Efectes de la proteïna Mitofusina 2 sobre el metabolisme muscular

Els mitocondris són orgànuls citoplasmàtics que tenen un paper fonamental en múltiples processos biològics com l’oxidació de substrats i la producció d’ATP, la senyalització cel•lular, l’apoptosi, el control del cicle cel•lular i l’homeòstasi del calci. Els mitocondris són orgànuls dinàmics, que pateixen canvis de morfologia regulats per processos de fusió i de fissió. Existeix un equilibri entre ambdós processos que és indispensable per a la correcta funció mitocondrial. Les proteïnes que participen directament en la fusió mitocondrial en mamífers són les mitofusines (Mfn1 i Mfn2), localitzades a la membrana mitocondrial externa i OPA1, situada a la membrana mitocondrial interna. Diferents estudis han demostrat que la proteïna Mfn2, a més de promoure la fusió dels mitocondris, també està implicada en la interacció entre els mitocondris i el reticle endoplasmàtic i que participa en la regulació del cicle cel•lular i del metabolisme mitocondrial. Per altra banda, l’expressió de Mfn2 es troba disminuïda en múscul esquelètic en situacions de resistència a la insulina, com l’obesitat o la diabetis de tipus 2, que a la vegada es caracteritzen per una alterada activitat mitocondrial. En base a aquestes observacions, l’objectiu principal de la present tesi doctoral ha estat estudiar els efectes de la modulació de l’expressió de Mfn2 sobre el metabolisme i la bioenergètica mitocondrial en múscul esquelètic. Amb aquest propòsit hem expressat una forma truncada de Mfn2 (hMfn2Δ614-757) o bé hem reprimit l’expressió de Mfn2 endògena en el model cel•lular C2C12 i en múscul esquelètic de ratolí. Per dur a terme aquest objectiu hem generat 3 models de ratolí diferents: el model d’expressió transitòria de la forma hMfn2Δ614-757; el model de repressió transitòria de Mfn2 i el ratolí knockdown de Mfn2. Els dos primers models han estat generats mitjançant la tècnica de l’electrotransferència d’ADN en múscul esquelètic. La sobreexpressió de la forma hMfn2Δ614-757 en cèl•lules C2C12 diferenciades incrementa el consum d’oxigen mitocondrial en situació basal i també en desacoblar la cadena de transport d’electrons de la síntesi d’ATP, suggerint una major capacitat respiratòria dels miotubs que expressen la hMfn2Δ614-757. En múscul esquelètic de ratolí, l’expressió d’aquesta forma de Mfn2 causa una estimulació de la taxa d’oxidació de glucosa així com un increment de la Respiratory Control Ratio (RCR). La inducció del metabolisme mitocondrial observada en sobreexpressar la forma hMfn2Δ614-757 no és deguda a un augment de la massa mitocondrial, sinó a un increment en l’expressió i l’activitat d’alguns dels complexes de la cadena respiratòria mitocondrial. La repressió de Mfn2 en miotubs C2C12 produeix un increment en la respiració no associada a la producció d’ATP o proton leak i una disminució en el potencial de membrana mitocondrial. Aquests resultats indiquen que la repressió de Mfn2 provoca el desacoblament de la cadena de transport d’electrons i la síntesi d’ATP, suggerint una disminució de l’eficiència de la fosforilació oxidativa. Els músculs dels ratolins knockdown de Mfn2 presenten una reducció de la taxa d’oxidació de glucosa i de la Respiratory Control Ratio. A més, la repressió de Mfn2 disminueix l’activitat del complex IV de la cadena respiratòria. En conjunt aquests resultats suggereixen que la disminució de l’expressió de Mfn2 origina una disfunció del sistema de transport electrònic mitocondrial. També cal remarcar que els ratolins knockdown de Mfn2 presenten una major susceptibilitat a desenvolupar resistència a la insulina en resposta a l’envelliment o a una dieta rica en greixos. La disfunció mitocondrial i l’augment en la producció d’espècies reactives d’oxigen (ROS) observats en el múscul esquelètic d’aquests ratolins podrien explicar aquesta major susceptibilitat.Mitochondria are cellular organelles that play a fundamental role in many cellular functions, such as substrates oxidation, ATP production, apoptosis and calcium economy. Mitochondria are dynamic organelles that can fuse and divide; the balance between both processes is crucial for a correct mitochondrial function. The most relevant proteins described to date involved in the regulation of mitochondrial fusion are mitofusins 1 and 2 (Mfn1 and Mfn2, respectively) and OPA1. Substantial data indicates that Mfn2 is also a key regulator of cell cycle and mitochondrial metabolism. On the other hand, Mfn2 expression is reduced in skeletal muscle of obese subjects and type 2 diabetic patients, situations characterized by altered mitochondrial activity. Based on these observations, the main objective of this thesis was the study of the metabolic role of Mfn2 in skeletal muscle. We have studied the metabolic effects caused by the manipulation of Mfn2 expression in mice skeletal muscle in vivo. By means of DNA electrotransfer technologies, we have expressed a truncated Mfn2 mutant in skeletal muscle and we have also repressed endogenous Mfn2 expression with microRNAs. We have also generated a skeletal muscle Mfn2 knockout mouse model (Mfn2 KO). The expression of truncated Mfn2 mutant in tibialis stimulated glucose oxidation and increased the Respiratory Control Ratio (RCR). It also increased the expression of subunits Cox4 of OXPHOS complex IV and Atp5a1 of complex V. We observed these metabolic effects in absence of changes in mitochondrial content. The repression of Mfn2 in mice skeletal muscle caused a marked reduction in the expression of subunit Cox4 of OXPHOS complex IV, accompanied with a 20% of decrease in COX activity. In this case we neither observed differences in mitochondrial content. Skeletal muscle from Mfn2 KO mice showed a decrease in glucose oxidation and in the RCR. In addition, Mfn2 KO mice showed a higher susceptibility to develop insulin resistance in response to aging or a high fat diet. Mitochondrial dysfunction and the increased ROS production observed in skeletal muscle of these mice could explain this higher susceptibility

The dystrophin glycoprotein complex regulates the epigenetic activation of muscle stem cell commitment

Asymmetrically dividing muscle stem cells in skeletal muscle give rise to committed cells, where the myogenic determination factor Myf5 is transcriptionally activated by Pax7. This activation is dependent on Carm1, which methylates Pax7 on multiple arginine residues, to recruit the ASH2L:MLL1/2:WDR5:RBBP5 histone methyltransferase complex to the proximal promoter of Myf5. Here, we found that Carm1 is a specific substrate of p38γ/MAPK12 and that phosphorylation of Carm1 prevents its nuclear translocation. Basal localization of the p38γ/p-Carm1 complex in muscle stem cells occurs via binding to the dystrophin-glycoprotein complex (DGC) through β1-syntrophin. In dystrophin-deficient muscle stem cells undergoing asymmetric division, p38γ/β1-syntrophin interactions are abrogated, resulting in enhanced Carm1 phosphorylation. The resulting progenitors exhibit reduced Carm1 binding to Pax7, reduced H3K4-methylation of chromatin, and reduced transcription of Myf5 and other Pax7 target genes. Therefore, our experiments suggest that dysregulation of p38γ/Carm1 results in altered epigenetic gene regulation in Duchenne muscular dystrophy.We thank Drs. Jeffrey Dilworth and Lynn Megeney for careful reading of the manuscript. We also thank Jennifer Ritchie for animal husbandry, Dr. Lawrence Puente for mass spectrometry analysis, Dr. Chloë van Oostende for microscopy and imaging analysis, Paul Oleynik for FACS, and Fan Xiao, Natasha Mercier, and David Wilson for technical assistance. N.C.C. is a recipient of the Centre for Neuromuscular Disease Scholarship in Translational Research Award from the University of Ottawa Brain and Mind Research Institute and was supported by Postdoctoral fellowships from the Canadian Institutes of Health Research (CIHR) and the Ontario Institute for Regenerative Medicine (OIRM). F.P.C. was supported by a Postdoctoral fellowship from the French Muscular Dystrophy Association (AFM)-Téléthon (380782). C.E.B. is supported by a Postdoctoral fellowship from OIRM. M.L. was supported by a Postdoctoral fellowship from CIHR. P.M.-C. acknowledges support from ERC-2016-AdG-741966 (STEM-AGING) and SAF2015-67369-R. M.A.R. holds the Canada Research Chair in Molecular Genetics. These studies were carried out with support of grants to M.A.R. from the US NIH (R01AR044031), the Canadian Institutes of Health Research (FDN-148387), the Muscular Dystrophy Association (USA), E-Rare-2: Canadian Institutes of Health Research/Muscular Dystrophy Canada (ERA-132935), and the Stem Cell Network

Chromatin-wide and transcriptome profiling integration uncovers p38α MAPK as a global regulator of skeletal muscle differentiation

Background: Extracellular stimuli induce gene expression responses through intracellular signaling mediators. The p38 signaling pathway is a paradigm of the mitogen-activated protein kinase (MAPK) family that, although originally identified as stress-response mediator, contributes to establishing stem cell differentiation fates. p38α is central for induction of the differentiation fate of the skeletal muscle stem cells (satellite cells) through not fully characterized mechanisms. Methods: To investigate the global gene transcription program regulated by p38α during satellite cell differentiation (myogenesis), and to specifically address whether this regulation occurs through direct action of p38α on gene promoters, we performed a combination of microarray gene expression and genome-wide binding analyses. For experimental robustness, two myogenic cellular systems with genetic and chemical loss of p38α function were used: (1) satellite cells derived from mice with muscle-specific deletion of p38α, and (2) the C2C12 murine myoblast cell line cultured in the absence or presence of the p38α/β inhibitor SB203580. Analyses were performed at cell proliferation and early differentiation stages. Results: We show that p38α binds to a large set of active promoters during the transition of myoblasts from proliferation to differentiation stages. p38α-bound promoters are enriched with binding motifs for several transcription factors, with Sp1, Tcf3/E47, Lef1, FoxO4, MyoD, and NFATc standing out in all experimental conditions. p38α association with chromatin correlates very well with high levels of transcription, in agreement with its classical function as an activator of myogenic differentiation. Interestingly, p38α also associates with genes repressed at the onset of differentiation, thus highlighting the relevance of p38-dependent chromatin regulation for transcriptional activation and repression during myogenesis. Conclusions: These results uncover p38α association and function on chromatin at novel classes of target genes during skeletal muscle cell differentiation. This is consistent with this MAPK isoform being a transcriptional regulator.The authors acknowledge funding from MINECO, Spain (SAF2012-38547, PLE2009-0124, SAF2015-67369-R, "María de Maeztu" Programme for Units of Excellence in R&D MDM-2014-0370), AFM, MDA, DDP-Netherlands, E-RARE, Fundació Marató TV3 and EU-FP7 (Myoage, Optistem and Endostem). JS is recipient of a Juan de la Cierva postdoctoral fellowship

Senescence atlas reveals an aged-like inflamed niche that blunts muscle regeneration

Tissue regeneration requires coordination between resident stem cells and local niche cells1,2. Here we identify that senescent cells are integral components of the skeletal muscle regenerative niche that repress regeneration at all stages of life. The technical limitation of senescent-cell scarcity3 was overcome by combining single-cell transcriptomics and a senescent-cell enrichment sorting protocol. We identified and isolated different senescent cell types from damaged muscles of young and old mice. Deeper transcriptome, chromatin and pathway analyses revealed conservation of cell identity traits as well as two universal senescence hallmarks (inflammation and fibrosis) across cell type, regeneration time and ageing. Senescent cells create an aged-like inflamed niche that mirrors inflammation associated with ageing (inflammageing4) and arrests stem cell proliferation and regeneration. Reducing the burden of senescent cells, or reducing their inflammatory secretome through CD36 neutralization, accelerates regeneration in young and old mice. By contrast, transplantation of senescent cells delays regeneration. Our results provide a technique for isolating in vivo senescent cells, define a senescence blueprint for muscle, and uncover unproductive functional interactions between senescent cells and stem cells in regenerative niches that can be overcome. As senescent cells also accumulate in human muscles, our findings open potential paths for improving muscle repair throughout life.We thank M. Jardí, A. Navarro, J. M. Ballestero, K. Slobodnyuk, M. González, J. López and M. Raya for their technical contributions; A. Harada and K. Tanaka for assistance in ATAC-seq; all of the members of the P.M.-C. laboratory for discussions; J. Campisi for p16-3MR mice; J. A. Fernández-Blanco (PRBB Animal Facility); O. Fornas (UPF/CRG FACS Facility); E. Rebollo (IBMB Molecular Imaging Platform); V. A. Raker for manuscript editing; and the members of the Myoage network (A. Maier) for human material. We acknowledge funding from MINECO-Spain (RTI2018-096068, to P.M.-C. and E.P.); ERC-2016-AdG-741966, LaCaixa-HEALTH-HR17-00040, MDA, UPGRADE-H2020-825825, AFM, DPP-Spain, Fundació La MaratóTV3-80/19-202021 and MWRF to P.M.-C.; Fundació La MaratóTV3-137/38-202033 to A.L.S.; María-de-Maeztu Program for Units of Excellence to UPF (MDM-2014-0370) and Severo-Ochoa Program for Centers of Excellence to CNIC (SEV-2015-0505). This work was also supported by JST-CREST JPMJCR16G1 and MEXT/JSPS JP20H00456/18H05527 to Y.O.; the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030502) to M.A.E.; V.M. and A.C. were supported by FPI and Maria-de-Maeztu predoctoral fellowships, respectively, and V.S. by a Marie Skłodowska-Curie individual fellowship. Parts of the figures were drawn using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licences/by/3.0/)

Mitochondrial dynamics maintain muscle stem cell regenerative competence throughout adult life by regulating metabolism and mitophagy

Skeletal muscle regeneration depends on the correct expansion of resident quiescent stem cells (satellite cells), a process that becomes less efficient with aging. Here, we show that mitochondrial dynamics are essential for the successful regenerative capacity of satellite cells. The loss of mitochondrial fission in satellite cells-due to aging or genetic impairment-deregulates the mitochondrial electron transport chain (ETC), leading to inefficient oxidative phosphorylation (OXPHOS) metabolism and mitophagy and increased oxidative stress. This state results in muscle regenerative failure, which is caused by the reduced proliferation and functional loss of satellite cells. Regenerative functions can be restored in fission-impaired or aged satellite cells by the re-establishment of mitochondrial dynamics (by activating fission or preventing fusion), OXPHOS, or mitophagy. Thus, mitochondrial shape and physical networking controls stem cell regenerative functions by regulating metabolism and proteostasis. As mitochondrial fission occurs less frequently in the satellite cells in older humans, our findings have implications for regeneration therapies in sarcopenia.Work in the PMC laboratory was supported by Spanish Ministerio de Ciencia e Innovación ( RTI2018-096068 to P.M.-C. and E.P), ERC - 2016-AdG-741966 , LaCaixa - HEALTH-HR17-00040 , MDA , UPGRADE-H2020-825825 , AFM-Telethon , DPP-Spain , Fundació La Marató TV3-80/19-202021 to P.M.-C; Fundació La Marató TV3-137/38-202033 to A.L.S.; partly supported by Milky Way Research Foundation (MWRF) to P.M.-C; Severo Ochoa Program for Centers of Excellence to CNIC ( SEV-2015-0505 ) and Maria de Maeztu Program for Units of Excellence to UPF ( MDM-2014-0370 ). Work in the JAE laboratory was supported by Ministerio de Ciencia e Innovacion ( RTI2018-099357-B-I00 , RED2018-102576-T ), Human Frontier Science Program HFSP ( RGP0016/2018 ), Centro de Investigación Biomédica en Red en Fragilidad y Envejecimento Saludable ( CIBERFES16/10/00282 ), and Leduq Foundation award ( REDOX-17CVD04 ). Work in JMV laboratory was supported by the Spanish Ministerio de Ciencia e Innovación ( RTI2018-100695-B-I00 ), Spanish Junta de Andalucía ( P18-RT-4264 , 1263735-R and BIO-276 ), the FEDER Funding Program from the European Union , and Universidad de Córdoba . The authors are indebted to the personnel from the Servicio Centralizado de Apoyo a la Investigación (SCAI; University of Córdoba) for technical support with the transmission electron microscope. Work in MS laboratory was funded by the Italian Assoc. for Cancer Research ( AIRC IG-D17388 and ID23257 ) and ASI (MARS-PRE, project DC-VUM-2017-006). X.H., S.C., I.R.-P, and A.C were supported by Severo Ochoa PFI , PI , FPI , and H2020 Marie Skłodowska-Curie Actions predoctoral fellowships, respectively. P.H.-A was supported by Juan de la Cierva-Incorporación fellowship