Mitochondrial modulation of amplified preconditioning influences of remote ischemia plus erythropoietin against skeletal muscle ischemia/reperfusion injury in rats

Abstract

Aims: Skeletal muscle ischemia and reperfusion (S-I/R) injury is relieved by interventions like remote ischemic preconditioning (RIPC). Here, we tested the hypothesis that simultaneous exposure to a minimal dose of erythropoietin (EPO) boosts the protection conferred by RIPC against S-I/R injury and concomitant mitochondrial oxidative and apoptotic defects.

Main methods: S-I/R injury was induced in rats by 3-h right hindlimb ischemia followed by 3-h of reperfusion, whereas RIPC involved 3 brief consecutive I/R cycles of the contralateral hindlimb.

Key findings: S-I/R injury caused (i) rises in serum lactate dehydrogenase and creatine kinase and falls in serum pyruvate, (ii) structural deformities like sarcoplasm vacuolations, segmental necrosis, and inflammatory cells infiltration, and (iii) decreased amplitude and increased duration of electromyography action potentials. These defects were partially ameliorated by RIPC and dose-dependently by EPO (500 or 5000 IU/kg). Further, greater repairs of S-I/R-evoked damages were seen after prior exposure to the combined RIPC/EPO-500 intervention. The latter also caused more effective (i) preservation of mitochondrial number (confocal microscopy assessed Mitotracker red staining) and function (citrate synthase activity), (ii) suppression of mitochondrial DNA damage and indices of oxidative stress and apoptosis (succinate dehydrogenase, myeloperoxidase, cardiolipin, and cytochrome c), (iii) preventing calcium and nitric oxide metabolites (NOx) accumulation and glycogen consumption, and (iv) upregulating EPO receptors (EPO-R) gene expression.

Significance: dual RIPC/EPO conditioning exceptionally mends structural, functional, and neuronal deficits caused by I/R injury and interrelated mitochondrial oxidative and apoptotic damage. Clinically, the utilization of relatively low EPO doses could minimize the hormone-related adverse effects.

Keywords: Erythropoietin; Gastrocnemius muscle; Ischemia-reperfusion injury; Mitochondria; Remote preconditioning.