P43-dependent mitochondrial activity regulates myoblast differentiation and slow myosin isoform expression by control of Calcineurin expression

We have previously shown that mitochondrial protein synthesis regulates myoblast differentiation, partly through the control of c-Myc expression, a cellular oncogene regulating myogenin expression and myoblast withdrawal from the cell cycle. In this study we provide evidence of the involvement of Calcineurin in this regulation. In C2C12 myoblasts, inhibition of mitochondrial protein synthesis by chloramphenicol decreases Calcineurin expression. Conversely, stimulation of this process by overexpressing the T3 mitochondrial receptor (p43) increases Calcineurin expression. Moreover, expression of a constitutively active Calcineurin (ΔCN) stimulates myoblast differentiation, whereas a Calcineurin antisense has the opposite effect. Lastly, ΔCN expression or stimulation of mitochondrial protein synthesis specifically increases slow myosin heavy chain expression. In conclusion, these data clearly suggest that, partly via Calcineurin expression, mitochondrial protein synthesis is involved in muscle development through the control of myoblast differentiation and probably the acquisition of the contractile and metabolic phenotype of muscle fibres.     

Thyroid hormone receptor/c-erbA: control of commitment and differentiation in the neuronal/chromaffin progenitor line PC12

The c-erbA proto-oncogenes encode nuclear receptors for thyroid hormone (T3), a hormone intimately involved in mammalian brain maturation. To study thyroid hormone receptor (TR) action on neuronal cells in vitro, we expressed the chicken c-erbA/TR alpha-1 as well as its oncogenic variant v-erbA in the adrenal medulla progenitor cell line PC12. In the absence of T3, exogenous TR alpha-1 inhibits NGF-induced neuronal differentiation and represses neuron-specific gene expression. In contrast, TR alpha-1 allows normal differentiation and neuronal gene expression to occur in the presence of T3. Finally, TR alpha-1- expressing cells become NGF-responsive for proliferation when T3 is absent, but NGF-dependent for survival in presence of T3. A similar differentiation induction by NGF plus T3 was observed in a central nervous system-derived neuronal cell line (E 18) expressing exogenous TR alpha-1. Together with the finding that TR alpha-1 constitutively blocked dexamethasone-induced differentiation of PC12 cells into the chromaffin pathway, these results suggest that TR alpha-1 plays an important role in regulating commitment and maturation of neuronal progenitors. In contrast, the v-erbA oncogene, a mutated, oncogenic version of TR alpha-1, partially but constitutively inhibited NGF- induced neuronal differentiation of PC12 cells and potentiated dexamethasone-induced chromaffin differentiation, giving rise to an aberrant "interlineage" cell phenotype.     

Triiodothyronine influences quail myoblast proliferation and differentiation*

The influence of triiodothyronine (T3) on avian myoblast proliferation and differentiation was studied in secondary cultures using plating densities of 2500 and 7000 cells/cm². Culture media were depleted of T3 (control myoblasts) and increasing amounts were then added to concentrations of 0.6, 3 and 15 nM T3 (treated myoblasts). Independent of the cell density, T3 induced a dose-related decrease in myoblast proliferation measured by cell number, doubling time and ³H-thymidine incorporation. However, with the lower plating density, this influence was delayed, occurring only after the third day of culture for 0.6 nM T3-treated myoblasts and simultaneous with the onset of myosin heavy chain accumulation. Moreover, when myoblasts were exposed to BrdU for 48 h, the T3 growth inhibitory effect disappeared, thus showing that this effect was clearly linked to differentiation. In addition, we have shown that T3 induced an early fusion of myoblasts: 65% of the maximal value of the fusion index was reached on day 3 in the T3-treated cells in comparison to 25% in the control myoblasts. This hormone also enhanced accumulation of muscle-specific proteins (connectin, acetylcholine receptors, myosin heavy chain), tested by cytoimmunofluorescence, ELISA, binding experiments and Western blot. All these results show that T3 increased myoblast differentiation through a pathway including myoblast withdrawal from the cell cycle. The influence of T3 could partly explain its previously reported positive effect on the number of muscle fibers.     

Changes in mitochondrial activity during avian myoblast differentiation: Influence of triiodothyronine or v-erbA expression

Numerous data suggest that mitochondrial activity is involved in the regulation of cell growth and differentiation. Therefore, we have studied the changes in mitochondrial activity in avian myoblast cultures (QM7 line) undergoing differentiation or in BrdU-treated, differentiation-deficient cells. As we have previously shown that triiodothyronine and v-erb A expression stimulate myogenic differentiation, we have also observed their influence upon mitochondrial activity. Comparison of control and BrdU-treated myoblasts indicated that precocious differentiation events were associated with a stimulation of citrate synthase and cytochrome oxidase activities. They also induced a transient decrease in mitochondrial membrane potential assessed by rhodamine 123 uptake. In control myoblasts, a general stimulation of mitochondrial activity was recorded at cell confluence, prior to terminal differentiation. These events did not occur in BrdU-treated myoblasts, thus indicating that they were tightly linked to myoblast commitment. Whereas no significant triiodothyronine influence could be detected upon mitochondrial activity, we observed that v-erb A expression significantly depresses the mitochondrial membrane potential in control myoblasts. This action was not observed in BrdU-treated myoblasts, thus suggesting that it involves an indirect pathway linked to differentiation. Moreover, the oncoprotein abrogated the decrease in E2-PDH subunit level observed at cell confluence. These data underline that changes in mitochondrial activity occurred prior to myoblast terminal differentiation and could be involved in the processes regulating myogenesis. In addition, they provide the first evidence that the v-erb A oncoprotein influences mitochondrial activity. © 1996 Wiley-Liss, Inc.     

An anti-sense c-erbA clone inhibits thyroid hormone-induced expression from the alpha-myosin heavy chain promoter.

The effects of thyroid hormone on expression of cardiac myosin heavy chain genes generally are thought to be mediated by nuclear 3,5,3'-triiodo-L-thyronine (T3) receptors that have been identified as the products of the protooncogene, c-erbA. This hypothesis has been tested by transfection of cardiomyocytes in primary culture with a plasmid, pRSVhEACAT-, expressing anti-sense c-erbA mRNA. Because only a low percentage of cells (20%) could be transfected in primary culture an alpha-myosin heavy chain-chloramphenicol acetyltransferase fusion construct was used as a reporter gene. The results indicate that the anti-sense plasmid almost completely blocks T3-induced activity of the reporter gene (less than 1% control) while transfection of a similar amount of the sense construct, pRSVhEACAT+, has no effect. When the c-erbA plasmids were cotransfected with constructs containing T3-independent promoters, no effects on expression were observed. The combined use of an anti-sense construct and a report gene provides a means of studying the role of c-erbA products in intracellular signal transduction even in differentiated, nondividing cells like those of the heart.     

Zc3h10 is a novel mitochondrial regulator

Mitochondria are the energy‐generating hubs of the cell. In spite of considerable advances, our understanding of the factors that regulate the molecular circuits that govern mitochondrial function remains incomplete. Using a genome‐wide functional screen, we identify the poorly characterized protein Zinc finger CCCH‐type containing 10 (Zc3h10) as regulator of mitochondrial physiology. We show that Zc3h10 is upregulated during physiological mitochondriogenesis as it occurs during the differentiation of myoblasts into myotubes. Zc3h10 overexpression boosts mitochondrial function and promotes myoblast differentiation, while the depletion of Zc3h10 results in impaired myoblast differentiation, mitochondrial dysfunction, reduced expression of electron transport chain (ETC) subunits, and blunted TCA cycle flux. Notably, we have identified a loss‐of‐function mutation of Zc3h10 in humans (Tyr105 to Cys105) that is associated with increased body mass index, fat mass, fasting glucose, and triglycerides. Isolated peripheral blood mononuclear cells from individuals homozygotic for Cys105 display reduced oxygen consumption rate, diminished expression of some ETC subunits, and decreased levels of some TCA cycle metabolites, which all together derive in mitochondrial dysfunction. Taken together, our study identifies Zc3h10 as a novel mitochondrial regulator.     

Mitochondrial activity regulates myoblast differentiation by control of c-Myc expression

We have previously shown that mitochondrial activity is an important regulator of myoblast differentiation, partly through processes targeting myogenin expression. Here, we investigated the possible involvement of c-myc in these processes. Inhibition of mitochondrial activity by chloramphenicol abrogated the decrease in c-myc mRNA and protein levels occurring at the onset of terminal differentiation. Conversely, stimulation of mitochondrial activity by overexpression of the T3 mitochondrial receptor (p43) down-regulated c-myc expression. In addition, c-myc overexpression mimicked the influence of mitochondrial activity inhibition on myoblast differentiation. Moreover, like chloramphenicol, c-myc overexpression strongly inhibited the myogenic influence of p43 overexpression. These data suggest that c-Myc is an important target of mitochondrial activity involved in the myogenic influence of the organelle. Lastly, we found that chloramphenicol influence is negatively related to the frequency of post-mitotic myoblasts in the culture at the onset of treatment, and cell cycle analyses demonstrated that the frequency of myoblasts in G0-G1 phase at cell confluence is increased by p43 overexpression and decreased by chloramphenicol or c-myc overexpression. These results suggest that irreversible myoblast withdrawal from the cell cycle is a target of mitochondrial activity by control of c-Myc expression.