Sharon L. Rowan

Mitochondrial functional impairment with aging is exaggerated in isolated mitochondria compared to permeabilized myofibers

Mitochondria regulate cellular bioenergetics and apoptosis and have been implicated in aging. However, it remains unclear whether 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 and permeabilized myofiber bundles, but the reliability of these measures in the context of sarcopenia has not been systematically assessed before. A key difference between these approaches is that contrary to isolated mitochondria, permeabilized bundles contain the totality of fiber mitochondria where normal mitochondrial morphology and intracellular interactions are preserved. Using the gastrocnemius muscle from young adult and senescent rats, we show marked effects of aging on three primary indices of mitochondrial function (respiration, H2O2 emission, sensitivity of permeability transition pore to Ca2+) when measured in isolated mitochondria, but to a much lesser degree when measured in permeabilized bundles. Our results clearly demonstrate that mitochondrial isolation procedures typically employed to study aged muscles expose functional impairments not seen in situ. We conclude that aging is associated with more modest changes in mitochondrial function in sarcopenic muscle than suggested previously from isolated organelle studies.

Denervation Causes Fiber Atrophy and Myosin Heavy Chain Co-Expression in Senescent Skeletal Muscle

Although denervation has long been implicated in aging muscle, the degree to which it is causes the fiber atrophy seen in aging muscle is unknown. To address this question, we quantified motoneuron soma counts in the lumbar spinal cord using choline acetyl transferase immunhistochemistry and quantified the size of denervated versus innervated muscle fibers in the gastrocnemius muscle using the in situ expression of the denervation-specific sodium channel, Nav1.5, in young adult (YA) and senescent (SEN) rats. To gain insights into the mechanisms driving myofiber atrophy, we also examined the myofiber expression of the two primary ubiquitin ligases necessary for muscle atrophy (MAFbx, MuRF1). MN soma number in lumbar spinal cord declined 27% between YA (638±34 MNs×mm−1) and SEN (469±13 MNs×mm−1). Nav1.5 positive fibers (1548±70 μm2) were 35% smaller than Nav1.5 negative fibers (2367±78 μm2; P<0.05) in SEN muscle, whereas Nav1.5 negative fibers in SEN were only 7% smaller than fibers in YA (2553±33 μm2; P<0.05) where no Nav1.5 labeling was seen, suggesting denervation is the primary cause of aging myofiber atrophy. Nav1.5 positive fibers had higher levels of MAFbx and MuRF1 (P<0.05), consistent with involvement of the proteasome proteolytic pathway in the atrophy of denervated muscle fibers in aging muscle. In summary, our study provides the first quantitative assessment of the contribution of denervation to myofiber atrophy in aging muscle, suggesting it explains the majority of the atrophy we observed. This striking result suggests a renewed focus should be placed on denervation in seeking understanding of the causes of and treatments for aging muscle atrophy.

Accumulation of severely atrophic myofibers marks the acceleration of sarcopenia in slow and fast twitch muscles

The age-related decline in muscle mass, known as sarcopenia, exhibits a marked acceleration in advanced age. Although many studies have remarked upon the accumulation of very small myofibers, particularly at advanced stages of sarcopenia, the significance of this phenomenon in the acceleration of sarcopenia has never been examined. Furthermore, although mitochondrial dysfunction characterized by a lack of cytochrome oxidase (COX) activity has been implicated in myofiber atrophy in sarcopenia, the contribution of this phenotype to the accumulation of severely atrophied fibers in aged muscles has never been determined. To this end, we examined the fiber size distribution in the slow twitch soleus (Sol) and fast twitch gastrocnemius (Gas) muscles between young adulthood (YA) and senescence (SEN). We also quantified the abundance of COX deficient myocytes and their size attributes to gain insight into the contribution of this phenotype to myofiber atrophy with aging. Our data showed that the progression of muscle atrophy, particularly its striking acceleration between late middle age and SEN, was paralleled by an accumulation of severely atrophic myofibers (≤ 1000 μm(2) in size) in both Sol and Gas. On the other hand, we observed no COX deficient myofibers in Sol, despite nearly 20% of the myofibers being severely atrophic. Similarly, only 0.17 ± 0.06% of all fibers in Gas were COX deficient, and their size was generally larger (2375 ± 319 μm(2)) than the severely atrophied myofibers noted above. Collectively, our results suggest that similar processes likely contribute to the acceleration of sarcopenia in both slow twitch and fast twitch muscles, and that COX deficiency is not a major contributor to this phenomenon.

Slow twitch soleus muscle is not protected from sarcopenia in senescent rats

Although most literature suggests a relative protection of slow twitch muscle with aging, there is limited data in senescence when muscle atrophy and functional decline markedly accelerate. To address this issue we examined age-related changes in muscle mass, contractile function, mitochondrial enzyme activities, and myosin heavy chain (MHC) expression in the slow twitch soleus (Sol) and fast twitch gastrocnemius (Gas) muscle of young adult (YA) and senescent (SEN) rats. Muscle mass declined between YA and SEN in the Sol by 35% compared to 55% in the Gas muscle. After normalizing for muscle mass, tetanic force per g of muscle declined by 58% in Sol and by 36% in Gas muscle. Time-to-peak tension was increased only in the Gas (30%), whereas time-to-half relaxation was increased by 70% in Sol and 51% in Gas. Citrate synthase and complex IV activity declined in homogenates of Sol (30-36%) and red oxidative region of Gas (46-51%), but not white glycolytic region of Gas. Strikingly, the shift away from the dominant adult MHC isoform with aging was much greater in Sol (fibers positive for MHC fast: 11+/-2% in YA versus 36+/-3% in SEN) than in Gas (fibers positive for MHC slow: 12+/-1% in YA versus 26+/-3% in SEN) muscle. Collectively, these results show that the slow twitch Sol muscle undergoes large phenotypic alterations in very old age and for several measures (tetanic tension per g, time-to-half relaxation and shift in adult MHC expression) that is of greater magnitude than fast twitch muscle, underscoring the importance of including age-related changes in slow twitch muscle in seeking potential treatments for sarcopenia.

Anatomic capillarization is elevated in the medial gastrocnemius muscle of mighty mini mice.

House mice selectively bred for high voluntary wheel running display a mini-muscle (MM) phenotype wherein mass-specific mitochondrial enzyme activities are double that of normal, but muscle mass is reduced by half. In addition, mini-muscles are characterized by small muscle fibers in the superficial region of the plantaris and medial gastrocnemius muscles. To determine the structural alterations facilitating aerobic metabolism in these mini-muscles, cross-sections of the medial gastrocnemius muscle of normal (N; n = 6) and mini-muscle (MM; n = 6) mice were histo- and immunochemically labeled and analyzed for fiber size, capillarization, and fiber type. On the basis of the higher mitochondrial enzyme activities in muscles of MM mice, we hypothesized that they would have greater fiber capillarization in the medial gastrocnemius than N mice. Furthermore, we hypothesized that augmented capillarization in MM would principally be a function of the smaller fibers in the superficial aspect of this muscle. On average, MM had higher capillary-to-fiber ratio and higher capillary density. Binning fibers according to size revealed that it was primarily the normal-sized fibers of the MM that had higher capillarity. The small fibers seen in the superficial region of MM were distinct from N mice in that they had heterogeneous myofibrillar ATPase staining and patchy succinate dehydrogenase staining in the interior of the fibers. These results support the hypothesis that the MM have higher indexes of capillarity, caused primarily by greater capillary number around normally sized fibers. These alterations are consistent with the superior mass-specific aerobic function of these muscles.

Severe atrophy of slow myofibers in aging muscle is concealed by myosin heavy chain co-expression.

Although slow myofibers are considered less susceptible to atrophy with aging, slow fiber atrophy may have been underestimated previously. First, the marked atrophy of the aging rat soleus (Sol) muscle cannot be explained by the atrophy of only the fast fibers, due to their low abundance. Second, the increase in small fibers co-expressing both fast and slow myosin heavy chains (MHC) in the aging rat Sol is proportional to a decline in pure MHC slow fibers (Snow et al., 2005), suggesting that these MHC co-expressing fibers represent formerly pure slow fibers. Thus, we examined the size and proportion of MHC slow, MHC fast, and MHC fast-slow co-expressing fibers in the Sol and mixed region of the gastrocnemius (Gas) muscle in young adult (YA) and senescent (SEN) rats. Our results suggest that formerly pure MHC slow fibers are the source of MHC co-expressing fibers with aging in both muscle regions. Accounting for the atrophy of these fibers in calculating MHC slow fiber atrophy with aging revealed that MHC slow fibers atrophy on average by 40% in the Sol and by 38% in the mixed Gas, values which are similar to the 60% and 31% atrophy of pure MHC fast fibers in the Sol and mixed Gas, respectively. Probing for the atrophy-dependent ubiquitin ligase, MAFbx (atrogin 1), it was suggested that former slow fibers acquire atrophy potential via the up-regulation of MAFbx coincident with the co-expression of fast MHC. These results redefine the impact of aging on slow fiber atrophy, and emphasize the necessity of addressing the atrophy of fast and slow fibers in seeking treatments for aging muscle atrophy.