Inhibition of NHE protects reoxygenated cardiomyocytes independently of anoxic Ca2+ overload and acidosis

C. Schäfer, Y. V. Ladilov, M. Schäfer, H. M. Piper
2000 American Journal of Physiology. Heart and Circulatory Physiology  
Schä fer, C., Y. V. Ladilov, M. Schä fer, and H. M. Piper. Inhibition of NHE protects reoxygenated cardiomyocytes independently of anoxic Ca 2ϩ overload and acidosis. Am J Physiol Heart Circ Physiol 279: H2143-H2150, 2000. -We investigated the question of whether inhibition of the Na ϩ /H ϩ exchanger (NHE) during ischemia is protective due to reduction of cytosolic Ca 2ϩ accumulation or enhanced acidosis in cardiomyocytes. Additionally, the role of the Na ϩ -HCO 3 Ϫ symporter (NBS) was
more » ... (NBS) was investigated. Adult rat cardiomyocytes were exposed to simulated ischemia and reoxygenation. Cytosolic pH [2Ј,7Ј-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)], Ca 2ϩ (fura 2), Na ϩ [sodium-binding benzolfuran isophthatlate (SBFI)], and cell length were measured. NHE was inhibited with 3 mol/l HOE 642 or 1 mol/l 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and NBS was inhibited with HEPES buffer. During anoxia in bicarbonate buffer, cells developed acidosis and intracellular Na and Ca (Na i and Ca i , respectively) overload. During reoxygenation cells underwent hypercontracture (44.0 Ϯ 4.1% of the preanoxic length). During anoxia in bicarbonate buffer, inhibition of NHE had no effect on changes in intracellular pH (pH i ), Na i , and Ca i , but it significantly reduced the reoxygenation-induced hypercontracture (HOE: 61.0 Ϯ 1.4%, EIPA: 68.2 Ϯ 1.8%). The sole inhibition of NBS during anoxia was not protective. We conclude that inhibition of NHE during anoxia protects cardiomyocytes against reoxygenation injury independently of cytosolic acidification and Ca i overload. hypercontracture; pH control; calcium; sodium-hydrogen exchanger IT HAS BEEN SHOWN that the ischemic-reperfused myocardium can be protected when the Na ϩ /H ϩ exchanger (NHE) is inhibited (27, 31). Pronounced protection is achieved, however, only if the inhibitor of NHE is applied before or with the onset of ischemia (7, 15, 17, 25) . The mechanism of this protection is still unknown, but the following hypotheses have been put forward. First, inhibition of NHE reduces cytosolic Na ϩ accumulation (2) and subsequent Ca 2ϩ overload, which may result from reduced activity of the Na ϩ /Ca 2ϩ exchanger (NCE) in forward mode operation or from inhibition of its reverse mode operation (37). Second, inhibition of NHE enhances acidification of the ischemic myocardial cell. Finally, reduced Ca 2ϩ overload and enhanced acidification is protective for the myo-cardial cell in ischemia-reperfusion (20, 21, 34). The present study was undertaken to test these hypotheses at the cellular level. Additionally, we investigated whether the protective effect of NHE inhibition can be reproduced or enhanced by inhibition of another Na ϩdependent H ϩ extrusion system: the Na ϩ /HCO 3 Ϫ symporter (NBS). The role of NBS in the pathophysiology of ischemic myocardium (22) is still poorly understood, and it is unknown if sole inhibition of NBS can be as protective as sole inhibition of NHE. As an experimental model we used isolated ventricular cardiomyocytes from the adult rat heart, which were exposed to a sequence of anoxia at extracellular pH (pH o ) 6.4 (simulated ischemia) and reoxygenation at pH o 7.4 (simulated reperfusion). This model has been characterized in previous studies (33, 35) . It was investigated, first, if the presence during simulated ischemic conditions of one of the chemically distinct NHE inhibitors HOE 642 (32) or 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) alters the development of intracellular acidosis, Na ϩ , or Ca 2ϩ overload. Second, the results were compared for circumstances permitting bicarbonate-dependent sarcolemmal H ϩ transport and for circumstances that do not, i.e., the role of NBS was analyzed. Third, effects of intraischemic NHE inhibition on ischemic cells were differentiated from its effects on reoxygenated cells, specifically on reoxygenation-induced hypercontracture. The last point of this analysis is based on previous investigations on this model, which demonstrated that reoxygenation-induced hypercontracture is elicited by the coincidence of Ca 2ϩ overload, accumulated during ischemic conditions, and recovery of energy production within the reoxygenated cardiomyocyte (34). MATERIALS AND METHODS Isolation of cardiomyocytes. Ventricular heart muscle cells were isolated from 200-to 250-g adult male Wistar rats and plated in medium 199 with 4% fetal calf serum on glass coverslips that had been preincubated overnight with 4% fetal calf serum (28, 29) . Four hours after plating the coverslips, the coverslips were washed with medium 199. As a result of the wash, damaged cells were removed, leaving a homogeneous population of rod-shaped quiescent cardiomyocytes (Ͼ95%) attached to the coverslip. From each isolation,
doi:10.1152/ajpheart.2000.279.5.h2143 pmid:11045947 fatcat:ucpglvlr6vgbzjl46mlp3nyorm