SS-31: A Mitochondrial-Specific Therapy that Provides Significant Protection against Ischemia-Reperfusion Injury in Muscle In Vitro and In Vivo
Nicola Bavinck, BA, Peter W. Henderson, MD MBA, Natalia Jimenez, BA, Yoann H. Millet, BA, Hazel H. Szeto, MD PhD, Jason A. Spector, MD FACS.
Weill Cornell Medical College, New York, NY, USA.
BACKGROUND: Every free tissue transfer procedure has an obligatory period of flap ischemia. Subsequent reperfusion has been clearly shown to have the potential to lead to multi-factorial tissue injury, collectively referred to as ischemia-reperfusion injury (IRI). SS-31 is a synthetic cell-permeable tetrapeptide that targets and concentrates in the mitochondrial inner membrane, where it inhibits opening of the mitochondrial transition pore, thus preventing the deleterious calcium ion flux, mitochondrial swelling, and subsequent cellular apoptosis. SS-31 is readily absorbed after subcutaneous administration and has been shown to inhibit myocardial, cerebral and renal IRI. Given this background, we sought to determine whether SS-31 would protect skeletal muscle and against the deleterious effects of IRI both in vitro and in vivo.
METHODS: In vitro, myotubes were exposed to media containing either 0 or 10nM SS-31. Twenty minutes later, the cells underwent 5 hours of anoxia (0% O2) in a custom-made anoxic chamber. After the anoxic period, the cells were placed in a standard incubator (21% O2) for 3 hours. Once the anoxia-normoxia cycle was completed, the cells were subjected to a TUNEL assay, and an apoptotic index (AI) was determined for each group. In vivo, 6 mice were divided into 2 groups; one group received an intravenous injection of saline, and the other group received an injection of 3mg/kg SS-31. Twenty minutes later, all mice underwent 3 hours of tourniquet-induced hindlimb ischemia. After the ischemic period, the tourniquet was removed and the hindlimbs were allowed to reperfuse for 3 hours. At the end of the ischemia-reperfusion cycle, the mice were sacrificed, the bilateral gastrocnemius and soleus muscles were harvested, processed for histology, and stained with hematoxylin and eosin (H&E) in order to evaluate the cellular architecture. There was no sign of cellular injury in SS-31-treated, non-ischemic tissue.
RESULTS: In vitro, myotubes that were not treated with SS-31 had an AI of 6.9% +/- 1.3%, while myotubes that were treated with SS-31 had an AI of 0.5% +/- 0.1% (p<0.001). In vivo, tissue from mice that were not treated with SS-31 showed a distinct loss of normal cellular architecture, with membranous discontinuity, intracellular vacuolization, and extracellular edema (Figure). Tissue from mice that had been treated with SS-31 showed a relative preservation of normal cellular architecture, closely resembling the non-ischemic tissue.
CONCLUSIONS: The results of this study indicate that pharmacologic preconditioning with SS-31 protects and skeletal muscle cells in both in vitro and in vivo models of IRI. Because of its ease of delivery and lack of toxicity, we believe SS-31 holds tremendous promise for use in the setting of anticipated ischemic intervals such as free tissue transfer, solid organ transplantation, and many other medical and surgical procedures.
Back to Program