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Am J Physiol Heart Circ Physiol 290: H613-H623, 2006. First published July 29, 2005; doi:10.1152/ajpheart.01219.2004
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Effects of caffeine, verapamil, lithium, and KB-R7943 on mechanics and energetics of rat myocardial bigeminies

E. Savio-Galimberti and J. E. Ponce-Hornos

Instituto de Investigaciones Cardiológica, School of Medicine, Universidad de Buenos Aires; Consejo Nacional de Investigaciones Científicas y Tecnicas; and Department of Biophysics, School of Dentistry, Universidad de Buenos Aires, Buenos Aires, Argentina

Submitted 3 December 2004 ; accepted in final form 26 July 2005


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We examined the effects of pharmacological alteration of Ca2+ sources on mechanical and energetic properties of paired-pulse ("bigeminic") contractions. The fraction of heat release that is related to pressure development and pressure-independent heat release were measured during isovolumic contractions in arterially perfused rat ventricles. The heat released by regular and bigeminic contractions showed two brief pressure-independent components (H1 and H2) and a pressure-dependent component (H3). We used the ratio of active heat (H'a) to pressure-time integral (PtI) and the ratio of H3 to PtI to estimate the energetic cost of muscle contraction (overall economy) and pressure maintenance (contractile economy), respectively. Neither of these ratios was affected by stimulation pattern. Caffeine (an inhibitor of sarcoplasmic reticulum function) significantly decreased mechanical responses and increased the energetic cost of contraction ({Delta} = 101 ± 12.6%). Verapamil (an L-type Ca2+ channel blocker) decreased pressure maintenance of extrasystolic ({Delta} = 43.4 ± 3.7%) and postextrasystolic ({Delta} = 37.5 ± 3.5%) contractions without affecting postextrasystolic potentiation, suggesting that a verapamil-insensitive fraction is responsible for potentiation. The verapamil-insensitive fraction was further studied in the presence of lithium (45 mM) and KB-R7943 (5 µM), inhibitors of the Na+/Ca2+ exchanger. Both agents decreased all mechanical responses, including postextrasystolic potentiation ({Delta} = 67.3 ± 3.3%), without altering overall or contractile economies, suggesting an association of the verapamil-insensitive Ca2+ fraction to the sarcolemmal Na+/Ca2+ exchanger. The effect of the inhibitors of the Na+/Ca2+ exchanger on potentiation suggests an increased participation of extracellular Ca2+ (and, thus, a redistribution of the relative participation of the Ca2+ pools) during bigeminic contractions in rat myocardium.

postextrasystolic potentiation; paired-pulse stimulation; excitation-contraction coupling; calorimetry; sodium/calcium exchanger

IN CARDIAC MYOCYTES, the coupling between depolarization of the sarcolemma (SL) and contraction is a complex phenomenon that results from the interaction of many ion-transporting systems (most of them Ca2+-dependent mechanisms) (52). The contraction itself is initiated by an increase in intracellular Ca2+ concentration ([Ca2+]i). The sequence begins when an action potential depolarizes the SL, inducing Ca2+ entry via L-type Ca2+ channels (Ca2+ current) and also (in a smaller amount) via Na+/Ca2+ exchange (NCX) (2). Ca2+ influx triggers the release of additional Ca2+ from the sarcoplasmic reticulum (SR) through ryanodine receptors by a mechanism known as "Ca2+-induced Ca2+ release," which amplifies the cytosolic Ca2+ signal (14, 15, 40, 57). In addition, Ca2+ influx can also directly activate the myofilaments. These events raise [Ca2+]i, allowing the binding of Ca2+ to different intracellular structures, such as the contractile protein troponin C (TnC), which activates contraction. The beginning of relaxation occurs when [Ca2+]i declines to resting levels (22). Removal of Ca2+ from the cytosol involves different transporting systems in the SL (NCX and the Ca2+ pump) and within the myocyte (the SR Ca2+ pump and "slow" transport into the mitochondria) (1, 29, 52). Whether Ca2+ is accumulated in intracellular stores or removed to the extracellular matrix is of energetic significance. This is important, because each removal mechanism has a particular stoichiometry between the quantity of Ca2+ molecules removed and the ATP expended for the process, which determines a particular energy balance. For instance, whereas the SR Ca2+ pump extrudes two Ca2+ per ATP hydrolyzed (54), the SL Ca2+ pump has a stoichiometry of one Ca2+ per ATP hydrolyzed (10). If the SL NCX removes Ca2+, the cell would take up three Na+ per Ca2+ removed. Because the Na+-K+-ATPase maintains the intracellular Na+ concentration by using one ATP per three Na+ transported, Ca2+ removal through the NCX would use one ATP per Ca2+ removed by this mechanism (36, 43).

Even though the sequence of events we have just described is generally accepted, the source of the contractile Ca2+ remains controversial and can vary depending on the species and the experimental conditions. For instance, it has been shown that the Ca2+ source for an extrasystole (ES) obtained by applying an extrastimulus 200 ms after a regular stimulus is extracellular (36). The Ca2+ that enters the cell during the ES is accumulated in the SR and released during the postextrasystole (PES), inducing postextrasystolic potentiation (PP) (36, 38, 39). The purpose of the present study is to further investigate Ca2+ sources that can participate during bigeminy, a condition characterized by the presence of sustained ES and PES contractions. Myothermic responses from hearts under steady stimulation yielded three components of energy released: H1 and H2 (pressure-independent) and H3 (pressure-dependent), which were simultaneously measured for each contraction. These energy fractions were previously related to processes that normally occur during the contractile event (44). The results obtained under sustained paired-pulse stimulation suggest that two fractions of extracellular Ca2+ enter during the ES: one is associated with the activity of SL Ca2+ channels and the other is associated with the NCX and is responsible for PP. As previously shown for isolated ES (36, 38), under bigeminic conditions the Ca2+ involved in PP is accumulated in the SR from the first ES and remains there, ensuring the potentiation during bigeminies.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Biological Preparation

Wistar rats (250–340 g) were heparinized (1,500 U) and anesthetized with an overdose of pentobarbital sodium. The beating hearts were excised and perfused by the Langendorff method at room temperature (20–24°C) with control solution. Atria and papillary muscles were dissected from the heart, and a small cut was made in the septal wall, close to the aorta, to prevent spontaneous contractions. A latex balloon (connected to a Statham P23Db pressure transducer) was placed inside the left ventricle, so that developed pressure (P) could be measured. The ventricles were mounted in the inner chamber of a calorimeter (48). Optimal P was functionally established under stimulation (0.16 Hz) by gradual inflation of the latex balloon until stable twitch P showed no detectable increase at regular gain. All the experiments were performed at 25°C. Under these experimental conditions, mechanical and energetic parameters remain reproducible for >6 h.

At the end of each experiment, the tissue was removed from the calorimeter, weighed in a preweighed vial, and dried at 110°C to constant weight so that the water content could be calculated. The average water content in the present experiments was 82 ± 0.3% (n = 20), which is consistent with values previously published (5, 36, 50, 51).

All animal procedures and experimental protocols were approved by the Ethics Committee for Research of the School of Dentistry of the University of Buenos Aires and in accordance with the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society and the National Institutes of Health Guide for the Care and Use of Laboratory Animals (41).

Solutions

The rat ventricles were perfused at a constant rate (4.5 ml/min) with a control solution containing (in mmol/l) 120 NaCl, 6.0 KCl, 1.0 CaCl2, 1.0 MgCl2, 25 NaHCO3, 0.5 NaH2PO4, and 6.0 dextrose (pH 7.3–7.4). For the evaluation of intracellular Ca2+ stores, the effect of caffeine (Caf), added to a final concentration of 1 mmol/l, was studied. For the evaluation of extracellular Ca2+ via the Ca2+ channel, verapamil (Ver, 0.2 µmol/l) was used. In addition, for the evaluation of extracellular Ca2+ via the NCX, the effects of lithium (LiCl, final concentration of 45 mmol/l added to a perfusion medium containing Ver at 0.2 µmol/l) and KB-R7943 (KBR, final concentration of 5 µmol/l added to a perfusion medium containing Ver at 0.2 µmol/l) were studied. The osmotic difference between lithium (LiCl at 45 mmol/l) and control solutions was compensated by addition of choline chloride (90 mmol/l) to the control. The solution containing KBR (Tocris Cookson, Ellisville, MO; 5 µmol/l) was prepared from a stock solution (12 mmol/l in DMSO) added to a Krebs solution containing Ver (0.2 µmol/l). In all cases, solutions were bubbled with 95% O2-5% CO2 (pH 7.3–7.4).

Protocol

The experimental protocol is shown in Fig. 1. The extrasystolic interval (ESI), defined as the period between two consecutive stimuli, was decreased in predetermined steps during paired-pulse stimulation. The ESI was followed by the postextrasystolic interval (PESI), which was increased so that the sum of ESI and PESI was held constant. After the muscle was placed in the inner chamber of the calorimeter and allowed to equilibrate, it was subjected to regular stimulation at a constant frequency (0.4 Hz) until a new steady state was reached. With this type of stimulation, the interval between two consecutive stimuli was held constant (Fig. 1, top). Then paired-pulse stimulation was applied (Fig. 1, bottom). In all cases, no measurements were performed until the new steady state was reached. The paired contractions obtained by paired-pulse stimulation are identified as the bigeminic couple (BC): the first contraction is PES, and the second is ES. After completion of the paired-pulse stimulation series (which included ES intervals of 0.6, 0.5, and 0.4 s and a 0.3-s interval for the Caf experiments), regular stimulation was resumed.



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  		Fig. 1. Experimental protocol. Contractions were obtained by regular stimulation, where the interval between 2 consecutive stimuli is held constant (1–2 interval = 2–3 interval) and bigeminic stimulation, where the interval between 2 consecutive stimuli is decreased (1–2 interval) while the following interval is increased (2–3 interval), so the number of stimuli per unit of time (min) is held constant. Three traces are shown for each type of contraction: heat production record (top trace), digitalized mechanical response (middle trace), and electrical stimuli represented in vertical bars (bottom trace). Vertical bar in heat records (top traces) represents 125 µW/g. Vertical bar in mechanical responses (middle traces) represents 10.4 mN/mm2. Horizontal bar in mechanical records (middle traces) represents time scale (1 s). ES, extrasystole; PES, postextrasystole; ESI, extrasystolic interval; PESI, postextrasystolic interval.

 
Mechanical and Heat Measurements

The technique for online measurement of heat production and mechanical activity of isolated heart muscle has been described in detail elsewhere (44, 48, 50). 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 different perfusate solutions were also equilibrated. By this method, it was possible to record continuously and simultaneously resting pressure, P, pressure-time integral (PtI), perfusion pressure, total heat production (Ht), and resting heat production (Hr). PtI was measured for regular, bigeminic, ES, and PES contractions (PtIRC, PtIBC, PtIES, and PtIPES, respectively). Heat and mechanical parameters were recorded in a Grass S7 eight-channel recorder and simultaneously digitized in a desk-top computer. The difference between PtIPES and PtIRC is PP. The muscle was placed inside the inner chamber of the calorimeter and allowed to equilibrate with control solution for 60 min before any experimental intervention. Reproducible P values throughout the experiment were used as an indication of muscle stability, which was checked several times (under control perfusion) during the experiment. The energy components released in a contraction were analyzed as described elsewhere (44). When the power is interrupted before the integration time, the calorimetric output from time 0 to its peak value can be fitted by the following equation

(1)
where A0 = (µ4{pi}–2{beta}–1)0.5tan[(µ4{pi}–2{beta}–1)0.5], µ is the cooling rate constant of the calorimeter, {beta} is the diffusion delay constant, Ai = 1/{(2i + 1)2[1 – (2i + 1)2µ{beta}–1]}, {alpha}i = (2i + 1)2µ, {tau} is the rate constant of the declining fraction, t is time, and H0 represents a fitted value of the power; {beta} and µ were determined as described previously (44). The area under the whole calorimeter output curve represents the total energy released during the event. 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 (44). Because of the delay associated with the calorimeter response, the time to peak of each component should not be considered the time to peak of the process associated with it (44). 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. The active heat per beat (H'a) was calculated as the difference between Ht and Hr, divided by the frequency of stimulation (44, 48). Inasmuch as bigeminic contractions include two consecutive contractions, the frequency of stimulation considered in the calculation of H'a was 0.2 Hz (i.e., half of the frequency applied). Then, H'a calculated for each paired-pulse stimulation is (H'a)BC and H'a calculated under regular stimulation is (H'a)RC. For each stimulation pattern, the overall economy was evaluated by the H'a-to-PtI ratio. The baseline used to measure the heat records of regular and PES contractions was obtained from the calorimeter signal on when the stimulation was interrupted (for ~15 s). The baseline for regular contractions was obtained by interrupting the stimulation during normal 0.4-Hz contractions, and the baseline for PES was obtained by interrupting the stimulation immediately after application of an ES. An example of a baseline obtained with this methodology is illustrated in Fig. 2A. The heat record associated with an ES was obtained from the difference between the heat record of the BC (which includes a PES with an ES) and the heat record of a PES without an ES (Fig. 2B). The ratio of the H3 component (heat fraction mainly related to actomyosin interaction) to the corresponding PtI was used to estimate the energetic cost per unit of pressure maintenance (contractile economy). All heat values are expressed per gram of wet weight.



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  		Fig. 2. Baselines used to measure heat released during ES and PES contractions and corresponding typical records. Baseline and contraction heat records are represented by continuous lines. A: baseline for PES was obtained by interrupting the stimulation immediately after application of an ES. B: heat record associated with an ES was obtained from the difference between the heat record of the bigeminic couple (BC) and the heat record of a PES contraction alone. Vertical bars, 240 µW/g. Horizontal bars, time scale (5 s).

 
Statistical analysis. Values are means ± SE, and statistical significance was settled at P < 0.05. For paired comparisons, the paired "t"-test was used. When more than two groups were compared, ANOVA was used. When the ANOVA was significant, Tukey's test was performed. The regression analysis of the thermograms was performed with a nonlinear regression technique that uses the Marquardt algorithm based on minimum square criteria (44) running on a desktop computer. Because in some of the parameters analyzed in this work there was no significant difference between the changes at 0.4-, 0.5-, and 0.6-s ESI, averaged values were obtained for pooled data.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of Paired-Pulse Stimulation on Myocardial Mechanics and Energetics

After 60 min of equilibration with control perfusate, the ventricles were kept at rest for ~20 min, yielding an average Hr of 4.4 ± 0.2 mW/g (n = 6). After this resting period, we obtained mechanical and myothermic records of regular contractions. In six experiments with regular contractions, P averaged 31.6 ± 3.2 mN/mm2, with maximum rates of contraction (+dP/dt) and relaxation (–dP/dt) of 250 ± 29 and 42.5 ± 4 mN·mm–2·s–1, respectively (Table 1). For regular contractions, the maintenance of P (PtIRC) averaged 10.3 ± 0.8 mN·mm–2·s–1 and total time of contraction was 0.7 ± 0.02 s. The average active heat per beat (H'aRC) was 9.6 ± 1.6 mJ/g. The heat signal associated with a regular contraction was best fitted to the three heat fractions, H1, H2, and H3 (Table 2).


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  		Table 1. Mechanical parameters related to regular, postextrasystolic, and extrasystolic contractions under control and experimental conditions

 

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  		Table 2. Heat components related to regular, postextrasystolic, and extrasystolic contractions under control and experimental conditions

 
The contractions resulting from paired-pulse stimulation were fused at all ESIs, and two peaks in the P record were always present. The transition from regular to bigeminic contractions showed a progressive increment of the first maximum in P (corresponding to PES) and an initial fall followed by an increment with a second maximum in P (corresponding to ES). In both cases, P reached its maximum level after about the sixth pair of stimuli. Under the new steady condition, PtIPES was significantly higher than PtIRC at all ESIs, suggesting an increase in Ca2+ availability to the myofilaments. In addition, PtIES was significantly lower than PtIRC, suggesting a decrease in Ca2+ availability to the contractile proteins. No significant differences were found between PtIES and PP. Because no differences were found between ESIs, the averaged values of pooled data are shown in Fig. 3 and Table 1.



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  		Fig. 3. Effect of paired-pulse stimulation on pressure-time integral (PtI). PP, postextrasystolic potentiation; RC, regular contraction. Values are means ± SE. PtI was significantly higher for PES than for ES and RC. PtI was significantly lower for ES than for RC, but no significant differences were found between ES and PP. Because ANOVA showed no significant difference between groups, for each parameter, total data from ESIs were pooled to obtain average values. *P < 0.05. NS, nonsignificant difference.

 
H'aBC for each ESI was compared with (H'a)RC (19.1 ± 3.1 mJ/g, n = 6). The ratio of H'a to the corresponding PtI was used to estimate the energetic cost of muscle contraction (and, thus, as an indirect indicator of overall economy). The H'a-to-PtI ratio was not significantly affected by the stimulation pattern (0.9 ± 0.1 and 0.9 ± 0.04 mJ·g–1·mN–1·mm2·s–1 for regular and bigeminic contractions, respectively; Fig. 4), which indicates that the overall economy remained unaffected.



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  		Fig. 4. Overall economy [evaluated as active heat per beat (H'a)-to-PtI ratio] for RC and BC at 0.4, 0.5, and 0.6-s ESIs. Because PtI for BC increased proportionally to the increment in H'a for BC, the H'a-to-PtI ratio for BC was not significantly affected compared with the value for RC (determined by ANOVA). Values are means ± SE.

 
As described for regular contractions, the thermograms associated with each of the bigeminic contractions were best fitted to the three components of heat released: pressure-independent H1 and H2 and pressure-dependent H3 (Fig. 5, top and middle). The absolute values of the components are shown in Table 2. In all cases, the energy components related to PES contractions were significantly higher than those related to ES contractions. The paired differences for each component are shown in Fig. 5, bottom. In all cases, the H3-to-PtI ratio remained unaltered, which indicates that the contractile economy is not affected by the stimulation pattern. Because there was no significant difference between the groups, data were pooled and the average value for 42 data points was 0.62 ± 0.02 mJ·g–1·mN–1·mm2·s–1.



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  		Fig. 5. Digitized data for rate of heat produced from a PES beat (top) and an ES contraction (middle). Experimental data points shown for PES contraction were obtained after subtraction of baseline ({circ}). Data points for ES were calculated as the difference between the heat released during a bigeminic couple and the heat released during a PES contraction. Continuous lines represent the fitting result of the digitized data (see Eq. 1, in which {beta} and µ, i.e., conduction delay and heat loss, were considered). In both cases, the myothermograms required fitting 3 components of heat release (H1, H2, and H3). Bottom: paired differences between PES and ES at each ESI. *P < 0.05.

 
Intracellular Ca2+ Stores: Effects of Caf

To further study the role of the SR in bigeminic contractions, the previously applied protocol (with an additional ESI of 0.3 s) was reproduced in the presence and absence of Caf (1 mmol/l, a condition in which the SR is expected to contribute with minimal Ca2+ for contraction). Caf was present for 30 min before any measurement was considered. In the presence of Caf, PtIES were maximally affected at the longest ESIs (0.5 and 0.6 s) but was not significantly affected at 0.3 s (Fig. 6). This suggests that SR participation during ES depends on the duration of the ESI: it decreases as the interval shortens and becomes negligible at 0.3 s. PtIPES and PP were significantly decreased by Caf at all ESIs, suggesting a major role for the SR during PP. With the exception of ES obtained at 0.3-s ESI (which was not affected by Caf), the mechanical parameters measured on ES and PES contractions (P, +dP/dt, and –dP/dt) were also significantly decreased by Caf (Table 1).



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  		Fig. 6. Effect of caffeine (Caf, 1 mmol/l) on PtI for ES, PES, and PP, shown as paired differences. Values are means ± SE. PtI for ES was maximally affected at 0.6 s but was not significantly affected at 0.3 s. PtI at PES and PP were significantly decreased by Caf at all ESIs. *P < 0.05.

 
The heat released by each contraction (H'a) decreased in the presence of Caf because of the fall of all three components (Table 2). The H'a-to-PtI ratio for bigeminic contractions was significantly increased by Caf at all ESIs, but we found no significant differences between the different groups. The average increment in the H'a-to-PtI ratio in the presence of Caf was 0.7 ± 0.1 mJ·g–1·mN–1·mm2·s–1 (n = 16). This increment in the H'a-to-PtI ratio (which indicates an increased energetic cost of muscle contraction and, therefore, a deterioration of overall economy) was similar to the increment calculated for regular contractions: {Delta}(H'a/PtI)RC = 0.8 ± 0.2 mJ·g–1·mN–1·mm2·s–1 (Table 3).


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  		Table 3. Overall economy related to regular and bigeminic couples under control and experimental conditions

 
The thermograms associated with each type of contraction were best fitted to the three components of energy released (H1, H2, and H3). In all cases, the three components were significantly decreased by the presence of Caf in the perfusate (Table 2). The ratio of (H1 + H2) to PtI increased with Caf, suggesting a relative increase in H2, the heat component related to Ca2+ cycling (44). The H3-to-PtI ratio for each type of contraction analyzed in the presence of Caf increased (Table 4). This result is consistent with the present work concerning overall economy and with previous findings of an increase in total active heat per unit of force development (6, 7, 36).


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  		Table 4. Contractile economy related to regular, postextrasystolic, and extrasystolic contractions under control and experimental conditions

 
Extracellular Ca2+ Source: Ca2+ Channels and NCX

Effects of Ver. Intracellular Ca2+ enters the cell through at least two mechanisms: Ca2+ channels and/or NCX. The role of the first mechanism during bigeminic contractions was studied in the presence of Ver (0.2 µmol/l), a Ca2+ channel blocking agent that affects the L-type Ca2+ current (37). After 60 min of perfusion in the presence of Ver, mechanical and energetic parameters were registered. Under resting conditions, the average Hr in the presence of Ver was 4.6 ± 1.2 mW/g (n = 6). In six experiments, all mechanical parameters related to a regular contraction were significantly decreased by Ver: P from 31.6 ± 3.2 to 15.3 ± 1.8 mN/mm, PtI from 10.2 ± 1.4 to 4.4 ± 0.6 mN·mm–2·s, +dP/dt from 250 ± 28.9 to 123 ± 14.3 mN·mm–2·s–1, and –dP/dt from 42.5 ± 3.9 to 26.7 ± 3.1 mN·mm–2·s–1 (in all cases, P < 0.05; Table 1). The average H'a calculated for two regular contractions was also significantly decreased from 19.1 ± 3.1 to 11.8 ± 2.5 mJ/g. Because the decrease in H'a was not proportional to the decrease in PtIRC, the corresponding H'a-to-PtI ratio increased in the presence of Ver (Table 3). The increased overall energetic cost of contraction suggests that the overall economy has deteriorated, which agrees with previous results (47).

The heat-released components associated with a regular contraction were also affected by Ver (Table 2). Because the decrease in the heat released related to actomyosin interaction (H3) was proportional to the decrease in PtI, the H3-to-PtI ratio was not significantly affected by Ver (Table 4) indicating that contractile economy remained unaffected.

Both bigeminic contractions were affected by the presence of Ver in the perfusate at all ESIs. P, PtI, +dP/dt, and –dP/dt were significantly decreased in the presence of the blocking agent. In the case of ES, maximum P decreased from 28.7 ± 2.2 to 14.3 ± 1.6 mN/mm2 and PtI decreased from 8.2 ± 0.6 to 4.6 ± 0.4 mN·mm–2·s. The decrease in P was accompanied by a decrease in +dP/dt (from 214 ± 16.7 to 120 ± 9.9 mN·mm–2·s–1 without and with Ver, respectively) and –dP/dt (from 42.3 ± 3.3 to 26.4 ± 2.3 mN·mm–2·s–1 without and with Ver, respectively). In the case of PES, maximum P decreased from 46 ± 1.8 to 29.6 ± 1.6 mN/mm2 and PtI decreased from 18.1 ± 0.9 to 11.4 ± 0.9 mN·mm–2·s. +dP/dt decreased from 397 ± 22 to 244 ± 18 mN·mm–2·s–1, and –dP/dt decreased from 52 ± 1.9 to 38 ± 2.4 mN·mm–2·s–1 (Table 1).

Although Ver decreased PtIES and PtIPES, PP was not significantly affected at any ESI (Fig. 7, top). This suggests that the Ca2+ involved in PP enters the cell during the ES contraction through a mechanism that is not affected by Ver (probably the NCX). H'aBC was also significantly decreased by Ver at all ESIs: {Delta}H'a = 7.3 ± 2.8, 7.5 ± 2.4, and 7.1 ± 2 mJ/g at 0.4, 0.5, and 0.6 s, respectively (in each case, P < 0.05, n = 6). As previously described for regular contractions, the H'a-to-PtI ratio for bigeminic contractions increased in the presence of Ver (Table 3), indicating that the overall economy deteriorated as the overall energetic cost of contraction increased. This result agrees with previous results (47).



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  		Fig. 7. Effects of verapamil (Ver, 0.2 µmol/l; top), LiCl (45 mmol/l; middle), and KB-R7943 (KBR, 5 µmol/l; bottom) on PtI for ES, PES, and PP, shown as paired differences. Values are means ± SE. PtI for ES and PES were significantly affected by Ver, LiCl and KBR. PP was not significantly affected by Ver but was significantly decreased by LiCl and KBR. *P < 0.05.

 
As in the control condition, in the presence of Ver the thermograms associated with each of the contractions were best fitted to the three components of energy released (H1, H2, and H3). In all cases, the three components were significantly decreased by Ver (Table 2). Because the fall in the pressure-dependent heat fraction H3 induced by Ver was proportional to the fall in the corresponding PtI, the H3-to-PtI ratio was not significantly affected by Ver. This result indicates that the contractile economy is not affected by Ver. Because no significant differences were found between the data groups for the ESIs, total data were pooled (Table 4).

Effects of LiCl. To further investigate the Ver-insensitive Ca2+ fraction, the effects of LiCl on mechanical and myothermic responses of regular and bigeminic contractions were studied. Lithium was used because it reduces Ca2+ uptake under intracellular Na+-overload conditions (46), and the bigeminic couple is an example of such a condition (16, 20, 26). In addition, lithium also reduces contractile Ca2+ availability under physiological conditions (5). Lithium was added to the perfusion medium in the presence of Ver (0.2/µmol/l) to a final concentration of 45 mmol/l. Four experiments were performed in which the same stimulation protocol was applied. Under resting conditions, the average Hr did not significantly differ from the Hr in the absence of LiCl: {Delta} = 0.01 ± 0.16 mW/g (n = 4). In four experiments, all mechanical parameters of a regular contraction were significantly decreased by lithium (Table 1). For a regular contraction, although the ratio of +dP/dt to P was not affected, the ratio of –dP/dt to P was significantly increased ({Delta} = 0.93 ± 0.1 s–1). The average H'a calculated for two regular contractions was also decreased by lithium, and the H'a-to-PtI ratio remained unchanged ({Delta} = 0.05 ± 0.05 mJ·g–1·mN–1·mm2·s–1), which indicates that overall economy was not affected (Table 3).

The heat released by a regular contraction was decomposed into three fractions, which were decreased by LiCl compared with the previous condition (Table 2), but the H3-to-PtI ratio (and, thus, the contractile economy) remained unchanged for a regular contraction: {Delta} = 0.003 ± 0.03 mJ·g–1·mN–1·mm2·s–1 (Table 4).

Bigeminic contractions were also affected by lithium. P and PtI were significantly decreased at all ESIs (Table 1), suggesting a diminished contractile Ca2+ availability. PP was also significantly decreased by lithium at the different ESIs, and the average decrease for the pooled data was 3.5 ± 0.5 mN·mm–2·s (Fig. 7, middle). The simultaneous decrease in PtIES and PP in the presence of lithium suggested that part of the ES contractile Ca2+ enters the cell through a lithium-sensitive mechanism, which would be responsible for at least part of PP.

+dP/dt and –dP/dt were also significantly decreased by lithium (Table 1). Because the fall in +dP/dt was proportional to the decrease in P, the ratio of +dP/dt to P was not significantly affected. In contrast, inasmuch as the effect of lithium was proportionally less on –dP/dt than on P, the ratio of –dP/dt to P increased: {Delta} = 2.3 ± 0.3 and 0.8 ± 0.05 s–1 for ES and PES, respectively (in both cases, P < 0.05, n = 12, pooled data). In both cases, the ratio of –dP/dt to +dP/dt significantly increased in the presence of lithium: {Delta} = 0.17 ± 0.03 and 0.11 ± 0.01 for ES and PES, respectively (average values for pooled data). The increases in the ratios of –dP/dt to P and –dP/dt to +dP/dt suggest that mechanical relaxation would be facilitated in the presence of lithium.

Lithium significantly decreased H'aBC at all ESIs: {Delta} = 4.4 ± 1.1, 4.4 ± 1.2, and 4.3 ± 1.1 mJ/g at 0.4, 0.5, and 0.6 s, respectively (in all cases, P < 0.05, n = 4). Inasmuch as the decrease in H'a for bigeminic contractions was associated with a proportionally similar decrease in PtI, the H'a-to-PtI ratio for bigeminic contractions was not significantly affected (Table 3), which indicates that the overall economy was not modified. On the other hand, the average H'a-to-PtI ratio was similar for bigeminic and regular contractions.

For regular contractions, the heat released by both bigeminic contractions was decomposed into three fractions of heat released (H1, H2, and H3), which decreased in the presence of lithium (Table 2). Because the fall in the H3 fraction induced by lithium was similar to the fall in the corresponding PtI, the H3-to-PtI ratio remained unchanged, which indicates that the contractile economy remained unaffected (Table 4).

Effects of KBR. To further investigate the contribution of NCX to bigeminic contractions, the effects of a specific inhibitor for NCX at a concentration that affects the Ca2+ entry mode (49) were analyzed. In six experiments, KBR was added to the perfusion medium, in the presence of Ver (0.2 µmol/l), to a final concentration of 5 µmol/l. Under resting conditions, the average Hr was not significantly affected by the presence of KBR in the perfusion medium: {Delta} = 0.14 ± 0.4 mW/g (n = 6). KBR induced a slight but not significant decrease in all mechanical parameters of a regular contraction (Table 1). Neither the H'a-to-PtI nor the H3-to-PtI ratio was significantly affected by KBR (Tables 3 and 4). These results indicate that neither the overall nor the contractile economy was affected by KBR.

In the case of bigeminies, both contractions were affected by KBR. P and PtI were significantly decreased at all ESIs (Table 1). KBR also significantly decreased PP (Fig. 7, bottom). The average decrease in this parameter for 18 data points was 2.1 ± 0.2 mN·mm–2·s. +dP/dt and –dP/dt were also significantly decreased by KBR (Table 1). Inasmuch as +dP/dt and –dP/dt decreased in proportion to the fall in P, the ratios of +dP/dt to P and –dP/dt to P for ES and PES remained unaffected. These results are consistent with results reported by Kurogouchi et al. (27).

At all ESIs, H'aBC was significantly decreased by KBR: {Delta} = 2.8 ± 0.8, 3.1 ± 0.8, and 3.5 ± 0.9 mJ/g at 0.4, 0.5, and 0.6 s, respectively. Inasmuch as the decrease in H'a was associated with a proportional decrease in PtI for bigeminic contractions, the H'a-to-PtI ratio for bigeminic contraction remained unaffected (Table 3), which indicates that the overall economy was not significantly modified. The average H'a-to-PtI ratio was similar for bigeminic and regular contractions: 0.92 ± 0.17 and 0.91 ± 0.16 mJ/g.

All three components of the thermograms were affected by the presence of KBR in the perfusate (Table 2). Because the fall in the H3 fraction induced by KBR was proportional to the fall in the corresponding PtI, the H3-to-PtI ratio remained unchanged (Table 4), indicating that the contractile economy was not affected.


   DISCUSSION
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Under resting conditions, the Hr in the presence of control perfusate was 4.3 ± 0.2 mW/g, which compares well with values previously reported (4.1 ± 0.2 and 4.5 ± 0.5 mW/g) (17–19, 34, 35, 44, 50). With regular stimulation (0.4 Hz), P was 31.6 ± 3.2 mN/mm2 and H'a was 9.6 ± 1.5 mJ/g. At a stimulation frequency of 0.2 Hz, H'a was 19.1 ± 3.1 mJ/g, which also compares well with previously published values (44).

The transition from regular to bigeminic (paired-pulse) stimulation showed a different pattern of P for each contraction of the pair that constitutes the bigeminic contraction. The contractions of the pair were always fused at all ESIs, so the ES always began before the relaxation of the previous contraction was completed. Consequently, ES begins under a muscle condition in which Ca2+-removing processes predominate, and myofilaments are partially occupied with Ca2+ that has participated in the previous contraction (which was initiated in a muscle in a full resting condition). This could explain the different pattern for the transition from regular to bigeminic (paired-pulse) stimulation observed in both contractions of the pair, reaching its maximum and steady level after about the sixth pair of stimuli. Once the new steady state was reached, paired-pulse stimulation showed a maintained potentiation of the PES beat and an ES with a reduced force generation compared with a regular contraction. These results are in agreement with those reported by Vassallo et al. (55) under similar coupling intervals. Because myofilaments can be considered sensors of cytosolic Ca2+, a different pattern of maximum P evolution (a progressive increment of the first maximum in P and an initial fall but posterior increment in the second maximum of P) suggests that a different pattern of Ca2+ is presented to the myofilaments for each contraction type. This difference in Ca2+ pattern could be attributed to a differential SR participation in both types of contractions. In the new steady state of bigeminic contractions, increased SR participation during PES could be explained by the longer period preceding this contraction, which would allow the SR to restore its Ca2+ content. An increased SR participation during PES and a decreased SR participation during ES were confirmed by Caf experiments (Fig. 6). In agreement with the findings of Morad and Goldman (39), PtIPES and PP decreased in the presence of Caf at all ESIs, suggesting a major role for the SR during PP. In addition, the Caf experiments suggested that SR participation during ES depends on ESI duration: it decreases as ESI shortens and becomes negligible at 0.3 s. During ES, a fraction of intracellular Ca2+ enters the cell, is recycled by the SR, and further increases the Ca2+ content of this organelle. All these observations support the results previously reported by Marengo et al. (36) for a single ES. Because ES interrupts the relaxation of the previous contraction, the lower PtIES could also be attributed to the already activated Ca2+ removal mechanisms at the time the ES stimulus reaches the muscle.

The altered overall economy of contractions with Caf could be explained in part by an increase in the energetic cost of Ca2+ removal associated with the reduced capacity of the SR to retain Ca2+. This finding is in agreement with results reported in the literature (3, 4, 6, 7, 13). Different mechanisms have been proposed to explain the effects of Caf on heart muscle. One of these effects is the alteration of the capacity of the SR to retain Ca2+. Ca2+ removal by SL mechanisms represents a higher energetic cost in terms of ATP hydrolyzed per Ca2+ removed (1 ATP per Ca2+ removed) than removal of Ca2+ by the SR Ca2+ pump (1 ATP per 2 Ca2+ removed). Thus inhibition of the SR Ca2+ pump by Caf would induce a higher rate of ATP hydrolysis with the increase in the H'a-to-PtI ratio as a result of recruitment of SL Ca2+ removal mechanisms (6, 43). This is consistent with the relative increase in the H2 component (the heat component related to Ca2+ cycling) (44) in the presence of Caf. We have found that the (H1 + H2)-to-PtI ratio increased with Caf, which is also in agreement with results published by Ponce-Hornos et al. (44). Another mechanism proposed to explain the effects of Caf is the increase in mitochondrial oxidative phosphorylation due to the increase in the free Ca2+ concentration (24). This increment in the oxidative phosphorylation could contribute to the increases in the H'a-to-PtI (Table 3) and H3-to-PtI (Table 4) ratios, because ~26% of the H3 component is related to oxidative metabolism (44).

The trans-SL inward movement of Ca2+ during the action potential seems to be mediated by voltage-operated channels (23, 37, 45) and by the NCX (25, 52). Depending on the Na+ and Ca2+ electrochemical gradients, the NCX can operate in a Ca2+-influx mode (8, 9, 28). The effect of Ver on ES and PES, i.e., decreasing all mechanical parameters (P, PtI, +dP/dt, and –dP/dt; Table 1), is in agreement with results found in the literature (11, 37–39). Our findings suggest that, under bigeminic contractions, a fraction of extracellular contractile Ca2+ enters the cell through a Ver-sensitive mechanism (most probably the SL Ca2+ channels). However, as shown in Fig. 7 (top), although a Ver-sensitive fraction of Ca2+ participates in both bigeminic contractions (ES and PES), the lack of effect of Ver on PP seems to indicate that this extracellular Ca2+ fraction is not related to this phenomenon. Therefore, the Ca2+ involved in PP should be entering the cell during the ES contraction through a mechanism that is not affected by Ver (probably the NCX) and stored by the SR. This notion is supported by our finding that lithium affected PtIES, PtIPES, and PP (Fig. 7, middle). The decrease in PtIES and PtIPES suggests a diminished contractile Ca2+ availability explained by an inhibitory effect of lithium on cell Ca2+ uptake through NCX (46). This inhibitory effect of lithium could lower the contractile Ca2+ offered to the myofilaments directly by a decrease in Ca2+ entry or indirectly by a reduction in the Ca2+-induced Ca2+ release mechanism. Some authors (2, 21, 25, 31–33, 42, 56) have suggested that the Ca2+ fraction entering through the NCX might participate in this triggering release mechanism. It has also been proposed that when L-type Ca2+ channels and NCX participate in the Ca2+ influx, Ca2+ entry via NCX appears to synergistically amplify the effect of triggering SR Ca2+ release via the L-type Ca2+ current (53). In fact, this lithium-sensitive Ca2+ fraction seems to be the Ca2+ fraction related to PP. The energetic effects of lithium on myocardial bigeminies are consistent with its effects on Ca2+ availability: reduced Ca2+ uptake (46) and, consequently, decreased Ca2+ availability for the myofilaments.

It is interesting to compare the effects of lithium and KBR on –dP/dt. Although –dP/dt decreased in the presence of KBR, the decrease was proportional to the decrease in P. So the ratio of –dP/dt to P remained constant. The same effect is found if, for instance, extracellular Ca2+ is decreased (30). It is striking that, in the presence of lithium [which decreases Ca2+ uptake and P (46)], –dP/dt remained unchanged or the decrease was minor compared with the decrease in P, so that the ratio changed (i.e., the ratio of –dP/dt to P increased). This indicates that, in the presence of KBR, for a given level of P, relaxation processes are maintained; in the presence of lithium, however, relaxation processes for a given level of P are enhanced. Although it is purely speculative, it can be hypothesized that whereas lithium might be inhibiting the forward and reverse mode of the NCX, on the outside of the cell it will be competing with 145 meq Na+/l and on the inside of the cell with perhaps ≤5 meq Na+/l. This agrees with the previous finding that reduction of extracellular Na+ (which should particularly influence the NCX) increases the ratio of –dT/dt to T (i.e., the speed of relaxation normalized to the maximum tension developed during a contraction) (45).

These results also suggest that lithium could be affecting a mechanism different from the SL NCX. This second mechanism could be related to the effects of lithium on isolated contractions [suppressing the oxygen-dependent H4 component (5)] and the cardioprotective effect proposed for this agent (51). The experiments performed in the presence of KBR [5 µmol/l, an NCX inhibitor in the intracellular Ca2+-extracellular Na+ mode (49)] further support the conclusions indicated above. The simultaneous decrease in PtIES and PP in the presence of KBR suggests that 1) during ES a fraction of the contractile Ca2+ would enter the cell through a KBR-sensitive mechanism, 2) this KBR-sensitive fraction would be responsible for PP, and 3) the KBR-sensitive fraction is the same as the previously characterized lithium-sensitive fraction. The fact that lithium and KBR significantly decreased PtIES, PtIPES, and PP strongly indicates that the mechanism should be the NCX (Fig. 7, middle and bottom).

Under all the experimental conditions tested, the thermograms associated with a regular contraction, ES and PES, were best fitted to the three components H1, H2, and H3. In the case of the ES contraction, the presence of H1 (which has been attributed to Ca2+ adsorption to TnC), together with the second maximum in P, suggests that there would be additional Ca2+ adsorption when the ES stimulus arrives at the muscle. In agreement with the mechanical differences described for both types of contraction, the PES heat components were always higher than the ES components (Fig. 5, Table 2). In the case of H1 and H2, these differences would be related to cycling of more Ca2+ and other ions during PES. In the case of the H3 component, the difference would be explained by the higher pressure developed during PES. Despite the increase in H3 and the mechanical and energetic differences, the H3-to-PtI ratio remained unaltered (Table 4). This is in agreement with the behavior of the H'a-to-PtIBC ratio, which also remained unaffected under bigeminic conditions (Fig. 4, Table 3). Because the use of different Ca2+ removal processes implies a different amount of energy release, the preservation of the energetic cost of muscle contraction (and, thus, the overall economy) suggests that, under regular and bigeminic stimulation patterns, the relative participation of SL and SR mechanisms for cytosolic Ca2+ removal remained unchanged.

The diminished contractile Ca2+ availability in the presence of lithium would decrease actomyosin interaction and explains at least part of the decline in H3. Even though the presence of Ver plus LiCl or KBR significantly decreased the magnitude of the heat components (Table 2), the H'a-to-PtI ratio remained unaffected (Table 3). Therefore, these results suggest that the relative participation of SL and SR Ca2+ removal processes is not affected by these agents.

The results presented in this work suggest that during bigeminic contractions the relative participation of the Ca2+ pools during the active state became redistributed. This means that this relative participation is "flexible," and it could represent an adaptive response of the myocardium. The results also suggest the participation of two fractions of extracellular Ca2+ entering during the ES: the Ver-sensitive fraction can be associated with the activity of SL Ca2+ channels and would not participate in PP, and the lithium- and KBR-sensitive fraction can be associated with the NCX, which seems related to PP. The identification of the second fraction supports the idea of the participation of the NCX as a Ca2+ influx mechanism under physiopathological conditions, such as bigeminies, and not only as a mechanism for Ca2+ extrusion. Despite the redistribution of the relative participation of contractile Ca2+ pools during the active state, the muscle economy was not affected under bigeminic stimulation (neither the overall nor the contractile economy). The unaffected muscle economy during bigeminies, together with PP, might be of potential clinical relevance. Clinical applications have been suggested for PP at the diagnostic (to identify a viable myocardium) and therapeutic (to improve ventricular function in the systolic failing heart) levels (12). Even though the present experiments were not performed in failing hearts, it could be hypothesized that the bigeminic stimulation might improve systolic ventricular function because of an increase in PtI without an effect on the muscle economy (neither overall nor contractile economy). Therefore, short ESIs might generate the same number of "effective" but potentiated contractions (PES) per minute, because even though the ES stimulus depolarizes the muscle, it does not generate an additional "effective" (i.e., ejective) contraction. Although promising, the application of short-interval paired-pulse stimulation should be further investigated in the context of its eventual clinical application, such as a resource for systolic heart failure.


   GRANTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas Grant Proyectos de Investigaciones Plurianuales 4564, University of Buenos Aires Grants OD-018/T and O-008, University of Buenos Aires-Secretaria de Ciencia y Tecnica Grant O(023), and Fondo Nacional para la Ciencia y la Tecnología Grant Proyectos de Investigación Científica y Tecnológica 2267, República Argentina.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. E. Ponce-Hornos, M. T. de Alvear 2142, 17th Fl., Dept. of Biophysics, C1122AAH Buenos Aires, Argentina (e-mail: pohornos{at}mail.retina.ar)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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