We used expression and reporter gene analysis to understand how changes in transcription factors impinge on mitochondrial gene expression during myogenesis of cultured murine myoblasts (C2C12 and Sol8). The mRNA levels for nuclear respiratory factor-1 (NRF-1) and NRF-2alpha increased 60% by the third day of myogenesis, whereas NRF-1 and NRF-2 reporter gene activity increased by fivefold over the same period. Although peroxisome proliferator activated receptor (PPARalpha) mRNA levels increased almost 10-fold, the activity of a PPAR reporter was unchanged during myogenesis. The PPAR coactivator PPAR-gamma coactivator-1alpha (PGC1alpha), a master controller of mitochondrial biogenesis, was not expressed at detectable levels. However, the mRNA for both PGC1alpha-related coactivator and PGC1beta was abundant, with the latter increasing by 50% over 3 days of differentiation. We also conducted promoter analysis of the gene for citrate synthase (CS), a common mitochondrial marker enzyme. The proximal promoter ( approximately 2,100 bp) of the human CS lacks binding sites for PPAR, NRF-1, or NRF-2. Deletion mutants, a targeted mutation, and an Sp1 site-containing reporter construct suggest that changes in Sp1 regulation also participate in mitochondrial biogenesis during myogenesis. Because most mitochondrial genes are regulated by PPARs, NRF-1, and/or NRF-2, we conducted inhibitor studies to further support the existence of a distinct pathway for CS gene regulation in myogenesis. Although both LY-294002 (a phosphatidylinositol 3-kinase inhibitor) and SB-203580 (a p38-MAPK inhibitor) blocked myogenesis (as indicated by creatine phosphokinase activity), only SB-203580 prevented the myogenic increase in cytochrome oxidase activity, whereas only LY-294002 blocked the increase in CS (enzyme and reporter gene activities). Collectively, these studies help delineate the roles of some transcriptional regulators involved in mitochondrial biogenesis associated with myogenesis and underscore an import role for posttranscriptional regulation of transcription factor activity.

Although striated muscles differ in mitochondrial content, the extent of fiber-type specific mitochondrial specializations is not well known. To address this issue, we compared mitochondrial structural and functional properties in red muscle (RM), white muscle (WM), and cardiac muscle of rainbow trout. Overall preservation of the basic relationships between oxidative phosphorylation complexes among fiber types was confirmed by kinetic analyses, immunoblotting of native holoproteins, and spectroscopic measurements of cytochrome content. Fiber-type differences in mitochondrial properties were apparent when parameters were expressed per milligram mitochondrial protein. However, the differences diminished when expressed relative to cytochrome oxidase (COX), possibly a more meaningful denominator than mitochondrial protein. Expressed relative to COX, there were no differences in oxidative phosphorylation enzyme activities, pyruvate-based respiratory rates, H2O2 production, or state 4 proton leak respiration. These data suggest most mitochondrial qualitative properties are conserved across fiber types. However, there remained modest differences ( approximately 50%) in stoichiometries of selected enzymes of the Krebs cycle, beta-oxidation, and antioxidant enzymes. There were clear differences in membrane fluidity (RM > cardiac, WM) and proton conductance (H+/min/mV/U COX: WM > RM > cardiac). The pronounced differences in mitochondrial content between fiber types could be attributed to a combination of differences in myonuclear domain and modest effects on the expression of nuclear- and mitochondrially encoded respiratory genes. Collectively, these studies suggest constitutive pathways that transcend fiber types are primarily responsible for determining most quantitative and qualitative properties of mitochondria.

Chronic treatment of cultured cells with very low levels of azide (I 50 <10 M) leads to slow (t1 ⁄2 ‫؍‬ 6 h), irreversible loss of cytochrome c oxidase (COX) activity. Azidemediated COX losses were not accompanied by inhibition of other mitochondrial enzymes and were not dependent upon electron flux through oxidative phosphorylation. Although azide treatment also reduced activity (but not content) of both CuZn superoxide dismutase and catalase, a spectrum of pro-oxidants (and anti-oxidants) failed to mimic (or prevent) azide effects, arguing that losses in COX activity were not due to resultant compromises in free radical scavenging. Loss of COX activity was not attributable to reduced rates of mitochondrial protein synthesis or declines in either COX subunit mRNA or protein levels (COX I, II, IV). Co-incubation experiments using copper (CuCl 2 , CuHis) and copper chelators (neocuproine, bathocuproine) indicated that azide effects were not mediated by interactions with either Cu A or Cu B . In contrast, difference spectroscopy and high performance liquid chromatography analyses demonstrated azide-induced losses in cytochrome aa 3 content although not to the same extent as catalytic activity. Differential azide effects on COX content relative to COX activity were confirmed using a refined inhibition time course in combination with blue native electrophoresis, and established that holoenzyme dissociation occurs subsequent to losses in catalytic activity. Collectively, these data suggest that COX deficiency can arise through enhanced holoenzyme dissociation, possibly through interactions with the structure or coordination of its heme moieties.

Mitochondrial content of skeletal muscle varies among fiber types, and changes in complex ways during aging. We evaluated the regulatory origins of differences in mitochondrial content among muscles of varied fiber type in F344xBNF1 rats, and how these regulatory patterns are altered with aging. In adult (12 month) animals we found that units citrate synthase (CS)/g tissue, a marker for mitochondrial content, varied approximately 3-fold among 10 skeletal muscles. Stoichiometric relationships between CS and isocitrate dehydrogenase, aconitase, and cytochrome c oxidase were generally preserved across fiber types. Among the 10 muscles of adult rats, CS content correlated with nuclear content (R2= 0.36). Muscles differed widely in CS messenger RNA (mRNA)/DNA (an index of variation in transcriptional regulations) and units CS/CS mRNA (an index of variation in posttranscriptional regulations). All muscles of aged rats (35 months) showed an increase in mg DNA/g, suggestive of atrophy. Age-dependent declines in units CS/DNA were accompanied by reductions in CS mRNA/DNA and/or units CS/CS mRNA, depending on muscle fiber type. Thus, declines in units CS/DNA with age appeared to be due to transcriptional as well as translational variations. Differences in mitochondrial content among muscle fiber types and age groups may arise from variations in nuclear content and posttranscriptional processes, as well as transcriptional regulation.

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