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.
li

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 4 2
Results 48
Discussion 59
iii

Page
EFFECTS OF RESPIRATORY ACIDOSIS ON THE TWITCH
CONTRACTION 6 8
Introduction 68
Methods 69
Results 70
Discussion 79
SUMMARY 89
O2 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 9 5
APPENDIX 9 6
BIBLIOGRAPHY 100
BIOGRAPHICAL SKETCH 106
IV

LIST OF FIGURES
Page
1. Excitation-Contraction-Coupling 7
2. Gastrocnemius-Plantaris Muscle Preparation . . 18
3. Sample Experiments; VOg versus Developed
Tension 28
4. VOg 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, POg, PCOg 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
v

Page
16. Arterial and Venous [H+] 76
17. PC>2 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
vi

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
Vll

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.
vii i

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 PC>2, PCC>2 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 O2 (arterial PO2 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 Ca^+
concentration during contraction or a reduced response
of the myofilaments at a given Ca^+ concentration.
x

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

2
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 mechanism(s) 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

4
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 mechanism(s) 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

6
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
Ca^+ released from the lateral sacs raises the sarcoplasmic

7
FIGURE 1. The sequence of events occurring in
excitation-contraction coupling are
listed below. The numbers refer to
numbered events shown in the diaqram
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
membrane, and causes depolarization
of the transverse tubules.
4. Depolarization of the transverse tubules
triqgers 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.

8
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
9

10
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

11
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 Ca^+ concentration and therefore
a reduced activation. Direct measurement of Ca 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

12
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
f
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+.

13
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

14
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 Ca^+.
Sequestering Ca^+ 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 Ca^+ may be
accompanied by a limitation of energy availability. This
situation would complicate the elucidation of the
mechanism(s) 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 38°C 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

FIGURE 2.
The in situ dog gastrocnemius-plantaris
preparation (58).
G - gastrocnemius-plantaris muscle
Gr- gracilis muscle
S - sartorius muscle
SM- two heads of semimembranosis muscle
ST- semitendinosis muscle
muscle

VENOUS
SAMPLE
FIGURE 2
6fY//////////////////// / /

19
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 37°C,
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

21
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 (V02) of muscle can increase more than
40 times resting levels during repetitive stimulation (57).
At low frequencies of stimulation, V02 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 V02 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 V02 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 V02 and isometric developed tension in
the in situ dog gastrocnemius muscle. Further experiments
have also been conducted to determine the VO2:AT relation¬
ship for muscle "fatigued" by curare infusion or ischemia
during repetitive stimulation.
22

23
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 VO2 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 O2 content with a
Lex O2 Con analyzer.
Series 1 and 2
Contractions began at the rate of 1/sec, and O2
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.

24
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 O2 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 O2 uptake rates calculated from the two pairs of
blood samples were averaged and the resting O2 uptake
rate was subtracted to give the net O2 uptake per minute.
This value was divided by the muscle weight and the
number of contractions per minute to give the O2 uptake
in microliters of O2 per gram of wet muscle per
contraction (pi O2 * g“l * C_1) . Developed tension was
expressed as grams of developed tension per gram of wet
muscle (g* g“l) .

25
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 C>2 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

26
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 O2 uptake for the gastrocnemius-plantaris
muscle averaged 7.7 y 1 O2 ' g-^ * min--*-. 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.

27
In the first series of experiments, analysis of
variance for repeated measures (8) on the ratios
between O2 uptake and developed tension observed before
and after fatigue revealed no significant difference
(p>.25). Table I shows the O2 uptake and developed
tension for each of the muscles both before and after
fatigue.
The results of the O2 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 O2 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

UPTAKE (Ml 02 g-I C-l)
28
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.

29
FIGURE
X
it
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 denotes (100%, 100%)
which is common to all of the experiments. The
line in this figure is the line of identity (X=Y).

TABLE I
02 UPTAKE AND DEVELOPED TENSION BEFORE AND AFTER FATIGUE IN SERIES 1
Pre-Fatigue (Single Impulses) Post-Fatigue (Twin Impulses)
Developed Developed
Experiment
Tension
(a-g-1)
O2 Uptake
(ul 09-g-1-C-1)
Tension
(g*q-1)
O2 Uptake
(y1 0?•g-1•C~
1
148
.688
125
. 361
2
196
. 314
177
.356
3
254
.579
250
.446
4
134
. 396
174
.435
5
207
.452
178
.258
6
219
. 382
197
.476
Mean ± SEM
193 ± 18
.468 + .057
184 ± 16
.389 ± .033
Units are as given for Figure 3
u>
o

TABLE II
RELATIONSHIP BETWEEN 02 UPTAKE AND DEVELOPED TENSION IN SERIES 2-5
*N equals the number of data points in each experiment
+ Units for O2 uptake are microliters of O2 per gram of wet
muscle per contraction
Units for tension are grams per gram of wet muscle.

Series #
Expt. #
N*
Type of Fatigue
2
7
4
10/sec for
30
min
8
4
10/sec for
30
min
9
4
10/sec for
30
min
10
4
10/sec for
30
min
11
4
14/sec for
40
min
12
4
20/sec for
30
min
13
4
20/sec for
30
min
14
4
15/sec for
30
min
3
15
7
Continuous
at
3/sec
16
8
Continuous
at
4/sec
17
10
Continuous
at
5/sec
18
10
Continuous
at
6/sec
4
19
5
Ischemia
20
6
Ischemia
21
8
Ischemia
5
22
7
Partial Curare Block
23
8
Partial Curare Block
24
8
Partial Curare Block
25
7
Partial Curare Block
% Variance
Regression Equation* Explained
VO 2
=
3-3-10-3
T
-
9-3.10-2
99.8
VO 2
=
4 - 2•10“3
T
+
9•0•10“2
83.4
VO 2
=
3-6-10-3
T
-
2-6-10-1
89.7
VO 2
=
3-9-10-3
T
-
4-8-10"1
89.5
VO 2
-
2-8-10-3
T
-
2-0-10"1
98.0
VO 2
=
5-1-10-3
T
-
1-9-10-1
99.6
vo2
=
2-7-10-3
T
-
1-2-10"1
98.4
vo2
=
2-2-10-3
T
-
4 -5-10-2
98.6
VO 2
=
4-0-10-3
T
-
2-8-10-1
85.0
vo2
=
5-0-10"3
T
-
8-2-10-3
93.7
VO 2
=
3 * 4"10“3
T
+
8-1-10-2
95.2
VO 2
=
3-0-10-3
T
-
9-1-10-2
90.2
vo2
=
1-3-10"3
T
-
1-3*10-2
91.6
VO 2
=
4 - 6 -10-3
T
-
7-6-10-2
88.9
vo2
=
5 * 1 • 10“3
T
-
2-2-10-1
91.6
VO 2
=
4•6•10“3
T
+
1-1-10"1
97.2
vo2
=
1-5-10-3
T
-
7-2-10-2
97.7
VO 2
=
5-5-10-3
T
-
1*9*10-1
97.7
VO 2
=
5•2.10“3
T
-
2-6-10-1
95.9
oj
to

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 O2 uptake is plotted as the
percent of the O2 uptake at the highest developed tension.
Most importantly, Figures 3 and 4, and Tables I and II
show that the relationship between O2 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 O2 uptake approached the pre-fatigue level.

34
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 O2
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

35
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

36
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

37
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 C>2 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

FIGURE 5. Tracing of a recording of tension and
differential of tension for single impulse
and twin impulse contractions before and
after fatigue. Contractions are superimpos
for ease of comparison. There is no
maintained plateau in this type of
contraction.

TWITCH and TWIN
TENSION
dP/dt
FIGURE 5
CONTRACT ION
Post - fatigue
OJ

40
stimulation, normal muscle fatigue, ischemic fatigue and
partial neuromuscular transmission block with curare.
During all of these treatments, the relationship between
O2 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
41

42
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 iri 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

43
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.

44
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,
PCC>2 and PO2 at 37°C 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)

46
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.
FIGURE 7.

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 Ca 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
mechanism(s) 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).

48
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, PvC>2 was
50.2 ± 2.0 mm Hg. During the contraction period PvC>2
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, PvC>2
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 PvC>2 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 PaCC>2 was
statistically significant but probably is of minimal
physiological significance. Venous PCO2 was high during
the contraction period when PvC>2 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.

50
FIGURE 8

51
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 t 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 1/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

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 (r^ = 0.82).
Muscle temperature rose during the fatiguing contrac¬
tions. The increase in temperature was only 1-2°C. A
similar or smaller rise was seen during the 5/sec and
2.5/sec contractions. Muscle temperature fell slowly to
37°C during recovery, but was not permitted to go below
37°C.
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.

TABLE III
PHOSPHORYLCREATINE ANALYSIS DURING CONTRACTIONS AND FOLLOWING THE RECOVERY PERIOD
Animal #
L: R ratio:
a) during contractions
b) following recovery
0.36 0.63 0.40 0.46
0.83 0.96 1.0 1.11 1.08 1.02 1.26 1.09 0.83 1.02
Phosphorylcreatine is presented above as a ratio:
PC • Creatine--'- (left): PC • Creatine--1- (right)
(See text for discussion of creatine as an index of muscle weight.)
U1
Ln

FIGURE 10. Peak rate of force development and peak rate
of relaxation. See Figure 5 for significance
of asterisks and numbers. Note similarity
between dP/dt versus time and AT versus time
(Figure 5).

100
DP/DT
(°/o)
60
20
TIME (min)
10 25 40
5,10
FIGURE 10
i i
55 70
T
Ln

58
i
I
I
TETANIC CONTRACTIONS
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.

59
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 AT. 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.

60
FIGURE
ischemia
2. 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.

61
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.

62
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.

63
TENSION
DIFFERENTIAL
l/sec after
10/sec
FIGURE 14. Following fatiguing contractions at 10/sec,
switching the stimulator to l/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 l/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

65
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, PvC>2 was not below minimum
critical PO2 (59) at any time that PvC>2 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
O i
periods. There is a possibility that Ca 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 factor(s) 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

67
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
94-
of Ca 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.
68

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 9 5% C>2
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).

70
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, PO2, ventilation and
arterial pH for one dog, from Group A.
Blood samples and fast traces obtained
at * were used for statistical analysis

72
200
DEV ELOPED
FIGURE 15

73
7.37 - .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 Í .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 PC>2 •
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

74
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 12
.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.

FIGURE 16. Arterial and venous H+ concentration during
ventilatory states. The three columns on
the left are from Group A. The three columns
on the right are from Group B. Open columns
are arterial [H+]. Vertical bars are one
standard error of the mean. Upper bar is
for venous, lower bar for arterial SEM.

VENTILATION ( b re a t h s / mi nu te )
FIGURE 16

FIGURE 17. Mean arterial and venous PO2 during the
different ventilatory states. The three
columns on the left represent Group A.
The three on the right represent Group B.
Open bars represent venous PO2
and combined (open and closed) bars
represent arterial PO2. Vertical bars
represent one SEM.

100
ART ERIAL
and
VENOUS 50
po2
(mm Hq)
10 4 20 10 20 4
VENTILATION (breaths/minute)
FIGURE 17

79
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

FIGURE 18. Mean developed tension, dP/dt and -dP/dt
during different ventilatory states.
Developed tension is % of highest in each
experiment. dP/dt and -dP/dt are % of
highest dP/dt in each experiment. Vertical
bars represent one standard error of the
mean.

A T ■■ dP/dt d -dp/dt Ed
10 4 20 10 20
VENTILATION ( bre a t h s / m i nu te )
FIGURE 18

FIGURE 19. Mean contraction time and Half relaxation
time during different ventilatory states.
Vertical bars represent one SEM.

Rt 1/2 O Ct
I 0
20
VENTILATION ( brea t h s/m i nute )
FIGURE 19

84
TABLE V
STATISTICS FOR TWITCH CHARACTERISTICS VERSUS BLOOD GASES
Group
T
Rt
1/2
dP/dt
-dP/dt
Ct
p= r2=
P=
r2=
p= r2=
P=
r2=
P= r2=
Art [H+]
A
. 4
.001
00
00
•
. 8
.09
.17
B
.25
.06
. 26
.07
.1
C
. 33
.29
.4
.15
.22
ven [H+]
A
. 78
.002
.85
. 72
.2
.31
B
. 77
.13
. 75
.23
.03 .56
C
. 59
.26
.11
.24
.26
PaC02
A
.25
.001
. 89
.57
.05
.52
.13
B
.04 .56
.04
.44
.04 .4
.01
.75
.25
C
.2
.13
. 15
.04
. 89
.06
Significance values are 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.

85
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 (PaCC>2 = 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

86
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

88
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 experiements.

SUMMARY
O2 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.
89

90
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 is not 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).

91
Acidosis and the Twitch Contraction
When acidosis was induced by hypoventilation, developed
tension did not decrease. It is likely that the intra¬
cellular pH reached a level in these experiments comparable
with that which was reached in the experiments summarized
above. This suggests that the reduced intensity of
activation observed as a result of fatiguing contractions
was not a result of intracellular acidosis.

PROPOSED HYPOTHESES FOR SKELETAL MUSCLE FATIGUE
Several hypotheses can be invoked to explain how there
may be less Ca2 + released from the lateral sacs. Following
is a discussion of a few hypothetical mechanisms to explain
the fatigue. These mechanisms are presented, not as an
explanation for the observed fatigue, but as hypotheses
which need to be tested to prove or disprove their
involvement in this fatigue. A detailed hypothesis of
the fatigue seen in these experiments is not justified
by the data available.
Depletion of Calcium at Lateral Sacs
Less Ca2+ would be released from the lateral sacs if
less Ca2+ were located at the lateral sacs. This situation
would occur if Ca2+ were lost from the cell or sequestered
by some other organelle within the cell. It is unlikely
that significant Ca2+ was lost from the cell during this
period of stimulation. The Ca concentration of the
interstitial fluid is high compared with the intracellular
concentration at rest or during contractions (18).
The amount of Ca within each muscle cell may be
unaltered in fatigue. Under these circumstances, the
amount of Ca^t available to the lateral sacs may be
limited if some organelle sequesters Ca2+. It has been
reported that mitochondria accumulate Ca during exercise
92

93
(5). It is conceivable that calcium accumulation by
mitochondria may reduce the amount of Ca2+ available
for recycling in the sarcoplasmic reticulum. This could
reduce the amount of Ca^ released with each impulse.
Other organelles may also sequester Ca , limiting the
amount of Ca2 + available to the sarcoplasmic reticulum.
The longitudinal tubules are known to accumulate
Ca2 + (67). It is unlikely that this accumulation would
permit the observed persistence of fatigue. Apparently
the time necessary for translocation of the Ca2+ from
the longitudinal reticulum to the lateral sacs is much
shorter (67) than the observed 40 minutes of recovery
during which no significant increase in AT occurred.
Compartmentalization Within the Lateral Sacs
A reduced Ca2+ release from lateral sacs would occur
if Ca were compartmentalized within the lateral sacs in a
manner which made it unavailable for release (i.e.f bound
vs free). There is no specific evidence available to
support this hypothesis. It is mentioned here only
because it is a hypothetical explanation of the data.
Attenuated Trigger Mechanism for Release of Calcium
A third potential mechanism which would result in
reduced Ca release would be an attenuation of the
2+
trigger mechanism for release of Ca . This mechanism
is poorly understood and therefore a discussion of how
this might be affected in fatigue is unwarranted. One
possibility, however, would be that conduction of the

muscle membrane depolarization into the transverse
tubular system is altered in a manner which affects the
trigger mechanism and therefore reduces Ca^ release.
Reduced Binding Sensitivity for Calcium
It is possible that the amount of Ca2 + released from
the lateral sacs is unaltered, but the degree of activation
accomplished at that concentration is reduced. It has been
observed that acidosis was not likely the cause of this
alteration, but other metabolic factors may be capable
of altering the binding relationship between Ca2+ and
troponin, thereby reducing the intensity of activation
• 9 +
at a given free Ca
concentration.

CONCLUSIONS
It is apparent that further research will be necessary
to elucidate the mechanism causing the fatigue observed
in the experiments reported herein. This research,
however, has eliminated several proposed mechanisms of
fatigue. A few hypotheses are presented which may
contribute to the fatigue observed in these experiments.
Each of these hypotheses is based on a central theme,
a reduced activation of the contractile proteins. It
remains to be seen which of these mechanisms (if any) is
the actual cause of the twitch fatigue observed in these
experiments.
95

APPENDIX
Method for Phosphorylcreatine Analysis
The method of Ennor and Stocken (25) was used to
determine phosphorylcreatine content of muscle samples.
This method determines creatine content before and after
a period of acid hydrolysis of phosphorylcreatine. The
key reaction for this determination is creatine with
diacetyl which yields a pink colored compound. The
intensity of the pink color is proportional to creatine
concentration when diacetyl is available in excess. The
optical density is determined with a spectrophotometer
at 525 nm.
Chemicals and Reagents for Deproteinization and Determination
I Perchloric Acid, stock 70-72% (Baker)
a) .8 M in 40% ethanol
b) .6 M (no ethanol)
II Potassium Carbonate, (Baker) 3 M containing
.5 M triethanolamine (Baker)
III Sodium Hydroxide (Malinkrodt)
a) 1 N
b) . 4 N
IV Hydrochloric acid (Fisher)
a) IN
b) . 4 N
V p Chloromercuric acid (Sigma) (also called p
Hydroxymercuric acid) .05 M purchased as
solution, or as powder then dissolved in
NaOH (1 N) then made to volume with H2O.
VI Naphthol (Sigma) 1% in stock alkali, mix new
daily
96

97
VII Diacetyl (Sigma)
a) stock solution 1%
b) mix 1:20 daily for use in assay
VIII Stock Alkali
for 1 liter: 160 g Na2CC>3 (Baker) and 60 g NaOH
(Malinkrodt)
IX Creatine (Sigma) (for standards)
16 yg/ml
Procedure for Deproteinization
Muscle samples weighing 30-80 mg were frozen in
situ with metal clamps pre-cooled in liquid N2* The
muscle samples were kept frozen until they were
homogenized in Perchloric acid. The samples were
homogenized (Vertis) for 60 seconds at high speed in
6 ml of ice cold .8 M Perchloric acid. The homogenate
was poured into a centrifuge tube (15 ml) and the blades
and bown were rinsed with 3 ml of .6 M Perchloric acid.
The .6 M Perchloric acid was retained in a separate
centrifuge tube.
The homogenate was spun down at 1200 g for 15 minutes
at 0°C. The supernatant was saved, and the pellet was
resuspended in the 3 ml rinse (.6 M Perchloric acid) .
This suspension was recentrifuged at 1200 g for 15
minutes and the supernatants were combined.
The combined supernatant was neutralized to pH
5.5-6.0 with addition of 3 M Na2CC>3. This was added
dropwise with constant mixing to avoid bubbling over.
Samples were subsequently certrifuged at room temperature
in a desk-top centrifuge at maximal rpm for 10 minutes.
The volume of the supernatant was determined and the

98
supernatant was stored in a freezer until further analysis
was done (usually same day, but a delay of 2-3 days made
no difference).
Determination of Free and Total Creatine
Duplicate analysis for free and total creatine were
made on each sample (4 aliquots per supernatant). Standards
also in duplicate were run with each determination.
Following, is a list of the procedures for free and total
determination:
1. Neutralized samples to pH = 7.0-7.3 with IN
NaOH (and IN HC1 if needed).
2. Measure 4 aliquots (.5-1 ml) of each supernatant
into graduated tubes.
3. Add H2O to 3 ml mark.
4. Place 2 (of 4) tubes in hot water bath (65°C)
to equilibrate.
5. Add 1 ml of .4 N HC1 to tubes which have
equilibrated to 65°C.
6. Replace in hot water bath for 9 minutes. These
are tubes for total creatine determination.
7. Remove the tubes from the bath and add 1 ml of
.4 N NaOH. Place in ice bath to cool quickly
to room temperature.
8. To each of the four tubes (2 free and 2 total),
add sequentially: 1 ml of p hydroxymercuribenzoate,
2 ml of Naphthol and 1 ml diacetyl.
9. Make volume to 10 ml, agitate and place in dark
for 20 minutes.
10.Read optical density at 525 nm.
Standards receive the same treatment as the samples
for free creatine (blank does not get 1 ml p hydroxy¬
mercuribenzoate. Creatine content of samples is

99
determined from the regression line obtained from the
standards. Phosphorylcreatine content is (total
creatine - free creatine)/total creatine.

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BIOGRAPHICAL SKETCH
Brian Robert Macintosh was born in Owen Sound,
Ontario, March 26, 1952. He graduated from the
Owen Sound Collegiate and Vocational Institute in
1971. He received his Bachelor of Science degree
in Human Kinetics in 1975 at the University of
Guelph.
Brian was accepted into the Ph.D. program in
the Department of Physiology at the University of
Florida in 1975, and was admitted to candidacy for
the Doctor of Philosophy degree in 1977.
Brian is married to the former Patricia Dale
Wilkie. They have two children. Jennifer Louise
and Robert John David.
106

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.
tt
(
JL
Irina
W. N. Stainsby, Chairman
Professor of Physiology
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.
Professor of Physiology
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.
C '•'l
P. Posner
Associate Professor
of Physiology

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.
Professor of Biochemistry
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.
C~! w7 2fauner
Professor of Professional
Physical Education
This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate Council,
and was accepted as partial fulfillment of the require¬
ments for the degree of Doctor of Philosophy.
June, 1979
Dean, College of Medicine
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