H. Albadawi1, A. A. Tzika3, J. D. Long1, G. M. Cach1, H. Yoo1, W. G. Austen2, M. T. Watkins1 1Massachusetts General Hospital,Department Of Surgery, Division Of Vascular And Endovascular Surgery,Boston, MA, USA 2Massachusetts General Hospital,Plastic And Reconstructive Surgery,Boston, MA, USA 3Massachusetts General Hospital And Shriners Burns Institute,NMR Surgical Laboratory,Boston, MA, USA
Introduction: Skeletal muscle necrosis following an acute ischemic event may lead to a limb threatening complication in the lower extremity. Mitochondrial dysfunction is a component of ischemic muscle necrosis, which leads to metabolic deficit and a subsequent inflammatory response during reperfusion. Recent studies have reported that targeting mitochondria with d-Arg-2′, 6′-dimethyltyrosine-Lys-Phe-NH2 peptide (SS-31) resets mitochondrial function, reduces apoptosis, and protects against tissue injury in models of acute renal, cardiac, and cerebral ischemia. Experiments were undertaken to assess whether SS-31 treatment can ameliorate myofiber injury and reduce metabolic deficit in a mouse model of acute hind limb ischemia reperfusion injury (IR).
Methods: Two groups of C57BL6 mice were subjected to 1.5 hours of hind limb tourniquet ischemia followed by 72 hours of reperfusion. Mice received intraperitoneal injections of either 3 mg/kg SS-31 (n=8) or normal saline (n=8) at 5 minutes before ischemia, 5 minutes before reperfusion, and at 2, 4, 6, 24, 48 and 70 hours following reperfusion. Assessment of ischemic limb function was serially performed using a rodent scoring system. After 72 hours of reperfusion, hind limb muscles were harvested for quantitative analysis of myofiber injury (histologic assessment), infiltrating leukocytes (Ly6b immunohistochemistry), ATP levels (chemiluminescent assay), pro-apoptotic marker (cleaved caspase-3 by western blot), and expression of the proinflammatory chemokine-keratinocyte chemoattractant (KC) protein (ELISA). Data were analyzed using a Student t-test.
Results: At 72 hours following IR, SS-31 treated mice had a greater recovery of limb function when compared to the saline group (p=.008), which corresponded with reduced myofiber injury (47±12 vs. 85±5 percent injured fibers per field, p=.049) and enhanced steady state levels of ATP (12±2.4 vs. 3±1 pmol/µg protein, p=.001). Furthermore, SS-31 treatment correlated with diminished inflammatory cell infiltration (35±23 vs. 115±13, average counted Ly6B+ leukocytes per field, p=.006), lower KC protein content (6.6±0.8 vs. 13.5±2 pg/mg protein, p=.001) and decreased caspase-3 activation (1.7±0.3 vs. 4.8±1 AU, p=.002) in skeletal muscle tissues.
Conclusion: Our results demonstrated that SS-31 treatment is protective against myofiber necrosis following acute IR. This effect may be attributed to the preservation of mitochondrial function and the modulation of apoptotic and pro-inflammatory responses. SS-31 may be useful as a therapeutic adjunct for limb salvage in patients following revascularization.