Dissecting apoptosis and intrinsic death pathways in the heart.

The limited regenerative capacity of postnatal ventricular myocytes coupled with their meager ability for genetic manipulation has presented a major technical obstacle for deciphering apoptosis initiation and execution signals in the heart. In this report, we describe the technical approaches used to study the intrinsic death pathways in postnatal ventricular myocytes during acute hypoxic injury. Discussed are methods for hypoxia, recombinant adenovirus-mediated gene transfer, cellular viability assays using the vital dyes calcein acetomethoxyester and ethidium homodimer-1, analysis of nuclear morphology by use of Hoechst dye 33258, and assessment of the state of the mitochondrial permeability transition pore. Our work has established that hypoxia triggers perturbations to mitochondria consistent with loss of mitochondrial membrane potential, permeability transition pore opening, and apoptotic cell death by the intrinsic pathway.