Androgens regulate mitochondrial cytochrome c oxidase and lysosomal hydrolases in mouse skeletal muscle.

The gastrocnemius, a fast-twitch white muscle, and the soleus, a slow-twitch red muscle, were studied in A/J mice. The specific activities of the lysosomal hydrolases, beta-D-glucuronidase, hexosaminidase, beta-D-galactosidase and arylsulphatase, the inner-mitochondrial-membrane enzyme cytochrome c oxidase, and the outer-mitochondrial-membrane enzyme monoamine oxidase, were greater in the soleus than in the gastrocnemius. The specific activities of the lysosomal hydrolases and cytochrome c oxidase in the gastrocnemius and soleus were substantially higher in male mice than in female mice. Orchiectomy abolished this sex difference. Testosterone increased the activities of the lysosomal hydrolases and cytochrome c oxidase and coincidentally induced muscle hypertrophy and an accretion of protein and RNA, but total DNA remained constant. Monoamine oxidase was unaffected by sex, orchiectomy and testosterone. These findings indicate that endogenous androgens regulate the activity of enzymes associated with lysosomes and the inner mitochondrial membrane, as well as muscle fibre growth in mouse skeletal muscle.     

Cmc1p is a conserved mitochondrial twin CX9C protein involved in cytochrome c oxidase biogenesis

Copper is an essential cofactor of two mitochondrial enzymes: cytochrome c oxidase (COX) and Cu-Zn superoxide dismutase (Sod1p). Copper incorporation into these enzymes is facilitated by metallochaperone proteins which probably use copper from a mitochondrial matrix-localized pool. Here we describe a novel conserved mitochondrial metallochaperone-like protein, Cmc1p, whose function affects both COX and Sod1p. In Saccharomyces cerevisiae, Cmc1p localizes to the mitochondrial inner membrane facing the intermembrane space. Cmc1p is essential for full expression of COX and respiration, contains a twin CX9C domain conserved in other COX assembly copper chaperones, and has the ability to bind copper(I). Additionally, mutant cmc1 cells display increased mitochondrial Sod1p activity, while CMC1 overexpression results in decreased Sod1p activity. Our results suggest that Cmc1p could play a direct or indirect role in copper trafficking and distribution to COX and Sod1p.     

Cyclin E is recruited to the nuclear matrix during differentiation, but is not recruited in cancer cells

Cyclin E supports pre-replication complex (pre-RC) assembly, while cyclin A-associated kinase activates DNA synthesis. We show that cyclin E, but not A, is mounted upon the nuclear matrix in sub-nuclear foci in differentiated vertebrate cells, but not in undifferentiated cells or cancer cells. In murine embryonic stem cells, Xenopus embryos and human urothelial cells, cyclin E is recruited to the nuclear matrix as cells differentiate and this can be manipulated in vitro. This suggests that pre-RC assembly becomes spatially restricted as template usage is defined. Furthermore, failure to become restricted may contribute to the plasticity of cancer cells.     

Changes in mitochondrial reactive oxygen species synthesis during differentiation of skeletal muscle cells

Myogenesis is accompanied by an intensive metabolic remodeling. We investigated the mitochondrial reactive oxygen species (ROS) generation at different levels of skeletal muscle differentiation: in C2C12 myoblasts, in C2C12 myotubes and in adult mouse skeletal muscle. Differentiation was accompanied by an increase in mitochondrial content and respiratory chain activity. The detected ROS production levels correlated with mitochondrial content, being the lowest in the myoblasts. Unlike the adult skeletal muscle, myoblast ROS production was significantly stimulated by the complex I inhibitor rotenone. Our results show that mitochondria are an important ROS source in skeletal muscle cells. The substantial changes in mitochondrial ROS synthesis during skeletal muscle differentiation can be explained by intensive bioenergetic remodeling.     

Caspase cleavage of cytochrome c1 disrupts mitochondrial function and enhances cytochrome c release

Mitochondrial catastrophe can be the cause or consequence of apoptosis and is associated with a number of pathophysiological conditions. The exact relationship between mitochondrial catastrophe and caspase activation is not completely understood. Here we addressed the underlying mechanism, explaining how activated caspase could feedback to attack mitochondria to amplify further cytochrome c (cyto.c) release. We discovered that cytochrome c1 (cyto.c1) in the bc1 complex of the mitochondrial respiration chain was a novel substrate of caspase 3 (casp.3). We found that cyto.c1 was cleaved at the site of D106, which is critical for binding with cyto.c, following apoptotic stresses or targeted expression of casp.3 into the mitochondrial intermembrane space. We demonstrated that this cleavage was closely linked with further cyto.c release and mitochondrial catastrophe. These mitochondrial events could be effectively blocked by expressing non-cleavable cyto.c1 (D106A) or by caspase inhibitor z-VAD-fmk. Our results demonstrate that the cleavage of cyto.c1 represents a critical step for the feedback amplification of cyto.c release by caspases and subsequent mitochondrial catastrophe.     

Overlapping specificities of the mitochondrial cytochrome c and c1 heme lyases

Heme attachment to the apoforms of fungal mitochondrial cytochrome c and c1 requires the activity of cytochrome c and c1 heme lyases (CCHL and CC1HL), which are enzymes with distinct substrate specificity. However, the presence of a single heme lyase in higher eukaryotes is suggestive of broader substrate specificity. Here, we demonstrate that yeast CCHL is active toward the non-cognate substrate apocytochrome c1, i.e. CCHL promotes low levels of apocytochrome c1 conversion to its holoform in the absence of CC1HL. Moreover, that the single human heme lyase also displays a broader cytochrome specificity is evident from its ability to substitute for both yeast CCHL and CC1HL. Multicopy and genetic suppressors of the absence of CC1HL were isolated and their analysis revealed that the activity of CCHL toward cytochrome c1 can be enhanced by: 1) reducing the abundance of the cognate substrate apocytochrome c, 2) increasing the accumulation of CCHL, 3) modifying the substrate-enzyme interaction through point mutations in CCHL or cytochrome c1, or 4) overexpressing Cyc2p, a protein known previously only as a mitochondrial biogenesis factor. Based on the functional interaction of Cyc2p with CCHL and the presence of a putative FAD-binding site in the protein, we hypothesize that Cyc2p controls the redox chemistry of the heme lyase reaction.     

Effect of body temperature during exercise on skeletal muscle cytochrome c oxidase content.

This study determined the role of body temperature during exercise on cytochrome-c oxidase (CytOx) activity, a marker of mitochondrial content, and mitochondrial heat shock protein 70 (mtHSP70), which is required for import of nuclear-coded preproteins. Male, 10-wk-old, Sprague-Dawley rats exercised identically for 9 wk in ambient temperatures of 23 degrees C (n = 10), 8 degrees C with wetted fur (n = 8), and 4 degrees C with wetted fur and fan (n = 7). These conditions maintained exercising core temperature (T(c)) at 40.4, 39.2, or 38.0 degrees C (resting temperature), respectively. During weeks 3-9, exercisers ran 5 days/wk up a 6% grade at 20 m/min for 60 min. Animals were housed at 23 degrees C. Gastrocnemius CytOx activity in T(c)=38.0 degrees C (83.5 +/- 5.5 microatoms O x min(-1) x g wet wt(-1)) was greater than all other groups (P < 0.05), exceeding sedentary (n = 7) by 73.2%. T(c) of 40.4 and 39.2 degrees C also were higher than sedentary by 22.4 and 37.4%, respectively (P < 0.05). Quantification of CytOx content verified that the increased activity was due to an increase in protein content. In extensor digitorum longus, a nonactive muscle, CytOx was not elevated in T(c) = 38.0 degrees C. mtHSP70 was significantly elevated in gastrocnemius of T(c) = 38.0 degrees C compared with sedentary (P < 0.05) but was not elevated in extensor digitorum longus (P > 0.05). The data indicate that decreasing exercise T(c) may enhance mitochondrial biogenesis and that mtHSP70 expression is not dependent on temperature.     

IKK/NF-kappaB regulates skeletal myogenesis via a signaling switch to inhibit differentiation and promote mitochondrial biogenesis

Nuclear factor kappaB (NF-kappaB) is involved in multiple skeletal muscle disorders, but how it functions in differentiation remains elusive given that both anti- and promyogenic activities have been described. In this study, we resolve this by showing that myogenesis is controlled by opposing NF-kappaB signaling pathways. We find that myogenesis is enhanced in MyoD-expressing fibroblasts deficient in classical pathway components RelA/p65, inhibitor of kappaB kinase beta (IKKbeta), or IKKgamma. Similar increases occur in myoblasts lacking RelA/p65 or IKKbeta, and muscles from RelA/p65 or IKKbeta mutant mice also contain higher fiber numbers. Moreover, we show that during differentiation, classical NF-kappaB signaling decreases, whereas the induction of alternative members IKKalpha, RelB, and p52 occurs late in myogenesis. Myotube formation does not require alternative signaling, but it is important for myotube maintenance in response to metabolic stress. Furthermore, overexpression or knockdown of IKKalpha regulates mitochondrial content and function, suggesting that alternative signaling stimulates mitochondrial biogenesis. Together, these data reveal a unique IKK/NF-kappaB signaling switch that functions to both inhibit differentiation and promote myotube homeostasis.     

Regulation of exercise-induced fiber type transformation, mitochondrial biogenesis, and angiogenesis in skeletal muscle

Skeletal muscle exhibits superb plasticity in response to changes in functional demands. Chronic increases of skeletal muscle contractile activity, such as endurance exercise, lead to a variety of physiological and biochemical adaptations in skeletal muscle, including mitochondrial biogenesis, angiogenesis, and fiber type transformation. These adaptive changes are the basis for the improvement of physical performance and other health benefits. This review focuses on recent findings in genetically engineered animal models designed to elucidate the mechanisms and functions of various signal transduction pathways and gene expression programs in exercise-induced skeletal muscle adaptations.     

Inhibiting Drp1-mediated mitochondrial fission selectively prevents the release of cytochrome c during apoptosis

Most cell death stimuli trigger the mitochondrial release of cytochrome c and other cofactors that induce caspase activation and ensuing apoptosis. Apoptosis is also associated with massive mitochondrial fragmentation and cristae remodeling. Dynamin-related protein 1 (Drp1), a protein of the mitochondrial fission machinery, has been reported to participate in apoptotic mitochondrial fragmentation. Several theories explaining the mechanisms of cytochrome c release have been proposed. One suggests that it relies on the activation of Drp1-mediated mitochondrial fission. Here, we report that downregulation of Drp1 inhibits fragmentation of the mitochondrial network and partially prevents the release of cytochrome c but fails to prevent the release of other mitochondrial factors such as second mitochondria-derived activator of caspase/direct IAP-binding protein with low pI, Omi/HtrA2, adenylate kinase 2 and deafness dystonia peptide/TIMM8a. An explanation for the prevention of cytochrome c release is provided by our observation that inhibiting Drp1-mediated mitochondrial fission prevents the mitochondrial release of soluble OPA1 that was proposed to regulate cristae remodeling and complete cytochrome c release during apoptosis. Finally, we observed that downregulation of Drp1 delays but does not inhibit apoptosis, suggesting that mitochondrial fragmentation is not a prerequisite for apoptosis.