Delayed recovery of intracellular acidosis during reperfusion prevents calpain activation and determines protection in postconditioned myocardium.
OBJECTIVE
Indirect data suggest that delayed recovery of intracellular pH (pHi) during reperfusion is involved in postconditioning protection, and calpain activity has been shown to be pH-dependent. We sought to characterize the effect of ischaemic postconditioning on pHi recovery during reperfusion and on calpain-dependent proteolysis, an important mechanism of myocardial reperfusion injury.


RESULTS
Isolated Sprague-Dawley rat hearts were submitted to 40 min of ischaemia and different reperfusion protocols of postconditioning and acidosis. pHi was monitored by (31)P-NMR spectroscopy. Myocardial cell death was determined by lactate dehydrogenase (LDH) and triphenyltetrazolium staining, and calpain activity by western blot measurement of alpha-fodrin degradation. In control hearts, pHi recovered within 1.5 +/- 0.24 min of reperfusion. Postconditioning with 6 cycles of 10 s ischaemia-reperfusion delayed pHi recovery slightly to 2.5 +/- 0.2 min and failed to prevent calpain-mediated alpha-fodrin degradation or to elicit protection. Lowering perfusion flow to 50% during reperfusion cycles or shortening the cycles (12 cycles of 5 s ischemia-reperfusion) resulted in a further delay in pHi recovery (4.1 +/- 0.2 and 3.5 +/- 0.3 min, respectively), attenuated alpha-fodrin proteolysis, improved functional recovery, and reduced LDH release (47 and 38%, respectively, P < 0.001) and infarct size (36 and 32%, respectively, P < 0.001). This cardioprotection was identical to that produced by lowering the pH of the perfusion buffer to 6.4 during the first 2 min of reperfusion or by calpain inhibition with MDL-28170.


CONCLUSIONS
These results provide direct evidence that postconditioning protection depends on prolongation of intracellular acidosis during reperfusion and indicate that inhibited calpain activity could contribute to this protection.
Inserte
Javier
J
Laboratorio de Cardiología Experimental, Servicio de Cardiología, Hospital Universitari Vall d'Hebron, Passeig Vall d'Hebron, 119-129, 08035 Barcelona, Spain.
Barba
Ignasi
I
Hernando
Víctor
V
Garcia-Dorado
David
D
D000138:Acidosis / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis; D002154:Calpain / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D004195:Disease Models, Animal; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism; D015428:Myocardial Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D010725:Phosphocreatine / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013049:Spectrin
D002154:Calpain; D002352:Carrier Proteins; D007770:L-Lactate Dehydrogenase; D008840:Microfilament Proteins; D010725:Phosphocreatine; D013049:Spectrin