Effect of temperature on myosin phosphorylation

in mouse skeletal muscle

RUSSELL L. MOORE, BRADLEY M. PALMER, SHERI L. WILLIAMS,

HIROYUKI TANABE, ROBERT W. GRANGE, AND MICHAEL E. HOUSTON
Departments of Medicine and of Cellular and Molecular Physiology, Division of Cardiology, The Milton
S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033;
and Department of Kinesiology, The University of Waterloo, Waterloo, Ontario NZL 3Gl, Canada

MOORE, RUSSELL L., BRADLEY M. PALMER, SHERI, L. WIL- osin (3,4,24,31). In view of the fact that skeletal muscle
LIAMS, HIROYUKI TANABE, ROBERT W. GRANGE, AND MI- P-light chain phosphorylation is highly regulated by the
CHAEL E. HOUSTON. Effect of temperature on myosin phos- concerted actions of MLCK and a myosin light chain
phorylation in mouse skeletalmuscle. Am. J. Physiol. 259 (Cell phosphatase activity (19, 31), it has been suggested that
Physiol. 28): C432-C438, 1990.-The effect of musclecontrac-
this covalent modification of myosin has a modulatory
tion on phosphorylatable myosin light chain (P-light chain)
phosphatecontent and isometric twitch tension was examined influence on skeletal muscle contraction. In purified
at 25, 30, and 35°C in intact mouseextensor digitorum longus myosin preparations, submaximal activation of myosin
muscle. Peak tetanic tension was unaffected by temperature, adenosinetriphosphatase (ATPase) activity by actin is
whereaspeak unpotentiated isometric twitch tension was in- enhanced by P-light chain phosphorylation, whereas
versely proportional to muscle incubation temperature. The maximal actin-activated myosin ATPase activity is not
extent of phosphate incorporation into P-light chain elicited affected by phosphate incorporation into P-light chain
by a 20-s train of twitches (5/s) was inversely proportional to (21, 23). In skinned fiber preparations, phosphate incor-
muscleincubation temperature, whereasthe fractional increase poration into P-light chain renders the contractile ele-
in twitch tension (twitch potentiation) elicited by repetitive
stimulation was directly proportional to muscle incubation ment more sensitive to activation by Ca2+ without af-
temperature. After the twitch train, the rate of decline of fecting maximal tension generation or unloaded short-
potentiated twitch tension and of P-light chain dephosphoryl- ening velocity (16, 22, 32, 33). This sensitization of the
ation was directly proportional to muscleincubation tempera- contractile element to activation by Ca2+ appears to be a
ture. The net result wasthat a significant and unique relation- manifestation of an increased probability of transition
ship betweenP-light chain phosphatecontent and contraction- of cross bridges from a nonforce-generating state to a
induced tension potentiation existed at each temperature ex- force-generating state (16, 33). These in vitro findings
amined. The slopeof the P-light chain phosphatevs. isometric are consistent with and provide a mechanistic explana-
twitch potentiation relationship varied directly as a function of tion for several aspects of intact skeletal muscle behavior.
muscleincubation temperature. The observationsthat the slope Specifically, phosphate incorporation into P-light chain
of this relationship increasesand that unpotentiated twitch
has no apparent effect on maximal tetanic tension (PJ
tension decreaseswhen muscleincubation temperature is in-
creasedsupport the hypothesis that contraction-induced ten- or unloaded shortening velocity (u,) (1, 5, 20), whereas a
sion potentiation in intact mammalian skeletal muscle is the quantitative relationship between the phosphate content
result of a sensitization of the contractile element to activation of P-light chain and the rate of force development and
by Ca*+that is brought about by P-light chain phosphorylation. extent of contraction-induced submaximal isometric ten-
sion potentiation has been observed in intact fast-twitch
phosphorylatablemyosin light chain; myosin light chain kinase; skeletal muscles of several mammalian species, including
twitch potentiation; positive staircase potentiation; extensor the rat (13, 15, 19), rabbit (18), mouse (20), and human
digitorum longusmuscle (12)
It’ is well known that many of the characteristics of
intact skeletal muscle contractility are profoundly af-
PRIMARY REGULATION of mammalian skeletal muscle fected by temperature. In mammalian fast-twitch skele-
contraction occurs at the level of the thin filament. When tal muscle, both the time course of and the peak tension
a sarcolemmal action potential triggers the release of generated by the isometric twitch are inversely related
Ca2’ from the sarcoplasmic reticulum (SR) and cytosolic to muscle incubation temperature (6, 8), whereas un-
free [Ca”‘] ([Ca”‘],) is elevated, Ca2+ binds to troponin loaded shortening velocity and the extent and rates of
thus reducing its inhibitory influence and allows myosin onset and decay of contraction-induced tension potentia-
to combine with actin to generate tension. Increased tion are directly proportional to temperature (8, 14, 29).
[Ca”‘], also results in the activation of a Ca2’-calmodu- Many of these observations can be explained by the
lin-dependent myosin light chain kinase (MLCK) and known effects of temperature on several intracellular
subsequent phosphate incorporation into the phospho- regulatory processes. The effects of temperature on the
rylatable light chain [P-light chain (ll)] subunit of my- time course and magnitude of the isometric twitch ten-
C432 0363-6143/90 $1.50 Copyright 0 1990 the American Physiological Society

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 TEMPERATURE DEPENDENCE OF MYOSIN
sion transient can be explained by the effects of temper-
ature on the amplitude and shape of the [Ca”‘], transient
(2, 10, 34) and the sensitivity of the contractile ele-
ment to activation by Ca2+ (30). Temperature-dependent
changes in unloaded shortening velocity are largely due
to a direct effect of temperature on actin-activated my-
osin ATPase activity (8, 29). Although the processes
responsible for the onset and reversal of contraction-
induced tension potentiation have not been established
with certainty, it appears that the best cellular correlate
of isometric tension potentiation is myosin P-light chain
phosphate content (13, 15, 18-20, 31). If P-light chain
phosphorylation is causally related to contraction-in-
duced tension potentiation, then it follows that the proc-
esses of P-light chain phosphorylation and dephospho-
rylation should vary as a function of muscle temperature
in a manner that is consistent with the known tempera-
ture-dependent characteristics of contraction-induced
tension potentiation in intact mammalian skeletal mus-
cle.
To further characterize the relationship between P-
light chain phosphate content and contraction-induced
tension potentiation in intact mammalian skeletal mus-
cle, the effect of temperature on both of these phenomena
was examined in mouse extensor digitorum longus (EDL)
muscle. The results obtained from these studies are
consistent with the hypothesis that P-light chain phos-
phorylation and contraction-induced tension potentia-
tion are causally related via a mechanism whereby phos-
phate incorporation into P-light chain has the sole effect
of rendering the contractile element more sensitive to
activation by Ca2’. The results of this study do not,
however, rule out the possibility that other mechanisms
may contribute to the phenomenon of contraction-in-
duced potentiation of isometric twitch tension.
METHODS
Muscle isolation and specific experimental protocols.
This study was conducted under the guidelines estab-
lished by the Milton S. Hershey Medical Center’s Animal
Research Committee concerning the care and use of
animals in research. Young adult female BALB/c mice
(12-16 g) were deeply anesthetized with pentobarbital
sodium (5 mg/lOO g body wt ip). The EDL muscle from
each hindlimb was surgically isolated, and a 5-O surgical
silk ligature was attached to the distal tendon. The
proximal tendon was then cut, and the muscle was care-
fully excised and placed into a temperature-controlled
(22°C) dissection dish containing oxygenated (95% 02-
5% C02) physiological saline solution (PSS) of the fol-
lowing composition (in mM): 118 NaCl, 1.2 KH2POd,
1.18 MgSO,, 1.9 CaC12, 25 NaHC03, and 11 glucose.
Animals were then killed by cervical dislocation. Under
PSS, the proximal tendon of the muscle was anchored
between Plexiglas clamps, and the muscle was mounted
in a jacketed oxygenating muscle bath containing PSS
maintained at 25, 30, or 35OC. The distal tendon was
attached to a Kulite BG-50 force transducer (1.8-kHz
frequency response, unloaded beam) via the silk ligature.
Each muscle was positioned between two platinum wire
stimulating electrodes, and electrical stimuli were deliv-
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PHOSPHORYLATION IN SKELETAL MUSCLE c433
ered using a Grass S-88 stimulator and Hewlett-Packard
direct-current amplifiers. Stimulus pulse duration was
0.2 ms, and optimal stimulus intensity was determined
according to a variation of the principles described by
Close and Hoh (7). At all muscle incubation tempera-
tures, a stimulus intensity equal to three times the
threshold resulted in a peak twitch tension that was
clearly on a “voltage vs. response” plateau (7). Muscle
length was adjusted so that optimal twitch tension was
produced. Before experimentation, muscles were given
quiescent equilibration periods of 30, 35, and 40 min at
35, 30, and 25OC, respectively. The longer equilibration
times at the lower temperatures were used to ensure that
the phosphate content of P-light chain attained steady-
state basal levels. The muscle stimulation frequency
required to elicit P, is temperature dependent. In deter-
mining P, in our preparation at 25, 30, and 35°C (Fig.
l), we found that maximal tension responses were elicited
at muscle stimulation frequencies of 92 t 4 (n = 5), 150
t 0 (n = 5), and 164 t 16 (SE) Hz (n = 5), respectively.
Gould transducer amplifiers were used to amplify trans-
ducer outputs, and muscle tension data were recorded on
a Gould 2600 chart recorder (140.Hz pen response time)
and/or were digitized and stored for subsequent analysis
on a laboratory microcomputer.
Experiments were conducted according to the follow-
ing experimental protocols. After stimulation and muscle
length optimization, muscles were allowed to remain
quiescent for 30 min. At this time, muscles were either
frozen between tongs prechilled in liquid N2 or were
stimulated to repetitively twitch contract at a frequency
of 5 Hz for 20 s. This protocol has previously been shown
to be effective in producing isometric tension potentia-
tion (7, 13, 14, 19) and in elevating P-light chain phos-
phate content (13, 19) in fast-twitch skeletal muscles.
Muscles were then frozen and/or single muscle twitch
contractions were elicited at various times after the 20-s
trains of twitches. Isometric twitch tension potentiation
was expressed as P*/P - 1, where P* and P correspond
to peak potentiated and unpotentiated twitch tensions,
respectively. The peak tension of the first twitch in the
20-s twitch train was always used to represent P.
Determination of muscle shortening velocity. The effect
of a 20-s ~-HZ twitch train on muscle shortening velocity
was determined at 25 and 35OC. Peak shortening velocity
before and after a 20-s twitch train was determined as
previously described (1, 5). Briefly, muscles were fixed
between a Plexiglas clamp and a Cambridge 300 H
transducer arm. Before experimentation, muscle length
was adjusted to yield optimal twitch tension. Muscle
length was then determined using a Bausch and Lomb
stereoscope equipped with reticule. Muscles were then
allowed to equilibrate for 30 and 40 min at 35 and 25OC,
respectively. Maximal tetanic contractions were then
elicited by stimulation at 150 Hz (25°C) and 167 Hz
(35°C) for 570 ms. A release to l-g load (<lo% P,) was
invoked during the last 70 ms of the tetanic stimulation,
and peak shortening velocity data were collected on-line
at 2,048 Hz. After a 2-min pause, muscles were stimulated
for 20 s at 5 Hz. Peak shortening velocity data were
c434 TEMPERATURE DEPENDENCE OF MYOSIN PHOSPHORYLATION IN SKELETAL MUSCLE

TABLE 1. Effect of muscle incubation temperature ily performed using data-average coordinates derived
on unpotentiated isometric twitch tension dynamics from different groups of muscles because under most
in mouseextensor digitorum longus muscle experimental circumstances, P-light chain phosphate
content data and isometric twitch tension data repre-
Temperature sentative of the same time point cannot be collected from
25°C 30°C 35°C Effect?
the same muscle. Muscle shortening velocity data gath-
P, mN 43.622.3 29.0t2.8 20.0t2.2 Yes ered before and after a 20-s ~-HZ twitch train were
TPT, ms 21.9t0.5 14.1k0.2 9.5kO.2 Yes analyzed using a paired Student’s t test; associated mor-
%RT, ms 21.4tl.O 9.5t0.3 6.OkO.3 Yes
phological data were analyzed using an unpaired Stu-
Values are means t SE of 15 observations. P, peak unpotentiated dent’s t test. A P value of ~0.05 was accepted to indicate
isometric twitch tension; TPT, time required to achieve P; %RT, time
required to achieve relaxation from P to a tension equal to 0.5P; Yes, significant differences, interactions, and correlations. All
parameter assessed was significantly (P c 0.01) affected by temperature values are presented as means t SE.
when data were subjected to a l-way ANOVA.
RESULTS
collected again 5 and 20 s after the twitch train at 35
and Z5”C, respectively. Consistent with previous observations (6, 14, 29), P,
Analysis of P-light chain phosphate content. Phosphate the time required to achieve P (TPT), and the time
incorporation into P-light chain was quantitated in a required to achieve relaxation to a tension equal to 0.5 P
“single-blind” fashion using the two-dimensional electro- (l/zRT) were all significantly reduced as a function of
phoretic technique of Silver and Stull (27) as modified increasing muscle incubation temperature (Table 1, Fig.
by Moore and Stull (19). Data are expressed in the units 1). Furthermore, the time course of P* after a ~-HZ
of moles phosphate per moles P-light chain (mol P/mol staircase for 20 s was significantly reduced at all muscle
PLC). incubation temperatures; this observation corroborates
Statistical analysis. The effect of temperature on char- previous findings regarding the effect of previous con-
acteristics of isometric tension and P-light chain phos- tractile activity on mammalian skeletal muscle twitch
phorylation was determined by an analysis of variance dynamics (8, l4,20). Muscle incubation temperature did
(ANOVA) procedure. Net P-light chain phosphate con- not significantly affect P, (Fig. 1). Consequently, the P/
tent vs. isometric tension potentiation ( AP-light chain P, was reduced as a function of increasing muscle tem-
phosphate vs. P*/P - 1) relationships at different muscle perature (Fig. 1).
temperatures were examined by regression analysis (e.g., The effect of a 20-s ~-HZ twitch train on relative twitch
Fig. 4) where AP-light chain phosphate = poststimula- tension potentiation at 25, 30, and 35°C is illustrated in
tion P-light chain phosphate content - basal P-light Fig. 2. Three striking features are apparent. First, the
chain phosphate content. These analyses were necessar- magnitude of the relative increase in twitch tension

10 111N

FIG. 1. Effect of muscle incubation

temperature on unpotentiated twitch
60 80 tension (P), maximal tetanic tension
0 20 40 (PO), and P/P,. A: unpotentiated twitch
time ( ms ) tension dynamics in the same muscle at
25, 30, and 35°C. B: P, generated by
C 0.3
mouse extensor digitorum longus (EDL)
muscles (n = 5) at 25, 30, and 35°C. C:
P/P0 resulting from mouse EDL muscles
(n = 5) at 25, 30, and 35OC. * Signifi-
cantly different from 25°C value at P <

2s” 3o” 3s” 2s” 3o” 3s”

temperature (“c> temperature (OC)

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 TEMPERATURE DEPENDENCE OF MYOSIN PHOSPHORYLATION IN SKELETAL MUSCLE c435
0.40
r immediately after the 20-s twitch train at 35”C, P-light
chain phosphorylation levels reached maxima at 10 and
20 s after the end of the ~-HZ stimulation train in muscle
incubated at 30 and 25OC, respectively. The rate of P-
light chain dephosphorylation after the 5 Hz twitch train
was directly proportional to muscle incubation temper-
ature. At 25, 30, and 35”C, estimated rates of return of
P-light chain phosphate contents back to basal values
were estimated to be 3.0 X 10D3, 9.0 X 10D3, and 4.2 X
1o-2 s-l, respectively.
The slope of the AP-light chain phosphate (x) vs. P*/
P - 1 (y) relationship resulting from a 20-s ~-HZ train
-0.10 ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ of twitches increased directly as a function of muscle
-50 0 50 100 200 300
incubation temperature (Fig. 4). At 25,30, and 35”C, this
time (s) relationship was described by y = 2.20 x lo-lx + 1.5 x
FIG. 2. Twitch tension potentiation in mouse EDL muscles after a 1O-2 (r = 0.81; P c O.O5),y = 7.43 x lo-lx: + 2.6 x 1O-4
~-HZ 20-s train of twitches at 25 (x), 30 (H), and 35°C (0). The 20-s (r = 0.95; P < O.Ol), and y = 1.906x + 8.8 X lOa (r =
train of twitches was initiated at t = -20 s on abscissa. All values 0.83; P C 0.05), respectively.
represent means t SE of 6-11 samples.
In the context of our experimental design, a 20-s ~-HZ
twitch train (which was preceded by a 570-ms tetanic
0.80 stimulation) did not significantly affect peak shortening
velocity at 25 or 35°C (Table 2).
2 0.60 DISCUSSION
a, The experiments described in this study were designed
0 to determine whether temperature-dependent changes in
E 0.40
the characteristics of contraction-induced tension poten-
n‘
0 0.20
0.40 -
E
0.30 -
0.00
-50 0 50 100 200 300
‘;- 0.20 -
time (s) 11
FIG. 3. P-light chain
after a ~-HZ 20-s train of
phosphate
twitches
content in mouse EDL mu&les
at 25 (x), 30 (m), and 35°C (0). The
h 0.10 -
20-s train of twitches was initiated at t = -20 s on abscissa. All values
represent means k SE of 4-6 samples. 0.00 -
elicited by repetitive stimulation was directly propor- I L I I I I ’ fi I
tional to muscle incubation temperature. Second, -0.10 ’
0.0 0.2 0.4 0.6 0.8
whereas the potentiated tension was maximal immedi-
ately after the 20-s twitch train at 35”C, twitch tension A mol P/mol PLC
did not reach a maximum for -10-20 s after the end of FIG. 4. AP-light chain phosphate vs. P*/P - 1 relationship in mouse
the ~-HZ stimulation train in muscles incubated at 30 EDL muscles at 25 (x), 30 (m), and 35°C (0). Data from Figs. 2 and 3
were used to construct these relationships.
and 25°C. Third, the rate of decline of potentiated twitch
tension after the ~-HZ twitch train was directly propor- TABLE 2. Effect of a 20-s ~-HZ twitch train on peak
tional to muscle incubation temperature. At 25, 30, and shortening velocity in mouse extensor digitorum longus
35OC, estimated rates of return of potentiated twitch musclesduring tetanic release to 1 g at 25 and 35OC
tensions back to unpotentiated values were 3.2 x 10m3,
6.2 X 10B3, and 1.6 X 10m2 s-‘, respectively. All of our 25°C 35°C
observations are qualitatively similar to the findings of Mouse mass, g 22.85k1.34 23.58t0.35
Krarup (14) in which the effect of temperature on iso- Muscle length, mm 12.40k0.34 12.68k0.35
metric twitch tension potentiation in rat EDL muscle Shortening velocity before twitch 3.71t0.21 4.24k0.45
was examined. train, muscle lengths/s
Shortening velocity after twitch 3.21k0.12 3.65t0.46
The effect of a 20-s ~-HZ twitch train on P-light chain train, muscle lengths/s
phosphate content at 25, 30, and 35°C is illustrated in n 4 5
Fig. 3. The magnitude of the elevation in P-light chain Values are means t SE; n, no. of observations. Relative to maximal
phosphate content elicited by repetitive stimulation was tetanic tension (PJ elicited before the 20-s ~-HZ twitch train, P, elicited
inversely proportional to muscle incubation temperature. after the twitch train was reduced by 35 and 41% at 25 and 35”C,
Whereas P-light chain phosphate content was maximal respectively.

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C436 TEMPERATURE DEPENDENCE OF MYOSIN
tiation in intact skeletal muscle were accompanied by
similar temperature-dependent changes in P-light chain
phosphate content. Our observations regarding the ef-
fects of temperature on mouse EDL muscle peak tetanic
tension and isometric twitch tension dynamics are in
agreement with the documented effects of temperature
on mammalian fast-twitch skeletal muscle (6, 14). Con-
sistent with previous findings (l4), a direct relationship
between temperature and the magnitude and rate of
decay of isometric tension potentiation following a 20-s
~-HZ twitch train was observed (Fig. 3). Furthermore,
our observations regarding the differential effects of tem-
perature on isometric twitch potentiation dynamics could
not be attributed to between-temperature differences in
the extent of fatigue produced by the conditioning stimuli
(20-s ~-HZ twitch train), since peak shortening velocity
was not significantly affected at 25 or 35°C (Table 2).
Unique findings of this study were that temperature
similarly affected the rates of P-light chain dephospho-
rylation and decay of potentiated twitch tension, whereas
it had dissimilar effects on the extents of contraction-
induced tension potentiation and P-light chain phos-
phorylation. The net effect was that the slope of the AP-
light chain phosphate vs. P*/P - 1 relationship varied
directly as a function of muscle incubation temperature
(Fig. 4). The latter observation is qualitatively similar to
the results of Manning and Stull (15) in which the
relationship between P-light chain phosphate content
and posttetanic twitch tension potentiation was exam-
ined in rat EDL muscle.
At least two possible explanations for the effect of
temperature on the slope of the AP-light chain phosphate
vs. P*/P - 1 relationship exist. The first explanation
assumes a causal relationship between P-light chain
phosphorylation and isometric tension potentiation
a mechanism whereby P-light chain phosphorylation
renders the contractile element more sensitive to acti-
vation by Ca2’ (16, 21-23, 32, 33). Under these condi-
tions, one would expect that the relative effect of P-light
chain phosphorylation on submaximal tension genera-
tion would become more profound under conditions in
which the extent of contractile element activation by
Ca2+ is reduced (20, 22, 32, 33), i.e., the slope of the AP-
light chain phosphate vs. P*/P - 1 relationship would
increase (20). Temperature-dependent reductions in the
sensitivity of the contractile element to activation bY
Ca2+ (30) and in the time course and amplitude of the
[Ca”‘], transient during a twitch may explain in part
why peak unpotentiated isometric twitch tension de-
creases when muscle incubation temperature increases.
Because temperature does not appear to influence the
intrinsic strength of the contractile element (i.e., P, is
temperature independent between 25 and 35"C), the
observation that the P/P0 decreases as temperature in-
creases (Refs. 6,8, Fig. 1) is consistent with the idea that
the extent of contractile element activation occurring
during a single unpotentiated twitch is less at 35 than at
25OC. If this is the case, and if it is assumed that P-light
chain phosphorylation similarly increases the sensitivity
of the contractile element to activation by Ca2+ at 25,30,
and 35°C (Fig. 5A), then it follows that the slope of the
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PHOSPHORYLATION IN SKELETAL MUSCLE
z 0.0 -
5.7 5.5 5.3 5.1 4.9
effective pCa
6
1.5 -
q 35”
0 30”
I A 25”
1.0 -
0.0 0.2 0.4 0.6 0.8
A mol P / mol PLC
FIG. 5. Hypothetical effect of muscle incubation temperature on
relative extent of contractile element activation by Ca2+ during a twitch
and on the AP-light chain vs. P*/P - 1 relationship. A: curves (from
right to left) representative of P-light chain phosphorylation states of
0.00,0.20,0.40, and 0.70 mol P/mol PLC were constructed as previously
described (20). The hypothesis that an increase in muscle incubation
temperature results in a reduction in the extent of contractile element
activation by Ca2’ during a twitch is illustrated by a temperature-
dependent increase in effective pCa achieved during a single twitch.
For clarity of presentation, a single pCa vs. tension curve representative
of the P-light chain unphosphoryiated state at 25, 30, and 35°C is
presented even though this relationship is known to move to the right
with increasing temperature (30). For this reason, and because effective
pCa is a function of both the amplitude, shape, and duration of the
via intracellular [Ca”‘] transient that occurs during a twitch (20), values
appearing on the abscissa are arbitrary and are intended for illustrative
rather than quantitative use. The use of an arbitrarily defined pCa axis
in no way influences the final outcome of this qualitative illustration.
The effective pCa values, and hence relative positions on the pCa vs.
tension curve, occurring at the different temperatures are identified on
abscissa and were chosen to correspond to percent maximal responses
(on ordinate) that were similar to the P/P,-observed at the 3 different
temperatures (see Fig. 1). B: at each of the 3 points identified on
abscissa of A, fractional increase in tension produced by the 3 different
levels of P-light chain phosphorylation was estimated and plotted as
previously described (20). It is apparent that the relative effect of P-
light chain phosphorylation on tension augmentation is greater when
effective pCa is increased. As a result, slope of AP-light chain phosphate
vs. P*/P - 1 relationship increases directly as a function of muscle
incubation temperature.
AP-light chain phosphate vs. P*/P - 1 relationship
should increase when temperature is increased (Fig. 5B).
This simple theoretical prediction is qualitatively similar
to and may provide an explanation for our experimental
observations (Fig. 4). It should be noted that an implicit
assumption of this simple theoretical argument, in the
context of a simple two-state cross-bridge model and of
recently refined theory regarding the effect of P-light
chain phosphorylation on cross-bridge dynamics (16,33),
is that twitch tension potentiation induced by P-light
chain phosphorylation occurs as a result of an increase
in the rate at which cycling cross bridges undergo a
transition from nonforce-generating to aforce-generat-
 TEMPERATURE DEPENDENCE OF MYOSIN
ing state (j&-J without affecting the rate at which cross
bridges leave the force-generating state (gap*) (16, 33).
Because the number of force-generating cross bridges is
dependent on the relative magnitude of fapp with respect
to gapp9it is apparent that twitch tension augmentation
could occur via an increase in fapp with respect to gapp or
a reduction in gapp with respect to fapp. Under certain
experimental conditions, prolonged and intense muscle
contraction has been shown to produce a marked reduc-
tion in peak shortening velocity (5, 9), and presumably
gapp, that is independent of the phosphorylation state of
P-light chain (1, 5). Our observation that the condition-
ing twitch train used to elicit potentiation did not signif-
icantly affect peak shortening velocity at either temper-
ature examined is consistent with, but does not prove,
the hypothesis that the temperature-dependent differ-
ences in the magnitude of contraction-induced twitch
tension potentiation were not due to differential effects
of prior contraction on gappat the different temperatures.
The second explanation for the temperature depend-
ence of the slope of the AP-light chain phosphate vs. P*/
P - 1 relationship is that the relationship is coincidental
rather than causal and that P-light chain phosphoryla-
tion-dephosphorylation kinetics display a different tem-
perature dependence than do the processes that are ac-
tually responsible for contraction-induced tension poten-
tiation. Our data do not rule out the possibility that other
cellular mechanisms contribute to or are primarily re-
sponsible for the phenomenon of contraction-induced
potentiation of isometric tension. For example, under
our 35°C conditions, 34% of the variation (? = 0.66) in
contraction-induced tension potentiation could not be
explained by changes in P-light chain phosphate content.
It could be argued that other processes were at least
partly responsible for the contraction-induced increase
in isometric twitch tension. However, it is difficult to
ascertain what these other processes might be. It is
known that contraction-induced tension potentiation
can occur in the absence of changes in P,, uO, and me-
chanochemical coupling efficiency (1, 5, 20, 22, 32, 33).
Consequently, alterations in the intrinsic strength of the
contractile element are not likely to account for the
phenomenon of contraction-induced tension potentia-
tion. There are also no data to support the hypotheses
that contraction-induced tension potentiation is due to
an increase in the magnitude of Ca2+ release from the
SR or to postcontraction Ca2+ buffering in the cytosol.
In amphibian skeletal muscle, previous contractile activ-
ity has actually been shown to decrease the amount of
Ca2’ that is released from the SR in subsequent contrac-
tions (2, 17, 26, 34). In addition, the removal of residual
Ca2+ from the sarcoplasm of amphibian skeletal muscle
occurs to completion under conditions in which the con-
traction-induced twitch tension potentiation phenome-
non persists (28).
Other unique findings of this study were that contrac-
tion-induced phosphate incorporation into P-light chain
was inversely proportional to muscle incubation temper-
ature, whereas the rate of P-light chain dephosphoryla-
tion following the twitch train was directly proportional
to muscle incubation temperature (Fig. 4). From the
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PHOSPHORYLATION IN SKELETAL MUSCLE c437
former observation, it is apparent that net P-light chain
phosphorylation was faster at 25 than at 35°C in spite of
the fact that MLCK displays a temperature coefficient
(Q& of -2 (4). Several temperature-dependent aspects
of MLCK activation-inactivation may provide an expla-
nation for this experimental result. For example, the
ability of the Ca2,“-calmodulin, n equal to three or four,
complex to produce steady-state activation of rabbit skel-
etal MLCK is increased as temperature is reduced (4).
There is also evidence to suggest that the absolute
amount of Ca2’ released from the SR during a single
muscle twitch is not affected by temperature (25) but
that the dynamics of Ca2’ release from and resequester-
ation by the SR during an excitation-contraction cou-
pling cycle are (2, 10, 34). It is likely that both the time
course and the amplitude of the [Ca”‘], transient during
a twitch are increased when temperature is decreased.
These types of changes in the [Ca”‘], transient would
favor a greater extent of Ca2+-calmodulin-dependent ac-
tivation of MLCK. Relative to the time required for Ca2+
to be removed from the sarcoplasm during the relaxation
phase of each contraction (ms), the rate of inactivation
of active MLCK after contraction is slow and occurs over
several seconds (15,19,31). The observation that P-light
chain phosphate content continues to increase following
the conditioning stimuli at the lower temperatures but
not at 35°C (Fig. 4) is consistent with the hypothesis
that the rate of MLCK inactivation is directly propor-
tional to muscle incubation temperature. This would also
favor a greater fractional activation (active MLCK/total
MLCK) of MLCK at the lower temperatures. Because
the rate of reaction catalysis by MLCK is a function of
“maximal catalytic rate X fractional activation” (19,31),
our data (Fig. 4) are supportive of the concept that the
extent of fractional activation of MLCK occurring at the
lower temperatures was sufficient to overcome the effect
of temperature on the maximal catalytic rate of active
MLCK. It should also be noted that the rate of P-light
chain dephosphorylation was -10 times faster at 35 than
at 25” C. Although this effect is striking and consistent
with the observed temperature-dependent differences in
net P-light chain phosphate incorporation, the rates of
P-light chain dephosphorylation are probably too slow
to be the primary determinants of the temperature-
dependent differences in the net rate of phosphate in-
corporation into P-light chain during repetitive twitch
contraction.
In summary, we found that a significant relationship
between P-light chain phosphate content and contrac-
tion-induced tension potentiation existed at all temper-
atures examined, whereas the slope of the AP-light chain
phosphate vs. P*/P - 1 relationship varied directly as a
function of m uscle incubation temperature. Increases in
the slope of this relationship occurred concomitantly
with temperature-dependent reductions in peak unpo-
tentiated isometric twitch tension. If the reduction in
unpotentiated isometric twitch tension was due to a
reduced extent of contractile element activation by Ca2+,
then the results of this study are consistent with the
hypothesis that contraction-induced twitch potentiation
is due to P-light chain phosphorylation and a resultant
C438 TEMPERATURE DEPENDENCE OF MYOSIN PHOSPHORYLATION IN SKELETAL MUSCLE

sensitization of the contractile element to activation by low frequency stimulation on myosin light chain phosphorylation
Ca2’ (20, 22, 23, 32, 33; Fig. 5). Although this hypothesis in skeletal muscle. J. Biol. Chem. 257: 4688-4690, 1982.
14. KRARUP, C. Temperature dependence of enhancement of diminu-
is attractive, it is also possible that other mechanisms tion of tension evoked by staircase and by tetanus in rat muscle.
may contribute to the phenomenon of contraction-in- J. Physiol. Land. 311: 373-387, 1981.
duced potentiation of isometric tension in mammalian 15. MANNING, D. R., AND J. T. STULL. Myosin light chain phosphoryl-
fast-twitch skeletal muscle. Toward the aim of better ation-dephosphorylation in mammalian skeletal muscle. Am. J.
Physiol. 242 (Cell Physiol. 11): C234X241, 1982.
understanding the cellular events underlying this phe- 16. METZGER, J. M., M. L. GREASER, AND R. L. Moss. Variations in
nomenon, it is clear that more information regarding the cross-bridge attachment rate and tension with phosphorylation of
effects of temperature and P-light chain phosphorylation myosin in mammalian skinned skeletal muscle fibers. J. Gen.
on the pCa vs. tension relationship and cross-bridge Physiol. 93: 855-883, 1989.
kinetics in skinned fibers and on the effects of tempera- 17. MILEDI, R., I. PARKER, AND
P. H. ZHU. Calcium transients in frog
skeletal muscle fibers following conditioning stimuli. J. Physiol.
ture and previous contractile activity on [Ca”‘], dynam- Lond. 339: 223-242,1983.
ics in intact mammalian skeletal muscle fibers will be 18. MOORE, R. L., M. H. HOUSTON, G. A. IWAMOTO, AND J. T. STULL.
required. Phosphorylation of rabbit skeletal muscle P-light chain in situ. J.
Cell. Physiol. 125: 301-305, 1985.
This work was supported in part by a Grant-in-Aid from the Penn- 19. MOORE, R.L., AND J. T. STULL. Myosin light chain phosphoryla-
sylvania Affiliate of the American Heart Association, the Sam Ostrow tion in fast and slow skeletal muscle in situ. Am. J. Physiol. 247
(CeZl Physiol. 16): C462-C471, 1984.
Cardiology Research fund, and National Heart, Lung, and Blood Insti-
20. PALMER, B. M., AND R. L. MOORE. Myosin light chain phosphoryl-
tute Grant HL-40306.
Address for reprint requests: R. L. Moore, Div. of Cardiology, The ation and tension potentiation in mouse skeletal muscle. Am. J.
Physiol. 257 (Cell Physiol. 26): C1012-C1019, 1989.
Pennsylvania State University, PO Box 850, Hershey, PA 17033.
21. PEMRICK, S. M. The phosphorylated L2 light chain of skeletal
Received 14 July 1989; accepted in final form 30 April 1990. myosin in a modifier of the actomyosin ATPase. J. Biol. Chem.
255: 8836-8841,198O.
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