Pascal Maire

AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity

AMP‐activated protein kinase (AMPK) is a sensor of cellular energy status that plays a central role in skeletal muscle metabolism. We used skeletal muscle‐specific AMPKα1α2 double‐knockout (mdKO) mice to provide direct genetic evidence of the physiological importance of AMPK in regulating muscle exercise capacity, mitochondrial function, and contraction‐stimulated glucose uptake. Exercise performance was significantly reduced in the mdKO mice, with a reduction in maximal force production and fatigue resistance. An increase in the proportion of myofibers with centralized nuclei was noted, as well as an elevated expression of interleukin 6 (IL‐6) mRNA, possibly consistent with mild skeletal muscle injury. Notably, we found that AMPKα1 and AMPKα2 isoforms are dispensable for contraction‐induced skeletal muscle glucose transport, except for male soleus muscle. However, the lack of skeletal muscle AMPK diminished maximal ADP‐stimulated mitochondrial respiration, showing an impairment at complex I. This effect was not accompanied by changes in mitochondrial number, indicating that AMPK regulates muscle metabolic adaptation through the regulation of muscle mitochondrial oxidative capacity and mitochondrial substrate utilization but not baseline mitochondrial muscle content. Together, these results demonstrate that skeletal muscle AMPK has an unexpected role in the regulation of mitochondrial oxidative phosphorylation that contributes to the energy demands of the exercising muscle.—Lantier, L., Fentz, J., Mounier, R., Leclerc, J., Treebak, J. T., Pehmøller, C., Sanz, N., Sakakibara, I., Saint‐Amand, E., Rimbaud, S., Maire, P., Marette, A., Ventura‐Clapier, R., Ferry, A., Wojtaszewski, J. F. P., Foretz, M., Viollet, B. AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity. FASEB J. 28, 3211–3224 (2014). www.fasebj.org

A combination of MEF3 and NFI proteins activates transcription in a subset of fast-twitch muscles.

The human aldolase A pM promoter is active in fast-twitch muscles. To understand the role of the different transcription factors which bind to this promoter and determine which ones are responsible for its restricted pattern of expression, we analyzed several transgenic lines harboring different combinations of pM regulatory elements. We show that muscle-specific expression can be achieved without any binding sites for the myogenic factors MyoD and MEF2 and that a 64-bp fragment comprising a MEF3 motif and an NFI binding site is sufficient to drive reporter gene expression in some but, interestingly, not all fast-twitch muscles. A result related to this pattern of expression is that some isoforms of NFI proteins accumulate differentially in fast- and slow-twitch muscles and in distinct fast-twitch muscles. We propose that these isoforms of NFI proteins might provide a molecular basis for skeletal muscle diversity.

Six1 and Six4 gene expression is necessary to activate the fast-type muscle gene program in the mouse primary myotome.

While the signaling pathways and transcription factors active in adult slow- and fast-type muscles begin to be characterized, genesis of muscle fiber-type diversity during mammalian development remains unexplained. We provide evidence showing that Six homeoproteins are required to activate the fast-type muscle program in the mouse primary myotome. Affymetrix transcriptomal analysis of Six1(-/-)Six4(-/-) E10.5 somites revealed the specific down-regulation of many genes of the fast-type muscle program. This data was confirmed by in situ hybridization performed on Six1(-/-)Six4(-/-) embryos. The first mouse myocytes express both fast-type and slow-type muscle genes. In these fibers, Six1 and Six4 expression is required to specifically activate fast-type muscle genes. Chromatin immunoprecipitation experiments confirm the binding of Six1 and Six4 on the regulatory regions of these muscle genes, and transfection experiments show the ability of these homeoproteins to activate specifically identified fast-type muscle genes. This in vivo wide transcriptomal analysis of the function of the master myogenic determinants, Six, identifies them as novel markers for the differential activation of a specific muscle program during mammalian somitic myogenesis.

Six1 regulates stem cell repair potential and self-renewal during skeletal muscle regeneration

Six1 in satellite cells is important for muscle regeneration and homeostasis of the stem cell niche by regulating MyoD, Myogenin, and Dusp6-ERK signaling.

Genesis of muscle fiber-type diversity during mouse embryogenesis relies on Six1 and Six4 gene expression

Adult skeletal muscles in vertebrates are composed of different types of myofibers endowed with distinct metabolic and contraction speed properties. Genesis of this fiber-type heterogeneity during development remains poorly known, at least in mammals. Six1 and Six4 homeoproteins of the Six/sine oculis family are expressed throughout muscle development in mice, and Six1 protein is enriched in the nuclei of adult fast-twitch myofibers. Furthermore, Six1/Six4 proteins are known to control the early activation of fast-type muscle genes in myocytes present in the mouse somitic myotome. Using double Six1:Six4 mutants (SixdKO) to dissect in vivo the genesis of muscle fiber-type heterogeneity, we analyzed here the phenotype of the dorsal/epaxial muscles remaining in SixdKO. We show by electron microscopy analysis that the absence of these homeoproteins precludes normal sarcomeric organization of the myofiber leading to a dystrophic aspect, and by immunohistochemistry experiments a deficiency in synaptogenesis. Affymetrix transcriptome analysis of the muscles remaining in E18.5 SixdKO identifies a major role for these homeoproteins in the control of genes that are specifically activated in the adult fast/glycolytic myofibers, particularly those controlling Ca(2+) homeostasis. Absence of Six1 and Six4 leads to the development of dorsal myofibers lacking expression of fast-type muscle genes, and mainly expressing a slow-type muscle program. The absence of restriction of the slow-type program during the fetal period in SixdKO back muscles is associated with a decreased HDAC4 protein level, and subcellular relocalization of the transcription repressor Sox6. Six genes thus behave as essential global regulators of muscle gene expression, as well as a central switch to drive the skeletal muscle fast phenotype during fetal development.

Six1 is not involved in limb tendon development, but is expressed in limb connective tissue under Shh regulation.

Mice deficient for the homeobox gene Six1 display defects in limb muscles consistent with the Six1 expression in myogenic cells. In addition to its myogenic expression domain, Six1 has been described as being located in digit tendons and as being associated with connective tissue patterning in mouse limbs. With the aim of determining a possible involvement of Six1 in tendon development, we have carefully characterised the non-myogenic expression domain of the Six1 gene in mouse and chick limbs. In contrast to previous reports, we found that this non-myogenic domain is distinct from tendon primordia and from tendons defined by scleraxis expression. The non-myogenic domain of Six1 expression establishes normally in the absence of muscle, in Pax3-/- mutant limbs. Moreover, the expression of scleraxis is not affected in early Six1-/- mutant limbs. We conclude that the expression of the Six1 gene is not related to tendons and that Six1, at least on its own, is not involved in limb tendon formation in vertebrates. Finally, we found that the posterior domain of Six1 in connective tissue is adjacent to that of the secreted factor Sonic hedgehog and that Sonic hedgehog is necessary and sufficient for Six1 expression in posterior limb regions.

Fast-muscle-specific DNA-protein interactions occurring in vivo at the human aldolase A M promoter are necessary for correct promoter activity in transgenic mice.

The human aldolase A tissue-specific M promoter (pM) has served as a model system for identifying pathways that lead to fast-muscle-specialized expression. The current study has delimited the sequences necessary and sufficient for fast-muscle-specific expression in transgenic mice to a short 209-bp fragment extending from bp -164 to +45 relative to the pM transcription start site. Genomic footprinting methods showed that in this proximal region, the same elements that bind muscle nuclear proteins in vitro are involved in DNA-protein interactions in intact muscle nuclei of transgenic mice. Furthermore, these experiments provided the first evidence that different DNA-binding activities exist between slow and fast muscles in vivo. Fast-muscle-specific interactions occur at an element named M1 and at a muscle-specific DNase I-hypersensitive site that was previously detected by in vitro methods. The formation of the muscle-specific DNase I-hypersensitive site reflects binding of proteins to a close element, named M2, which contains a binding site for nuclear factors of the NF1 family. Mutational analysis performed with transgenic mice confirmed the importance of the M1 element for high-level fast-muscle-specific pM activity and suggested that the M2/NF1 element is differently required for correct pM expression in distinct fast muscles. In addition, two other protein binding sites, the MEF3 motif and the USF site, seem to act as stage-specific activators and/or as participants in the establishment of an active chromatin configuration at pM.

An opportunistic promoter sharing regulatory sequences with either a muscle-specific or a ubiquitous promoter in the human aldolase A gene.

The human aldolase A gene is transcribed from three different promoters, pN, pM, and pH, all of which are clustered within a small 1.6-kbp DNA domain. pM, which is highly specific to adult skeletal muscle, lies in between pN and pH, which are ubiquitous but particularly active in heart and skeletal muscle. A ubiquitous enhancer, located just upstream of pH start sites, is necessary for the activity of both pH and pN in transient transfection assays. Using transgenic mice, we studied the sequence controlling the muscle-specific promoter pM and the relations between the three promoters and the ubiquitous enhancer. A 4.3-kbp fragment containing the three promoters and the ubiquitous enhancer showed an expression pattern consistent with that known in humans. In addition, while pH was active in both fast and slow skeletal muscles, pM was active only in fast muscle. pM activity was unaltered by the deletion of a 1.8-kbp region containing the ubiquitous enhancer and the pH promoter, whereas pN remained active only in fast skeletal muscle. These findings suggest that in fast skeletal muscle, a tissue-specific enhancer was acting on both pN and pM, whereas in other tissues, the ubiquitous enhancer was necessary for pN activity. Finally, a 2.6-kbp region containing the ubiquitous enhancer and only the pH promoter was sufficient to bring about high-level expression of pH in cardiac and skeletal muscle. Thus, while pH and pM function independently of each other, pN, remarkably, shares regulatory elements with each of them, depending on the tissue. Importantly, expression of the transgenes was independent of the integration site, as originally described for transgenes containing the beta-globin locus control region.

APC is required for muscle stem cell proliferation and skeletal muscle tissue repair

In muscle stem cells, APC dampens canonical Wnt signaling to allow cell cycle progression.

Six Homeoproteins and a linc-RNA at the Fast MYH Locus Lock Fast Myofiber Terminal Phenotype

Abstract Thousands of long intergenic non-coding RNAs (lincRNAs) are encoded by the mammalian genome. However, the function of most of these lincRNAs has not been identified in vivo. Here, we demonstrate a role for a novel lincRNA, linc-MYH, in adult fast-type myofiber specialization. Fast myosin heavy chain (MYH) genes and linc-MYH share a common enhancer, located in the fast MYH gene locus and regulated by Six1 homeoproteins. linc-MYH in nuclei of fast-type myofibers prevents slow-type and enhances fast-type gene expression. Functional fast-sarcomeric unit formation is achieved by the coordinate expression of fast MYHs and linc-MYH, under the control of a common Six-bound enhancer.