Isolated Mitochondria from Skeletal Muscle Show Exaggerated Impairments with Aging Compared to Mitochondria from Permeabilized Myofibers

Abstract

PURPOSE: Mitochondria regulate cellular bioenergetics and apoptosis and have been implicated in the pathogenesis of several diseases. However, it is unclear if age-related loss of muscle mass, known as sarcopenia, is associated with abnormal mitochondrial function. Two technically different approaches have mainly been used to measure mitochondrial function: isolated mitochondria (IsoMitos) and permeabilized myofibers (PermMyo) but the utility of these measures in the context of sarcopenia has not been previously assessed. We hypothesized that isolated mitochondria from skeletal muscle would show more subtle impairments with aging compared to permeabilized myofibers, due to selective harvest of the healthiest mitochondria during isolation procedures. METHODS: Using young adult (YA) and senescent (SEN) rats, we prepared IsoMitos and PermMyo from the mixed region of gastrocnemius muscle. Oxidative capacity, mitochondrial reactive oxygen species (ROS) production, and calcium retention capacity (CRC) were measured from both preparations. CRC was used as an indicator of sensitivity to permeability transition pore (PTP) opening, a physiological trigger for apoptosis. Between-group differences were analyzed using T-tests. RESULTS: Muscle mass was 38% lower in SEN gastrocnemius muscle (1.28±0.08 g; mean±SE) compared to YA (2.05±0.09 g). In PermMyo, no age differences were detected in oxidative capacity, ROS production, or CRC, except for a 30% shorter time to opening of the PTP in SEN compared to YA (p<0.05). In contrast, IsoMitos from SEN had a 65% lower oxidative capacity, 58% lower ROS production, 39% lower CRC, and 48% shorter time to PTP opening (all p<0.05). CONCLUSIONS: Contrary to our hypothesis, mitochondria from aged muscles show functional impairments that are exaggerated in IsoMitos preparations compared to PermMyo. The data suggest mitochondrial isolation reveals vulnerabilities in aged mitochondria that are not manifest under conditions where mitochondrial network structure is preserved. Care should be taken in interpreting data on aged muscle from isolated mitochondrial preparations.