DIMETHYLTHIOUREA, AN OXYGEN RADICAL SCAVENGER, PROTECTS ISOLATED CARDIAC MYOCYTES FROM HYPOXIC INJURY BY INHIBITION OF NA-CA2+ EXCHANGE ANDNOT BY ITS ANTIOXIDANT EFFECTS()

Myocardial reoxygenation injury may be attenuated by oxygen free radic al scavengers, arguing for a role of oxygen radicals in this process. To determine whether free radical scavengers affect reoxygenation inju ry 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 mM, n = 46), human recombinant superoxide dismut ase (SOD, 1,000 units/ml, n = 40), or the combination of these agents (n = 41). All cells responded by undergoing contracture to a rigor for m. Hypoxia was then continued for a second period (T2), the duration o f which correlates inversely with survival. After reoxygenation, cells either retained their rectangular shape (survival) or hypercontracted 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 a ddition of SOD to DMTU offered no advantage to DMTU alone. The protect ive effect of DMTU was not observed when it was added at reoxygenation , suggesting that this agent has an important effect during the hypoxi c 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 wer e studied. DMTU significantly blunted the [Ca2+] rise during the hypox ic period. When normoxic, electrically stimulated cells were exposed t o this agent, they displayed a progressive rise in diastolic [Ca2+], a n increase in the amplitude of the Ca2+ transient, and a parallel incr ease in contractility. These findings could be explained by inhibition of Na+-Ca2+ exchange. To test the hypothesis that DMTU inhibits Na+-C a2+ exchange, myocyte Ca2+ loading via the exchanger was induced by ex posing cells to normoxic buffer with Na+ fully replaced by choline. Ce lls exposed in this fashion displayed an intracellular [Ca2+] rise tha t was nearly abolished by DMTU, consistent with pharmacological inhibi tion of the exchanger. We conclude that Na+-Ca2+ exchange inhibition i s responsible for an important part of the effect of DMTU on preventio n of hypoxic injury of isolated cardiac myocytes. Although free radica l scavenging may play a more important role in the intact heart than i n isolated myocytes, the establishment of the role of DMTU as an inhib itor of the Na+-Ca2+ exchanger suggests that previous reports of impro ved postischemic myocardial function with DMTU attributed to free radi cal scavenging should be interpreted cautiously.