Dimethylthiourea, an oxygen radical scavenger, protects isolated cardiac myocytes from hypoxic injury by inhibition of Na+-Ca2+ exchange and not by its antioxidant effects

Abstract

Myocardial reoxygenation injury may be attenuated by oxygen free radical scavengers, arguing for a role of oxygen radicals in this process. To determine whether free radical scavengers affect reoxygenation injury in isolated cardiac myocytes, resting rat ventricular myocytes were exposed to hypoxic (Po2 <0.02 mm Hg) glucose-free buffer alone (n=50) or with the addition of the oxygen radical scavengers 1,3-dimethyl-2-thiourea (DMTU, 25 niM, n=46), human recombinant superoxide dismutase (SOD, 1,000 units/ml, n=40), or the combination of these agents (n=41). All cells responded by undergoing contracture to a rigor form. Hypoxia was then continued for a second period (T2), the duration of which correlates inversely with survival. After reoxygenation, cells either retained their rectangular shape (survival) or hypercontraded to a rounded form (death). For the group of cells with a T2 period >30 minutes, no cell exposed to buffer alone (n=20) or to SOD (n=16) survived, in contrast to 15 of 24 (63%) cells exposed to DMTU. The addition of SOD to DMTU offered no advantage to DMTU alone. The protective effect of DMTU was not observed when it was added at reoxygenation, suggesting that this agent has an important effect during the hypoxic period when intracellular Ca2+ is known to rise, most likely because of the reversal of Na+-Ca2+ exchange. Therefore, the effects of DMTU on Ca2+ regulation (indexed by indo-1 fluorescence) during hypoxia were studied. DMTU significantly blunted the [Ca2+] rise during the hypoxic period. When normoxic, electrically stimulated cells were exposed to this agent, they displayed a progressive rise in diastolic [Ca2+], an increase in the amplitude of the Ca2+ transient, and a parallel increase in contractility. These findings could be explained by inhibition of Na+-Ca2+ exchange. To test the hypothesis that DMTU inhibits Na+-Ca2+ exchange, myocyte Ca2+ loading via the exchanger was induced by exposing cells to normoxic buffer with Na+ fully replaced by choline. Cells exposed in this fashion displayed an intracellular [Ca2+] rise that was nearly abolished by DMTU, consistent with pharmacological inhibition of the exchanger. We conclude that Na+-Ca2+ exchange inhibition is responsible for an important part of the effect of DMTU on prevention of hypoxic injury of isolated cardiac myocytes. Although free radical scavenging may play a more important role in the intact heart than in isolated myocytes, the establishment of the role of DMTU as an inhibitor of the Na+-Ca2+ exchanger suggests that previous reports of improved postischemic myocardial function with DMTU attributed to free radical scavenging should be interpreted cautiously.