INTRODUCTION

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STUDIES USING CURRENT CLAMP techniques have shown that increases in either the duration or amplitude of the action potential enhance cardiac contractility by increasing transarcolemmal Ca²⁺ entry (1, 15). This results in sarcoplasmic reticular Ca²⁺ loading and an increase in the peak of the intracellular Ca²⁺ transient (15). It has recently been reported (5) that extracellularly applied electric fields can prolong action potential duration, influence action potential amplitude and similarly enhance myocardial contractility in isolated papillary muscles. When applied locally to intact canine hearts, such electric impulses enhance myocardial contractility in the region of signal application, an effect that results in enhanced global contractility (10). These signals also appear effective in enhancing contractility in animals and humans with heart failure (11-13). However, the mechanism of regional myocardial contractility enhancement with extracellular electrical impulses has not been clarified.

The purpose of the present study was to test the hypothesis that the primary inotropic mechanism of cardiac contractility modulating (CCM) electrical impulses, analogous to voltage-clamp techniques, is by enhancing peak intracellular Ca²⁺. The effects of CCM signals on myofilament Ca²⁺ sensitivity were tested by measuring CCM effects at different extracellular Ca²⁺ concentration ([Ca²⁺]_o). The contribution of -adrenergic stimulation to inotropic effects of CCM signals were investigated through studies performed in the presence of high-dose -blockers.

	METHODS

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The animals involved in this study received humane care in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1985). This study was approved by the Institutional Animal Care and Use Committee of Columbia University.

Surgical preparation. In each of 10 experiments, an 8- to 14-wk-old ferret (1.2-1.4 kg body wt) was heparinized (3,000 U ip), anesthetized with pentobarbital sodium (50 mg/kg ip), and studied during Langendorff perfusion, as detailed previously (2, 14). In brief, a bilateral sternotomy was performed and the heart was rapidly excised and submerged in oxygenated, warmed, modified Tyrode solution (36°C; composition provided below). The severed end of the aorta was fed over a 16-gauge needle that was connected to a modified Langendorff perfusion system, and perfusate flow was adjusted to provide a perfusion pressure of ~70-80 mmHg. Complete heart block was achieved by formalin injection (10%, 0.1-0.3 ml) into the atrioventricular nodal region. The left atrium was opened and a latex balloon attached to the end of a stiff polyethylene tube was inserted into the left ventricle (LV) and held in place by a 2-0 silk purse-string suture placed around the mitral annuls. The balloon and tubing were filled with water and connected to a Statham pressure transducer for measurement of isovolumic LV pressure (LVP). Balloon volume was then adjusted with the use of a calibrated 2-ml syringe. Two subepicardial pacing electrodes were inserted into the apex of the right ventricular free wall. A pair of platinum wire electrodes (each 5 mm in length, ~5 mm apart) was inserted into the LV free wall for delivering CCM signals.

Measurement of Ca²+ transients. Techniques for measuring Ca²⁺ transients from the epicardial surface of crystalloid-perfused hearts were similar to those described previously (2, 8, 14). An aequorin solution composed of (in mM) 154 NaCl, 5.4 KCl, 1 MgCl₂, 12 HEPES, 11 glucose, and 0.1 EDTA, and 1 mg/ml aequorin (adjusted to pH 7.40) was prepared. After the isolated heart was stabilized, 3-5 µl of this solution were injected just below the epimysium in the inferoapical region with the use of a low-resistance glass micropipette with an inner diameter of ~30 µm. This was accomplished by performing six separate injections (0.5-1.0 µl each) into an ~3 mm² area. It has been shown that a fraction of this aequorin is "loaded" into myocytes by an unknown mechanism with minimal damage to the cells near the injection site (8).

Experimental protocol and data analysis. All hearts were paced at a rate of 120 beats/min. After stabilization, baseline LVP and aequorin luminescence signals were recorded for 3 min. CCM signals were biphasic (15 ms per phase) square-wave current pulses with amplitude ±20 mA that were delivered 40 ms after delivering the right ventricular pacing spike. After a stable contractile state was reached (~30-60 s), data were recorded for 3 min. In six of the animals, the same procedure was repeated while the heart was perfused with Tyrode solutions having four different [Ca²⁺]_o concentrations (1, 2, 4, and 8 mM). In four experiments, CCM signals were delivered after the addition of propranolol (1 mg/l) to Tyrode solution with 1 mM [Ca²⁺]_o. This concentration of propranolol was shown in preliminary experiments to completely block the inotropic response of isoproterenol. All data were recorded on a digital computer at a sampling rate of 500 Hz for offline analysis.

Statistics. Data are expressed as means ± SD. Student's t-test was applied to compare paired mean values between control and CCM. The relationship between LVP and [Ca²⁺]_i was compared by analysis of covariance.

	RESULTS

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Figure 1 shows original LVP and aequorin light emission tracings under baseline conditions and during the application of CCM signals (with [Ca²⁺]_o of 2 mM). On initiation of CCM signals, there was a rise in peak pressure and aequorin light emission, which reached plateau levels after ~10-15 s.

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Simultaneously measured isovolumic left ventricular pressure (LVP) and aequorin light emission tracings from an isolated ferret heart before and during application of a cardiac contractility modulating (CCM) electrical signal in the antero-apical region of the heart. Arrows indicate the onset of CCM signal delivery. Signal-averaged (SA) signals (from within the specified bracketed regions) show the significant increase in aequorin light emission indicative of a rise in peak intracellular Ca2+ during CCM signal application, which parallels the increase in peak isovolumic pressure. Biphasic CCM signal was recorded on the ECG (bottom).

Effects of CCM signals on LVP and signal-averaged [Ca²⁺]_i transients at different [Ca²⁺]_o are shown in the examples of Fig. 2. Baseline peak LVP and [Ca²⁺]_i both increased as [Ca²⁺]_o was increased. The degree of increase in LVP in response to CCM signals decreased as [Ca²⁺]_o was increased. For example, CCM signals on average induced a 20.1 ± 10.3 mmHg (41 ± 20%, P = 0.0051) increase in LVP at a [Ca²⁺]_o of 1 mM, but at 8 mM [Ca²⁺]_o CCM increased LVP by only 7.2 ± 5.4 mmHg (8.1 ± 3.6%, P = 0.0220). This observation is further summarized for all [Ca²⁺]_o values in Fig. 3A.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   LVP and signal-averaged intracellular Ca2+ concentration ([Ca2+]i) transients from a single experiment measured at different perfusate extracellular Ca2+ concentrations ([Ca2+]o) between 1 and 8 mM. As [Ca2+]o is increased, baseline peak pressure and peak [Ca2+]i increase. At each [Ca2+]o, CCM signals increase peak [Ca2+]i, but the effects on peak LVP are most pronounced at low [Ca2+]o.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   A: average results from 6 experiments showing the relationships between [Ca2+]o and LVP showing that the inotropic effects of CCM signals are most effective at normal and subphysiological [Ca2+]o. B: relationship between [Ca2+]o and peak intracellular Ca2+ ([Ca2+]i) showing that CCM signals are equally effective at all [Ca2+]o. C: relationship between [Ca2+]i and LVP showing a nonlinear relationship that plateaus at higher Ca2+ levels, which explains why LVP responses to CCM signals are smaller at higher baseline contractilities. See text for further details.

In contrast, the effects of CCM signals on the absolute change in peak [Ca²⁺]_i were independent of [Ca²⁺]_o despite the rise in baseline [Ca²⁺]_i. For example, at a [Ca²⁺]_o of 1 mM, peak [Ca²⁺]_i increased by 0.84 ± 0.25 µM (P = 0.0004), whereas at a [Ca²⁺]_o of 8 mM, the increase was 0.82 ± 0.22 µM (P = 0.0003). These findings are further summarized in Fig. 3B.

The relationship between LVP and [Ca²⁺]_i (Fig. 3C) shifted slightly toward higher [Ca²⁺], but this difference was not statistically significant (analysis of covariance) between baseline and CCM conditions indicating that there was no significant effect of CCM signals on myofilament Ca²⁺ affinity. The nonlinearity of this relationship, however, explains the blunted inotropic effects at high [Ca²⁺]_o; the higher the baseline Ca²⁺, the smaller the inotropic effect of a given increase in [Ca²⁺]_i.

In terms of the dynamics of contraction (Table 1), CCM signals decreased the time to peak pressure slightly but significantly by an amount that depended on the extracellular [Ca²⁺]. There was no effect on the time constant of relaxation.

CCM signals may stimulate release of endogenous norepinephrine from nerve terminals which could contribute to their inotropic effects. To explore this possibility, CCM signals were applied to ferret hearts during exposure to high-dose propranolol. The results, summarized in Fig. 4, show that the changes in [Ca²⁺]_i were indistinguishable from those observed in the original set of experiments (an increase in [Ca²⁺]_i of 0.56 ± 0.16 µM compared with a 0.86 ± 0.10 µM increase without -blockers) and that there was a slight decrease in the change in pressure development in response to CCM signals (an increase in LVP of 8.6 ± 5.2 mmHg compared with a 20.0 ± 10.3 mmHg increase without -blockers).

View larger version (8K): [in this window] [in a new window]   Fig. 4.   Effects of CCM signals on LVP (A) and [Ca2+]i (B) in isolated ferret hearts exposed to a high concentration of propranolol to block potential effects of endogenously released norepinephrine with perfusate [Ca2+] of 1 mM. The observed effects are similar to those observed without propranolol. * P < 0.01.

	DISCUSSION

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It has long been recognized that cardiac contractility can be modulated by actively controlling the duration and profile of the transmembrane potential, which in turn influences Ca²⁺ entry, sarcoplasmic reticular Ca²⁺ loading and ultimately Ca²⁺ release to the myofilaments (1, 15). Extracellular electric currents have recently been demonstrated to have similar inotropic effects in isolated papillary muscles (5), intact normal (10) and failing hearts (13), and in patients with heart failure (11, 12), though the mechanism of inotropic action has not been elucidated. The results of the present study indicate that application of extracellular electrical fields during the refractory period result in increased peak Ca²⁺ delivery to the myofilaments, which increases myocardial contractility in isolated ferret hearts with little effect on myofilament Ca²⁺ sensitivity.

The electric currents used in the present study, CCM signals, are applied to a region of the heart and affect contractility in the region of application (10). Therefore, relatively large changes in local contractility result in smaller, though substantial changes at the global level.

The effect of CCM signals on the locally measured intracellular Ca²⁺ transients were similar at all [Ca²⁺]_o tested, but the effect on global contractility was blunted when [Ca²⁺]_o concentrations higher than physiological levels (4 and 8 mM) were studied. This is compatible with the nonlinear relationship between peak intracellular Ca²⁺ and myocardial force of contraction observed in isolated myocardium (16) and intact hearts (2). As [Ca²⁺]_i increases, myofilament force production plateaus both in isolated muscles and intact ferret hearts. Thus, when starting at a state in which peak intracellular Ca²⁺ is already high, an intervention, which further increases Ca²⁺, will not significantly augment pressure generation. On the other hand, heart failure is a state in which a reduced peak of the intracellular Ca²⁺ transient is believed to contribute to contractile dysfunction (7). In this setting, CCM signals would be applied under favorable conditions for achieving an inotropic effect.

Myocardial tissue is richly innervated with nerve fibers, and inotropic effects of CCM signals could be mediated by release of norepinephrine. However, high-dose -blockers did not influence the ability of CCM signals to augment peak intracellular Ca²⁺, and there was only a relatively small reduction on the overall inotropic effect. This suggests that release of endogenous catecholamines is not the mechanism of positive inotropism in this setting.

Prior studies (1, 5) of extracellular CCM signals applied to isolated papillary muscle in vitro have suggested that one mechanism for increased Ca²⁺ cycling is an effect of the signals on action potential duration and/or amplitude, which in turn, influences entry of Ca²⁺ during contraction. This in turn leads to Ca²⁺ loading of the sarcoplasmic reticulum so that in the steady state, the amount of Ca²⁺ released to the myofilaments is increased compared with baseline conditions. One limitation of the present study is that the effects of CCM signals on transmembrane potentials have not been investigated and therefore the mechanism by which Ca²⁺ cycling is influenced in the intact heart is not yet defined. However, Fast et al. (6) recently showed that electric shocks (10 ms duration and 2-50 V/cm) applied through bipolar electrodes during the action potential plateau prolonged action potential duration and increased amplitude. In the porcine isolated anterior wall segment used in that study, mechanical activity was abolished by perfusion with 2,3-butanedione monoxime so the effects on contraction were not directly studied. Compared with the signals used by Fast et al. (6), CCM signals used in the present study are of similar amplitude but applied earlier during the action potential. Nevertheless, these data show that electric signals can influence the action potential amplitude and duration in support of the proposed mechanism.

In summary, the primary mechanism by which CCM signals enhance contractility is to increase in systolic intracellular Ca²⁺. Myofilament Ca²⁺ affinity (indexed by the relationship between peak intracellular Ca²⁺ and peak developed pressure) is not significantly influenced. The inotropic effects of CCM signals are most pronounced at physiological and reduced levels of extracellular Ca²⁺, though their ability to augment peak intracellular Ca²⁺ is preserved at all concentrations. These findings suggest that further study of CCM signals as a potential therapy for heart failure are warranted.