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Effects of caffeine, verapamil, lithium, and KB-R7943 on mechanics and energetics of rat myocardial bigeminies

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 ( = 101 ± 12.6%). Verapamil (an L-type Ca²⁺ channel blocker) decreased pressure maintenance of extrasystolic ( = 43.4 ± 3.7%) and postextrasystolic ( = 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 ( = 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

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.

View larger version (16K): [in this window] [in a new window]   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.

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 (H_t), and resting heat production (H_r). PtI was measured for regular, bigeminic, ES, and PES contractions (PtI_RC, PtI_BC, PtI_ES, and PtI_PES, 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 PtI_PES and PtI_RC 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^–2–1)^0.5tan[(µ4^–2–1)^0.5], µ is the cooling rate constant of the calorimeter, is the diffusion delay constant, A_i = 1/{(2i + 1)²[1 – (2i + 1)²µ^–1]}, _i = (2i + 1)²µ, is the rate constant of the declining fraction, t is time, and H₀ represents a fitted value of the power; 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 H_t and H_r, 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.

View larger version (8K): [in this window] [in a new window]   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).

	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 H_r 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 (PtI_RC) 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).

View larger version (20K): [in this window] [in a new window]   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.

View larger version (11K): [in this window] [in a new window]   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.

View larger version (17K): [in this window] [in a new window]   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 (). 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 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.

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, PtI_ES 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. PtI_PES 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).

View larger version (24K): [in this window] [in a new window]   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.

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 H_r 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 PtI_RC, 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 PtI_ES and PtI_PES, 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: 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).

View larger version (30K): [in this window] [in a new window]   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.

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 H_r did not significantly differ from the H_r in the absence of LiCl: = 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 ( = 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 ( = 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: = 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 PtI_ES 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: = 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: = 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: = 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 H_r was not significantly affected by the presence of KBR in the perfusion medium: = 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: = 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

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION GRANTS REFERENCES

 
Under resting conditions, the H_r 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), PtI_PES 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 PtI_ES 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 PtI_ES, PtI_PES, and PP (Fig. 7, middle). The decrease in PtI_ES and PtI_PES 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 PtI_ES 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 PtI_ES, PtI_PES, 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-PtI_BC 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

TOP 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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