Inhibition of the Na+–H+ exchanger with cariporide abolishes stretch-induced calcium but not sodium accumulation in mouse ventricular myocytes
Introduction
Recently we have shown that local axial stretch of stimulated mouse ventricular myocytes induces an inward current ISAC and a significant increment in the sodium and calcium concentrations of cytosol and other cell compartments within 2 min, as measured with sodium sensitive dyes and with electron probe microanalysis (EPMA) [1], [2]. Both ISAC and Na-accumulation were prevented by streptomycin, a blocker of non-selective stretch-activated channels. Hence, we postulated that the increase in intracellular sodium was due to sodium influx through stretch-activated channels [3], [4].
It is known from the literature, that stretch of myocardial trabeculae increases developed contractile force in two phases: a first component due to an increase in the Ca-responsiveness of the myofilaments and a second slower phase due to a rise in cytosolic calcium [5]. The mechanism underlying the slow force response and the rise in the Ca2+ transient is not yet clear. Since ICaL does not increase with stretch [6], it has been suggested that the increase in the cytosolic Ca2+ transients may be mediated by stretch-activation of the Na+–H+ exchanger (NHE) [7], the resulting reduction of the transsarcolemmal Na+ gradient increasing intracellular calcium via Na+/Ca2+ exchanger (NCX) ([8], for review). These authors described that both the rise in [Na] and the slow force response were abolished by NHE inhibitors. A new study [9] on the effect of cariporide (a blocker of NHE) on cultured neonatal myocytes under oxidative stress has shown that blockade of NHE with cariporide protects cardiomyocytes against oxidant-induced cell death by preserving intracellular ion homeostasis and mitochondrial integrity. Here, we were interested to quantify the effects of inhibition of the NHE on the stretch-induced Na and Ca accumulation we have measured recently in mouse ventricular myocytes. We also wanted to answer the question whether, and to what extent, the NHE-dependent mechanism may be involved in the Na-accumulation in stretched cardiac myocytes.
Since NHE-blockers have found application in the protection for reperfusion damages of ischemic myocardium (prevention of Ca-overload) and for ventricular arrhythmia [10], [11], [12], [13], it is important to understand the mechanisms underlying the effects of NHE blockers on the Na and Ca homoeostasis. The protective effects of cariporide [11] suggest that the stretch-induced activation of the sodium–proton exchanger may be an initial step in the activation of signal cascades involved in load-induced cardiac hypertrophy and possibly heart failure. In this study we present data suggesting that the Na-accumulation induced by local axial stretch in isolated ventricular myocytes is not due to Na+ influx through the NHE. The data also suggest that the protective effect against Ca-overload induced by the NHE-blocker cariporide may be due to an additional effect on the Ca-homeostasis independent from the activation of NHE.
Section snippets
Methods
Myocytes were enzymatically dissociated from ventricles of the mouse according to [1]. Briefly, hearts were excised from anesthetized mice of approximately 20 g weight. The hearts were mounted on a Langendorff apparatus and retrogradely perfused at 37 °C and constant flow (2 ml min−1), first with a physiological salt solution (PSS) containing (in mM) 150 NaCl, 5.4 KCl, 1.2 MgCl2 (6 H2O), 20 glucose, 5 HEPES/NaOH, 1.8 CaCl2, (pH 7.4, 37 °C) for 5 min, then with the same but nominally Ca2+-free solution
Results
The total elemental concentrations of Na, Mg, P, S, Cl, K and Ca in central cytosol are given in Table 1 for controls and for stretched myocytes, both superfused and stimulated in the presence of cariporide. The significant increase in cytosolic [Na] with stretch goes in parallel with a significant decrease in [K] and [Ca].
Discussion
The results of this study show that block of the sodium–proton exchanger with cariporide does not prevent either the stretch-activation of ISAC or the stretch-induced rise in sodium in mouse ventricular myocytes, but it does reduce the stretch-induced diastolic [Ca] accumulation in cytosol. Our results give evidence that (1) activation of the NHE does not represent a major mechanism for the stretch-induced increment in intracellular sodium in the mouse ventricular myocytes stretched with the
Acknowledgements
The authors are very grateful to Nicole Walter for excellent technical assistance and to G. Isenberg for reading and discussing the manuscript. The work was supported by the Deutsche Forschungsgemeinschaft Grant SFB Transregio TR 02 TP A1 to M.F.G. and by NBL3 Roux-Ergänzungsprojekt to M.F.G. Cariporide (HOE 642) was kindly provided by the Aventis Company, Frankfurt, Germany.
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