Myocardial energetics during isometric contractions of cat papillary muscle

twitch

GEORGE COOPERIV Department of Environmental Biosciences, Naval Medical Research Institute, National Naval Medical Center, Bethesda, Maryland 20014; and the Cardiovascular Division, Department of Internal Medicine and Cardiovascular Center, University of Iowa College of Medicine, Iowa City, Iowa 52242

COOPER, GEORGE, IV. Myocardial energetics during isometric twitch contractions of catpapillary muscle. Am. J. Physiol. 236(2): H244-H253, 1979 or Am. J. Physiol.: Heart Circ. Physiol. 5(2): H244-H253, 1979.-During myocardial tetanus, when activation is maximum and constant, there is a linear relationship throughout contraction between oxygen consumption (MVo& and the cumulative product of active tension and time (SAT). The goal of this study was to determine the relation of MVo, to SAT during isometric myocardial twitch contractions. Ten right ventricular cat papillary muscleswere studied in a flow respirometer. MVo, was determined during contractions unloadedfrom Lax to a slack length at successive 100.msintervals after stimulation. In contrast to the linear relationship observed during tetanus, MVo,lSAT varied during twitch contractions: when the muscleswere made slack 100 ms after stimulation MVo&AT was 389 * 51 (SE) (nl of O$mg of dry muscle)/(N of active tension/mm2s of active tension). This value was 94 t 7 at peak active tension and was constant thereafter. There was a continuous increase in cumulative MVo, as SAT increased; before SAT began, MVo, was 0.41 t 0.04 (nl of 02/mg)/contraction at L,, and 0.22 ,t 0.04 at a slack length; at peak isometric tension MVo2 was 1.84 * 0.19; for a complete contraction MVo, was 2.89 t 0.25. These data support two concepts1) activation energy is small and dependent on initial length and tension; and 2) SAT is variably energy dependent throughout the entire isometric twitch contraction.

myocardial mechanics;polarography; activation

THE

IMPORTANCE

OF

UNDERSTANDING

THE

CONTROL

Of

myocardial oxygen consumption (MVo,) has made insight into this control a major goal of cardiovascular research. The relation of muscle mechanics to energetits has received particular attention; as a result, the greater importance of tension development and the lesser importance of shortening in determining MVo, are now well recognized (2, 32). However, current knowledge of the mechanical determinants of MVo, is largely descriptive. The possible interaction during each contraction between changing mechanical state of the myocardium and variable rate of the energy-dependent events supporting contraction is unknown. It would seem reasonable to suggest that such an interaction may be the basic mechanism underlying the mechanical determinants of M’Vo,. The purpose of this investigation was to elucidate the

interaction between active tension and metabolism during the normal isometric myocardial twitch contraction. That is, what is the relation of ongoing metabolism (MVo,) to the increasing product of active tension and time, here expressed as active tension summed or integrated in time (JAT), during the course of contraction from the onset through complete relaxation. Three specific questions were asked 1 ), what is the ratio of MVo, to JAT at specified times during contraction; 2), what is the activation energy of cardiac muscle and its distribution in time, and 3) is activation energy length and tension dependent? To answer these questions, we used a method of controlled release to examine cumulative MVo, at specified times during contraction. The experiments showed I), the ratio of MVo, to JAT is highest early in a contraction and then reaches a constant value by the time of peak active tension, which persists through complete relaxation; 2), activation energy is relatively small for cardiac muscle and is most important early in a contraction; and 3), activation energy is length and tension dependent. In contrast, during tetanus (9), when activation is maximum, the ratio of MVo, to JAT is constant, and the effect of changing mechanical conditions on MVo, is minor. The difference between these two sets of data has led to the hypothesis that the interaction during contraction between changing mechanical conditions and the level of activation regulates myocardial energy demands. This regulation may well be the unifying mechanism for the mechanical determinants of MVo,.

Experimental

Apparatus

The flow respirometer, the associated equipment, and the attendant methods have been described fully in previous papers (8, 9, 24). The principal feature ofthis respirometer is that it allows the simultaneous determination of mechanical behavior and oxygen consumption for the isolated, superfused cat papillary muscle preparation. For the particular purposes of the present study the respirometer and the other devices were modified in the following ways. The first modification allowed muscle length to be measured and controlled from above. Simultaneous determination of length and tension had not been pos-

H244

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sible previously with this system. To accomplish this, the plug at the bottom of the respirometer was replaced by a mercury seal through which passed a steel rod with a C-shaped metal clip on its upper end. When this clip, with the ventricular end of the papillary muscle within it, was tightened and enclosed by screwing a cylindrical sleeve up over it, the muscle was held rigidly, and the ventricular end was occluded by the sleeve from the circulating superfusate in the respirometer. The distal end of the rod was screwed directly onto a strain gauge. With the chordal end of the muscle fixed to the lever above the muscle by a tie just above the chorda-muscle junction, tension generated by the muscle was measured with very little stray compliance (~0.7 pM/mN) over the range of force studied. In addition, the enclosed clip produced’ discrete end-segment damage and excluded damaged tissue from the metabolic measurements of interest. The second modification allowed muscle length, and therefore tension, to be varied during and between contractions in a controlled manner in an attempt to prevent variable length and load effects on activation (11). The lever above the muscle was equipped with a very small dashpot on the end toward the papillary mu&le in a manner analogous to that reported by Edman and Nilsson (11). Because the results obtained by standard quick-release techniques depend on lengthand time-dependent muscle elasticity and viscosity, it was instead elected to actively lower the lever to shorten the muscle in a more precisely controlled manner. Vacuum was supplied to a rigid hollow tube resting beneath the lever by opening a solenoid. A second small piston attached to the muscle end of the le ver rested within the lumen of this tube so that the lever was drawn down when the solenoid was energized. The rate at which the lever descended to actively shorten the papillary muscle was a function of the viscosity of the oil in the dashpot and the degree of vacuum. The time when the solenoid was activated was determined by the output of a second stimulator. A variable delay was adjusted so that at a O-ms interval between muscle stimulus and solenoid activation (0-ms stimulus-release interval) the lever was pulled down and the muscle released just at the end of the latent period. Other intervals are defined as the time from stimulus-release the end of the latent period to the beginning of release. Thus, the controlled release consisted of lowering the lever holding up the chordal end of the muscle at a fairly constant rate for each stimulus-release interval. This resulted in each case in a decrease in muscle length from Lmax, that muscle length at which developed tension was greatest, to less than Lo, a length at which the muscle was slack, with no active or passive tension during contraction (Lo as used here is that point on the ascending limb of the length-tension curve where active tension is first seen upon stimulation). Further details of the sensors, signal cond .itioners , and recorders are provided in a previous report (9) . Measurement of Mechanical Activity Consumption

Rapid cardiectomy

was performed

2.7 kg) after anesthesia

was induced with sodium pentobarbital (30 mg/kg ip). Ten right ventricular papillary muscles 4.69-8.33 mm in length (6.53 t 0.38, mean t SE) and 0.45-1.11 mm2 in cross-sectional area (0.90 t 0.06) were excised and mounted in the flow respirometer. The metabolism of muscles of these dimensions is not diffusion limited (8, 9, 29). The muscles were superfused at 29*C by a solution of the following millimolar composition: CaC12, 2.5; KCl, 4.7; MgS04, 1.2; KH2POd, 1.1; NaHC03, 24.0; Na acetate, 20.0; NaCl, 98.0; and glucose, 10.0; with 10 units of insulin added per liter. This solution was equilibrated with 95% 02-5% CO, with a resultant pH of 7.4 and .rculated past the muscle from a l-liter reservoir. After the muscles were mounted in the respirometer, each muscle was lightly preloaded and stimulated at 0.2 Hz until a stable mechanical response was obtained. Field stimuli, 5-10% above threshold and of alternating _ polarity with zero net current flow between the electrodes, were employed to minimize electrolytic contamination. The muscles were then brought to L,,,. At this length resting tension was 13.26 t 2.29% of total tension (active plus resting), with a resting tension of 10.54 t 1.38 mN/mm2 (milliNewtons per square millimeter muscle cross section). Thus, according to criteria developed by Brutsaert et al. (3), the muscles had physiologically appropriate dimension .a1 and mechani cal characteristics. Muscle mechanics and energetics were then measured during controlled release at various stimulusrelease intervals. This procedure is illustrated in Fig. 1 and explained in its legend. An important feature of this method is that it allows muscle load and length to be altered in a controlled manner. Table 1 summarizes the data for controlled release and lengthening for the nine stimulus-release intervals in the 10 muscles studied. Because of variable active tension during contraction, the unloading rate at which force was removed from the muscles during contraction differed at each stimulus-release interval. However, the maximum rate at which the muscles could potentially shorten during contraction did not vary. The bottom half of this table shows that after each contraction, when active tension was no longer present, both the rate of lengthening back to Lax and the rate of passive tension reimposition that produced this length change were the same at the varying stimulus-release intervals. Oxygen consumption was measured polarographitally in a flow respirometer. This measurement requires the assumption that oxygen consumption measured over long periods of time is an accurate reflection of metabolism occurring during the contractions themselves. This would appear to be a valid assumption if the work of Challoner (6) on the aerobic nature of myocardial respiration is correct and if a properly designed flow respirometer is used to study a stable muscle preparation. This flow respirometer was designed from criteria originally defined by Carlson et al. (5); these criteria have been used in the study of and Oxygen papillary muscle respiration first by Lee (22), then by McDonald (24)) Coleman (8), and Cooper (9), and in the of this respirometer on adult cats (1.7- present study. The dimensions

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G. COOPER

IV

FIG. 1. Isometric contractions released at two stimulusrelease intervals from L,,, to a slack length. Calibration bar for each pair of tracings is on far leR, and identification of each pair is on far right; each applies to tracings in both panels. J Tension denotes the continuously recorded integral of the tension signal. No deflection of the J Tension tracing is seen for resting tension at Lax (far leR on both panels); the cumulative upward deflection (note that the integrator resets to the middle of its range at a fixed upper and lower limit) during active tension generation quantifies the product of active tension and time (JAT). Following a brief latent period after the stimulus, active tension was developed. Tension was then unloaded from the muscle by shortening it by an amount and at a rate shown by the top two tracings. This placed the muscle at a slack length, L < Lo, as shown by a decrease in tension to zero external tension. Since the muscle was now slack, the tension integrator shows a progressive negative deflection. Finally, on the far right, the muscle was returned to L, prior to the next stimulus.

300 mrec

TABLE

1. Controlled Controlled

Release

500 msec

release and lengthening from L = L,-

to L < Lo during

muscle length at L,, and cross-sectional area. Thus, tension was expressed as millinewtons per square millimeter, shortening as muscle lengths, and oxygen consumption as nanoliters per milligram of dry muscle per contraction.

data

Each Contraction

Experimental Controlled Stimulus-release interual, m.8 Controlled lengthening rate, muscle lengths/s Reloading rate, (N/mm*)/s

Lengthening

from L < b to L = 4,

after Each Contraction

0

100

200

300

400

500

600

700

800

4.49

kO.71

4.50 20.72

4.60 20.81

4.30 20.79

4.35 20.81

4.48 ~0.84

4.51 +0.86

4.55 +0.85

4.58 20.88

0.32 -to.03

0.31 20.03

0.32 -to.03

0.30 +0.03

0.30 20.03

0.30 40.03

0.29 20.03

0.28 rto.02

0.28 20.02

Release and lengthening rates specify rate of change in muscle length; unloading and * There were significant reloading rates specify rate of change in muscle tension. differences among these values.

(Fig. 1 of Ref. 8) are the same as those used before (8,9, muscle chamber is 41 mm long, 3.5 mm wide, and has a volume of 0.4 ml. The sampling capillary at the bottom of the muscle chamber is 0.8 mm wide with the oxygen cathode 39 mm distal to its origin. The superfusate flow rate past the muscle and then through this capillary is 6.25 ml/h. These conditions assure uniform difision of 0, across the sampling capillary at the electrode site (Eq. 17 of Ref. 5). The stability of the muscle preparation over a 24-h period was demonstrated in a prior study (9). Figure 2 demonstrates the stability and sensitivity of the respirometer in its present form. After each experiment, muscle length was measured by a micrometer with a known preload attached to the muscle. This length, along with the passive tension portion of the length-tension curve, allowed calculation of muscle length at L,,,. Assuming a wet-to-dry weight ratio of 4.O:l.O (10) muscle cross-sectional area was calculated from the length at Lmax and the dry weight as before (9). Results were normalized in terms of 24): the central tubular

Protocols

There were four experimental protocols, the order of which was randomized. 1) m02 and muscle mechanics during isometric contractions at L,,, unloaded at specific times during contraction. These experiments were done to define cumulative MVo, at specified times during the isometric myocardial twitch contraction. With the muscles at L max9 groups of 120 contractions (stimulus rate of 0.5 Hz over a 4-min period for each group of contractions) were produced, and the associated MVoz was measured. Tension generation during each contraction in a group was interrupted by controlled release of the muscles at a particular stimulus-release interval. The stimulusrelease interval was determined by solenoid activation starting either 0 ms after stimulus (mechanical release beginning at the end of the latent period) and progressing in NO-ms increments through 800 ms or, altematively, at 800 ms after stimulus and progressing through 0 ms. The mechanical and metabolic data were similar with either sequence of studies. Extrabasal MVoz produced by the 120 contractions was quantified as the time integral of Po2. After basal superfusate P% was once again reached with the muscle quiescent, another series of contractiohs at a new stimulus-release interval was initiated. MVo, and JAT were expressed as follows. For each stimulus-release interval, MVo, and SAT were summed for the group of 120 contractions; the cumulative increase in SAT is shown in Fig. 4A. The ratio of MVo2 to JAT was then defined for each stimulus-release interval (Fig. 4B). Finally, oxygen consumption was expressed as MVoJcontraction (Fig. 5); this was done by dividing total MVo, by 120 at e&h stimulus-release interval. Because of a brief force trepne observed for the

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++-p02

FIG. 2. Photograph of calibration record from flow respirator in its present form. Bottom record shows on left a stable Poll record when superfusate is equilibrated with 100% 0,. Upward deflection to a new value is produced when gas mixture is changed to one having 5% less 0,. This is followed on right by a return to the same stable PO, level after 100% 0, is reintroduced. Upper tracing, the output of an integrator, gives area under lower calibration curve, where no deflection is produced at PO, of 760 mmHg, and cumulative upward deflection (integrator resets to zero at top of each of its upward deflections) quantifies area under the bottom tracing. Calibration box on original record was 4.0 cm2. Range of Po, and time variation during muscle contraction was within limits of changes in these two variables shown on this calibration record.

=722mmHg

I

1

1

I= 4.0 cm*

first 4 or 5 contractions at the beginning of each group of 120 contractions, a precisely average-contraction for each group did not exist. However, within the precision of these methods this did not pose a problem, as the duration of the force treppe was the same at each stimulus-release interval, and MVoJcontraction showed no consistent change at the various stimulus-release intervals when 60 contractions over 2 min were compared to 120 contractions over 4 min. 2) M'Vo2 during contractions at a slack length, L < LW ‘&se experiments were done to define anY possible length and tension dependence of activation energy. The muscles were allowed to remain at the slack length (L c LJ determined by their intrinsic elastic and viscous properties. The MVoz produced by 120 contractions at a rate of 0.5 Hz was then measured without release. This MVo, value was compared to that observed for the 0-ms stimulus-release interval in the first section of studies, where the muscles were at L,, only during the latent period and slack thereafter. 3) MVO, and muscle mechanics during contractions at Lnax without release. These experiments were done on 4 of the 10 muscles to determine whether the controlled release of contracting muscles affected potential mechanical or metabolic performance. Either before or afIer the above two sections of studies, the muscles were held at L max and stimulated to contract isometrically without being released at any time. Again, 120 contractions during 4 min were studied. The-mechanical data were compared to those observed for the stimulus-release intervals in the first. section of studies where an appropriate comparison could be made. The metabolic data were compared to those observed for the 800.ms stimulus-release interval in the first section of studies. 4) MVO, during muscle release without. stimulation. To further assess the possible effect .of the release intervention itself on MVoz, 4 of the 10 muscles were released from L = L,, to L < Lo and then returned to L = Lmax 120 times during 4 min without being stimulated to contract.

Statistical

Analysis

All values are expressed as mean t SE. For paired comparisons, Student’s paired t test was employed. For comparisons of greater numbers of variables, one-way analysis of variance and multiple intergroup comparisons with Tukey’s test were employed (1). A significant difference was said to exist when P was less than 0.05. RESULTS MVO, and Muscle Mechanics during Isometric Contractions at L,,, Unlo&ed at Specified Times during Contraction Figure 3 shows the externally measured active tension of these muscles at the specified times ofcontrolled 80

t

0

200

400

600

800

Stimulus-Release Interval (msec) FIG. 3. Average active tension values at time of release of 120 contractions at each of the 9 isometric stimulus-release intervals studied in 10 muscles. Point at 360 + 10 ms is at time of and shows amount of peak active tension; this is not a point at which the muscles were released.

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G. COOPER IV

release. The point at 360 -+ 10 ms is at the time of peak active tension and is not a time at which the muscles were released; the value for peak active tension was 69.0 t 4.7 mN/mm2. The contractions were essentially complete at the 800.ms stimulus-release interval. Table 2 shows that the maximum rate of tension generation did not vary with differing stimulus-release intervals. Thus, this particular estimate of inotropic state, an important determinant of MVo, (2), did not vary for the eight stimulus-release intervals where it could be measured. The Figure 4A shows the cumulative increment in the area under the active tension curve of Fig. 3 as the active tension generation of these contractions was terminated at progressively later times. At the time of peak active tension indicated by the arrow, the SAT was 57% of that for a complete contraction. Figure 4B defines the ratio of MVo, to JAT at the times during these contractions specified on the abscissa. It should be compared to Fig. 4A. This ratio has a relatively high value early in the contraction ‘when JAT is small. It

TABLE

2. Maximum

Stimulusrelease interval, ms

rate of tension development

100 200 300 400 500 600 700 600

dT/dt, (mN/ 241 248 229 227 220 222 238 mm2)/s 227 224 219 +20 219 221 224 There was no significant difference among these values.

233 227

4

reaches a fairly constant value by the time of peak active tension, shown by the arrow and does not change appreciably thereafter. Thus no significant differences exist among the 400- to NO-ms points for the ratio of MVo, to SAT, but they are all significantly greater than zero. Figure 5 displays the cumulative oxygen consumption associated with contractions in which active tension generation was terminated at the times specified. There was a progressive increase in oxygen consumption as the duration of active tension increased. At the time of peak active tension, indicated by the vertical arrow, MVo, was 64% of that for a complete contraction. MVo2 during Contractions at a Slack Length, L < LO

Referring to the two points on the ordinate of Fig. 5, there is in absolute terms a small, but in relative terms, a large and significant difference between the oxygen consumption at the two muscle lengths indicated. For contractions in which the muscles were completely unloaded to 0 resting and active tension just before active tension generation began (0 ms, L = L,,,) the MVo, was 14% of that observed for the contractions released at the 800.ms stimulus-release interval. For contractions in which the muscles were not released but were at a slack length (0 ms, L < L,) at which they generated no active tension and demonstrated no resting tension at any time during the 4-min period of stimulation, the MVo, was 8% of the value for the 800. ms stimulus-release interval. 500 .

B

N = 10

200

Stimulus-Release

0

400

Interval

(msec)

4. A: average JAT values for 120 contractions at each of 9 isometric contraction intervals studied in 10 muscles. Arrow indicates the time of peak active tension. B: average values for the ratio FIG.

200

Stimulus-Release

400

Interval

600

(msec)

of MVo, to JAT during 120 contractions at contraction intervals studied in 10 muscles. There is no finite point for the 0-ms stimulusrelease interval. Arrow indicates time of peak active tension.

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Stimulus-Release Interval (msec) 5. Average values of MVo, per contraction for 9 isometric contraction intervals studied in 10 &uscles. Arrow parallel to ordinate indicates time of peak active tension. Arrows parallel to abscissa indicate specified muscle lengths. FIG.

and Muscle Mechanics during Contractions Release aGna~ Without . The MVoz for 120 full isometric contractions at L,,, without release at any time was 2.88 t 0.62 (nl of 02/ mg)/contraction; this was not significantly different from the value of 2.89 t 0.25 for the contractions released at 800 ms after stimulus. Similarly, peak active tension, the time to peak tension, and the maximum rate of tension development did not differ significantly for the unreleased contractions when compared to those released at 400-800 ms, and the JAT was the same for the unreleased contractions and those released at the 800-ms stimulus-release interval.

M'VO,

M'Vo2

during

Muscle Release without Stimulation

When the quiescent muscles were released 120 times without being stimulated, there was no detectable deflection in the MVo, record. These last two types of experiments demonstrate 1) that the release intervention itself altered neither MVo, nor muscle mechanics and 2) that different muscle lengths and resting tensions, rather than the release intervention, were responsible for the different MVo, values shown on Fig. 5 at L = Ltlx and L < Lo. DISCUSSION

The *four principal findings of this study are I ) active tension requires energy throughout the isometric myocardial twitch contraction, 2 ) the energy requirements for those activation events that initiate contraction are small, 3) there is a variable ratio of MVo, to JAT during contraction, and 4) activation energy is length dependent in cardiac muscle. Experimental

Preparation

Because of several recent questions about the usefulness of the isolated super-fused papillary muscle as an

H249 appropriate preparation for the study of normal myocardial behavior, these questions must be addressed before the physiological significance of these findings can be discussed. The questions have centered first on the mechanical suitability of the papillary muscle preparation and second on the adequacy of metabolic support for this preparation. Mechanical suitability. The papillary muscle has often been used as a simple mechanical analog of the intact heart, and this analogy has also been extended to the level of the sarcomere (30). Possible problems with the latter analogy are inherent in the study of Krueger and Pollack (20). This study showed that during contractions in which the whole papillary muscle was isometric, there was 7% central segment shortening at the expense of lengthening of both damaged end segments. They concluded from this finding that direct correlations between whole papillary muscle mechanics and simultaneous behavior on the sarcomere level may be inappropriate. The recent study of Suga et al. (31), suggests that end-segment damage does not introduce major difficulties into the interpretation of whole panillarv muscle mechanics. Their data show that the&rfusea in situ dog papillary muscle exhibits mechanical characteristics very similar to those of the superfused excised cat papillary muscle. When normalized for dimensional differences, their Fig. 3 shows both a value for developed force and a relationship between relative length and developed force that are very similar to those observed in the present study and in many other previous studies of the isolated cat papillary muscle preparation. Series elasticity also mimicked that of excised preparations. Thus the contribution of the single damaged end segment to the overall mechanical characteristics of the papillary muscles used in the present study was probably minor. It is possible that in both skeletal (7, 26) and cardiac muscle (17.20,33), a purely isometric twitch contraction on the segment or the sarcomere level does not exist. However, this potential limitation does not significantly interfere with the interpretation of these data, as the purpose of the present study was to determine how welldefined mechanical conditions of the whole muscle interact with metabolic behavior during the course of isometric contractions at a single whole muscle length. Metabolic support. The excised, superfused papillary muscle depends on diffusion for metabolic support. Lee (22) initiated the study of cat papillary muscle oxygen consumption. Because of Hill’s work (15) regarding delivery of metabolites by diffision, Lee limited his study to muscles of a diameter cl.20 mm (cylinder cross-sectional area < 1.13 mm2). Coleman (8) later developed a technique of stretching muscles by progressive increases in preload; in muscles that were too thick a hypoxic core was unmasked resulting in increased resting MVo, during stretch. In the present study of muscles of 0.45-l. 11 mm2 cross-sectional area, a similar technique showed that resting MVo, did not change as the muscles were stretched and thus thinned while being brought from a length determined by a light preload to L,,, . For contracting myocardium, Snow and

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Bressler (29) have recently demonstrated directly, using intramitochondrial NADH fluorescence of muscles during isometric contractions, an adequate oxygen level for normal oxidative metabolism throughout the entire cross section of superfused papillary muscles with a cross-sectional area of Cl. 16 mm2 and a contraction frequency of up to 0.8 Hz. Conditions in the present study fall well within these constraints.

(11). Because this information was not available at the time of the prior study (25), it is unlikely that the variables of muscle length and rate of length change were carefully controlled. In the present study these variables were controlled. First, the muscles were released from the same overall length at each stimulus-release interval. Second, the top half of Table 1 shows that the maximum rate at which the muscles were allowed to shorten did not varv during contraction for any of the stimulus-release interSignificance of the Results vals, although variable degrees of active tension during Tension generation, for a given heart rate and inothe course of contraction necessarily caused the rate of tension unloading to vary. Third, the bottom half of tropic state, is generally accepted as the major determiTable 1 shows that the rate of tension and length nant of MVo, (2, 32). In this study in which heart rate (stimulus frequency) and inotropic state (rate of tension reimposition prior to each succeeding contraction did not vary for the different stimulus-release intervals. development from constant initial muscle length) did not vary, tension generation was again shown to be a Therefore, the variable effects of length and rate of length change on activation were controlled both during major determinant of MVo, in isometric twitch contraceach contraction and between contractions. tions at L,,, . Specifically, Fig. 5 shows that the generation of peak active tension obligates 64% of the oxygen In Monroe’s study (25) the left ventricular myocarrequired for a full isometric contraction. This confirms dium must have shortened progressively as the conthe basic relation of MVo, to the tension-time integral tained air was compressed during the portion of the first elucidated in detail by Sarnoff et al. (28). There contractions that preceded ventricular venting. Thereare, however, four significant new findings that re- fore, muscle length must have varied as a function of sulted from this study. changing muscle tension at the different stimulus-reTime course of MVO, during contraction. The first lease intervals. Progressive deactivation during connew finding, in distinct contrast to the currently ac- traction would be expected as the ventricles compressed the enclosed air prior to release. Moreover, without cepted concept, is that active tension is an important determinant of MVo, throughout the isometric myocarcontrolled release rates, further incremental deactivadial contraction, even after peak active tension has tion would be expected at progressively later venting been reached. Referring to Fig. 5, it is clear that MVo, times up to the time of peak active tension as the continues to increase for contractions in which release ventricles developed increasing wall tension; i.e., with occurs a&r peak active tension is reached. Thus, active increasing active wall tension the ventricles would be tension is energy dependent at all phases of isometric expected to shorten at progressively greater rates upon venting. Therefore, these progressive deactivation efcontraction. The currently accepted concept is that the relaxation fects during contraction may well explain, at least in part, Monroe’s finding that by the time peak active phase of cardiac contraction is an essentially energy independent, passive process (2, 32). This is based tension (and shortening) was reached, the energetic largely on an earlier study (25) of the time course of requirement of this particular type of contraction was MVo, during contractions of an isolated heart preparaessentially complete (25). In contrast, the present data demonstrate an energy dependency of active tension tion, in which the left ventricle was allowed to compress air and was then vented at various times during con- throughout the isometric myocardial twitch contraction traction. In that study Monroe (25) found that 91% of when muscle length and its rate of change are conthe total MVo, for a contraction was accounted for by trolled. the time peak active tension was reached. This is quite Myocardial activation energy. The second new finddifferent from the value of 64% found in the present ing is the low value for activation MVo, of heart muscle indicated in Fig. 5 by the arrow at L = L,, . This value study. of 0.41 t 0.04 (nl of 02/mg dry wt)/contraction is 14% of There are a variety of possible reasons for the appar-ent discrepancy between the present study and that of the total MVo, for a complete contraction at L = L,,, . Monroe (25). The more obvious ones involve differences In terms of the metabolic relationships formulated in in experimental preparations and protocols. A less ob- detail in a previous paper (9), this is the amount of MVo, associated with activation at L = L,,, . Potential vious but more basic one is that information about the the interrelation of. muscle length w ith activation ( 18) and sources for this activation MVo, include primarily the importance of the rate of length change during energy-dependent accumulation of the calcium that initiates and regulates ongoing contraction by the sarrelease in modifying this relationship (11) has only recently become available. A review of this information coplasmic reticulum, and possibly by mitochondria (4), of the (18) suggests that the level of activation, thought of and to a lesser extent the energy require’ments ionic pumps that produce repolarization (21). here as reflecting myoplasmic calcium concentration, This is not directly comparable to the tension-indevaries directly with initial muscle length at lengths pendent activation heat of slack heart muscle described below L,,, . Further, the ongoing level of actin-myosin interactions that maintain contraction probably varies by Gibbs and Vaughan (13). Instead, this represents a tension- and length-dependent activation MVo2, as the inversely with the rate of shortening during release Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (130.241.016.016) on December 15, 2018.

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muscles were released from L = L,,, to a slack length, L c Lo, just before the occurrence of externally apparent active tension generation, i.e., at the end of the latent period. Further, because the energetic requirements of activation events and actin-myosin interaction events cannot be rigorously separated here, the intercept of the ordinate must be considered a maximum value for activation MVo, at Lax, with an unknown contribution from internal shortening and tension generation during the subsequent contraction of the slack muscle. Time course of activation energy. The third new finding is that the small amount of oxygen consumption required for activation is not evenly distributed during contraction but is greatest during the initial portions of the isometric myocardial twitch contraction. This conclusion is based on the large and changing ratio of MVoz to SAT early in the contraction, which reaches a lower and constant value by the time of peak active tension, as shown in Fig. 4B. This conclusion must necessarily be somewhat inferential, as there is no way to completely separate activation MVo2 from that required for generating and maintaining active tension during the course of these contractions. In addition, any temporal dispersion of sarcomere lengths resulting in internal shortening by some segments prior to release could be greatest early in contraction and, even after release, at all of the stimulus-release intervals up to and including 700 ms, there is probably MVo, related to internal shortening and tension generation in the slack muscle following release, with no assurance that this MVo, varies directly with the duration of active tension prior to release. Further, it is not certain that the tension generation produced by a given amount of ATP breakdown at the crossbridges is always the same during contraction. However, the constant ratio of MVo, to SAT seen later in these contractions suggests that activation MVo, is at least relatively more significant and more variable in the early phases of these contractions. This is in contrast to the situation during isometric myocardial tetanus (Fig. 4 of Ref. 9), when activation and active tension are maximum throughout contraction. The sum of activation MVo, plus active tensiondependent MVoJs of tetanus is constant, so that a fixed ratio of MVo, to JAT obtains throughout tetani of variable duration. Were this the case throughout these twitch contractions, Fig. 4B would show a line parallel to the abscissa during the entire contraction. While these data do not preclude the probability that some oxygen consumption related to activation events is going on throughout and even after contraction, they do suggest that activation MVo, is relatively more important early in an isometric twitch contraction. To summarize the last two findings, the intercept of the ordinate at L = L,,, in Fig. 5 quantifies the total MVo, associated with activation energy at L,,, as well as other possible energy utilization that does not result in external tension development, whereas Fig. 4 provides a description of the relative distribution of total myocardial activation MVo, in time during contraction. Length dependence of activation energy. The differ-

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ence between the MVo, values at L = L,,, and L < Lo in Fig. 5 is the fourth new finding. This difference demonstrates a dependence of myocardial activation energy on initial muscle length and resting tension. The MVo, value at L < Lo was obtained when the muscles were not released but were at a slack length throughout the 4-min period of 120 contractions; this slack length is the same length to which they were released at the specified stimulus-release intervals for the other points in Fig. 5. The MVo, at L < Lo is 8% of that for a full contraction at Lmax. This confirms in energetic terms the length-dependent activation effects on muscle behavior described in mechanical terms for heart and skeletal muscle (18). Specifically, this shows for cardiac muscle that the processes that initiate contraction are energetically more costly when the latent period occurs at a greater muscle length and resting tension. The value for activation MVo, in cardiac muscle has been corroborated by myothermal techniques. The activation MVo, at L < Lo is 0.22 t 0.04 (nl of OJmg dry wt)/contraction in the present study. Using a caloric equivalent of oxygen of 4.86 kcal/l of 0, for myocardium (23), and a wet-to-dry weight ratio of 4.O:l.O (lo), the caloric value for activation energy at L c Lo would be 0.27 (meal/g wet wt)/contraction. This corresponds rather closely to the value for the tension-independent heat of slack muscle, given as 0.24 (mcal/g)/contraction by Gibbs and Vaughan (13) for rabbit myocardium at 32OC. In view of the directional temperature dependence of activation heat that they reported (13), the correspondence at 29OC might have been even closer. The identification of MVo, at L < Lo primarily with the energy required for calcium cycling is strengthened by information that Langer has reviewed (21). Drawing from a variety of data, he calculated that an enthalpy change of 0.25 meal/g woul .d be required to accumulate the Ca2+ needed to produce relaxation in a single cardiac cycle. The closeness of the agreement of this theoretical predicted value with the caloric equivalent (0.27 meal/ g) of the experimentally determined MVo, at L < Lo is remarkable. Further, when Langer (21) applied his theoretical construct to the data of Gregg et al. (14) for the MVo, of the awake, resting dog, he predicted that 6.5% of the total energy required for a normal heartbeat would be accounted for by Ca2+ cycling by the sarcoplasmic reticulum. The very near agreement between that predicted value and the ratio of MVo, at L < Lo, 0 ms to total MVo, at L = L,,, ,800 ms (8% in the present data) both bears out his prediction and suggests that the percentage value obtained in this in vitro preparation may well have relevance to the intact organism. These values for activation energy of cardiac muscle apparently differ from the value for activation energy of skeletal muscle, reported to be 1.24 meal/g at 20°C in the frog semitendinosus muscle (Table 2 of Ref. 16); this was 30% of the total heat for a full contraction at L,, The disparity between the ca rdiac and skeletal muscle values is enhanced by the fact that the figure for skeletal muscle may be considered a minimal value when compared to the present figure for heart muscle, because in the study of Homsher et al. (16) actin-myosin

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interactions were mechanically excluded by extending the muscles to such an extent that thick and thin filament overlap was prevented. However, the effects of muscle length on activation energetics shown in Fig. 5 must raise some doubt about the relevance of the value of 30% of total energy for the activation energy of even skeletal muscle in a more physiological length range. A relatively lower activation energy for cardiac as opposed to fast skeletal muscle might be expected in view of the lesser quantity and activity of sarcoplasmic reticulum in cardiac compared to skeletal muscle (4). Indeed, since it is likely that slow, tonic skeletal muscle is mechanically and energetically more nearly analogous to cardiac muscle than is fast, phasic skeletal muscle (9), it is of interest that Rail and Schottelius (27) have shown that although activation energy normalized to maximum twitch tension is similar in the two skeletal muscle types, the absolute value of activ&ion heat per gram per contraction is much less for twitch contractions of tonic skeletal muscle than it is for those of phasic skeletal muscle. In fact, in that study (27) the absolute value of activation heat in the tonic anterior latissimus dorsi of the chicken at 21°C was 0.19 t 0.01 (mcal/g)/contraction; this is similar to the value of 0.27 (mcal/g)/contraction for the caloric equivalent of MVo, found in the present study of cardiac muscle. The major point of this section of the discussion is that these data identify a small value for activation energy in cardiac muscle that depends on initial muscle length and resting tension. Since diastolic muscle length in the intact, normal heart must lie between Lo and bnax as defined herein, myocardial activation probably has a maximum value between 8% and 14% of the energy for a full isometric contraction and an actual value, because of the energy required by probable residual actin-myosin interactions during contraction of the slack muscl .e. incl uded in this estimate, which is very likely even less. CONCLUSION

In a previous paper (9), the energy requirements of tetanic myocardial contraction were related to an energetic active state defined as a measure of the underlying actin-myosin interactions that must largely form the biochemical basis of contraction. When thought of in this way, MVo, is a measure of the time course and extent of activation and resultant mechanical activity during contraction. In that study (9) it was found that during myocardial tetanus, when activation is maxi-

G. COOPER IV

mum and constant throughout contraction, the mechanical determinants of oxygen consumption that apply during twitch contractions are no longer important. Further, MVo, was linearly related to contraction duration during either isometric or isotonic tetanus. This represents an approach to a fully developed, unmodulated level of myocardial activation throughout contraction. The present study demonstrates that during isometric myocardial twitch contractions, active tension generation is energy dependent throughout contraction, and myothermal techniques have a.lso shown that myocardial heat output varies directly Wi th the total SAT of a contraction (12). However, the linear relationship between JAT and MVo, seen during tetanus was not observed in the present data, and further, activation energy was length and tension dependent. This energetic difference between myocardial twitch and tetanus is apparently based largely on variable modulation of the level of activation during these isometric twitch contractions by changing conditions of length, tension, and contraction duration. The contrast between myocardial twitch and tetanus has suggested the hypothesis that changing mechanical and temporal relationships during the normal heartbeat may modulate both the initial level of activation and the ongoing level of activation during contraction. The anatomic site or biochemical step at which any such feedback might occur remains speculative; however, this modulation may be a basic regulatory mechanism for the control of myocardial metabolism. The preparation of the graphics by Linda Godfrey, Tom Gillon, and Carolyn Hammer of the Medical Graphics Department of the University of Iowa and the preparation of the manuscript by Marilyn G. Jordan, Ruth Bonar, and Debra Mansure are gratefully appreciated. This study was supported in part by Naval Medical Research and Development Command, Research Work Unit No. MR000.01.1168, by Grant 77-G-41 from the Iowa Heart Association and by Biomedical Research Support Grant RR 05372 from the Biomedical Research Support Branch, Division of Research Facilities and Resources, National Institutes of Health. The experiments conducted herein were performed according to the principles set forth in the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Resources, National Research Council, DHEW, Pub. No. (NIH) 74-23. This study was presented in part before the 50th Scientific Sessions of the American Heart Association, Miami Beach, Florida, December 1, 1977. Address for reprints: George Cooper IV, M.D., Department of Internal Medicine, University of Iowa Hospitals and Clinics, Iowa City, IA 52242. Received 16 January 1978; accepted in final form 14 September 1978.

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Myocardial energetics during isometric twitch contractions of cat papillary muscle.

Myocardial energetics during isometric contractions of cat papillary muscle twitch GEORGE COOPERIV Department of Environmental Biosciences, Naval Me...
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