Inhibition of cytochrome c release by 10 - N - nonyl acridine orange , a 1 cardiolipin - specific dye , during myocardial ischemia / reperfusion in the rat 2 3

26 Release of cytochrome c from mitochondria to the cytosol is a critical step for 27 downstream caspase-mediated apoptotic signal transduction in ischemia/reperfusion 28 (I/R)-induced myocardial tissue injury. 10-N-nonyl acridine orange (NAO), a 29 cardiolipin-specific dye, has been shown to inhibit Bid-mediated cytochrome c release 30 from isolated mitochondria in vitro; however, the possible protective effects of NAO 31 and the mechanisms underlying protection from myocardial I/R-induced tissue injury in 32 a rat model are unknown. Male Sprague-Dawley rats were subjected to a 30-min 33 coronary arterial occlusion followed by reperfusion. All rats received either vehicle or 34 NAO (100 μg/kg, i.v.) 10 min before the occlusion. The infarct size in the heart at 24 h 35 after reperfusion was significantly reduced in NAO-treated rats compared to that in 36 vehicle-treated rats. NAO treatment significantly reduced the cytosolic cytochrome c 37 contents and caspase-9 activity in the ischemic region, but did not affect caspase-8 38 activity. Furthermore, NAO treatment markedly suppressed the translocation of 39 truncated Bid, a pro-apoptotic Bcl-2 family member, to the mitochondrial fraction. 40 NAO also suppressed the mitochondrial swelling and oxygen uptake stimulated by 41 calcium overloading. The results suggest that NAO possesses protective effects against 42 myocardial I/R injury, which may be due to suppression of cytochrome c release 43 through blockade of truncated Bid translocation to mitochondria and inhibition of the 44 opening of mitochondrial permeability transition pores. 45 46