| Aging results in a progressive loss of skeletal muscle, a condition termed sarcopenia which can have significant effects on physical function and quality of life as aging commences. At the cellular level, the aging process can activate stress-associated signal transduction pathways that result in mitochondrial dysfunction and apoptosis. Because the mitochondrion contains its own DNA, a central role for mitochondrial DNA (mtDNA) mutations in mammalian aging has been postulated. In fact, mtDNA mutations have been shown to accumulate with aging in skeletal muscle fibers of various species. The purpose of my dissertation project was to determine whether mtDNA mutations are causal to sarcopenia. The central hypothesis tested was that mutations in mitochondrial DNA, known to be associated with aging in many post mitotic tissues, play a causal role in skeletal muscle loss, possibly by inducing mitochondrial dysfunction, leading to the activation of a mitochondrial-mediated apoptotic program. In order to demonstrate a causal relationship between mtDNA mutations and skeletal muscle loss with age, we used a transgenic mouse model that expresses a proofreading-deficient version of the mitochondrial DNA polymerase gamma (PolgD257A), resulting in increased spontaneous mutation rates in mtDNA. The causal role of mtDNA mutations in mammalian aging is supported in this mouse model by the observation that mice with the PolgD257A (D257A) phenotype develop several aging phenotypes among which, is skeletal muscle loss.;We specifically hypothesized that the accumulation of mtDNA mutations in skeletal muscle will lead to compromised mitochondrial bioenergetics. We found that D257A mice have decreased protein content of complexes I, III and IV, all of which contain subunits encoded by mitochondrial DNA, compared to wild type (WT) mice at 11-mo of age. Mitochondrial dysfunction was also evident in D257A mice by decreased mitochondrial oxygen consumption, lower membrane potential during both state 3 (phosphorylative state) and state 4 (resting state), and lower ATP content. However, this dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage. In fact, we detected a decrease in the rate of H2O 2 production by intact D257A mitochondria and no difference in mtDNA oxidative modification measured by 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), compared to WT. This is in contrast to the mitochondrial "Vicious Cycle" theory of aging which suggests that mtDNA mutations may lead to mitochondrial dysfunction via further increases in mitochondrial ROS production. We further hypothesized that mitochondrial dysfunction will result in mitochondrial-mediated apoptosis, which would be responsible for the loss of skeletal muscle mass we have observed in D257A mice. We detected DNA laddering and an increase in the amount of cytosolic mono- and oligo-nucleosomes in D257A mice compared to WT, indicative of apoptosis. Concurrently, we demonstrated increased activity of both, the initiator caspase-9, and the effector caspase-3, as well as an increase in cleaved (activated) caspase-3 content. This suggests that apoptosis in mutant mice is mitochondrial-mediated, and is conferred upon mitochondrial dysfunction. Thus, mutations in mtDNA play a causal role in sarcopenia, through enhancing apoptosis induced by mitochondrial dysfunction. |
