Journal of Applied Physiology
Vol. 83, No. 2, pp. 652-660, August 1997
EXERCISE AND MUSCLE

Effect of inotropic interventions on contraction and Ca²⁺ transients in the human heart

Klara Brixius, Marcus Pietsch, Susanne Hoischen, Jochen Müller-Ehmsen, and Robert H. G. Schwinger

Medizinische Klinik III, Universität zu Köln, D-50924 Cologne, Germany

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Brixius, Klara, Marcus Pietsch, Susanne Hoischen, Jochen Müller-Ehmsen, and Robert H. G. Schwinger. Effect of inotropic interventions on contraction and on Ca²⁺ transients in the human heart. J. Appl. Physiol. 83(2): 652-660, 1997.The present study investigated the influences of inotropic intervention on the intracellular Ca²⁺ transient {intracellular Ca²⁺ concentration ([Ca²⁺]_i)} and contractile twitch. Isometric twitch and [Ca²⁺]_i (fura 2 ratio method) were measured simultaneously (1 Hz, 37°C) after stimulation with Ca²⁺ (0.9-3.2 mM), the cardiac glycoside ouabain (Oua; 0.1 µM), the ₁- and ₂-adrenoceptor-agonist isoprenaline (Iso; 1-10 nM), and the Ca²⁺ sensitizer EMD-57033 (30 µM) by using isolated human nonfailing right auricular trabeculae (n = 19). Inotropic interventions increased force of contraction and peak rate of tension rise (+T) significantly. Only Iso stimulated peak rate of tension decay (T) higher than +T (P < 0.05), thereby reducing time of contraction (T_twitch). EMD-57033 increased +T more effectively than T and prolonged T_twitch (P < 0.05). Ca²⁺, Oua, and Iso, but not EMD-57033, increased systolic Ca²⁺. Diastolic Ca²⁺ increased after stimulation with Oua or Ca²⁺, but not in the presence of EMD-57033. Iso shortened the Ca²⁺ transient and did not influence diastolic Ca²⁺. In conclusion, positive inotropic agents differently affect force and [Ca²⁺]_i depending on their mode of action. Inotropic interventions influence diastolic Ca²⁺ and thus may be less advantageous in a situation with altered intracellular Ca²⁺ homeostasis (e.g., heart failure due to dilated cardiomyopathy).

fura 2; heart failure; isoprenaline; ouabain; EMD-57033

INTRODUCTION

INTRACELLULAR CA²⁺ homeostasis plays a central role in regulating excitation-contraction coupling in the mammalian heart. The contractile state of the heart is dependent on the availability of intracellular free Ca²⁺ to the myofilaments. Ca²⁺ enters the cytosolic compartment through the sarcolemma from the extracellular space or through the sarcoplasmic reticulum (SR) from intracellular compartments, i.e., Ca²⁺-triggered Ca²⁺ release (19). Increasing the systolic intracellular free Ca²⁺ concentration runs parallel to changes in maximal force development (13). Even in the terminally failing human myocardium, elevation of the concentration of intracellular Ca²⁺ results in an increase in force of contraction (FOC) similar to that in nonfailing tissue (12, 22). Therefore, inotropic enhancement has been suggested to still be an effective means of increasing force maximally even in diseased human hearts, i.e., terminally failing myocardium due to dilated cardiomyopathy (12, 22).

The high-systolic Ca²⁺ concentrations may be decreased to low-diastolic Ca²⁺ levels by three main mechanisms: 1) Ca²⁺ will be taken up into the SR by activation of SR or endoplasmic reticulum Ca²⁺-adenosinetriphosphatase (SERCA); 2) activation of the Na⁺/Ca²⁺ exchanger will lead to Ca²⁺ extrusion out of the cytoplasm; and 3) Ca²⁺ buffer proteins will bind free Ca²⁺ ions. Interventions that lead to an identical increase in force after augmentation of intracellular Ca²⁺ concentration may affect parameters of contraction and relaxation in a different mode depending on their actions on the intracellular Ca²⁺-lowering systems. This mode of action of an inotropic compound has to be kept in mind, especially when the compound is being used to increase force in diseased human myocardium with an already increased diastolic Ca²⁺ level (6).

Force development and maximal systolic free Ca²⁺ concentration have been extensively studied in numerous excellent investigations by using the Ca²⁺ indicators aequorin (12, 18), quin 2 (14) and indomethacin 1 (27). Because alterations of contraction-coupling may be largely influenced by changes of diastolic Ca²⁺, Ca²⁺ indicators focusing on changes in low-diastolic Ca²⁺ levels have been developed. Fura 2 is an example (11). Because of its low dissociation constant (228 nM), fura 2 is a Ca²⁺ fluorescence indicator that has been reported to be more suitable in measuring changes of diastolic Ca²⁺ than aequorin (11). Use of fura 2 has shown that peak Ca²⁺ transients were reduced, diastolic Ca²⁺ levels were increased, and the diastolic Ca²⁺ decay was significantly prolonged in cardiomyocytes from patients with terminal heart failure due to dilated cardiomyopathy (6). In addition, the fura 2 ratio method has been used as a suitable tool for studying intracellular Ca²⁺ transients and force in isolated muscle strip preparations of rat myocardium (3).

In the present study, intracellular free Ca²⁺ concentrations were measured simultaneously with parameters of FOC by using the fura 2 ratio method after inotropic interventions. Because different inotropic interventions may influence force development and intracellular Ca²⁺ homeostasis, depending on their mode of action, the purpose of the present study is to investigate the effect of inotropic stimulation on force development and intracellular Ca²⁺ simultaneously. Parameters of contraction and the intracellular Ca²⁺ transient were measured after inotropic stimulation with 1) adenosine 3,5-cyclic monophosphate (cAMP)-independent inotropes [the cardiac glycoside ouabain (Oua)] or elevation of extracellular Ca²⁺; 2) the cAMP-dependent inotrope, the -adrenoceptor-agonist isoprenaline (Iso); and 3) the Ca²⁺ sensitizer EMD-57033.

MATERIALS AND METHODS

RESULTS

Table  1.   Parameters of contractile twitch and of Ca2+ transient of isolated right auricular muscle strip preparations from nonfailing human myocardium (0.9 mM Ca2+, 1 Hz, pH 7.4) Parameters Contractile twitch FOC, mN/mm2 12.6 ± 1.9  TPT, ms 109.6 ± 3.6* T1/2T, ms 110.2 ± 8.4* +T, mN/s 25.0 ± 3.8   T, mN/s 13.5 ± 2.2  Ttwitch, ms 433.3 ± 20.2* Ca2+ transient R340/380Ca 0.94 ± 0.15  RPR, ms 52.5 ± 3.1  R1/2R, ms 177.5 ± 9.0 TCa, ms 539.1 ± 31.0 Values are means ± SE; n = 24. FOC, force of contraction; TPT, time to peak tension; T1/2T, time to half peak relaxation; +T, maximal rate of tension increase; T, maximal rate of tension decrease; Ttwitch, duration of contraction; R340/380Ca, peak Ca2+; RPR, time to peak Ca2+; R1/2R, time to half peak Ca2+ relaxation; TCa, duration of Ca2+ transient. * P < 0.05 vs. Ca2+ transient. Signal was not quantified.

Table  2.   Effects of inotropic interventions on contractile force and Ca2+ transient Ca2+ Oua Iso EMD-57033 Contractile twitch FOC         TPT = = = = T1/2T = =     +T          T       = Ttwitch = =     Ca2+ transient R340/380Ca       = RPR = = = = R1/2R = =   = R340/380sys   =   = R340/380ED     = = TCa = =   = Oua, ouabain; Iso, isoprenaline; , increase; , decrease; double , large increase; =, no change.

DISCUSSION

Abnormalities in intracellular Ca²⁺ handling may become exaggerated under stimulated conditions, i.e., after inotropic stimulation of FOC with agents increasing systolic Ca²⁺. Thus it seems worthwhile to simultaneously study the influence of various inotropic interventions on the Ca²⁺ transient (systolic and diastolic Ca²⁺) and the contractile twitch in human myocardium. In the present study, inotropic stimulation was achieved by an elevation of extracellular Ca²⁺ concentration, by application of the cardiac glycoside ouabain, the -adrenoceptor-agonist Iso, and EMD-57033, which has been suggested to increase the Ca²⁺ sensitivity of the contractile filaments. Because attention has to be focused on diastolic Ca²⁺ changes, the Ca²⁺ indicator fura 2 was used. Fura 2 was shown to be suitable to measure changes of low Ca²⁺ concentrations (8, 11).

Under basal conditions (1 Hz, 37°C), the T_Ca, as measured by fura 2, was significantly prolonged compared with T_twitch. These findings are in contrast with observations obtained with the indicator aequorin in right ventricular myocardium of failing and nonfailing human hearts (12, 18). In these studies the Ca²⁺ transient declined toward baseline before the mechanical event was finished. However, differences might exist in Ca²⁺ handling and sensitivity depending on the origin of the muscle, e.g., right vs. left ventricle or atrium (23). Furthermore, there are important differences in the experimental conditions from those in the studies of Morgan (18) and Gwathmey et al. (12). In the present study the isolated right auricular trabeculae were perfused at a temperature of 37°C and electrically stimulated at a frequency of 1 Hz. The experiments of Morgan (18) and Gwathmey et al. (12) were performed at a temperature of 30°C and a stimulation frequency of 0.33 Hz. Frequency-dependent changes of FOC depend on experimental temperature and frequency range investigated (7). In addition, intracellular Ca²⁺ transients may change in a frequency-dependent mode as well (21).

The Ca²⁺ transients in the studies of Morgan (18) and Gwathmey et al. (12) were measured by the Ca²⁺ indicator aequorin. In the present study fura 2 was used as Ca²⁺ indicator. Fura 2 has a high affinity for Ca²⁺, with an in vitro dissociation constant close to 200 nM (11). The binding kinetics of fura 2 have been calculated to be much slower in the myofibrillar environment (23 s¹) (4) than in salt solutions (84 s¹) (15). In addition, the Ca²⁺ indicator aequorin is very sensitive to changes in peak systolic Ca²⁺ changes but less sensitive in the low-Ca²⁺ concentration range during diastole (8). The magnitude of the Ca²⁺ transient measured by fura 2 (or other indicators) can only be evaluated after calibration of the signals (5). The present study demonstrates the prolongation of the duration of the fura 2 signal compared with T_twitch, which is in agreement with the study of Baylor and Hollingwood (4).

The various inotropic agents used (Ca²⁺, ouabain, Iso, EMD-57033) increased contractile force but differently affected the contractile cycle and the intracellular Ca²⁺ transient (Table 2). Ouabain and Ca²⁺ increased systolic and diastolic intracellular Ca²⁺ in parallel to force development. Iso increased intracellular Ca²⁺, but not diastolic Ca²⁺, and initiated a fast decline of the high systolic Ca²⁺ levels. The Ca²⁺ sensitizer EMD-57033 significantly increased force but had no influence on parameters of the intracelllular Ca²⁺ transient.

The Ca²⁺-induced increase of intracellular Ca²⁺ concentration in human myocardium confirms the findings of Tatsukawa et al. (28) and Frampton et al. (10) by using fura 2 in isolated rat ventricular myocytes. It is also consistent with data observed by using the bioluminescent aequorin in quiescent ferret ventricular muscle preparation (1) or in cultured chick embryonic heart cells by using indomethacin 1 as Ca²⁺ indicator (20). Therefore, an increase in extracellular Ca²⁺ will increase the systolic as well as the diastolic Ca²⁺ as measured by the fura 2 ratio method by using multicellular or single-cell preparations.

The cardiac glycoside ouabain influenced the time course of contraction similarly to Ca²⁺. Ouabain increased systolic and diastolic Ca²⁺ without effect on the time parameters of the Ca²⁺ transient. These results are confirmed by studies of cultured rat ventricular cells (16, 28).

In contrast to Ca²⁺ and ouabain, Iso accelerated cardiac relaxation more than contraction. This increase of relaxation was paralleled by an increase in the decay of the intracellular Ca²⁺ transient. The -adrenoceptor-agonist Iso stimulates adenylate cyclase, leading to an elevation of intracellular cAMP, which activates intracellular protein kinases phosphorylating a number of subcellular sites. Phosphorylation of the voltage-dependent Ca²⁺ channel increases intracellular Ca²⁺ influx during each depolarization and thereby the amount of Ca²⁺ in the cell. As a result, systolic force is increased. Because cAMP-dependent protein kinase also leads to phosphorylation of troponin I, the Ca²⁺ sensitivity of the contractile apparatus will be reduced. Thus -adrenoceptor agonists should increase intracelullar Ca²⁺ concentration to a greater extent than force to initiate the same effectiveness as an elevation of the extracellular Ca²⁺ concentration. Phosphorylation of phospholamban, the regulatory subunit of the Ca²⁺ pump of the SR, enhances the rate of resequestration of intracellular Ca²⁺ during diastole, thus also leading to an increased Ca²⁺ uptake and relaxation during diastole.

A new class of inotropic compounds has been developed that enhances the sensitivity of the myofilaments to Ca²⁺, rather than increasing the Ca²⁺ availability to the myofilaments. EMD-57033 is the (+) enantiomer of the inotropic agent EMD-53998 (23). It has been found to possess potent Ca²⁺-sensitizing action with little effect on phosphodiesterases (26). The mechanism by which EMD-57033 increases the Ca²⁺ sensitivity of the myofilaments is unknown. Preliminary reports have indicated that EMD-57033 has no effect on Ca²⁺ binding to troponin C and is unlikely to affect thin-filament interactions, suggesting that it affects the cross-bridge cycling mechanism itself. In the human myocardium EMD-57033 increased FOC (26) and slowed cardiac relaxation but did not affect systolic or diastolic intracellular Ca²⁺ concentrations, as shown in the present study.

The various inotropic interventions examined in this study differently influenced diastolic Ca²⁺. At low concentrations the cAMP-dependent inotrop Iso accelerated relaxation (24) and did not enhance diastolic Ca²⁺ levels or diastolic tension. Diastolic Ca²⁺ increased after stimulation with ouabain or Ca²⁺, but not in the presence of EMD-57033. In human heart failure, changes in diastolic Ca²⁺ have been demonstrated, which have been suggested to be at least partly responsible for the altered force-frequency relationship. Low concentrations of Iso reversed the negative force-frequency relationship to a positive one (24). This has been suggested to be due to an enhanced activity of SERCA II after phospholamban phosphorylation (22, 24). This mode of action, to stimulate Ca²⁺ reuptake, accelerating diastolic Ca²⁺ decrease, demonstrates the usefulness of the Ca²⁺-handling sites as targets to influence contractility and the altered Ca²⁺ handling in heart failure. Because parameters of relaxation and the Ca²⁺-lowering systems may be altered in heart failure (25), the fura 2 method may be a suitable tool to study the mechanism leading to these intracellular Ca²⁺ changes.

ACKNOWLEDGEMENTS

We are indebted to all colleagues in the Department of the Cardiothoracic Surgery of the University of Cologne (Dr. R. E. de Vivie, Director) for providing us with human myocardial samples. We are also indebted to I. Lues, Merck, Darmstadt, Germany, for the kind gift of EMD-57033. The authors thank A. Herber, A. Gross, and T. Schewior for excellent technical help.

FOOTNOTES

Address for reprint requests: R. H. G. Schwinger, Universität zu Köln, Medizinische Klinik III, Joseph-Stelzmann-Str. 9, D-50924 Cologne, Germany.

Received 29 October 1996; accepted in final form 25 March 1997.

REFERENCES