DNA methylation dynamics in muscle development and disease

DNA methylation is an essential epigenetic modification for mammalian development and is crucial for the establishment and maintenance of cellular identity. Traditionally, DNA methylation has been considered as a permanent repressive epigenetic mark. However, the application of genome-wide approaches has allowed the analysis of DNA methylation in different genomic contexts revealing a more dynamic regulation than originally thought, since active DNA methylation and demethylation occur during cellular differentiation and tissue specification. Satellite cells are the primary stem cells in adult skeletal muscle and are responsible for postnatal muscle growth, hypertrophy, and muscle regeneration. This review outlines the published data regarding DNA methylation changes along the skeletal muscle program, in both physiological and pathological conditions, to better understand the epigenetic mechanisms that control myogenesis

uPA deficiency exacerbates muscular dystrophy in MDX mice

Duchenne muscular dystrophy (DMD) is a fatal and incurable muscle degenerative disorder. We identify a function of the protease urokinase plasminogen activator (uPA) in mdx mice, a mouse model of DMD. The expression of uPA is induced in mdx dystrophic muscle, and the genetic loss of uPA in mdx mice exacerbated muscle dystrophy and reduced muscular function. Bone marrow (BM) transplantation experiments revealed a critical function for BM-derived uPA in mdx muscle repair via three mechanisms: (1) by promoting the infiltration of BM-derived inflammatory cells; (2) by preventing the excessive deposition of fibrin; and (3) by promoting myoblast migration. Interestingly, genetic loss of the uPA receptor in mdx mice did not exacerbate muscular dystrophy in mdx mice, suggesting that uPA exerts its effects independently of its receptor. These findings underscore the importance of uPA in muscular dystrophy

MacroH2A1.1 regulates mitochondrial respiration by limiting nuclear NAD+ consumption

Histone variants are structural components of eukaryotic chromatin that can replace replication-coupled histones in the nucleosome. The histone variant macroH2A.1.1 contains a macrodomain able to bind NAD+ derived metabolites. Here, we report that macroH2A.1.1 is rapidly induced during myogenic differentiation through a switch in alternative splicing. Importantly, myotubes lacking macroH2A.1.1 display a defect in mitochondrial respiratory capacity. We find that the metabolite-interacting macrodomain is essential for sustaining optimal mitochondrial function, but dispensable for gene regulation. Through direct binding, macroH2A.1.1 inhibits basal poly-ADP ribose polymerase 1 activity and thus reduces nuclear NAD+ consumption. Consequentially, accumulation of the NAD+ precursor NMN allows the maintenance of mitochondrial NAD+ pools critical for respiration. Our data indicate that macroH2A.1.1-containing chromatin regulates mitochondrial respiration by limiting nuclear NAD+ consumption and establishing a buffer of NAD+ precursors in differentiated cells

Different lipid transfer protein mRNA accumulate in distinct parts of Prunus amygdalus flower

cDNA clones for three lipid transfer proteins (LTPI, LTPII and LTPIII) have been isolated from Prunus amygdalus, Batsch. The degree of similarity between these three LTPs from almond is around 70% and it allows a study of the mRNA accumulation for each one using specific probes. Although the three genes are highly expressed during floral development, significant differences in mRNA levels can be observed in distinct floral organs and in the developing fruit. Southern analysis indicates that in the genus Prunus, genes coding for LTP appear as belonging to a small multigenic family where at least three members differ significantly each one forming a distinct subfamily.The present work was supported by grant BIO93/0901 from Plan Nacional de Investigación Cientı́fica and Técnica. M.S. has a fellowship from Plan de Formación de Personal Investigador. This work has been carried out within the framework of Centre de Referència de Biotecnologia de la Generalitat de Catalunya.Peer reviewe

Phosphorylation of MRF4 transactivation domain by p38 mediates repression of specific myogenic genes

Skeletal myogenesis is associated with the activation of four muscle regulatory factors (MRFs): Myf5, MyoD, Myogenin and MRF4. Here we report that p38 mitogen-activated protein kinase represses the transcriptional activity of MRF4 (involved in late stages of myogenesis), resulting in downregulation of specific muscle genes. MRF4 is phosphorylated in vitro and in vivo by p38 on two serines (Ser31 and Ser42) located in the N-terminal transactivation domain, resulting in reduced MRF4-mediated transcriptional activity. In contrast, nonphosphorylatable MRF4 mutants display increased transcriptional activity and are able to advance both myoblast fusion and differentiation. We also show that expression of desmin and α-actin, but not muscle creatin kinase, decreased at late stages of muscle differentiation, correlating with the induction of MRF4 and p38 activation. Accordingly, inhibition of p38 during late myogenesis results in the upregulation of both desmin and α-actin. We propose that repression of MRF4 activity by p38 phosphorylation may represent a new mechanism for the silencing of specific muscle genes at the terminal stages of muscle differentiation

Muscle cell identity requires Pax7-mediated lineage-specific DNA demethylation

Altres ajuts: Minnesota Regenerative Medicine grant (MRM 2015 PDSCH 003)BACKGROUND: Skeletal muscle stem cells enable the formation, growth, maintenance, and regeneration of skeletal muscle throughout life. The regeneration process is compromised in several pathological conditions, and muscle progenitors derived from pluripotent stem cells have been suggested as a potential therapeutic source for tissue replacement. DNA methylation is an important epigenetic mechanism in the setting and maintenance of cellular identity, but its role in stem cell determination towards the myogenic lineage is unknown. Here we addressed the DNA methylation dynamics of the major genes orchestrating the myogenic determination and differentiation programs in embryonic stem (ES) cells, their Pax7-induced myogenic derivatives, and muscle stem cells in proliferating and differentiating conditions. RESULTS: Our data showed a common muscle-specific DNA demethylation signature required to acquire and maintain the muscle-cell identity. This specific-DNA demethylation is Pax7-mediated, and it is a prime event in muscle stem cells gene activation. Notably, downregulation of the demethylation-related enzyme Apobec2 in ES-derived myogenic precursors reduced myogenin-associated DNA demethylation and dramatically impaired the expression of differentiation markers and, ultimately, muscle differentiation. CONCLUSIONS: Our results underscore DNA demethylation as a key mechanism driving myogenesis and identify specific Pax7-mediated DNA demethylation signatures to acquire and maintain the muscle-cell identity. Additionally, we provide a panel of epigenetic markers for the efficient and safe generation of ES- and induced pluripotent stem cell (iPS)-derived myogenic progenitors for therapeutic applications

HDAC11 is a novel regulator of fatty acid oxidative metabolism in skeletal muscle

Skeletal muscle is the largest tissue in mammalian organisms and is a key determinant of basal metabolic rate and whole‐body energy metabolism. Histone deacetylase 11 (HDAC11) is the only member of the class IV subfamily of HDACs, and it is highly expressed in skeletal muscle, but its role in skeletal muscle physiology has never been investigated. Here, we describe for the first time the consequences of HDAC11 genetic deficiency in skeletal muscle, which results in the improvement of muscle function enhancing fatigue resistance and muscle strength. Loss of HDAC11 had no obvious impact on skeletal muscle structure but increased the number of oxidative myofibers by promoting a glycolytic‐to‐oxidative muscle fiber switch. Unexpectedly, HDAC11 was localized in muscle mitochondria and its deficiency enhanced mitochondrial content. In particular, we showed that HDAC11 depletion increased mitochondrial fatty acid β‐oxidation through activating the AMP‐activated protein kinase‐acetyl‐CoA carboxylase pathway and reducing acylcarnitine levels in vivo, thus providing a mechanistic explanation for the improved muscle strength and fatigue resistance. Overall, our data reveal a unique role of HDAC11 in the maintenance of muscle fiber‐type balance and the mitochondrial lipid oxidation. These findings shed light on the mechanisms governing muscle metabolism and may have implications for chronic muscle metabolic disease management.We thank Rosa Mª Ampudia and Dr. Sara Capdevila for excellent technical assistance with mice. We also thank Dr. Pilar Armengol for its technological support with the confocal microscopy. We thank Drs. Dolors Serra and Coral Sanfeliu for kindly providing ACC/pACC and TOM20, and COXIV antibodies, respectively. We thank Drs. Mercè Jardí and Monserrat Batlle for sharing with us the grip strength and treadmill equipments, respectively. Finally, we are also very grateful to Drs. Dolors Serra for her advice and helpful discussions. This work was supported by Ministerio de Economía y Competitividad (BFU2016‐80748 to MS and BFU2017‐89408‐R to AMP) and Ministerio de Ciencia, Innovación y Universidades (RTI2018‐094009‐B‐I00 to MAP), Feder funds, Generalitat de Catalunya (2017 SGR969 and 2017 SGR206), and Junta de Castilla y Leon (CSI239P18). EH was supported in part by BFU2016‐80748, and YNA was supported by Ministerio de Educación, Cultura y Deport (FPU12/05668).Peer reviewe

Loss of HDAC11 accelerates skeletal muscle regeneration in mice

Histone deacetylase 11 (HDAC11) is the latest identified member of the histone deacetylase family of enzymes. It is highly expressed in brain, heart, testis, kidney, and skeletal muscle, although its role in these tissues is poorly understood. Here, we investigate for the first time the consequences of HDAC11 genetic impairment on skeletal muscle regeneration, a process principally dependent on its resident stem cells (satellite cells) in coordination with infiltrating immune cells and stromal cells. Our results show that HDAC11 is dispensable for adult muscle growth and establishment of the satellite cell population, while HDAC11 deficiency advances the regeneration process in response to muscle injury. This effect is not caused by differences in satellite cell activation or proliferation upon injury, but rather by an enhanced capacity of satellite cells to differentiate at early regeneration stages in the absence of HDAC11. Infiltrating HDAC11‐deficient macrophages could also contribute to this accelerated muscle regenerative process by prematurely producing high levels of IL‐10, a cytokine known to promote myoblast differentiation. Altogether, our results show that HDAC11 depletion advances skeletal muscle regeneration and this finding may have potential implications for designing new strategies for muscle pathologies coursing with chronic damage.This work was supported by Ministerio de Economía y Competitividad (BFU2016‐80748 to MS and BFU2017‐89408‐R to AMP) and Ministerio de Ciencia, Innovación y Universidades (RTI2018‐094009‐B‐I00 to MAP), FEDER funds, Generalitat de Catalunya (2017 SGR969 and 2017 SGR206), and Junta de Castilla y Leon (CSI239P18). YN‐A was supported by FPU12/05668, and EH was supported in part by BFU2016‐80748 project.Peer reviewe

Amelioration of Duchenne muscular dystrophy in mdx mice by elimination of matrix-associated fibrin-driven inflammation coupled to the αMβ2 leukocyte integrin receptor

In Duchenne muscular dystrophy (DMD), a persistently altered and reorganizing extracellular matrix (ECM) within inflamed muscle promotes damage and dysfunction. However, the molecular determinants of the ECM that mediate inflammatory changes and faulty tissue reorganization remain poorly defined. Here, we show that fibrin deposition is a conspicuous consequence of muscle-vascular damage in dystrophic muscles of DMD patients and mdx mice and that elimination of fibrin(ogen) attenuated dystrophy progression in mdx mice. These benefits appear to be tied to: (i) a decrease in leukocyte integrin α(M)β(2)-mediated proinflammatory programs, thereby attenuating counterproductive inflammation and muscle degeneration; and (ii) a release of satellite cells from persistent inhibitory signals, thereby promoting regeneration. Remarkably, Fib-gamma(390-396A) (Fibγ(390-396A)) mice expressing a mutant form of fibrinogen with normal clotting function, but lacking the α(M)β(2) binding motif, ameliorated dystrophic pathology. Delivery of a fibrinogen/α(M)β(2) blocking peptide was similarly beneficial. Conversely, intramuscular fibrinogen delivery sufficed to induce inflammation and degeneration in fibrinogen-null mice. Thus, local fibrin(ogen) deposition drives dystrophic muscle inflammation and dysfunction, and disruption of fibrin(ogen)-α(M)β(2) interactions may provide a novel strategy for DMD treatment.This work was supported by the European Commission, Seventh Framework Programme (Myoage, Optistem and Endostem