Loss of cardiac mitochondrial function with age may cause increased cardiomyocyte death through mitochondria-mediated release of apoptogenic factors. We investigated ventricular subsarcolemmal (SSM) and interfibrillar (IFM) mitochondrial bioenergetics and susceptibility towards Ca(2+)-induced permeability transition pore (mPTP) opening with aging and lifelong calorie restriction (CR). Cardiac mitochondria were isolated from 8-, 18-, 29- and 37-month-old male Fischer 344 x Brown Norway rats fed either ad libitum (AL) or 40% calorie restricted diets. With age, H(2)O(2) generation did not increase and oxygen consumption did not significantly decrease in either SSM or IFM. Strikingly, IFM displayed an increased susceptibility towards mPTP opening during senescence. In contrast, Ca(2+) retention capacity of SSM was not affected by age, but SSM tolerated much less Ca(2+) than IFM. Only modest age-dependent increases in cytosolic caspase activities and cytochrome c levels were observed and were not affected by CR. Levels of putative mPTP-modulating components: cyclophilin-D, the adenine nucleotide translocase (ANT), and the voltage-dependent ion channel (VDAC) were not affected by aging or CR. In summary, the age-related reduction of Ca(2+) retention capacity in IFM may explain the increased susceptibility to stress-induced cell death in the aged myocardium.