Overexpression of the Mitochondrial T3 Receptor p43 Induces a Shift in Skeletal Muscle Fiber Types

et al. (2008) Overexpression of the Mitochondrial T3 Receptor p43 Induces a Shift in Skeletal Muscle Fiber Types. PLoS ONE 3(6): e2501. doi:10.1371/journal.pone.0002501 Overexpression of the Mitochondrial T3 Receptor p43 Induces a Shift in Skeletal Muscle Fiber Types Fran cois Casas 0 Laurence Pessemesse 0 Ste phanie Grandemange 0 Pascal Seyer 0 Nag 0 Gueguen 0 Olivier Baris 0 Laurence Lepourry 0 Ge rard Cabello 0 Chantal Wrutniak-Cabello 0 Jacques Samarut, Universite de Lyon, CNRS, INRA, Ecole Normale Superieure de Lyon, France 0 1 INRA, UMR866 Diffe renciation cellulaire et croissance, Montpellier, France, 2 Universite Montpellier 1, Montpellier, France, 3 Universite Montpellier 2 , Montpellier , France In previous studies, we have characterized a new hormonal pathway involving a mitochondrial T3 receptor (p43) acting as a mitochondrial transcription factor and consequently stimulating mitochondrial activity and mitochondrial biogenesis. We have established the involvement of this T3 pathway in the regulation of in vitro myoblast differentiation.We have generated mice overexpressing p43 under control of the human a-skeletal actin promoter. In agreement with the previous characterization of this promoter, northern-blot and western-blot experiments confirmed that after birth p43 was specifically overexpressed in skeletal muscle. As expected from in vitro studies, in 2-month old mice, p43 overexpression increased mitochondrial genes expression and mitochondrial biogenesis as attested by the increase of mitochondrial mass and mt-DNA copy number. In addition, transgenic mice had a body temperature 0.8uC higher than control ones and displayed lower plasma triiodothyronine levels. Skeletal muscles of transgenic mice were redder than wild-type animals suggesting an increased oxidative metabolism. In line with this observation, in gastrocnemius, we recorded a strong increase in cytochrome oxidase activity and in mitochondrial respiration. Moreover, we observed that p43 drives the formation of oxidative fibers: in soleus muscle, where MyHC IIa fibers were partly replaced by type I fibers; in gastrocnemius muscle, we found an increase in MyHC IIa and IIx expression associated with a reduction in the number of glycolytic fibers type IIb. In addition, we found that PGC-1a and PPARd, two major regulators of muscle phenotype were up regulated in p43 transgenic mice suggesting that these proteins could be downstream targets of mitochondrial activity. These data indicate that the direct mitochondrial T3 pathway is deeply involved in the acquisition of contractile and metabolic features of muscle fibers in particular by regulating PGC-1a and PPARd. - Funding: This work was supported by grants from the Institut National de la Recherche Agronomique (INRA), the Association Francaise contre les Myopathies (AFM). Stephanie Grandemange, Pascal Seyer, Nag Gueguen and Olivier Baris are recipient of fellowships from, respectively, the Ministe`re de la Recherche et de lEnseignement (SG and MB) and INRA (PS, NG and OB). Competing Interests: The authors have declared that no competing interests exist. Skeletal muscle of vertebrates contain myofibers differing in contractile function, mitochondrial content and metabolic properties. Slow-twitch fibers are characterized by type I myosin heavy chain (MHC) expression and a high mitochondrial density leading to a prominent oxidative metabolism. Fast-twitch fibers express type II MHCs including three subtypes: IIa, IIx and IIb. Type IIb fibers display a reduced mitochondrial density associated with a principally glycolytic metabolism. The oxidative capacity of type IIa and IIx fibers are intermediate between that recorded in fibers Type I and IIb [1,2]. In addition to its metabolic activity, triiodothyronine (T3) affects developmental processes, and is in particular considered as a major regulator of in vivo muscle development. This hormone not only stimulates growth of this tissue by increasing the number and diameter of muscle fibers [3,4], but also regulates the transition between neonatal and adult myosin isoforms [5] and influences the contractile features of adult muscle fibers [6]. Thyroid hormone acts through nuclear receptors (T3Rs) encoded by the TRa and TRb genes (NR1A1 and NR1A2 according to nuclear hormone receptor nomenclature) [7,8]. These receptors are ligand-dependent transcription factors that constituvely bind to specific sequences called thyroid hormone response elements (T3RE) located in the promoter of T3 target genes. More recently, we have identified in mitochondria two Nterminally truncated forms of the nuclear receptor TRa1, with molecular weight of 43 and 28 kDa (p43 and p28) [9,10]. These proteins are synthesized by the use of internal initiation sites of translation occuring in the TRa1 transcript. Despite the occurence of a nuclear localization signal, p43 is specifically imported into the mitochondria according to an atypical process [11]. In gel shift experiments, p43 binds as dimeric complexes involving at least two other truncated forms of nuclear receptors located in mitochondria, mt-RXR and mt-PPAR, to specific sequences of the mitochondrial genome, sharing strong homologies with nuclear T3RE [11,12,13]. Consequently, on isolated mitochondria, p43 stimulates mitochondrial transcription and protein synthesis in the presence of T3 [11]. Lastly, in CV1 cells, p43 overexpression stimulates mitochondrial biogenesis and respiratory chain activity [9]. We have previously shown that mitochondrial activity is an important regulator of myoblast differentiation. While inhibition of mitochondrial protein synthesis by chloramphenicol impaired myoblast differentiation, stimulation of mitochondrial activity by p43 overexpression induced a potent stimulation of terminal differentiation [14,15]. This regulation which does not involve changes in ATP stores, allows the expression of nuclear genes involved in the regulation of cell proliferation and differentiation. In particular, in myoblasts, p43 overexpression stimulates terminal differentiation, by down-regulating c-Myc expression and up regulating myogenin expression [14,15]. In addition, it also induces a preferential expression of the slow myosin isoform, through increasing calcineurin levels [16]. These data led us to examine a potential role of the direct mitochondrial pathway in in vivo muscle development and phenotype. To assess the influence of this mitochondrial receptor, we have generated transgenic mice overexpressing p43 under the control of the human a-skeletal actin promoter (HSA), a skeletal muscle specific promoter [17,18]. Here we report, that this overexpression increases mitochondrial gene expression and mitochondrial biogenesis. In addition, p43 induces a shift toward the oxidative phenotype: in soleus, an oxidative muscle, MyHC IIa fibers were partly replaced by type I fibers; in quadriceps, an oxido-glycolytic muscle, we found an increase in the (...truncated)