seems unlikely that neuromuscular junction failure

was a significant component of the fatigue observed in

Series 1-3. Testing for neuromuscular transmission

failure by direct stimulation of the dog gastronemius-

plantaris muscle group is not easy since its large size








makes constant field stimulation difficult. However,

several studies on other mammalian muscles (3, 41, 52)

have indicated that the possibility of neuromuscular

transmission failure at stimulation rates of less than

10/sec is minimal. In two experiments, nerve stimulation

at 20 impulses / sec for 30 minutes when muscle

contraction was blocked by curare or succinylcholine

caused less than a 10% decrease in developed tension.

Decreases in developed tension of 50-70% occurred under

the same stimulation conditions when muscle contraction

was not blocked. These findings indicate that presynaptic

failure of impulse propagation and inadequate release of

acetylcholine probably did not cause the fatigue observed

in our experiments. Desensitization of the endplate is

not ruled out by these results. However, neuromuscular

depression is presently believed to result from a

reduced number of released transmitter quanta and a

reduction of quantal size (42, 48).

In Series 4, the cause of fatigue might have been

muscular, neuromuscular, or a combination of the two

since ischemia can affect both the muscle and the

neuromuscular junction (16, 49). In Series 5, developed

tension was decreased by partial curare block which

presumably simulates neuromuscular junction failure.

These experiments do not allow identification of the

specific cause of fatigue. However, our results do

indicate that the oxygen uptake per unit of isometric








force production is unchanged by either muscle fatigue

or neuromuscular fatigue. This suggests that fatigue,

whether muscular, ischemic, neuromuscular, or a

combination of these three, does not cause any change in

the efficiency of energy transduction from ATP to

external tension development by the muscle, without

concommitant changes in the opposite direction for

energy transduction from foodstuffs to ATP. This is

not likely the case.

These results differ from those of Bronk (6),

Feng (28), Edwards and Hill (20) and Edwards, Hill

and Jones (21) in that they found that the energy

expenditure per unit of force production (or of tension-

time) decreased during fatigue. Unlike our experiments,

however, these earlier studies used stimulus parameters

which caused partially to completely fused tetanic

contractions of relatively long duration. The present

study is of twitch or very brief tetanic contractions,

for which no plateau in developed tension occurs (see

Figure 5). There is little if any tension maintenance

involved.

The data presented in this study along with those

of Wilson and Stainsby (66) demonstrate a constant

coupling between 02 uptake and developed tension in

isometric twitch contractions. In these two studies,

developed tension has been changed by stimulation

frequency, potassium ion infusions, twin impulse
























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40

stimulation, normal muscle fatigue, ischemic fatigue and

partial neuromuscular transmission block with curare.

During all of these treatments, the relationship between

02 uptake and developed tension has been unaltered.

The data also show that although resting metabolic

rate following fatigue approaches pre-fatigue levels after

30 minutes, developed tension is still quite low.

Phosphorylcreatine and ATP levels should be fully

recovered following 30 minutes of rest (38, 51). Edwards

and coworkers (23) have also identified a long lasting

element of fatigue in humans that is not due to

depletion of high-energy phosphates. Further study

is warranted to determine whether or not there really

is a causative relationship between phosphorylcreatine

depletion and fatigue, as suggested by Spande and

Schottelius (56).














EVALUATION OF CHANGES IN THE TWITCH CONTRACTION
ASSOCIATED WITH FATIGUE

Introduction

Skeletal muscle has an attenuated response to a

single impulse following a prolonged period of twitch

contractions due to repetitive single impulse

stimulation (34, 66). This response is not necessarily

indicative of a reduced capacity of the muscle to develop

tension (23, 30). It is,however, a type of muscular

fatigue and warrants further investigation concerned

with determination of mechanisms responsible for this

"twitch fatigue."

Wilson and Stainsby (66) reported that twitch

fatigue occurs in the gastrocnemius-plantaris muscle

of the dog following 30-40 minutes of isometric

contractions (10-14/sec). They monitored recovery with

periods of low frequency stimulation over the course of

3-4 hours. An attenuation of developed tension was still

present following this recovery period. Little is known

of the mechanism responsible for this fatigue.

The purpose of the present investigation was to

study alterations in twitch contractions caused by

repetitive stimulation at three frequencies; 2.5, 5

and 10/sec. A twitch contraction can be characterized








by measurements of the magnitude and time-course of

tension development seen in the isometric myogram (3, 9).

These measurements are: developed tension (AT),

contraction time (Ct), half relaxation time (Rt 1/2),

peak rate of force development (dP/dt), and peak rate

of relaxation (-dP/dt) (see Figure 6). Sandow and Brust

(54) have named the changes in these measurements that

occur with repetitive stimulation the "fatigue patterns.'!

Fatigue patterns have been determined for single muscle

cells and whole mammalian and amphibian skeletal muscles

in vitro (9, 10, 35). Measurements on in situ muscle

where direct determination of muscle force during

repetitive stimulation can be made while the muscle

maintains a normal circulation have not yet been

reported.

Methods

Twenty mongrel dogs of either sex weighing 9-18 kg

were used in this study. The gastrocnemius-plantaris

muscle group was prepared as described in the General

Methods chapter.

Fatigue as a result of 30 minutes of stimulation at

three frequencies 2.5, 5 or 10/sec was studied. Five

animals were used at each frequency. To study the fatigue

patterns of muscle, it is necessary to obtain fast traces

of contractions, before, during and after the fatiguing

contractions. To evaluate twitch contractions before

fatigue, the muscles were stimulated at either 1/sec or







































dP/d t


- dP/dt


.5 AT


DIFFERENTIAL
OF
TENSION


Figure 6. Tracings of: Tension and dP/dt are presented to
demonstrate the manner by which the measurements
were made. See text for verbal description of
these terms.








2.5/sec for 2 minutes. After a fast trace (100 or 200

mm/sec paper speed) was obtained, contraction frequency

was either left at 2.5/sec or increased from 1/sec to 5

or 10/sec. Relaxation was not complete between

contractions when stimulation was 5/sec or 10/sec. To

facilitate measurement of the characteristics of a twitch,

the frequency of stimulation was reduced briefly, while

fast traces were obtained, then the fatiguing frequency

was restored (see Figure 7). During contractions at

2.5/sec complete relaxation occurs between contractions,

so fast traces were obtained without altering the

frequency of stimulation. Besides the contractions at

2 minutes, fast traces were obtained after 10 and 30

minutes of fatiguing contractions and after 10 and 40

minutes of recovery (see Figure 7). To get fast traces

during the recovery period which followed contractions

at 5/sec or 10/sec, the stimulator was turned on briefly

at 1/sec. Following the 30 minute period of contractions

at 2.5/sec, contractions were continued at a frequency

of 0.2/sec. Fast traces were obtained without altering

the frequency of stimulation. It has been reported that

contractions at this low frequency do not alter the

recovery process (66). In one experiment, a tetanic

contraction (200 msec duration, 100 impulses / sec) was

obtained, before and after the 10/sec fatiguing contractions.

This was done to permit evaluation of the contractile

capacity of the muscle. All fast traces were evaluated






45

for the characteristics of a twitch. These characteristics

are illustrated in Figure 6.

Arterial and venous blood samples (0.6 ml) were

obtained at regular intervals throughout the experiment.

Samples were drawn into glass tuberculin syringes, sealed

with mercury-containing caps, and placed in ice until

they were analyzed. These samples were analyzed for pH,

PCO2 and P02 at 370C with a radiometer (Copenhagen) blood

gas machine. These measurements permit evaluation of

viability of the animal and provide descriptive data

concerning metabolic status of the muscle.

At the end of each experiment, the fatigued muscle

was excised, trimmed of visible fat and connective tissue,

blotted and weighed. The force transducer was calibrated

after each experiment by hanging pre-weighed lead weights

on the lever.

In a few additional experiments, twitch contractions

were evaluated when AT was reduced by: ischemia, dantrolene

sodium or reduced stimulation voltage. Comparison of

these contractions with those obtained during and/or

following fatiguing contractions may provide some insight

into the mechanism of fatigue. To study the effects of

ischemia, the femoral artery was occluded while contractions

continued at 1/sec. Fatigue would not occur at this

frequency with an intact blood flow, but does occur with

ischemia. The occlusion was removed after AT fell to

about 50% of the pre-occlusion value (10-20 minutes)

















* I/sec


TENSION


TIME


FIGURE 7.


Tension developed versus time. Contractions
in this case were 10/sec except as indicated.
Fast traces (not illustrated) were obtained
during the 1/sec stimulation.






47

and recovery was observed. Dantrolene sodium, dissolved

in propylene glycol (25 mg/ml) was injected I.V. during

contractions at 0.2/sec. Dantrolene impairs release

of Ca2+ from the lateral sacs (24). This is accomplished

without changes in the action potential and is apparently

a direct effect on the lateral sacs. Sufficient drug

was given to reduce AT at least 50% (2-5 mg/kg). To

study the effects of reducing the number of motor units

contracting, the stimulation voltage was reduced while

the muscle contracted 0.2/sec. This results in excitation

of fewer motor neurons and their motor units. Consequently

less tension is developed. Comparing the twitch

characteristics of a normal versus a fatigued muscle may

provide information leading to an understanding of the

mechanisms) of fatigue.

Also, in a few experiments, samples of muscles were

obtained immediately following the 30 minute stimulation

period, and/or after 40 minutes of recovery. Samples

were frozen in situ with metal clamps pre-cooled in

liquid nitrogen. Small samples (30-80 mg) were then

homogenized (Vertis homogenizer) in perchloric acid

(8% in 40% ethanol) and analyzed for phosphorylcreatine

by the method of Ennor and Stocken (25) (see Appendix).

Statistical analysis was by the two way analysis of

variance for repeated measures. Differences between means

were determined by Duncan's multiple range test (2).







Results

Blood samples were obtained before the contractions

began and at t = 10, 30, 40 and 70 minutes. Arterial PO2

was 87 2.3 mm Hg (mean SEM) before contractions and

did not change significantly throughout the experiments

(see Figure 8). Before contractions began, PvO2 was

50.2 2.0 mm Hg. During the contraction period PvO2

was lower, but none of the blood samples measured had

a PO2 less than 12 mm Hg. Except for the experiments

where fatigue was caused by 2.5/sec contractions, PvO2

was back to pre-fatigue values early in the recovery

period. Contractions were continued, 0.2/sec, during the

recovery period of these (2.5/sec) experiments; therefore,

it might be expected that PvO2 would not be at rest levels.

Arterial PCO2 began at 31.6 0.8 mm Hg and fell

slowly during the experiments. The decrease in PaCO2 was

statistically significant but probably is of minimal

physiological significance. Venous PCO2 was high during

the contraction period when PvO2 was low, and returned

to pre-contraction levels early in the recovery period.

Arterial pH was 7.40 0.01 before contractions began,

and did not change significantly throughout the experiments.

Venous pH decreased from 7.37 at t = 0 minutes to 7.32

(for 2.5/sec) or 7.28 (for 5 or 10/sec) at t = 10 minutes.

By 10 minutes of recovery, venous pH had returned to pre-

fatigue values (see Figure 8).



























FIGURE 8.


Blood flow and the blood gas measurements
versus time. The horizontal bar
indicates where fatiguing contractions
occurred. When means at a given time
(for different frequencies) were not
significantly different, means were
combined. Numbers refer to the
fatiguing frequency. Asterisks
indicate where measurements are
significantly different from the
original value (time = 0 minutes).
Vertical bars are SEM.















150
BLOOD FLOW
(ml min IOOg )
90


30F


P 02 (mm


40F


PCO2 (mm









-8H
( O-eq


40
Hg )

20





4.8

2-I
I2)
2.4


P--I --a 02

5, I0

Ti 1 Pv 02




2 --5'-- PPv- C2O2

5.1- ---- --- PaC-022





5,10 O
x-_ x_--- VEN
...t--- ---- -------- ART
FATIGUING
CONTRACTIONS


10 25 40 55 7
TIME (min )


FIGURE 8








Although blood flow was measured continuously, only

those measurements corresponding to times when blood

samples were obtained are presented (see Figure 8).

Blood flow was higher during the contractions, but was

back to pre-fatigue values by 10 minutes of recovery.

There were no significant differences between frequencies

for blood flow response.

The first 2 minutes of contractions were at 1/sec

or 2.5/sec. There were no significant differences for

AT between these frequencies at t = 2 minutes. Mean AT

for all experiments was 2.3 15 g/g (wet wt) at this

time. The muscles weighed 48.5 3.3 g (wet wt).

Developed tension fell more rapidly during 10/sec

contractions than during 2.5/sec or 5/sec contractions

(see Figure 9). By 30 minutes all frequencies of

stimulation resulted in significant reductions in AT.

There was no significant recovery of AT during the 40

minutes following the fatiguing contractions.

Contraction time decreased during the fatiguing

contractions at 10/sec, but not during the contractions

at 2.5 or 5/sec. There was no significant difference

for Ct between recovery and pre-fatigue measurements at

any fatiguing frequency (see Figure 9).

Half relaxation time did not change during the

contractions or during the recovery except for recovery

of 2.5/sec fatigue. The Rt 1/2 was longer for contractions

at 0.2/sec than for I/sec. If contractions during recovery






























FIGURE 9.


Developed tension, contraction time
and half relaxation time versus time.
The horizontal bar indicates when
fatiguing contractions occurred.
Where there was no significant
difference between means, all
frequencies are combined. Numbers
refer to the fatiguing frequency.
Asterisks indicate where measure-
ments are significantly different
from the original value (at time =
2 minutes). Vertical bars are SEM.












100

A T (%)
60



20



96
Rt 1/2
(msec )
48


80
Ct
(msec)
40


-II
J2.5
125


FATIGUING
FATIGUING
CONTRACTIONS






I I -- TI------I 5 10


2 5~lI _


Iz- -I 5 10


10 25 40 55 7
TIME (min )


FIGURE 9






54

for this frequency of fatiguing contractions had been 1/sec

(at t = 40 and 70 minutes only) then no difference from

pre-fatigue contractions would be expected.

Figure 10 illustrates the changes in dP/dt and -dP/dt

seen in these experiments. The changes seen for dP/dt

closely parallel those observed for AT. A positive and

significant correlation exists between dP/dt and AT

(r2 = 0.93) and between -dP/dt and AT (r2 = 0.82).

Muscle temperature rose during the fatiguing contrac-

tions. The increase in temperature was only 1-2C. A

similar or smaller rise was seen during the 5/sec and

2.5/sec contractions. Muscle temperature fell slowly to

370C during recovery, but was not permitted to go below

370C.

Muscle samples obtained during a few of the experi-

ments were analyzed for phosphorylcreatine (PC). Analysis

revealed that PC is low at t = 30 minutes (during

contractions), but is back to resting levels by t = 70

minutes (see Table III). The values of PC given in the

table are left:right ratios. PC was determined relative

to total creatine in the muscle sample. Harris (38) has

shown that total muscle creatine content does not change

during exercise and therefore can be used as an index of

muscle weight.

In one experiment, a tetanic contraction was obtained

before and after fatiguing contractions at 10/sec. Figure

11 illustrates the lack of change seen for this contraction.













IX v o

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TETANIC CONTRACTIONS


Pre- fatigue
* and
SPost-fatigue


TENSION


dP/ dt


FIGURE 11.


Tracings of recordings of tension and
differential of tension for tetanic
contractions (100/sec for 200 msec).
Developed tension following 30 minutes
of fatiguing contractions (10/sec) was
only slightly lower than that before
the 10/sec contractions. The differential
tracer were virtually superimposable.








The tetanic contraction is recovered at a time when

twitch AT is still reduced.

Contractions observed during ischemia demonstrated

a reduced AT and a prolonged Rt 1/2. Following restoration

of blood flow, recovery of both AT and Rt 1/2 was 50%

complete in 30 minutes. Figure 12 shows recordings from

one muscle for contractions pre-ischemia, during ischemia

and post-ischemia. These recordings are typical for

what was seen.

Comparing the effects of dantrolene sodium, ischemia

and reduced stimulation voltage on Rt 1/2 vs AT (see

Figure 13), indicates that ischemia and reduced voltage

cause large alterations in Rt 1/2 with concomitant

reductions in ST. With administration of dantrolene

sodium, the attenuation of AT is not accompanied by a

substantial change in Rt 1/2. This pattern seen with

dantrolene is similar to the changes seen during the

fatiguing contractions.

Discussion

In these experiments, twitch fatigue has resulted

from 30 minutes of contractions at 2.5, 5 or 10/sec.

Forty minutes after the fatiguing contractions were

ended, no significant recovery had occurred. Recovery

would eventually have occurred following several hours

of relative inactivity (66). Twitch fatigue, then,

results from a relatively persistent alteration in the muscle

which affects the contractile response to a single impulse.
















TENSION pre-ischemia

dP/dt



-ischemia


post-ischemia


FIGURE 12.


Tension and dP/dt (upper and lower curves
of each pair respectively) are shown.
1) a control contraction before occlusion
of blood flow
2) contraction during ischemia, 5 minutes
after occlusion of blood flow
3) a post-ischemia contraction, 50 minutes
after release of occlusion, contraction
frequency 1/sec.











1.75

Rt ISCHEMIA
1.5 (Rt/Rti)


1.25


I. DANTROLENE


.75 REDUCED
SSTIMULUS
VOLTAGE

20 40 60 80 100
AT (*/')



FIGURE 13. Half relaxation time versus AT is presented
to illustrate the relative changes in Rt 1/2
when AT is reduced by ischemia, dantrolene
or reduced stimulation voltage. Each line
represents one dog. Lines were determined
by the least squares method common to all
three lines.








The response to tetanic stimulation is not altered.

Analysis of the fatigue patterns for this muscle group

may provide some insight into the mechanisms responsible

for this long-lasting fatigue.

The extent of comparisons between frequencies for

the fatigue patterns observed in these experiments is

limited. In the experiments where fatigue was caused

by contractions at 5/sec or 10/sec, the frequency of

stimulation was reduced to 1/sec to obtain fast traces.

This procedure was implemented because full relaxation

does not occur between contractions at 10/sec and 5/sec.

The measurements which have been made on these contractions

are affected by the resting tension. It was hoped that

by reducing the frequency to 1/sec for these contractions,

a true representation of the characteristics of a twitch

could be obtained. Although a common frequency is used,

it is evident that the preceding contractions did have

an effect on the measurements (see Figure 14). The

measurements made for the 2.5/sec series were made on

contractions at 2.5/sec during the 30 minute fatigue

period and at 0.2/sec during recovery. Due to the effect

of frequency and preceding contractions on the time

course of a twitch, caution must be exercised when

comparing values between frequencies. Comparing the trend

within one frequency with the trend within another

frequency is valid.


















TENSION






DIFFERENTIAL



I/sec after
10/sec


FIGURE 14.


Following fatiguing contractions at 10/sec,
switching the stimulator to 1/sec results
in a negative staircase. The top tracing
is tension, and the lower one is dP/dt.
This demonstrates the inotropic effects
of previous contractions, and illustrates
the mechanisms responsible for the decrease
in developed tension seen following the 30
minute period of contractions. The single
contraction on the left immediately follows
10/sec contractions; the others (at a reduced
paper speed) were continued at I/sec.






64

If contractions of the fatigued muscle are compared

with contractions before the muscle was fatigued, the

following characteristics are noted. Developed tension

is reduced. This reduction appears to be greater when

a higher frequency of stimulation occurred during the

fatigue period. Contraction time and Rt 1/2 are not

different in the fatigued muscle in comparison with the

rested muscle for muscles fatigued at 10/sec and 5/sec.

The prolongation of contraction time in recovery from

fatiguing contractions at 2.5/sec is not due to fatigue,

but due to the stimulation frequency during recovery.

Contraction time gets shorter as frequency of stimulation

is increased. For the same reason, Ct is shorter during

the fatiguing contractions at 10/sec. This is an effect

of previous contractions (10/sec) altering the time-

course cf contractions at 1/sec. A decrease was seen for

dP/dt which was proportional to the decrease in AT.

Brust (10) reported similar fatigue patterns for the

mouse soleus muscle in vitro. The same author (9)

reported different fatigue patterns for frog semitend-

inosis muscles. The major difference was that Rt 1/2 was

prolonged in the fatigued frog muscle but not in the

mammalian muscles. This difference is apparently not

species specific. Edwards et al. (22) reported that

there is a slowing of relaxation in mouse muscle

following a fatiguing effort. This slowing of relaxation

was correlated with low levels of ATP (22). This can







explain the increase in Rt 1/2 seen in ischemic fatigue

(see Figure 13). Since Rt 1/2 was not prolonged following

a period of fatiguing contractions with an intact blood

supply, it would appear that ATP was available within the

muscle.

The evidence presented above suggests that the fatigue

observed in these experiments did not occur as a result

of reduced ATP levels. Other evidence supports this

suggestion. Although PC levels were low during the

contraction period, (see Table III), resynthesis had

occurred before significant recovery of AT. Rapid

resynthesis of PC during recovery from a period of

contractions was also observed by Piiper and Spiller (51).

There seems to be no direct relationship between PC levels

and AT. This is contrary to a report by Spande and

Schottelius (56), but supports the observations of Fitts

and Holloszy (29). It should also be pointed out that

glycogen was still available after 30 minutes of stimulation

at 10/sec (14) and lactate production had declined (or

even reversed lactate uptake) (60). It would seem that

energy was available, but demand for energy was reduced.

In support of this conclusion, PvO2 was not below minimum

critical PO2 (59) at any time that PvO2 was measured.

The muscle is capable of developing more force than

that seen in a twitch. Twin impulse stimulation causes

developed tension to be double that seen with single





66

impulse stimulation (34). This relationship is maintained

for rested as well as fatigued muscles. It would appear

that the reduced AT of the fatigued muscle was due to

reduced activation.

A reduced activation of skeletal muscle could,

theoretically be the result of: a) reduced neuromuscular

transmission, b) reduced Ca2+ release, or c) reduced

responsiveness of the contractile elements to Ca2+.

Neuromuscular transmission appears to be intact (34).

This agrees with others (3, 35, 37) who have found only

minimal changes in muscle action potentials or electro-

myographic response following comparable stimulation

periods. There is a possibility that Ca2+ release has

been attenuated. This could cause a reduced AT and

dP/dt without altering Ct or Rt 1/2 (15). Brust (10),

who observed similar fatigue patterns with the mouse

soleus muscle, suggests that the fatigue he observed was

a result of reduced Ca2+ release (see also (63) for the

converse). The factors) responsible for the reduced

Ca2+ release is/are not known. Bonner et al. (5) have

reported that muscle mitochondria accumulate Ca2+ during

exercise. This would reduce the pool of Ca2+ available

for recycling in the sarcoplasmic reticulum (67) and

consequently limit the amount available for release.

Dantrolene sodium apparently reduces the amount of

Ca2+ released per impulse (24). This drug was used in

these experiments to determine the effects of reduced








Ca2+ release on a twitch. A dantrolene treated muscle

contracts with a time-course similar to the fatigued

muscle. Ischemia or reduced stimulation voltage caused

a reduction in AT, but this was accompanied by changes

in Rt 1/2.

An alternative hypothesis involves a reduced binding

of Ca2+ to the regulatory proteins. During contractions,

muscle pH probably falls (33). Fuchs et al. (32) has

shown that binding of Ca2+ to troponin is inhibited by

lower pH. Fitts and Holloszy (30) has suggested this

may be a mechanism responsible for the fatigue seen in

their experiments.

The experiments reported herein do not permit

discrimination between these two potential mechanisms

of fatigue. The fatigue observed following 30 minutes

of stimulation at 2.5, 5 or 10/sec appears to be a

result of reduced activation. Further experiments will

need to be conducted to determine which of the theories

described above applied to these muscles.














EFFECTS OF RESPIRATORY ACIDOSIS ON THE TWITCH CONTRACTION

Introduction

Twitch developed tension is attenuated following a

period of repetitive stimulation. This attenuation is

apparently the result of a reduced activation, due either

to a reduced release of Ca2+ from the lateral sacs or

from a reduced responsiveness of the contractile proteins.

It is not due to a reduced availability of energy (ATP,

PC). Fitts and Holloszy (30) have suggested that the

reduced twitch response is a result of inhibition of the

contractile process due to a reduced intracellular pH.

Steinhagen et al. reported evidence supporting this

hypothesis (61). He reported that dog gastrocnemius

muscle fatigues more quickly during respiratory acidosis

than during normal pH balance.

Specific mechanisms which may contribute to this

acidosis-induced fatigue have been proposed. Nakamura

and Schwartz (46) reported that uptake of Ca2+ by

sarcoplasmic reticulum is accelerated in low pH medium.

This could reduce the duration of activation for a twitch

by reducing the Ca2+ concentration more quickly. Fuchs

et al. (32) have reported that Ca2+ binding to troponin

is inhibited by H+. These molecular mechanisms, if

effective under physiological conditions would cause a

reduction in AT, fatigue.






69

The purpose of this study was to observe the effects

of acidosis on the developed tension and the time-course

of a twitch contraction of the in situ dog gastrocnemius-

plantaris muscle. Intracellular pH can be reduced more

easily via respiratory acidosis than by metabolic acidosis

(12). For this reason, acidosis was induced by reducing

the ventilatory rate. The results of this study indicate

that acidosis is unlikely a direct cause of twitch fatigue.
Methods

The gastrocnemius-plantaris muscle preparation as

described in the General Methods section was used in this

series of experiments.

In each experiment, the nerve was stimulated at a

frequency of 0.2/sec. In three experiments, ventilation

was controlled to maintain arterial pH near 7.4 for the

duration of the experiment (2 hours). In four experiments,

after the muscle had been contracting (0.2/sec) for

20 minutes, ventilation was reduced to 3-4 breaths/minute.

The mixture of inspired gas was adjusted (with 95% 02

and 5% CO2) to maintain normal arterial PO2 during the

hypoventilation. The period of hypoventilation was

continued until arterial pH was less than 7.1 (60-90

minutes). This period of hypoventilation was followed

by a period of hyperventilation (20 breaths/minute). The

hyperventilation was continued for 40-60 minutes (see

Figure 15).







With an additional four dogs, the sequence of

ventilatory adjustment was reversed (control, hyper-

ventilation, hypoventilation) to permit evaluation of

a possible order effect.

At ten minute intervals throughout each experiment,

arterial and venous blood samples were obtained (0.8 ml)

in glass tuberculin syringes (1 ml capacity). The samples

were sealed with mercury containing caps and kept in ice

until they were analyzed for PO2, PCO2 and pH (Radiometer,

Copenhagen).

Fast traces of contractions were also obtained at 10

minute intervals (200 mm/sec on Gould-Brush Model 2400

recorder). The fast traces were measured for developed

tension (AT), half relaxation time (Rt 1/2), contraction

time (Ct), peak rate of force development (dP/dt) and

peak rate of relaxation (-dP/dt) (see Figure 6).

Statistical analysis was by a two way analysis of

variance for repeated measures (2). For statistical

analysis, the last two measurements (fast traces or blood

gases) before alteration of the ventilation were utilized

to represent the state in which they occurred (see

Figure 15).

Results

After 20 minutes of contractions, ventilation was

reduced to 3-4 breaths per minute (Group A) or increased

to 20 breaths per minute (Group B). This adjustment in

ventilation resulted in a reduction in arterial pH from





































FIGURE 15. Developed tension, P02, ventilation and
arterial pH for one dog, from Group A.
Blood samples and fast traces obtained
at were used for statistical analysis.















200
DEV ELOPED
TENS ION
175



100

PO2


50






20

VENTILATION


10




7.5


7.25


a


V


.




* *


100
TIME (min)


150


FIGURE 15







7.37 1 .01 (mean SEM) during the control period to

7.08 .03 in Group A and an increase in Group B from

7.36 .02 to 7.52 .02. After 60-90 minutes, ventilation

was increased to 20 breaths per minute in Group A and

reduced to 3-4 breaths per minute in Group B. This

second alteration in ventilatory frequency resulted in

an increase in pH to 7.4 in Group A and a reduction in

Group B to 7.2 (see Figure 16). In Group C, ventilation

was not altered, and arterial pH did not vary during the

experiments. The reduction in ventilation did not

significantly decrease arterial PO2.

When arterial pH remained constant for 2 hours

(Group C), AT increased with time. By the end of 2 hours,

AT was greater than it had been at the first 20 minute

period. As illustrated in Figure 17, AT increased with

time in Group A also but not in Group B. The only

significant difference in Groups A and B was that AT

during hyperventilation was greater than AT at any other

time period in Group A and greater than control in Group

B (see Table IV).

Despite the apparent effect of hyperventilation on AT,

there was no significant correlation of AT with arterial

or venous H+ concentration (see Table V). Furthermore,

AT was not reduced during hypoventilation. When arterial

pH fell to 7.09 .03, AT did not change significantly.

In Group C, dP/dt did not change significantly. In

Group A and Group B, however, dP/dt was higher during

















TABLE IV

STATISTICS FOR DIFFERENCES BETWEEN
VENTILATION STATES FOR EACH GROUP


Group A Group B Group C

Rank Rank Rank

AT .006 3 2 1 .05 3 2 1 .04 3 2 1

Rt 1/2 .18 NS .003 1 2 3 .99 NS

Ct .86 NS .005 1 3 2 .98 NS

dP/dt .001 3 2 1 .001 3 2 1 .122 NS

-dP/dt .002 3 1 2 .02 3 2 1 .91 NS


The p values are given for AT, Rt 1/2, Ct, dP/dt and
-dP/dt for Group A (control, hypoventilation, hyper-
ventilation), Group B (control, hyperventilation,
hypoventilation) and Group C (control). Means are
listed under the rank order. Rank indicates the
order from largest to smallest for Control, 1,
Acidosis, 2, and Alkalosis, 3. For Group C, rank
1, 2 and 3 indicate time periods; first 20 minutes,
1, next 60 minutes, 2, and last 40 minutes, 3.
Horizontal bars across ranks indicate means which
are not significantly different. NS indicates that
means are not significantly different.


























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hyperventilation than during the initial control period

(see Table I). There was no significant correlation

between arterial pH and dP/dt. This suggests, as seen

for AT, that the alterations in dP/dt associated with

ventilation patterns are not directly related to pH

(see Figure 18).

The peak rate of relaxation was greatest during

hyperventilation. In Group A, this was significantly

greater than both the control period and the period of

hypoventilation. In Group B, however, where arterial

pH increased to 7.52 during hyperventilation (as opposed

to 7.4 in Group A), -dP/dt during hyperventilation was

greater than that during normal ventilation, but not

significantly different from that during hypoventilation.

There was no significant correlation between -dP/dt and

arterial [H+].

In Groups A and C there were no significant changes

in Ct or Rt 1/2. In Group B, however, Ct was shorter

during hypoventilation than at other times. Rt 1/2 was

shorter during hyperventilation than at other times in

Group B (see Figure 19).

Discussion

It has been suggested that intracellular acidification

is the cause of skeletal muscle fatigue (30). If there

is a direct influence of pH on the force of contraction,

this phenomenon would be independent of the manner in

which the pH change was obtained. It is apparent in























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TABLE V

STATISTICS FOR TWITCH CHARACTERISTICS VERSUS BLOOD GASES


Group T Rt 1/2 dP/dt -dP/dt


p= R2= p= R2=


p= R2=


Art [H+]


ven [H+]


PaCO2


A .4
B .25
C .33


.78
.77
.59


A .25
B .04 .56
C .2


.001 .88 .8
.06 .26
.29 .4


.002 .85
.13
.26

.001 .89
.04 .44
.13


.72
.75
.11


.57
.04 .4
.15


.09
.07
.15

.2
.23
.24


.05 .52
.01 .75
.04 .89


.17
.1
.22


.31
.03 .56
.26


.13
.25
.06


Significance valuesare given for correlations between
twitch characteristics and blood gases. R2 values indicating
the percent of variability explained by the variable are
given for significant correlations (p<.05). Groups are
as defined in the legend for Table IV.


p= R2=


p= R2=








these experiments that the changes in Ct, Rt 1/2 and

-dP/dt are not directly associated with arterial pH.

This complication will be discussed further below.

However, to give consideration to the possibility that

acidosis causes fatigue, the results from Group A will

be discussed with respect to the likelihood that the

hypoventilation in these experiments resulted in alterations

in intracellular pH comparable to what might be expected

from contractions at 10/sec for 30 minutes.

In these experiments, arterial pH was reduced from

7.37 to 7.08 by hypoventilation. The acidosis accompanying

the hypercapnia was not associated with any change in AT,

Rt 1/2 or Ct. It would appear that the fatigue described

earlier cannot be a result of acidosis, unless intra-

cellular pH during the fatiguing contractions changes

more than it did during hypoventilation.

The magnitude of the intracellular pH change occurring

in these experiments can be estimated from the results of

Burnell (12). Hypercapnia in dogs (PaCO2 = 55 mm Hg)

resulted in a reduction of intracellular pH of neck

muscles from 6.85 to 6.57. Intracellular pH was

determined by the DMO method (12). Burnell (12)

observed that the maximal response had occurred within

15 minutes. In the experiments reported herein, arterial

PCO2 increased to 57 3.7 mm Hg. The measurements

presented were taken 60-90 minutes after the alteration

in ventilation. It is reasonable to assume that a very







similar change in intracellular pH occurred in these

experiments as was observed by Burnell, under almost

identical circumstances. It can therefore be concluded

that a change in intracellular pH values from normal

resting values (approximatley 6.85) to 6.57 does not

result in a change in developed tension.

The important point, however, is whether or not

intracellular pH fell to this level or lower during the

fatiguing contractions reported in earlier chapters.

Estimates of intracellular pH changes under these

circumstances can only be tentative. Venous PCO2 during

the fatiguing contractions was never greater than 57 mm Hg

(mean = 49 mm Hg at t = 10 minutes for 10/sec). This

hypercapnia would not cause an acidosis sufficient to reduce

AT (if reduced pH will cause reduced AT!). However, there

is lactic acid production during the first 5-20 minutes of

this type of stimulation (60). This is likely to contribute

to an intracellular acidification. Sahlin et al. (53)

report that in humans, performing maximal exercise to

exhaustion, intracellular pH drops from 7.08 to 6.6. This

is comparable to that seen by Hermanson and Osnes (39).

This decrease in pH was associated with an increase in

lactic acid production. Following the bout of exercise,

intracellular pH recovered to 7.0 within twenty minutes.

It is unlikely that intracellular pH changed as much in

the 10/sec fatigue as it did during the exhausting exercise

reported by Sahlin et al. (53). Furthermore, it seems






87

likely that intracellular pH would have returned to resting

levels after 40 minutes of recovery. Developed tension

at this time is still very much reduced (i.e., no significant

recovery has occurred). It seems reasonable to conclude

that the persistent fatigue caused by contractions at

10/sec for 30 minutes does not result directly from

acidosis. There may, however, be indirect ways in which

intracellular pH may affect the contractile process,

resulting in a change within the muscle which persists

beyond the time when pH has returned to control (26).

It is evident that changes in ventilatory pattern

do affect muscle contraction. During hyperventilation, AT

was increased. This was the case whether hypoventilation

preceded the period of hyperventilation or if normal

ventilation preceded it. In the former case (hypo-

ventilation preceding) arterial pH returned to 7.4, so

there was no absolute arterial alkalinization. The

increase in AT under these circumstances was greater than

the increase seen in the latter case (hyperventilation

preceded by normal ventilation), despite the fact that

this procedure resulted in an increase in arterial pH

to 7.52. It seems that hyperventilation increases AT,

but hypoventilation only reduces AT when it is preceded

by hyperventilation.

The high p values reported in Table V reflect the

lack of relationship between AT and [H+]. The fact that

significant differences were observed between ventilation







periods is due not to absolute pH changes, but relative

changes possibly in conjunction with some other change

(ion distribution?i.e.,see 26, 31) associated with

changes in ventilation.

The changes seen in Ct, dP/dt, -dP/dt and Rt 1/2

also suggest that alterations in ventilatory pattern

can affect contraction. The mechanisms responsible for

these changes are not clear.

In this study, it is likely that hypercapnia resulted

in intracellular acidosis. This acidification was not

accompanied by a reduction in AT. It can be concluded

that fatigue is not caused by acidosis if fatiguing

contractions do not cause any greater acidification than

that which occurred in these experiments.














SUMMARY

02 Uptake and Developed Tension

The amount of oxygen used by a muscle was proportional

to the amount of tension developed. This occurred over a

wide range of forces when AT was altered by any of the

following:

a) Fatigue, after 30 minutes of stimulation at 14-20/sec

b) Fatigue, during fatiguing contractions at 3-6/sec

c) Twin impulse stimulation, before and after fatigue
at 14-20/sec

d) Fatigue, during contractions at 1/sec without blood
flow (ischemic fatigue)

e) Attenuated contraction, caused by administration of
curare in sufficient doses to reduce the force of
contraction by as much as 70%.

These results suggest that the major determinant of

energy utilization during an isometric contraction is the

magnitude of the developed tension. It should be emphasized

that these were twitches or very brief tetanic contractions

in which developed tension rose then fell, but did not

maintain a plateau of tension and can therefore be considered

to have a minimal "tension maintenance" component to the

determinants of energy utilization. The possibility that

neuromuscular junction failure may have contributed to

the observed fatigue was tested. It was demonstrated that

transmitter release was normal after 30 minutes of

stimulation at 20/sec.







Time-course of the Twitch Contraction in Fatigue

The time-course (Ct and Rt 1/2) and the rate of change

of force (dP/dt and -dP/dt) were observed when muscles were

fatigued with contractions at 2.5, 5 and 10/sec for 30

minutes. The pertinent observations are listed below.

a) Developed tension fell more rapidly during 10/sec
stimulation than 2.5 or 5/sec.

b) In the 40 minutes following the fatiguing
contractions, no significant recovery of
AT occurred.

c) The Ct and Rt 1/2 of a fatigued muscle are no
different from those of a rested muscle.

d) The dP/dt is greatly reduced in fatigue and is
significantly correlated with AT. A reduction
in -dP/dt is also seen in the fatigued muscle.

e) Despite the persistence of fatigue, the following
parameters have returned to pre-contraction values
after 40 minutes of recovery: venous pH, PO2, Q
and muscle phosphorylcreatine concentration.

It has been concluded from the above observations

that fatigue is not caused by a lack of availability of

energy. Fatigue appears to be the result of a reduced

activation of the muscle cells. This may be due to either

a reduced amount of Ca2+ released or a reduced sensitivity

of the contractile proteins. Since AT of a tetanic

contraction of the fatigued muscle was not different

from that of a rested muscle, it appears that the capacity

of the muscle to generate force isnot reduced. Fatigue,

then must be a result of lower Ca2+ release or a change in

the binding relationship between Ca2+ and troponin (less

Ca2+ bound at a given Ca2+ concentration).




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