Mitochondrial Modulation of Amplified Preconditioning Influences of Remote Ischemia Plus Erythropoietin Against Skeletal Ischemia/Reperfusion Injury in Rats

Abstract

<b>Abstract ID 18929</b> <b>Poster Board 421</b> Skeletal muscle ischemia and reperfusion (S-I/R) is frequently associated with aggravated inflammatory response and tissue injury that could be relieved by interventions like remote ischemic preconditioning (RIPC) or the hormonal growth factor erythropoietin (EPO). Here, we tested the hypotheses that (i) the simultaneous exposure to a low dose of EPO boosts the protection conferred by RIPC against S-I/R injury, and (ii) this privileged EPO/RIPC effect is prompted by mitochondrial and extra-mitochondrial machineries. The effects of RIPC, EPO (500 or 5000 IU/kg), or combined RIPC/EPO-500 regimen on S-I/R injury induced by right hindlimb 3-hr ischemia for followed by 3-hr reperfusion were investigated. RIPC was induced by 3 brief consecutive I/R cycles (10-min ischemia and 10-min reperfusion each) of the contralateral hindlimb. S-I/R injury was verified by the (i) rise in serum lactate dehydrogenase, pyruvate, and creatine kinase, (ii) histopathological signs of sarcoplasm vacuolations, segmental necrosis, and inflammatory cells infiltration, and (iii) increments and decrements in electromyographic amplitude and duration, respectively. These defects were partially ameliorated by RIPC or by EPO in a dose-related fashion. Further, greater repairs of functional/structural anomalies of S-I/R were seen in rats pre-exposed to the combined RIPC/EPO-500 intervention. The latter therapy also caused more effective preservation of the number of MitoTracker red-stained mitochondria assessed by confocal microscopy and suppression of mitochondrial DNA damage and indices of oxidative stress and apoptosis like cardiolipin, succinate dehydrogenase, citrate synthase, and cytochrome c. Additionally the combined RIPC/EPO therapy was more influential than individual treatments in ameliorating rises in muscular myeloperoxidase and nitric oxide metabolites and preventing excessive calcium accumulation and glycogen consumption. Overall, the dual RIPC/EPO therapy additively mends mitochondrial dysfunction and interrelated skeletal structural and functional deficits induced by S-I/R injury. The use of relatively low EPO doses might serve to minimize adverse effects often encountered with the clinical use of the hormone