INTRODUCTION

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THE RELATIONSHIP BETWEEN developed pressure, Ca²⁺ homeostasis, and energy release during the extrasystole (ES) is an intriguing aspect in heart muscle physiology. Direct evidence of an increased Ca²⁺ mobilization during an ES was reported by Wier and Yue (29), who found under these circumstances an increase in the aequorin signal. Whether the additional Ca²⁺ is accumulated in the sarcoplasmic reticulum (SR) or it is removed to the extracellular matrix is an aspect of energetic relevance. This is because the energy expended for Ca²⁺ removal depends on the mechanisms involved. For instance, whereas the SR Ca²⁺ pump has a stoichiometry of two Ca²⁺ extruded per ATP hydrolyzed (27), the sarcolemmal Ca²⁺ pump has a stoichiometry of 1 Ca²⁺:1 ATP (5). If Ca²⁺ were to be removed by the Na⁺/Ca²⁺ exchanger, the cell would take up 3 Na⁺ per 1 Ca²⁺ removed. Because the Na⁺-K⁺ pump maintains a steady intracellular Na⁺ concentration by consuming 1 ATP per 3 Na⁺ transported, Ca²⁺ removal through the Na/Ca²⁺ exchanger would use 1 ATP per 1 Ca²⁺.

As it has been reported, an unambiguous measurement of various components of heart muscle energy utilization has been a desirable but difficult task to achieve (2, 9, 11, 25). The various approaches used have frequently required the independent, nonsimultaneous measurement of 1) basal metabolism, 2) tension-dependent energy released, and 3) tension-independent energy released (1, 2, 11, 12). From the difference between the total energy released and that elicited without force, the force-dependent energy release could be calculated (3, 9, 11, 13, 22). More recently, four components of heat released (H1, H2, H3, and H4) were simultaneously measured in a single contraction (21), and these energy fractions were related with processes known to accompany different periods of the twitch myogram. The most relevant aspect of these measurements is that the pressure-independent heat (we measured isovolumic pressure generation instead of force) was evaluated simultaneously with the pressure-dependent heat component (H3) in the presence of pressure development (P) (21). Pressure-independent heat was further divided into two fractions (H1 and H2) of short evolution (similar to the fraction classically identified as the activation heat) and another one (H4) of long duration (21). Whereas H1 was tentatively associated (at least in part) with Ca²⁺ binding to troponin C (TnC), H2 was proposed to be mainly related to Ca²⁺ cycling processes (21). The H4 component showed a high dependence on mitochondrial respiration and Ca²⁺, and it is associated with a verapamil-sensitive process different from that related to force generation (6, 21). In the present study this approach was used to further investigate the energetic and mechanical consequences of an ES. The ES was induced 200 ms after the beginning of the regular beat, and the energetic and mechanical consequences of the postextrasystolic contraction (PES) were also studied. Additionally, the calcium sources associated with energy release and P for ES and PES were investigated. The results suggest an increased contribution of the SR for PES as compared with the normal contraction while the source of Ca²⁺ for ES would be entirely extracellular.

	METHODS

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Biological preparation. Experiments were performed on 10 Wistar rats of either sex, weighing 250-300 g at the time of death. Each animal was heparinized (2,000 U) and anesthetized with a pentobarbital sodium overdose. The beating hearts were rapidly excised, and retrograde perfusion was initiated with a low-Ca²⁺ (0.5 mM) and 7 mM K⁺ perfusate at room temperature (20-24°C). The right atrium was carefully dissected from the heart, and a small cut in the septal wall, close to the aorta, was made to prevent spontaneous contractions. A latex balloon was placed into the left ventricle and the muscle mounted in the inner chamber of a calorimeter. The latex balloon was connected to a Statham P23-Db pressure transducer so that pressure developed during isovolumic contractions could be measured. Optimal P was functionally established under stimulation (at ~0.1 Hz) by gradually inflating the latex balloon (resting pressure was increased in steps of ~10-15 mmHg) until stable twitch P showed no detectable increase at regular gain (188-375 mmHg full scale). All the results are quoted per gram of wet weight.

Solutions. The heart was perfused at a constant rate (5 ml/min) with a solution (control solution) containing (in mM) 1.3 MgCl₂, 110 NaCl, 1.2 NaH₂PO₄, 0.5 CaCl₂, 25 NaHCO₃, 7 KCl, and 6.0 dextrose. The solutions were bubbled with 95% O₂-5% CO₂ to achieve a pH of 7.3-7.4 (25°C). Under these experimental conditions, mechanical and energetic parameters remain reproducible for more than 6 h. In those experiments in which Ca²⁺ concentration in the perfusate ([Ca²⁺]_o) was changed, no corrections for changes in osmolarity or in ionic strength were performed. The caffeine experiments were performed at 0.5 mM Ca²⁺ because at this Ca²⁺ concentration the muscles paced at 0.16 Hz remained without spontaneous contractions. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Mechanical and heat measurements. The technique for online measurement of heat production and mechanical activity of isolated heart muscle has been described previously in detail (8, 21, 24, 25). Briefly, the calorimeter was submerged in a constant-temperature bath. The temperature of the calorimeter bath (25°C) was controlled with a cooling-heating bath (±0.003°C) in which the perfusate was also equilibrated. Calorimeter calibration was accomplished by passing a 2.1-kHz sine wave through the muscle by means of the stimulating electrodes (25). The minimum output of the thermosensitive units (two thermoelectric modules each containing 127 thermosensitive junctions) recorded in the present experiments was higher than 40 µV, whereas the electrical noise was between 1 and 2 µV at a maximum gain (1 µV/mm). With this method it was possible to record continuously and simultaneously rest pressure, P, pressure time integral (PTI), perfusion pressure, total heat production (H_t), and resting heat production (H_r). Once the muscle was placed in the inner chamber of the calorimeter, a 60-min equilibration period with control solution was allowed to elapse before any experimental intervention. A muscle was accepted for study only if during the 60-min equilibration period a minimum of 105 mmHg steady pressure was developed at 0.16-Hz stimulus frequency at a resting pressure of 26 mmHg and remained quiescent in the absence of stimulation. Reproducible P values throughout the experiment were used as an indication of muscle stability, and this was checked several times (under control perfusate) during the experiment. The analysis of the energy components released in a contraction was performed as described elsewhere (21). Briefly, when power applied is interrupted before the integration time, the calorimetric output from zero time to its peak value can be fitted by the following equation (21)

(1)

where µ is the cooling rate constant of the calorimeter,  is the diffusion delay constant, A_i = 1/{(2 · i + 1)² · [1(2 · i + 1)² · µ · ¹]}, _i = (2 · i + 1)² · µ,  is the rate constant of the declining fraction, t is time, A_o = (µ · 4 · ² · ¹)^0.5 · tan[(µ · 4 · ² · ¹)^0.5], and _o represents a fitted value of the power. The magnitudes of  and µ were determined as described previously (21). The area under the whole calorimetric output curve represents the total energy released during the event. Therefore, from the fitted parameters, the total amount of energy released can be calculated, but no information on the time course of that release was presented. The various fractions of energy released during a contraction were fitted as a linear combination of components each described by Eq. 1 as shown elsewhere (21). Note that due to the delay associated to the calorimetric response, the time to peak of each component should not be considered as the time to peak of the process associated with it (for detailed explanation, see Ref. 21). Therefore, from this analysis, the amount of energy released by each component can be calculated, but no information is obtained on the time course of that release. From the differences between H_r and H_t, divided by the frequency of stimulation, the active heat per beat was also calculated (21, 25).

The baseline used to measured the heat rate records of control and postextrasystolic beats was obtained from the calorimetric signal of the subsequent stimulus interval in which the stimulation was interrupted. In particular, the baselines for control beats (C) were obtained interrupting the stimulation during normal 0.16-Hz beating, and the baselines for PES were obtained interrupting the stimulation immediately after the application of an ES. Examples of baselines obtained with this methodology are illustrated in Fig. 5 (note dashed lines in Fig. 5, A and C).

The heat record associated with ES (Fig. 2B) was obtained from the difference between the heat record of the beat with ES (Fig. 1B) minus the heat record of the preceding C (Fig. 1A) (for further details see Ref. 21).

Statistical analysis. Data are presented as means ± SE, and statistical significance was set at P < 0.05. For paired comparisons, the paired t-test was used. Regression analysis was performed with the use of a nonlinear regression technique that uses the Marquardt algorithm running on an AT-386 compatible desk computer (21). The differences between the estimated and the hypothetical value (e.g., estimated slope against 1, correlation coefficients, and zero abscissa values against zero) as well as between two different correlation coefficients were analyzed as described elsewhere (7). Testing for symmetry and systematic deviations of the fitted curve from the data points was performed with the sign test (7). The comparison between fitted curves obtained with different number of terms for a given set of data points was performed with the Fisher test (7).

	RESULTS

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Constant [Ca²⁺]_o. We have recently shown that during steady stimulation of a heart muscle, an additional stimulus (200 ms after the regular one) releases an additional fraction of heat that can be decomposed into two components (21). To further investigate this phenomenon, muscles perfused with 0.5 mM Ca²⁺ control solution were paced at 0.16 Hz until steady mechanical and energetic parameters were observed. Under this condition, the mechanical and energetic effects of an extrasystolic stimulus (200 ms apart) on both the contraction that includes the extrasystolic stimulus and the PES were investigated. Figure 1 shows the simultaneous myothermal and mechanical records from a C, a beat with ES, and a PES contraction. ES stimulus systematically induced an increase in the PTI [65.3 ± 5.3 vs. 86.3 ± 7.5 mmHg · s for C (n = 10) and contractions with ES (n = 10), respectively, P < 0.0005], with no changes in peak pressure (P) (197 ± 14 vs. 200 ± 14 mmHg for C and contractions with ES, respectively). On the other hand, for PES (n = 10) both P and PTI were potentiated compared with the C (+47 ± 11 mmHg and +15.8 ± 2.9 mmHg · s, P < 0.05). The heat released by each control contraction was fitted to a linear combination of three components of energy release, two of them independent of the development of pressure (H1 and H2) and a third one dependent on pressure generation (H3) (Fig. 2A). The average energy values measured for each component under control conditions were 1.9 ± 0.2, 4.3 ± 0.7, and 5.3 ± 0.7 mJ/g (n = 10) for H1, H2, and H3, respectively. Under this condition, a fourth component (also independent of pressure generation) of energy released (H4) was measured as the difference between active heat per beat and the sum of H1, H2, and H3 components, yielding an average value of 2.07 ± 0.67 mJ/g. When a second stimulus was applied 200 ms after the regular one (ES), additional heat was released above the control heat record (Fig. 2B). This additional fraction of heat could be fitted to only two components (H'₂ and H'₃) with similar time courses to those observed for H2 and H3 during the control twitch (Fig. 2B). The average values for the energy released by these two components of additional heat were 0.7 ± 0.1 and 1.7 ± 0.3 mJ/g for H'₂ and H'₃, respectively (n = 10). It is of interest to note that, whereas H'₃ linearly correlated with the increase in PTI associated with the extrasystole (PTI_ES  PTI_C) (r = 0.7483, for n = 10 at 0.5 mM Ca²⁺), the correlation of H'₂ with the same parameter was not significant (r = 0.05196, n = 10). These results also suggest a strong similarity in the origin of H'₃ and H'₂ in the extrasystolic contraction with H3 and H2 in a regular contraction. PES contractions could also be fitted to a minimum of three components, H1, H2, and H3 (Fig. 2C), and their average energy values (2.6 ± 0.3, 5.0 ± 0.7, and 7.7 ± 1.3 mJ/g for H1, H2, and H3, respectively; n = 10) were higher than the values for control contractions (+0.7 ± 0.2 mJ/g, +0.7 ± 0.2 mJ/g, and +2.48 ± 0.7 mJ/g for H1, H2, and H3, respectively, P < 0.05).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Typical record of rate of heat production (upper traces) and pressure development (lower traces), from a control beat (A), a beat with an extrasystole (ES; B), and a postextrasystolic beat (PES; C). Time scale is shown as a horizontal bar representing 1 s. Vertical bar in upper trace of A represents 40 µJ/s and vertical bar in lower trace of A represents 188 mmHg.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Digitized data of rate of heat production (µJ · s1 · g wet wt1) released from control beat (A), additional heat fraction (above control) associated with ES (B), and PES contraction (C). Experimental data points shown were obtained after subtraction of baseline as indicated in METHODS and are represented as open circles. Data points in B were calculated as the difference between heat released under control conditions and overall heat released during contraction with ES. Continuous lines represent the fitting result of the digitized data as described in METHODS using Eq. 1, in which  and µ parameters (i.e., conduction delay and heat loss) were considered. Note that whereas control and PES required 3 components of heat release (H1, H2, and H3) to be fitted, the additional heat fraction of ES required only 2 (H'2 and H'3).

Variable [Ca²⁺]_o. To further investigate the origin of the calcium source associated with the energy released by ES, the dependence of mechanical and energetic parameters on [Ca²⁺]_o was studied. In each muscle, mechanical and energetic records from C, ES, and PES were obtained at four calcium concentrations (0.3, 0.5, 1, and 2 mM). The experiments were performed so that the initial Ca²⁺ concentration was always 0.5 mM Ca²⁺ but the changes in [Ca²⁺]_o were performed at random. In two experiments the increase in [Ca²⁺]_o to 2 mM induced spontaneous activity; therefore, those measurements (at 2 mM) were not considered. No measurements were considered for analysis until steady mechanical and energetic parameters for each calcium concentration were observed. The changes in additional heat components (H'₂ and H'₃) and in the PTI associated with ES (PTI_ES  PTI_C) with [Ca²⁺]_o variation are summarized in Fig. 3. The three parameters (i.e., H'₂, H'₃, and PTI_ES  PTI_C) increased with increasing [Ca²⁺]_o. Figure 3 also shows the [Ca²⁺]_o dependence of the postextrasystolic potentiation (i.e., the difference between PES and C) for H2, H3, and PTI. The energetic and mechanical dependencies on [Ca²⁺]_o associated on the potentiation of PES were different from those observed for the ES. At low [Ca²⁺]_o the potentiation of PTI and H3 (the energy component related to P) for PES was higher than the increase in PTI and H'₃ observed for the ES (Fig. 3). For PES, the potentiation in both PTI and H3 showed a peak at 0.5 mM [Ca²⁺]_o, followed by a decrease at higher Ca²⁺ concentrations (Fig. 3). In fact, at 2 mM [Ca²⁺]_o, these two parameters (H3 and PTI from PES  C; see Fig. 3) become significantly smaller than the corresponding parameters (PTI_ES  PTI_C and H'₃) associated with ES (Fig. 3). When Ca²⁺ is increased to 2 mM Ca²⁺, the force generation was close to saturation with no margin for PES potentiation. It should be noted that the decline in PTI and H3 for PES  C at [Ca²⁺]_o > 0.5 mM was due to an increase with Ca²⁺ in heat components and P of control contractions.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Mechanical and energetic dependencies on Ca2+ concentration ([Ca2+]o) for ES and PES. Filled bars, dependency of H'2 (A), H'3 (B), and pressure-time integral (PTI) (C) on Ca2+ concentration for the contraction associated with ES. Open bars, dependency of PES potentiation [(PES)  control (C)] for H2 (A), H3 (B), and PTI (C) with Ca2+ concentration in the perfusate. * Significant difference between columns (P < 0.05). a Not significant (NS) vs. 0.

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View larger version (14K): [in this window] [in a new window]   Fig. 4.   Energy released by the pressure-dependent component (H3 or H'3) as a function of PTI from control contraction (represented by regression line and the confidence interval, n = 25), ES (PTIES-PTIC, ), and PES (). Regression coefficient and P value for control contractions are included in the graph. Regression coefficients for ES and PES are 0.8144 and 0.9056, respectively. The graph includes data obtained at 0.3, 0.5, 1, and 2 mM Ca2+ concentrations.

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View larger version (15K): [in this window] [in a new window]   Fig. 5.   Typical record of control beat (A), ES (B), and PES (C) in presence of 1 mM caffeine. For each condition, heat production (upper traces) and pressure development (lower traces) are shown. Vertical bars represent heat production scale (20 µJ/s) and pressure development scale (75 mmHg). Dashed line represents the baseline of the heat record (for further explanation, see text).

	DISCUSSION

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In the present work, resting heat values averaged 4.28 ± 1.14 mJ · s¹ · g¹. These values compare well with those previously reported by our laboratory and by other investigators (3, 10, 11, 23, 24). Under steady stimulation, the total activity-related heat liberation per beat averaged 14.5 ± 1.5 mJ/g, and that also agreed with values previously reported (14, 15, 21). It is of interest to note that the observed H1 and H2 values are within the range expected for the processes involved during the activation process. With the use of an enthalpy for the binding of Ca²⁺ to TnC between 32 and 26 kJ/mol Ca²⁺ (16, 19), the H1 component found in the present work (1.9 mJ/g) would represent between 59 and 73 nmol Ca²⁺/g. If all this Ca²⁺ is to be removed by the sarcolemmal or sarcoreticular pumping systems (1 and 2 Ca²⁺ removed per 1 ATP, respectively), the heat expected (using 44 kJ/mol creatine phosphate and using an intermediate Ca²⁺-to-ATP ratio of 1.5) from these processes would be ~1.7-2.1 mJ/g. Although these values are lower than the H2 measured (4.3 mJ/g), it is reasonable to expect that the total Ca²⁺ released would be higher than that actually bound to TnC. Consequently, the heat output for Ca²⁺ removal should be higher than that expected for the removal of the Ca²⁺ bound to TnC. In addition, this difference leaves energy to be related to other ionic movements such as Na⁺ and K⁺ (21).

The energetic and mechanic parameters measured for ES and PES indicated that the economy of pressure maintenance remained similar for the three types of contractions studied. Although the energy associated to ES was smaller than that obtained for the C, the relationship between H'₃ and the associated increase in PTI was similar to that found in control contractions for H3 versus PTI. Because a similar H3-to-PTI ratio was found for PES, it can be concluded that the economy of pressure maintenance has not been affected by the extra stimulus. It is of interest to note that, during ES, if H'₂ (0.7 mJ/g) is fully attributed to SR Ca²⁺ removal, it would represent ~32 nmol of Ca²⁺ associated with the second stimulus (using now a Ca²⁺-to-ATP ratio of 2). The increase in H1 component observed for PES was ~0.7 mJ/g, which is the heat expected for the binding of 22-27 nmol Ca²⁺ to TnC. This agrees with a process in which most of the Ca²⁺ associated with the second stimulus (and captured by the SR) would have been released during the PES and used to generate force.

The results obtained in the presence of caffeine support the idea that the mechanical event related to ES depends on an extracellular Ca²⁺ source. Although the P decreased to 50% in the presence of 1 mM caffeine, the increase in PTI induced by ES was similar to that observed for control conditions. Also, caffeine failed to affect the second (Ca²⁺ dependent) component of energy release associated with ES. In this regard, an additional Ca²⁺ influx through sarcolemmal Ca²⁺ channels has been suggested to occur during ES (18). As it happens with H2 component [an energy component that has been related to pumping Ca²⁺ (21)], the H'₂ component released during ES is also dependent on [Ca²⁺]_o. The saturation of H'₂ component and PTI associated with ES at Ca²⁺ concentrations higher than 1 mM suggests that the amount of Ca²⁺ cycled during this event is saturated (Fig. 3). These results could be explained by saturation at the level of Ca²⁺ influx. This interpretation is in agreement with Wang et al. (28), who found that, in the absence of EGTA in the patch pipette (to mimic physiological conditions), the area under Ca²⁺ current saturates at ~2 mM Ca²⁺.

The absence of a postextrasystolic pressure enhancement with the caffeine pretreatment suggests that all the Ca²⁺ that entered the cell during ES was removed before the next twitch. In the presence of caffeine the capacity of the SR to retain Ca²⁺ is reduced. Consequently, the "extra" Ca²⁺ that entered during ES should have been removed to a site different from the SR and should not be available for the next twitch. It can be hypothesized that under control conditions the additional Ca²⁺ that entered the cell during ES could be accumulated by the SR and released by the next contraction. Caffeine impairs the accumulation of Ca²⁺ by the SR and consequently prevents PES pressure enhancement. On the other hand, H2 potentiation (PES  C) was similar to the H'₂ component in the entire range of [Ca²⁺]_o tested. This suggests that the whole amount of Ca²⁺ accumulated by SR as a result of ES stimulus was released to the cytosol during PES. Therefore, the saturation of H2 potentiation during PES would be an indirect consequence of Ca²⁺ influx saturation during ES. On the other hand, even though H2 potentiation either increased or remained unchanged with the increase in extracellular Ca²⁺ (suggesting a higher amount of Ca²⁺ cycling), the potentiation observed for PTI, H3, and H1 became smaller as extracellular Ca²⁺ increased. This could be due to a progressive saturation of the myofilaments under the control contraction yielding a smaller difference with the PES contraction.

It is of interest to note that the ES in the presence of caffeine induced an increase in peak P that was absent under control perfusion. This increased P was accompanied by an early component of heat similar to the H1 found in the regular contraction and supports the origin of H1 as related to binding Ca²⁺ to TnC (21). The results seem to indicate that the additional Ca²⁺ entry induced by ES in control conditions fails to increase the Ca²⁺ binding to TnC (note that there is no increase in P, but the duration of the twitch is prolonged). In contrast, in the presence of caffeine, a similar additional Ca²⁺ entry induces a clear increase in P, probably because the rate of Ca²⁺ removal from myofilaments is decreased (the maximal rate of relaxation was markedly reduced by caffeine). In consequence, the additional Ca²⁺ entry in the presence of caffeine should produce a net increase in Ca²⁺ binding to TnC. This additional Ca²⁺ binding would then release the H'₁ described under this condition.