The novel inhibitor of the reverse mode of the Na+/Ca2+exchanger (NCE) KB-R7943 (KB) was tested in isolated rat cardiomyocytes exposed to 80 min of simulated ischemia [substrate-free anoxia, extracellular pH (pHo) of 6.4] and 15 min of reoxygenation (pHo 7.4). At pHo 6.4, 20 μmol/l KB was required for complete inhibition of the reverse mode of NCE. Treatment with 20 μmol/l KB only during anoxia did not influence the onset of rigor contracture and intracellular pH (pHi) (monitored with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) but significantly reduced the cytosolic accumulation of Ca2+ (monitored with fura 2) and Na+ (monitored with sodium-binding benzofuran isophthalate). During reoxygenation, cardiomyocytes developed hypercontracture. This was significantly reduced by anoxic KB treatment. To investigate this protection against reoxygenation-induced injury in the whole heart, we exposed Langendorff-perfused rat hearts to 110 min of anoxia (pHo 6.4) and 50 min of reoxygenation (pHo 7.4). Application of 20 μmol/l KB during anoxia significantly reduced the reoxygenation-induced enzyme release. We conclude that KB offers significant protection of cardiomyocytes against Ca2+ and Na+ overload during anoxia and hypercontracture or enzyme release on reoxygenation.
1,2DOG-pretreatment delayed the time to extreme ATP depletion, but had no effect on lactate production and cytosolic pH. The accumulation of cytosolic Ca2+ was markedly accelerated in pretreated cells that developed rigor shortening, but reoxygenation-induced hypercontracture was significantly reduced. 1,2DOG, therefore, completely abolished Ca(2+)-dependence of hypercontracture. The effects of pretreatment were fully abolished with 1 mumol/l bisindolylmalcimide (PKC inhibitor). We conclude that PKC preactivation leads to (1) reduction of energy demand, (2) acceleration of Ca2+ overload during anoxia and (3) prevention of reoxygenation-induced hypercontracture independent of anoxic changes in cytosolic Ca2+ and pH.
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