Effects of temperature and concomitant change in pH on muscle

Effects of temperature in pH on muscle

and concomitant

change

E. DON STEVENS AND R. E. GODT Department of Zoology, University of Guelph, Guelph, Ontario, Canada AU G 2 Wl; and Department of Physiology and Endocrinology, Medical College of Georgia, Augusta, Georgia 30912

STEVENS, E. DON, AND R. E. GODT. Effects of temperature and concomitant change in pH on muscle. Am. J. Physiol. 259

(Regulatory Integrative Comp. Physiol. 28): R204-R209, 1990.-Contractile performance decreases with a decrease in temperature and increases with an increase in pH. In general, a decrease in ambient temperature is associated with an increase of the pH of the intracellular and extracellular fluids of ectotherms. Thus the concomitant increase in pH will to some extent counteract the effect of the decrease in temperature. We review the magnitude of this effect and show that it is modest for force (24%) but is small or negligible for speed or for variables involving time. Experiments with skinned fibers yield similar results to those with intact fibers. We argue that one important effect of the concomitant increase in pH is that it causes an increase in calcium sensitivity and that there may be a considerable metabolic saving associated with releasing less calcium at lower temperatures. ectotherm; skinned fiber; calcium

with changes in temperature, ectotherms do not maintain constant pH, rather they tend to regulate the pH of their intracellular and extracellular fluids in such a manner that the ratio of OH- concentration ([OH-]) to H+ concentration ([H+]) is maintained relatively constant. For instance, the plasma pH of the bullfrog is 7.7 at 25”C, but increases to 8.1 if the animal is moved to 5OC. Reeves (20) argued that such a pattern of regulation protects the charge state of proteins, the net charge of which is primarily a function of proton association to peptide-linked histidine imidazolium groups. He called it alphastat regulation, where alpha is the ratio of unprotonated to total imidazole. The dissociation constant of water is different at different temperatures (Table 1). At neutrality, the [H+] equals the [OH-], and their product equals the dissociation constant. (For simplicity, we are ignoring the difference between concentration and activity.) Thus water is neutral at a pH of 7.0 only at 24°C; the [H+] (and the pH) of water at neutrality changes with temperature. The pH at neutrality is denoted pN, and the way it changes with temperature is often called the “Buffalo curve” (Fig. 1). It is called the Buffalo curve, because its importance was realized and much studied by the group in the Physiology IN GENERAL,

This paper was presented at the symposium entitled “Influence of Temperatures on Muscle and Locomotor Performance” held in two parts: at the Spring Meeting of the Federation of American Societies for Experimental Biology, New Orleans, Louisiana, March 19-24,1989, and at the meeting of the International Union of of Physiological Sciences/Helsinki, Finland, July 9-14, 1989. R204

Department of the State University of New York at Buffalo. If an animal maintains pH in such a manner that pH parallels the pN curve with changes in temperature, then net charge on proteins is maintained and presumably protein function is maintained. It is important to note that this change is a result of active regulation on the part of the animal. There is some evidence to support the alphastat hypothesis in that the pH at which maximum activity of some enzymes occurs is higher at lower temperatures (Fig. 1). One of these enzymes, actomyosin Ca2+-adenosinetriphosphatase (ATPase), is important in muscle function, the topic of the present paper. A decrease in temperature causes a decrease in almost all aspects of muscle function. An increase in pH over a biologically relevant range causes an increase in almost all aspects of muscle function (22). When an ectotherm experiences a decrease in temperature, e.g., from 25 to 5”C, it regulates respiratory and excretory control of acid-base balance, so that there is an increase in pH of intracellular and extracellular fluids. Thus the concomitant increase in pH to some extent counteracts the effects of the decrease in temperature. The present paper deals with the magnitude of the effect of the concomitant change in pH in counteracting the effect of a decrease in temperature on muscle contraction. We first review experiments on isolated whole muscle and then present some new experiments on skinned single fibers. Whole Muscle Experiments

We carried out a number of experiments to study this pH-temperature interaction in isolated amphibian sartorius muscle (21-24). The effects are illustrated in Figs. 2 and 3. For this summary, only the data for temperatures of 25 and 5°C are illustrated, and in each case the effect is illustrated for two species (a frog, Rana pipiens, and a toad, Bufo americanus). The pH on the abscissa is that of the superfusing physiological saline and was controlled by changing carbon dioxide concentration. For the purposes of illustrating the effect, we have assumed that when the animal is held at 25°C the in vivo pH is 7.0 and when held at 5°C the pH increases to 7.5 (that is, we have intentionally used the maximum possible concomitant change in pH to estimate the extent to which its change can compensate for the change in temperature, dpH/dT = -0.025. The slopes of the curves in Fig. 1 are -0.021 for blood and -0.015 for muscle). The dashed line in Figs. 2 and 3 indicates the compensation; if

0363-6119/90 $1.50 Copyright 0 1990 the American Physiological

Society

TEMPERATURE

AND

pH

compensation were perfect (i.e., lOO%),then the parameter would have the same value at 5°C and pH 7.5 as it had at 25°C and pH 7.0. The top panel in Fig. 2 shows that the decrease in isometric tetanic force when temperature is decreased from 25 to 5°C is partially compensated by the concomitant change in pH (24.4% for frog and 24.8% for toad), but Fig. 2, bottom, shows that compensation for the maximum velocity of unloaded shortening ( Vmax) is negligible (1.1% for frog and 2.8% for toad). Figure 3 illustrates the force-velocity curves (left) and the force-power curves (right) for the two species. Power is calculated as the product of force and velocity at each load. The compensation of velocity as a result of the concomitant pH change at any load is negligible, because temperature modifies speed of contraction much more than pH does. Power is also much more modified by temperature than pH, but there is a small degree of compensation at higher loads. The time for contraction and relaxation is also much more sensitive to temperature than to pH. The time to reach one-half maximum isometric force and the time for force to decrease to one-half maximum are both prolonged by a decrease in temperature. The degree of compensation due to the concomitant pH change is small (compensation for time to one-half maximum isometric force is 0.0% for frog and 4.9% for toad, for time to onehalf relaxation it is 8.0% for frog and 8.4% for toad). The conclusions from the experiments on isolated whole muscle are that 1) there is modest compensation for force (24%) and 2) there is small or negligible compensation for speed or variables involving time. AddiTABLE

1. Effect of temperature Temperature,

on pK,, pN, and [H+]

“C

PKW

PN

[H+] x lo-‘, M

0

14.94 14.53 14.17 13.83

7.47 7.27 7.08 6.92

0.34 0.54 0.83 1.21

10 20 30

constant of water (26); pN, PKWY -log of dissociation neutrality; [H+], H+ concentration of water at neutrality.

pH of water

7.0

I 0

I

lo Temphature

I

30

40

1. Effect of temperature on pH of water at neutrality (pN), blood plasma, and intracellular muscle pH of bullfrog (13). Also shown are the pH values for maximal activity of 2 enzymes: Na+-K+-ATPase from toad skin (16) and actomyosin Ca2+-ATPase from frog muscle (12). All decrease in a similar fashion with an increase in temperature. FIG.

ON

R205

MUSCLE

FROG n cu < E 0 \ m x

3.c

w

1.6

2.: 2.c

aI F

1.a

0 LL

0.6 0

X 0 t

1III ’r

.

4

> 2

0

7

8pH

7

8

FIG. 2. Combined effect of pH and temperature on isometric tetanic force and unloaded speed of shortening ( Vmax)on frog and toad isolated sartorius muscle (22-24). In each panel, top line is at 25°C (open circles), bottom line at 5°C (closed squares). Compensation for effect of a decrease in temperature from 25°C (pH 7) to 5°C (pH 7.5) is indicated by dashed horizontal line; it joins point on the 5°C curve at constant pH to that showing concomitant change in pH that actually occurs when temperature decreases.

tionally, most experiments on amphibian muscle reported in the literature are done at low temperature and thus some underestimate actual values, because they are done at unphysiologically low pH values for the low temperatures used. These underestimates may be considerable for isometric force but are small for speed of contraction or variables involving time. This point applies to experiments in which the buffer system in the physiological solution can easily permeate the muscle membrane (e.g., carbon dioxide). If the muscle is relatively impermeable to the buffer system in the physiological solution (e.g., phosphate), then muscle behaves as a closed system, and intracellular pH should change as predicted by the alphastat theory. However, the [H+] gradient across the cell membrane will increase with a decrease in temperature. Skinned Fiber Experiments

6.8 6.6-

at

EFFECTS

Temperature and pH may affect a number of steps in muscle contraction, e.g., membrane excitation, excitation-contraction coupling, activation of contraction by Ca2+, cross-bridge mechanics, and metabolic supply of ATP. Using the simplified, mechanically skinned fiber preparation, one can control the free-Ca2+ concentration [free Ca2+], and hence the level of activation, and can

R206

TEMPERATURE

Force

AND

pH

ON

Ckg/cmA2>

‘Fo’rce

C kg/cm”2

I

toad

0.6

2.6

C k&mA2

MUSCLE

2.6

toad

Fo rck”

EFFECTS

5’”

0.0

0.5

2.6

Forck’

C kgji6cm”2

5.”

FIG. 3. Combined effect on pH and temperature on force-velocity curve and power calculated as product of force and velocity on frog and toad isolated sartorius muscle (22, 24). In each panel, top line is at 25”C, bottom 2 lines at 5°C. Of bottom 2 lines, lower indicates effect of temperature at constant pH, and upper indicates effect of temperature taking concomitant increase in pH into account.

study the activation and cross-bridge properties directly. Godt and Lindley (8) used this preparation to study the effects of temperature on skinned frog semitendinosus muscle at a constant pH of 7.0.They showed that skinned fibers require slightly less Ca2+ for activation at lower temperatures and that the fibers developed less maximum force. Their results on Ca2+ sensitivity were consistent with the observation that temperature alters the Ca2+ binding to Ca2+-specific sites on troponin C. Fabiato and Fabiato (6) studied the effect of pH on skinned semitendinosus frog muscle at constant temperature of 22°C. They showed that skinned fibers required less Ca2’ for activation and developed more maximum force at higher pH values. We conducted experiments to examine the combined effects of pH and temperature on skinned fibers to estimate the role of the compensatory effect of the concomitant change in pH that occurs in vivo when temperature changes. Single fiber segments from the semitendinosus muscle of the frog (R.pipiens) were mechanically skinned at room temperature in a low-Ca2+ skinning solution. One end of the skinned segment was placed in a clamp attached to a micromanipulator, the other in a clamp attached to a photoelectric force transducer based on a design by Hellam and Podolsky (10). Further details of skinning and mounting are given in Godt (7) and Godt and Lindley (8). To obviate length effects on the acti-

vation curves, fibers were stretched after mounting to a striation spacing of 2.6 pm (determined by laser diffraction). The bathing solutions contained (in PM): 1 M$+, 3.0 MgATP, 5 ethylene glycol-bis(P-aminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA), 20 imidazole buffer, 15 phosphocreatine, 16-30 KC1 (so that ionic strength was 0.150 M), 0.5 mg/ml creatine phosphokinase (100-150 units/mg), and varying amounts of CaC12. The skinning solution had a similar composition but contained no CaC12, phosphocreatine, or creatine phosphokinase. Calculation of the total concentration of ingredients needed to produce the desired free concentration of constituents in the bathing solutions was accomplished using a computer program similar in principle to that published by Fabiato and Fabiato (5). The stability constants used at the two temperatures investigated are given in Godt and Lindley (8). The binding constants for protons to imidazole are taken from Datta and Grzybowski (4). The value at 10°C was 2.09 x 107. A value for 22°C (1.14 x lo7 M-l) was interpolated by fitting the given binding constants to a Van’t Hoff isochore, K = a exp(b/T), where T is absolute temperature (isochore is the line representing variation of pressure with temperature at constant volume). Further details of the assumptions and calculations are given in Godt and Lindley (8).

TEMPERATURE

AND pH EFFECTS

ON MUSCLE

R207

of the form %force = 100 [Ca”+]NI(P

+ [Ca”‘]“) using a nonlinear least squares fitting program, where K is [Ca”‘] required to elicit half maximal force and 2v is the indicator of the steepness of the force-pCa curve. This relation was used primarily as a convenient description of the general pattern of the relation to aid in comparison of data under different conditions and to relate our data to that of others. The influence of a decrease in temperature from 22 to 10°C at pH 7 and at pH 6.5 is shown in Fig. 4. The curves are normalized to the maximum at pH 7 and 22°C. The i , I t I I Hill equation was fitted to the data and gave the param6 5.8 5.6 eters in Table 2. At pH 7, the decrease in temperature from 22 to 10°C causes the curve to shift to the left 0.141 FIG. 4. Effect of tempera&e on activation of skinned fibers (frog semitendinosus) at pH 7.0 and pH 6.5 (n = 6-8 for all points). Smooth pCa units, and at pH 6.5 it shifts to the left 0.124 pCa curves are nonlinear least square fits of Hill equation, %relative force units. That is, the increase in sensitivity to Ca2+ as a = 100 [Ca2’]“I(KN + [Ca”‘]“), to the points; parameters N and K of result of the decrease in temperature is similar at the Hill equation are given in Table 2. Each curve is normalized to two test pH values. At both pH 6.5 and pH 7, when maximum that occurs at 22°C and pH 7.0. Difference between maximal temperature decreases from 22 to 10°C, there is a -30% force for the 2 temperatures was taken from data of Godt and Lindley (8). Vertical lines are drawn at negative log of Ca2+ concentration (pCa) decrease in the maximum force that is developed, but at 6 (1 PM [free Ca”‘]) to facilitate comparisons at different pH values. low [Ca”‘] a given [Ca”‘] gives approximately the same force at both temperatures. TABLE 2. Coefficients of Hill equation of the form However, in v&o, when the temperature decreases percent force = 100 [Ca2+lN/(KN + [Ca2+lN) using a from 22 to lO”C, there is a concomitant increase in nonlinear least squares fitting program to force pCa data intracellular pH of -0.02 pH units per 1°C (Fig. 1). Thus the curves that should be compared are those at 22°C Temperature, pH K and pH 7 with those at 10°C and pH 7.25. Those curves n N “C PM appear in Fig. 5. Now we see that taking the pH change 22 7.0 9 1.33kO.032 3.06t0.205 that occurs in vivo into account is imnortant to the 22 6.5 10 2.04t0.095 2.73kO.324 animal. There is an increase in sensitivity to Ca2+. Also 10 7.0 7 0.964kO.030 6.61t1.191 the fiber can develop more force than if the pH change 10 6.5 9 1.53t0.024 4.29kO.255 10 7.25 7 0.491*0.018 4.82kO.765 did not occur. The increase in sensitivity to Ca2’ may be beneficial, Values are means t SE; n, no. of points on curve used in the fit; K, because the frog can get maximal force at a lower Ca2’ concentration of Ca2+ required to elicit half maximal force; N, indicator of the steepness of force-negative logarithm of Ca2+ concentration (pCa) concentration. Thus less Ca2+ needs to be released and curve. thus less Ca2+ will have to be pumped back into the sarcoplasmic reticulum to cause relaxation. Because a Before an experiment, the pH of each solution was large portion of energy cost in muscle is required to pump adjusted within to.01 pH units at the test temperature. Ca2+ [34-56% of total in frog muscle (18)], energy could Solutions were placed in 6-ml troughs in a temperaturebe saved if less Ca2’ is released during excitation and controlled solution changer (t0.25 “C). To minimize sur12clr face tension and functionally to compromise the sarcofrog 22 c plasmic reticulum in the fibers a drop of Triton X-100, PH 7 100 a nonionic detergent, was added to each trough (25). Between experiments, solution troughs were kept covered with glass slides to prevent evaporation. The relation between force and negative logarithm of [free Ca2+] (pCa) was determined at a fixed temperature, either 10 or 22°C. Because force tends to decrease when fibers are repeatedly activated, each fiber was subjected to only one submaximal Ca2+ concentration followed immediately by a supramaximal Ca2+ concentration at one pH, and then the same procedure was repeated at 0’ 6-8I--I - 6.4 ’ ’ 5.6’ ’ 5-2’ ’ 4-8’ ” another pH. In all cases, one of the pH values was 7.00; 6pCC3 the others were 6.50, 7.25, or 7.50. The order of pH exposures was changed, so that one-half of the fibers FIG. 5. Combined effect of pH and temperature on Ca2+ activation simulating overall in vivo effect of a decrease in temperature from 22 were exposed to pH 7 first and the other one-half to pH to 10°C. Curve on left is fitted Hill curve for 10°C and pH 7.25, the pH 7 last. Each fiber was equilibrated in a relaxing solution to which inside of cell changes in vivo when temperature is decreased for at least 3 min at the test pH before activation. from 22 to 10°C. Curves at pH 7 same as in Fig. 4; all curves are The force-free Ca2+ data were fitted to a Hill equation normalized to maximum that occurs at 22°C and pH 710. 80 -

.-I

R208

TEMPERATURE

AND pH EFFECTS

cod

cod

200

160

L

. rp

0 6.6

ON MUSCLE

7.0

PH

I

.

7.6

8.0

I 6.6

PH

7.6

8.0

I

I

.

7.0

PH

7.6

7.0

4c

3

t

X ii2 >

60 1 Isculpin I 1.6

1

sculpin I 7.0

PH

. 7.6

I 8.0

I

0

d.6

I

FIG. 6. Combined effect of temperature and pH on small bundles of chemically skinned muscle fibers from fish white muscle (from data in Ref. 15). Force is maximal in high [free Ca”‘], and V,,, is maximum speed of shortening estimated by slack test method. Compensation afforded by concomitant pH change when temperature is decreased from 10 to 0°C is indicated by horizontal line, and number below it indicates %compensation.

less pumped back during relaxation. The intriguing aspect of this hypothesis is whether the compensatory increase in pH actually is involved in less Ca2’ being released per excitation. That is, the energy benefit would only be realized if less Ca2+ was released on excitation. Less Ca2+ is released, because twitches of frog fibers injected with the Ca2+-sensitive bioluminescent protein aequorin are associated with a decreased peak light response as temperature decreases (Fig. 3 in Ref. 2). The decreased peak light is due to a decrease in peak Ca2+, because the peak aequorin light output decreased more than expected simply from the effect of temperature on aequorin itself (3). Rall(18) reported results that appear to contradict our hypothesis. Activation heat is temperature independent, and activation heat is thought to be a measure of Ca2+ cycling. However, he used a CO2 buffer and made his measurements at constant pH, exemplifying the point we made earlier regarding experiments on temperature effects. If activation heat is temperature independent at constant pH, the concomitant change in pH becomes even more important. The effect of temperature in vivo will be reflected totally by the effect of pH on Ca2+ cycling. Unfortunately, pH has a complex effect on Ca2’ cycling (6). The pH optimum for Ca2+ loading depends on [free Ca2+]; it is more alkaline when [free Ca2+] used to load is lower. Our hypothesis awaits a more direct test. Activation heat needs to be measured using the appro-

priate temperature-pH combination rather than at constant pH. The purpose of our experiments was to compare the effects of temperature at different pH values on skinned fibers with those that had been previously obtained with isolated frog muscle. Inasmuch as the pH-temperature effects on the maximum developed force are similar in the two preparations, we can ascribe the effects to the contractile machinery and can exclude participation of the sarcolemma, the sarcoplasmic reticulum, and the metabolic machinery. The mechanisms responsible for the pH effect are not known. Fabiato and Fabiato (6) listed a number of places the protons could act: a decrease i the affi .nity t of troubunits; the conforPan .in for Ca2+ or between troponin mation of tropomyosin; the force per cross bridge; or the number of active cross bridges. Other pH-Temperature

Studies

There are other studies that attempt to address the problem of temperature-pH interaction on skinned fibers. A study on Bufo used single fibers (19), whereas the other two (11, 15) used small bundles of fibers. Two of the studies used chemically skinned fibers (11, 15), whereas we and the study on toad (19) used mechanically skinned ones. The results on cod and sculpin muscle are unusual, because they show an extremely small effect on pH on force and Vmax (Fig. 6). The small pH effect in

TEMPERATURE

AND pH EFFECTS

these studies may be related to the [free Mg+] in the contracting solutions. The magnitude to the effect of pH decreases with an increase in [free M$+] and is abolished at [free Mg2+] = 5 PM in rabbit psoas muscle (17). [Free Mg2+] in living frog muscle is estimated to be -1 PM (1, 9, l4), and we calculate that it ranged from 0.93 to 1.17 in the studies on fish muscle. It is possible that the pH effect in fish muscle is inhibited at a lower [free M$+] than in rabbit muscle and thus explains the difference in results. The results of the study on sculpin are similar to others in that there is more compensation for force than Vmax.In contrast, results of the study on cod muscle are unusual because compensation for Vmax is greater than that for force. Single or small bundles of chemically skinned muscle fibers from turtle iliofibularis muscle showed small compensation; 5.6% for force and 5.3% for Vmax when comparing 20 and 10°C (11). The results of the turtle study are the only ones in the literature that show a decrease in force when pH is increased from pH 7.2 to pH 8.0. The magnitude of the compensation seen in the results of the study using mechanically skinned fibers from Bufo iliofibularis (19) is almost identical to what we found in Rana fibers. The compensation afforded by the concomitant pH change is 18.5% when temperature is decreased from 35 to 25°C and is 27.7% when temperature is increased from 25 to 15°C. Results from all studies on amphibian muscle are consistent in showing a modest compensation afforded by the concomitant increase in pH with a decrease in temperature. The studies on fish and turtle muscle may reflect true species differences or may result from methodological differences.

Locomotory

In conclusion, the concomitant increase in pH associated with a temperature decrease always causes a change in the correct direction to compensate for the effects caused by the decrease in temperature. The magnitude of the compensatory effect is appreciable for isometric force but is