Michael F. N. O'Leary

Adaptive plasticity of autophagic proteins to denervation in aging skeletal muscle

Aging muscle exhibits a progressive decline in mass and strength, known as sarcopenia, and a decrease in the adaptive response to contractile activity. The molecular mechanisms mediating this reduced plasticity have yet to be elucidated. The purposes of this study were 1) to determine whether denervation-induced muscle disuse would increase the expression of autophagy genes and 2) to examine whether selective autophagy pathways (mitophagy) are altered in aged animals. Denervation reduced muscle mass in young and aged animals by 24 and 16%, respectively. Moreover, young animals showed a 50% decrease in mitochondrial content following denervation, an adaptation that was not matched by aged animals. Basal autophagy protein expression was higher in aged animals, whereas young animals exhibited a greater induction of autophagy proteins following denervation. Localization of LC3II, Parkin, and p62 was significantly increased in the mitochondrial fraction of young and aged animals following denervation. Moreover, the unfolded protein response marker CHOP and the mitochondrial dynamics protein Fis1 were increased by 17- and 2.5-fold, respectively, in aged animals. Lipofuscin granules within lysosomes were evident with aging and denervation. Thus reductions in the adaptive plasticity of aged muscle are associated with decreases in disuse-induced autophagy. These data indicate that the expression of autophagy proteins and their localization to mitochondria are not decreased in aged muscle; however, the induction of autophagy in response to disuse, along with downstream events such as lysosome function, is impaired. This may contribute to an accumulation of dysfunctional mitochondria in aged muscle.

Relationship between Sirt1 expression and mitochondrial proteins during conditions of chronic muscle use and disuse

Sirt1 is a NAD(+)-dependent histone deacetylase that interacts with the regulatory protein of mitochondrial biogenesis PGC-1alpha and is sensitive to metabolic alterations. We assessed whether a strict relationship between the expression of Sirt1, mitochondrial proteins, and PGC-1alpha existed across tissues possessing a wide range of oxidative capabilities, as well as in skeletal muscle subject to chronic use (voluntary wheel running or electrical stimulation for 7 days, 10 Hz; 3 h/day) or disuse (denervation for up to 21 days) in which organelle biogenesis is altered. PGC-1alpha levels were not closely associated with the expression of Sirt1, measured using immunoblotting or via enzymatic deacetylase activity. The mitochondrial protein cytochrome c increased by 70-90% in soleus and plantaris muscles of running animals, whereas Sirt1 activity remained unchanged. In chronically stimulated muscle, cytochrome c was increased by 30% compared with nonstimulated muscle, whereas Sirt1 activity was increased modestly by 20-25%. In contrast, in denervated muscle, these markers of mitochondrial content were decreased by 30-50% compared with the control muscle, whereas Sirt1 activity was increased by 75-80%. Our data suggest that Sirt1 and PGC-1alpha expression are independently regulated and that, although Sirt1 activity may be involved in mitochondrial biogenesis, its expression is not closely correlated to changes in mitochondrial proteins during conditions of chronic muscle use and disuse.

Mitochondria in skeletal muscle: adaptable rheostats of apoptotic susceptibility.

Apoptosis is an essential process that plays a critical role in both tissue development and maintenance. Apoptosis has been shown to be involved in skeletal muscle atrophy resulting from chronic muscular disuse, sarcopenia, and mitochondrial myopathies. Exercise may attenuate some of the proapoptotic adaptations that occur during these conditions. This review will focus on the factors influencing mitochondrially mediated apoptosis in skeletal muscle.

Effect of prior chronic contractile activity on mitochondrial function and apoptotic protein expression in denervated muscle

Skeletal muscle is highly adaptable in response to increases and decreases in contractile activity. The purpose of this study was to determine whether the preconditioning of skeletal muscle has a protective effect against subsequent denervation-induced apoptotic protein expression. To investigate this, we chronically stimulated the tibialis anterior and extensor digitorum longus muscles for 7 days (10 Hz, 3 h/day) before 7 days of denervation. Denervation reduced total cytochrome-c oxidase activity by 39%, which was likely a consequence of a decrease in subsarcolemmal (SS) mitochondria. This decrease in the SS subfraction was prevented by prior chronic stimulation and, as a result, maintained total mitochondrial content at control levels. The expression of Bax was elevated 2.2-fold by denervation, and prior chronic stimulation did not attenuate this increase. This produced a increase in the Bax-to-Bcl-2 ratio, indicating greater muscle apoptotic susceptibility. Denervation also decreased state 3 respiration in SS and intermyofibrillar mitochondria and elevated state 4 reactive oxygen species production within both mitochondrial subfractions. These changes were not prevented by prior chronic stimulation. Furthermore, the antioxidant protein MnSOD was also reduced by denervation, whereas Beclin-1 was markedly elevated. This suggests that autophagic cell death could also play a significant part in denervation-induced muscle atrophy. Thus, despite prior chronic stimulation, denervation increases the apoptotic susceptibility of skeletal muscle by altering the Bax-to-Bcl-2 ratio, by increasing reactive oxygen species production, and by reducing the expression of MnSOD. Whether a more extensive stimulation paradigm would be more effective in attenuating apoptosis before muscle disuse remains to be determined.

Altered mitochondrial morphology and defective protein import reveal novel roles for Bax and/or Bak in skeletal muscle

The function Bax and/or Bak in constituting a gateway for mitochondrial apoptosis in response to apoptotic stimuli has been unequivocally demonstrated. However, recent work has suggested that Bax/Bak may have unrecognized nonapoptotic functions related to mitochondrial function in nonstressful environments. Wild-type (WT) and Bax/Bak double knockout (DKO) mice were used to determine alternative roles for Bax and Bak in mitochondrial morphology and protein import in skeletal muscle. The absence of Bax and/or Bak altered mitochondrial dynamics by regulating protein components of the organelle fission and fusion machinery. Moreover, DKO mice exhibited defective mitochondrial protein import, both into the matrix and outer membrane compartments, which was consistent with our observations of impaired membrane potential and attenuated expression of protein import machinery (PIM) components in intermyofibrillar mitochondria. Furthermore, the cytosolic chaperones heat-shock protein 90 (Hsp90) and binding immunoglobulin protein (BiP) were markedly increased with the deletion of Bax/Bak, indicating that the cytosolic environment related to protein folding may be changed in DKO mice. Interestingly, endurance training fully restored the deficiency of protein import in DKO mice, likely via the upregulation of PIM components and through improved cytosolic chaperone protein expression. Thus our results emphasize novel roles for Bax and/or Bak in mitochondrial function and provide evidence, for the first time, of a curative function of exercise training in ameliorating a condition of defective mitochondrial protein import.

The effects of chronic muscle use and disuse on cardiolipin metabolism.

Cardiolipin (CL) is a phospholipid that maintains the integrity of mitochondrial membranes. We previously demonstrated that CL content increases with chronic muscle use, and decreases with denervation-induced disuse. To investigate the underlying mechanisms, we measured the mRNA expression of 1) CL synthesis enzymes cardiolipin synthase (CLS) and CTP:PA-cytidylyltransferase-1 (CDS-1); 2) remodeling enzymes tafazzin and acyl-CoA:lysocardiolipin acyltransferase-1 (ALCAT1); and 3) outer membrane CL enzymes, mitochondrial phospholipase D and phospholipid scramblase 3 (Plscr3), during chronic contractile activity (CCA)-induced mitochondrial biogenesis and denervation. With CCA, CDS-1 expression increased by 128%, parelleling CL levels. Surprisingly, denervation also led to large increases in CDS-1 and CLS, despite a decrease in mitochondria, possibly due to a compensatory mechanism to restore lost CL. ALCAT1 decreased by 32% with CCA, but increased by 290% following denervation, indicating that both CCA and denervation alter CL remodeling. CCA and denervation also elicited 60-90% increases in Plscr3, likely to facilitate CL movement to the outer membrane. The expression of these genes was not affected by aging, but changes due to CCA and denervation were attenuated compared with young animals. The absence of PPARγ coactivator-1α in knockout animals led to a decrease in CDS-1 and an increase in ALCAT1 mRNA levels, implicating PGC-1α in regulating both CL synthesis and remodeling. These data suggest that chronic muscle use and disuse modify the expression of mRNAs encoding CL metabolism enzymes. Our data also illustrate, for the first time, that PPARγ coactivator-1α regulates the CL metabolism pathway in muscle.