Inhibition of the Na⁺–H⁺ exchanger with cariporide abolishes stretch-induced calcium but not sodium accumulation in mouse ventricular myocytes

Abstract

We address the question whether activation of the sodium–proton exchanger (NHE) does contribute to the stretch-induced accumulation of intracellular sodium and calcium in mouse ventricular myocytes.

NHE-blocker cariporide (10 μM) were applied to the bath for 10 min. Axial stretch was applied for 2 min by increasing the distance between an adherent glass stylus and the patch pipette by 20%. Myocytes (stimulated at 3 Hz) were shock-frozen in diastole and the membrane currents monitored till cryofixation. Controls were treated identically, but not stretched. Total sodium and calcium concentrations ([Na], [Ca] = sum of free and bound Na and Ca) were measured by electron probe microanalysis (EPMA) in peripheral and central cytosol, mitochondria, nucleus and nuclear envelope.

Cariporide did not reduce the stretch-activated negative current. The stretch-induced rise in [Na] was not different in the presence and in the absence of cariporide. Cariporide significantly reduced diastolic [Ca] in the cytosol of stretched myocytes.

Since cariporide does not prevent the stretch-induced [Na] accumulation, we suggest that not NHE but the stretch-activated streptomycin-sensitive current I_SAC causes the well documented stretch-induced [Na] accumulation. The discovery that cariporide prevents the stretch-induced rise in cytosolic [Ca] demonstrates an important additional effect of the drug on calcium handling.

Introduction

Recently we have shown that local axial stretch of stimulated mouse ventricular myocytes induces an inward current I_SAC 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 I_SAC 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 I_CaL 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.