Bnip3 is a pro-apoptotic BH3-only protein which is associated with mitochondrial 
dysfunction and cell death. Bnip3 is also a potent inducer of autophagy in many 
cells. In this study, we have investigated the mechanism by which Bnip3 induces 
autophagy in adult cardiac myocytes. Overexpression of Bnip3 induced extensive 
autophagy in adult cardiac myocytes. Fluorescent microscopy studies and 
ultrastructural analysis revealed selective degradation of mitochondria by 
autophagy in myocytes overexpressing Bnip3. Oxidative stress and increased 
levels of intracellular Ca(2+) have been reported by others to induce autophagy, 
but Bnip3-induced autophagy was not abolished by antioxidant treatment or the 
Ca(2+) chelator BAPT A-AM. We also investigated the role of the mitochondrial 
permeability transition pore (mPTP) in Bnip3-induced autophagy. Although the 
mPTP has previously been implicated in the induction of autophagy and selective 
removal of damaged mitochondria by autophagosomes, mitochondria sequestered by 
autophagosomes in Bnip3-treated cardiac myocytes had not undergone permeability 
transition and treatment with the mPTP inhibitor cyclosporine A did not inhibit 
mitochondrial autophagy in cardiac myocytes. Moreover, cyclophilin D (cypD) is 
an essential component of the mPTP and Bnip3 induced autophagy to the same 
extent in embryonic fibroblasts isolated from wild-type and cypD-deficient mice. 
These results support a model where Bnip3 induces selective removal of the 
mitochondria in cardiac myocytes and that Bnip3 triggers induction of autophagy 
independent of Ca(2+), ROS generation, and mPTP opening.