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.

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.

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