RESUMO
Athletes increasingly engage in repeated sprint training consisting in repeated short all-out efforts interspersed by short recoveries. When performed in hypoxia (RSH), it may lead to greater training effects than in normoxia (RSN); however, the underlying molecular mechanisms remain unclear. This study aimed at elucidating the effects of RSH on skeletal muscle metabolic adaptations as compared to RSN. Sixteen healthy young men performed nine repeated sprint training sessions in either normoxia (FIO2 = 0.209, RSN, n = 7) or normobaric hypoxia (FIO2 = 0.136, RSH, n = 9). Before and after the training period, exercise performance was assessed by using repeated sprint ability (RSA) and Wingate tests. Vastus lateralis muscle biopsies were performed to investigate muscle metabolic adaptations using proteomics combined with western blot analysis. Similar improvements were observed in RSA and Wingate tests in both RSN and RSH groups. At the muscle level, RSN and RSH reduced oxidative phosphorylation protein content but triggered an increase in mitochondrial biogenesis proteins. Proteomics showed an increase in several S100A family proteins in the RSH group, among which S100A13 most strongly. We confirmed a significant increase in S100A13 protein by western blot in RSH, which was associated with increased Akt phosphorylation and its downstream targets regulating protein synthesis. Altogether our data indicate that RSH may activate an S100A/Akt pathway to trigger specific adaptations as compared to RSN.
Assuntos
Adaptação Fisiológica , Hipóxia , Músculo Esquelético , Proteínas S100 , Transdução de Sinais , Humanos , Masculino , Hipóxia/metabolismo , Músculo Esquelético/metabolismo , Adaptação Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Adulto Jovem , Proteínas S100/metabolismo , Adulto , Proteínas Proto-Oncogênicas c-akt/metabolismo , Exercício Físico/fisiologiaRESUMO
The roles of CDK4 in the cell cycle have been extensively studied, but less is known about the mechanisms underlying the metabolic regulation by CDK4. Here, we report that CDK4 promotes anaerobic glycolysis and represses fatty acid oxidation in mouse embryonic fibroblasts (MEFs) by targeting the AMP-activated protein kinase (AMPK). We also show that fatty acid oxidation (FAO) is specifically induced by AMPK complexes containing the α2 subunit. Moreover, we report that CDK4 represses FAO through direct phosphorylation and inhibition of AMPKα2. The expression of non-phosphorylatable AMPKα2 mutants, or the use of a CDK4 inhibitor, increased FAO rates in MEFs and myotubes. In addition, Cdk4-/- mice have increased oxidative metabolism and exercise capacity. Inhibition of CDK4 mimicked these alterations in normal mice, but not when skeletal muscle was AMPK deficient. This novel mechanism explains how CDK4 promotes anabolism by blocking catabolic processes (FAO) that are activated by AMPK.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Ácidos Graxos/metabolismo , Músculo Esquelético/metabolismo , Condicionamento Físico Animal , Proteínas Quinases Ativadas por AMP/genética , Animais , Quinase 4 Dependente de Ciclina/genética , Embrião de Mamíferos/metabolismo , Ácidos Graxos/genética , Fibroblastos/metabolismo , Camundongos , Camundongos Knockout , Mutação , OxirreduçãoRESUMO
Regular exercise is associated with pronounced health benefits. The molecular processes involved in physiological adaptations to exercise are best understood in skeletal muscle. Enhanced mitochondrial functions in muscle are central to exercise-induced adaptations. However, regular exercise also benefits the brain and is a major protective factor against neurodegenerative diseases, such as the most common age-related form of dementia, Alzheimer's disease, or the most common neurodegenerative motor disorder, Parkinson's disease. While there is evidence that exercise induces signalling from skeletal muscle to the brain, the mechanistic understanding of the crosstalk along the muscle-brain axis is incompletely understood. Mitochondria in both organs, however, seem to be central players. Here, we provide an overview on the central role of mitochondria in exercise-induced communication routes from muscle to the brain. These routes include circulating factors, such as myokines, the release of which often depends on mitochondria, and possibly direct mitochondrial transfer. On this basis, we examine the reported effects of different modes of exercise on mitochondrial features and highlight their expected benefits with regard to neurodegeneration prevention or mitigation. In addition, knowledge gaps in our current understanding related to the muscle-brain axis in neurodegenerative diseases are outlined.
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Encéfalo/metabolismo , Suscetibilidade a Doenças , Músculo Esquelético/metabolismo , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Transdução de Sinais , Animais , Biomarcadores , Citocinas/metabolismo , Metabolismo Energético , Exercício Físico , Humanos , Mitocôndrias , Dinâmica Mitocondrial , Doenças Neurodegenerativas/patologia , Neuroproteção , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
PURPOSE: Mechanisms underlying the efficacy of sprint interval training (SIT) remain to be understood. We previously reported that an acute bout of SIT disrupts the integrity of the sarcoplasmic reticulum (SR) Ca2+ release channel, the ryanodine receptor 1 (RyR1), in recreationally active human subjects. We here hypothesize that in addition to improving the exercise performance of recreationally active humans, a period of repeated SIT sessions would make the RyR1 protein less vulnerable and accelerate recovery of contractile function after a SIT session. METHODS: Eight recreationally active males participated in a 3-week SIT program consisting of nine sessions of four-six 30-s all-out cycling bouts with 4 min of rest between bouts. RESULTS: Total work performed during a SIT session and maximal power (Wmax) reached during an incremental cycling test were both increased by ~ 7.5% at the end of the training period (P < 0.05). Western blots performed on vastus lateralis muscle biopsies taken before, 1 h, 24 h and 72 h after SIT sessions in the untrained and trained state showed some protection against SIT-induced reduction of full-length RyR1 protein expression in the trained state. SIT-induced knee extensor force deficits were similar in the untrained and trained states, with a major reduction in voluntary and electrically evoked forces immediately and 1 h after SIT (P < 0.05), and recovery after 24 h. CONCLUSIONS: Three weeks of SIT improves exercise performance and provides some protection against RyR1 modification, whereas it does not accelerate recovery of contractile function.
Assuntos
Exercício Físico/fisiologia , Resistência Física/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Adaptação Fisiológica/fisiologia , Adulto , Teste de Esforço/métodos , Treinamento Intervalado de Alta Intensidade/métodos , Humanos , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Consumo de Oxigênio/fisiologia , Adulto JovemRESUMO
Besides its crucial role in the pathogenesis of Alzheimer's disease, the knowledge of amyloid precursor protein (APP) physiologic functions remains surprisingly scarce. Here, we show that APP regulates the transcription of the glial cell line-derived neurotrophic factor (GDNF). APP-dependent regulation of GDNF expression affects muscle strength, muscular trophy, and both neuronal and muscular differentiation fundamental for neuromuscular junction (NMJ) maturation in vivo In a nerve-muscle coculture model set up to modelize NMJ formation in vitro, silencing of muscular APP induces a 30% decrease in secreted GDNF levels and a 40% decrease in the total number of NMJs together with a significant reduction in the density of acetylcholine vesicles at the presynaptic site and in neuronal maturation. These defects are rescued by GDNF expression in muscle cells in the conditions where muscular APP has been previously silenced. Expression of GDNF in muscles of amyloid precursor protein null mice corrected the aberrant synaptic morphology of NMJs. Our findings highlight for the first time that APP-dependent GDNF expression drives the process of NMJ formation, providing new insights into the link between APP gene regulatory network and physiologic functions.-Stanga, S., Zanou, N., Audouard, E., Tasiaux, B., Contino, S., Vandermeulen, G., René, F., Loeffler, J.-P., Clotman, F., Gailly, P., Dewachter, I., Octave, J.-N., Kienlen-Campard, P. APP-dependent glial cell line-derived neurotrophic factor gene expression drives neuromuscular junction formation.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Fibroblastos/fisiologia , Regulação da Expressão Gênica/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Junção Neuromuscular/fisiologia , Animais , Células Cultivadas , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Camundongos , Camundongos Knockout , Músculo Esquelético/fisiologiaRESUMO
KEY POINTS: Increase in blood pressure in the renal afferent arteriole is known to induce an increase in cytosolic calcium concentration ([Ca2+ ]i ) of juxtaglomerular (JG) cells and to result in a decreased secretion of renin. Mechanical stimulation of As4.1 JG cells induces an increase in [Ca2+ ]i that is inhibited by HC067047 and RN1734, two inhibitors of TRPV4, or by siRNA-mediated repression of TRPV4. Inhibition of TRPV4 impairs pressure-induced decrease in renin secretion. Compared to wild-type mice, Trpv4-/- mice present increased resting plasma levels of renin and aldosterone and present a significantly altered pressure-renin relationship. We suggest that TRPV4 channel participates in mechanosensation at the juxtaglomerular apparatus. ABSTRACT: The renin-angiotensin system is a crucial blood pressure regulation system. It consists of a hormonal cascade where the rate-limiting enzyme is renin, which is secreted into the blood flow by renal juxtaglomerular (JG) cells in response to low pressure in the renal afferent arteriole. In contrast, an increase in blood pressure results in a decreased renin secretion. This is accompanied by a transitory increase in [Ca2+ ]i of JG cells. The inverse relationship between [Ca2+ ]i and renin secretion has been called the 'calcium paradox' of renin release. How increased pressure induces a [Ca2+ ]i transient in JG cells, is however, unknown. We observed that [Ca2+ ]i transients induced by mechanical stimuli in JG As4.1 cells were completely abolished by HC067047 and RN1734, two inhibitors of TRPV4. They were also reduced by half by siRNA-mediated repression of TRPV4 but not after repression or inhibition of TRPV2 or Piezo1 ion channels. Interestingly, the stimulation of renin secretion by the adenylate cyclase activator forskolin was totally inhibited by cyclic stretching of the cells. This effect was mimicked by stimulation with GSK1016790A and 4αPDD, two activators of TRPV4 and inhibited in the presence of HC067047. Moreover, in isolated perfused kidneys from Trpv4-/- mice, the pressure-renin relationship was significantly altered. In vivo, Trpv4-/- mice presented increased plasma levels of renin and aldosterone compared to wild-type mice. Altogether, our results suggest that TRPV4 is involved in the pressure-induced entry of Ca2+ in JG cells, which inhibits renin release and allows the negative feedback regulation on blood pressure.
Assuntos
Sistema Justaglomerular/metabolismo , Mecanotransdução Celular/fisiologia , Renina/antagonistas & inibidores , Canais de Cátion TRPV/fisiologia , Aldosterona/sangue , Animais , Cálcio/fisiologia , Linhagem Celular Tumoral , Masculino , Camundongos Knockout , Pressão , Renina/sangue , Renina/metabolismo , Canais de Cátion TRPV/genéticaRESUMO
Increased plasma osmolarity induces intracellular water depletion and cell shrinkage (CS) followed by activation of a regulatory volume increase (RVI). In skeletal muscle, the hyperosmotic shock-induced CS is accompanied by a small membrane depolarization responsible for a release of Ca(2+) from intracellular pools. Hyperosmotic shock also induces phosphorylation of STE20/SPS1-related proline/alanine-rich kinase (SPAK). TRPV2 dominant negative expressing fibres challenged with hyperosmotic shock present a slower membrane depolarization, a diminished Ca(2+) response, a smaller RVI response, a decrease in SPAK phosphorylation and defective muscle function. We suggest that hyperosmotic shock induces TRPV2 activation, which accelerates muscle cell depolarization and allows the subsequent Ca(2+) release from the sarcoplasmic reticulum, activation of the Na(+) -K(+) -Cl(-) cotransporter by SPAK, and the RVI response. Increased plasma osmolarity induces intracellular water depletion and cell shrinkage followed by activation of a regulatory volume increase (RVI). In skeletal muscle, this is accompanied by transverse tubule (TT) dilatation and by a membrane depolarization responsible for a release of Ca(2+) from intracellular pools. We observed that both hyperosmotic shock-induced Ca(2+) transients and RVI were inhibited by Gd(3+) , ruthenium red and GsMTx4 toxin, three inhibitors of mechanosensitive ion channels. The response was also completely absent in muscle fibres overexpressing a non-permeant, dominant negative (DN) mutant of the transient receptor potential, V2 isoform (TRPV2) ion channel, suggesting the involvement of TRPV2 or of a TRP isoform susceptible to heterotetramerization with TRPV2. The release of Ca(2+) induced by hyperosmotic shock was increased by cannabidiol, an activator of TRPV2, and decreased by tranilast, an inhibitor of TRPV2, suggesting a role for the TRPV2 channel itself. Hyperosmotic shock-induced membrane depolarization was impaired in TRPV2-DN fibres, suggesting that TRPV2 activation triggers the release of Ca(2+) from the sarcoplasmic reticulum by depolarizing TTs. RVI requires the sequential activation of STE20/SPS1-related proline/alanine-rich kinase (SPAK) and NKCC1, a Na(+) -K(+) -Cl(-) cotransporter, allowing ion entry and driving osmotic water flow. In fibres overexpressing TRPV2-DN as well as in fibres in which Ca(2+) transients were abolished by the Ca(2+) chelator BAPTA, the level of P-SPAK(Ser373) in response to hyperosmotic shock was reduced, suggesting a modulation of SPAK phosphorylation by intracellular Ca(2+) . We conclude that TRPV2 is involved in osmosensation in skeletal muscle fibres, acting in concert with P-SPAK-activated NKCC1.
Assuntos
Canais de Cálcio/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Membro 2 da Família 12 de Carreador de Soluto/fisiologia , Canais de Cátion TRPV/fisiologia , Animais , Cálcio , Tamanho Celular , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Concentração Osmolar , Pressão Osmótica , FosforilaçãoRESUMO
Adult skeletal muscle can regenerate in response to muscle damage. This ability is conferred by the presence of myogenic stem cells called satellite cells. In response to stimuli such as injury or exercise, these cells become activated and express myogenic regulatory factors (MRFs), i.e., transcription factors of the myogenic lineage including Myf5, MyoD, myogenin, and Mrf4 to proliferate and differentiate into myofibers. The MRF family of proteins controls the transcription of important muscle-specific proteins such as myosin heavy chain and muscle creatine kinase. Different growth factors are secreted during muscle repair among which insulin-like growth factors (IGFs) are the only ones that promote both muscle cell proliferation and differentiation and that play a key role in muscle regeneration and hypertrophy. Different isoforms of IGFs are expressed during muscle repair: IGF-IEa, IGF-IEb, or IGF-IEc (also known as mechano growth factor, MGF) and IGF-II. MGF is expressed first and is observed in satellite cells and in proliferating myoblasts whereas IGF-Ia and IGF-II expression occurs at the state of muscle fiber formation. Interestingly, several studies report the induction of MRFs in response to IGFs stimulation. Inversely, IGFs expression may also be regulated by MRFs. Various mechanisms are proposed to support these interactions. In this review, we describe the general process of muscle hypertrophy and regeneration and decipher the interactions between the two groups of factors involved in the process.
Assuntos
Músculo Esquelético/patologia , Fatores de Regulação Miogênica/metabolismo , Somatomedinas/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Hipertrofia , Inflamação , Desenvolvimento Muscular , Músculos/patologia , Proteína MyoD/metabolismo , Regeneração , Células Satélites de Músculo Esquelético/citologiaRESUMO
Among the inherited myopathies, a group of muscular disorders characterized by structural and metabolic impairments in skeletal muscle, Duchenne muscular dystrophy (DMD) stands out for its devastating progression. DMD pathogenesis is driven by the progressive degeneration of muscle fibers, resulting in inflammation and fibrosis that ultimately affect the overall muscle biomechanics. At the opposite end of the spectrum of muscle diseases, age-related sarcopenia is a common condition that affects an increasing proportion of the elderly. Although characterized by different pathological mechanisms, DMD and sarcopenia share the development of progressive muscle weakness and tissue inflammation. Here, the therapeutic effects of Cyclo Histidine-Proline (CHP) against DMD and sarcopenia are evaluated. In the mdx mouse model of DMD, it is shown that CHP restored muscle contractility and force production, accompanied by the reduction of fibrosis and inflammation in skeletal muscle. CHP furthermore prevented the development of cardiomyopathy and fibrosis in the diaphragm, the two leading causes of death for DMD patients. CHP also attenuated muscle atrophy and functional deterioration in a mouse model of age-related sarcopenia. These findings from two different models of muscle dysfunction hence warrant further investigation into the effects of CHP on muscle pathologies in animal models and eventually in patients.
Assuntos
Modelos Animais de Doenças , Camundongos Endogâmicos mdx , Músculo Esquelético , Distrofia Muscular de Duchenne , Sarcopenia , Animais , Camundongos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Músculo Esquelético/metabolismo , Sarcopenia/patologia , Sarcopenia/prevenção & controle , Masculino , Camundongos Endogâmicos C57BLRESUMO
Mitochondrial respiration extends beyond ATP generation, with the organelle participating in many cellular and physiological processes. Parallel changes in components of the mitochondrial electron transfer system with respiration render it an appropriate hub for coordinating cellular adaption to changes in oxygen levels. How changes in respiration under functional hypoxia (i.e., when intracellular O2 levels limit mitochondrial respiration) are relayed by the electron transfer system to impact mitochondrial adaption and remodeling after hypoxic exposure remains poorly defined. This is largely due to challenges integrating findings under controlled and defined O2 levels in studies connecting functions of isolated mitochondria to humans during physical exercise. Here we present experiments under conditions of hypoxia in isolated mitochondria, myotubes and exercising humans. Performing steady-state respirometry with isolated mitochondria we found that oxygen limitation of respiration reduced electron flow and oxidative phosphorylation, lowered the mitochondrial membrane potential difference, and decreased mitochondrial calcium influx. Similarly, in myotubes under functional hypoxia mitochondrial calcium uptake decreased in response to sarcoplasmic reticulum calcium release for contraction. In both myotubes and human skeletal muscle this blunted mitochondrial adaptive responses and remodeling upon contractions. Our results suggest that by regulating calcium uptake the mitochondrial electron transfer system is a hub for coordinating cellular adaption under functional hypoxia.
Assuntos
Cálcio , Consumo de Oxigênio , Humanos , Cálcio/metabolismo , Consumo de Oxigênio/fisiologia , Respiração Celular , Hipóxia/metabolismo , Músculo Esquelético/metabolismo , Oxigênio/metabolismoRESUMO
The physical connection between mitochondria and endoplasmic reticulum (ER) is an essential signaling hub to ensure organelle and cellular functions. In skeletal muscle, ER-mitochondria calcium (Ca2+) signaling is crucial to maintain cellular homeostasis during physical activity. High expression of BCL2L13, a member of the BCL-2 family, was suggested as an adaptive response in endurance-trained human subjects. In adult zebrafish, we found that the loss of Bcl2l13 impairs skeletal muscle structure and function. Ca2+ signaling is altered in Bcl2l13 knockout animals and mitochondrial complexes activity is decreased. Organelle fractioning in mammalian cells shows BCL2L13 at mitochondria, ER, and mitochondria-associated membranes. ER-mitochondria contact sites number is not modified by BCL2L13 modulation, but knockdown of BCL2L13 in C2C12 cells changes cytosolic Ca2+ release and mitochondrial Ca2+ uptake. This suggests that BCL2L13 interaction with mitochondria and ER, and its role in Ca2+ signaling, contributes to proper skeletal muscle function.
RESUMO
We previously showed in vitro that calcium entry through Trpc1 ion channels regulates myoblast migration and differentiation. In the present work, we used primary cell cultures and isolated muscles from Trpc1(-/-) and Trpc1(+/+) murine model to investigate the role of Trpc1 in myoblast differentiation and in muscle regeneration. In these models, we studied regeneration consecutive to cardiotoxin-induced muscle injury and observed a significant hypotrophy and a delayed regeneration in Trpc1(-/-) muscles consisting in smaller fiber size and increased proportion of centrally nucleated fibers. This was accompanied by a decreased expression of myogenic factors such as MyoD, Myf5, and myogenin and of one of their targets, the developmental MHC (MHCd). Consequently, muscle tension was systematically lower in muscles from Trpc1(-/-) mice. Importantly, the PI3K/Akt/mTOR/p70S6K pathway, which plays a crucial role in muscle growth and regeneration, was down-regulated in regenerating Trpc1(-/-) muscles. Indeed, phosphorylation of both Akt and p70S6K proteins was decreased as well as the activation of PI3K, the main upstream regulator of the Akt. This effect was independent of insulin-like growth factor expression. Akt phosphorylation also was reduced in Trpc1(-/-) primary myoblasts and in control myoblasts differentiated in the absence of extracellular Ca(2+) or pretreated with EGTA-AM or wortmannin, suggesting that the entry of Ca(2+) through Trpc1 channels enhanced the activity of PI3K. Our results emphasize the involvement of Trpc1 channels in skeletal muscle development in vitro and in vivo, and identify a Ca(2+)-dependent activation of the PI3K/Akt/mTOR/p70S6K pathway during myoblast differentiation and muscle regeneration.
Assuntos
Diferenciação Celular/fisiologia , Mioblastos Esqueléticos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Canais de Cátion TRPC/metabolismo , Animais , Cálcio/metabolismo , Ativação Enzimática/fisiologia , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Knockout , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Proteína MyoD/biossíntese , Proteína MyoD/genética , Mioblastos Esqueléticos/citologia , Fator Regulador Miogênico 5/biossíntese , Fator Regulador Miogênico 5/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Canais de Cátion TRPC/genéticaRESUMO
BACKGROUND: Treatment of advanced prostate cancer (PCa) relies on pharmacological or surgical androgen deprivation. However, it is only temporarily efficient. After a few months or years, the tumor relapses despite the absence of androgenic stimulation: a state referred to as hormone-refractory prostate cancer (HRPCa). Although autophagy confers chemoresistance in some cancers, its role in the development of HRPCa remains unknown. METHODS: Autophagic flux was assayed by GFP-LC3 clustering, by LC3-I to LC3-II conversion and transmission electron microscopy. Cell death was detected by sub-G1 quantification and concomitant measurement of transmembrane mitochondrial potential and plasma membrane permeabilization. Inhibition of autophagy was achieved by siRNAs and pharmacological inhibitors. RESULTS: Androgen deprivation or treatment with the anti-androgen bicalutamide promoted autophagy in HRPCa-derived LNCaP cells. This effect was dramatically reduced after depletion of Atg5 and Beclin-1, two canonical autophagy genes, and was associated with an inhibition of the androgen-induced mTOR pathway. The depletion of Atg5 and Beclin-1 significantly increased the level of cell death induced by androgen deprivation or bicalutamide. Finally, the safe anti-malarial drug chloroquine, an inhibitor of autophagy, dramatically increased cell death after androgen deprivation or bicalutamide treatment. CONCLUSION: Taken together, our data suggest that autophagy is a protective mechanism against androgen deprivation in HRPCa cells and that chloroquine could restore hormone dependence. This set of data could lead to the development of new therapeutic strategy against HRPCa.
Assuntos
Antagonistas de Androgênios/farmacologia , Anilidas/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Nitrilas/farmacologia , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Compostos de Tosil/farmacologia , Antagonistas de Androgênios/uso terapêutico , Anilidas/uso terapêutico , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cloroquina/farmacologia , Humanos , Masculino , Nitrilas/uso terapêutico , Próstata/efeitos dos fármacos , Próstata/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Serina-Treonina Quinases TOR/metabolismo , Compostos de Tosil/uso terapêuticoRESUMO
The muscle molecular adaptations to different exercise intensities in combination with hypoxia are not well understood. This study investigated the effect of low- and supramaximal-intensity hypoxic training on muscle metabolic gene expression in mice. C57BL/6 mice were divided into two groups: sedentary and training. Training consisted of 4 weeks at low or supramaximal intensity, either in normoxia or hypoxia (FiO2 = 0.13). The expression levels of genes involved in the hypoxia signaling pathway (Hif1a and Vegfa), the metabolism of glucose (Gys1, Glut4, Hk2, Pfk, and Pkm1), lactate (Ldha, Mct1, Mct4, Pdh, and Pdk4) and lipid (Cd36, Fabp3, Ucp2, Hsl, and Mcad), and mitochondrial energy metabolism and biogenesis (mtNd1, mtNd6, CytC, CytB, Pgc1a, Pgc1ß, Nrf1, Tfam, and Cs) were determined in the gastrocnemius muscle. No physical performance improvement was observed between groups. In normoxia, supramaximal intensity training caused upregulation of major genes involved in the transport of glucose and lactate, fatty acid oxidation, and mitochondrial biogenesis, while low intensity training had a minor effect. The exposure to hypoxia changed the expression of some genes in the sedentary mice but had a moderate effect in trained mice compared to respective normoxic mice. In hypoxic groups, low-intensity training increased the mRNA levels of Mcad and Cs, while supramaximal intensity training decreased the mRNA levels of Mct1 and Mct4. The results indicate that hypoxic training, regardless of exercise intensity, has a moderate effect on muscle metabolic gene expression in healthy mice.
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BACKGROUND: Decreased ryanodine receptor type 1 (RyR1) protein levels are a well-described feature of recessive RYR1-related myopathies. The aim of the present study was twofold: (1) to determine whether RyR1 content is also decreased in other myopathies and (2) to investigate the mechanisms by which decreased RyR1 protein triggers muscular disorders. METHODS: We used publicly available datasets, muscles from human inflammatory and mitochondrial myopathies, an inducible muscle-specific RYR1 recessive mouse model and RyR1 knockdown in C2C12 muscle cells to measure RyR1 content and endoplasmic reticulum (ER) stress markers. Proteomics, lipidomics, molecular biology and transmission electron microscopy approaches were used to decipher the alterations associated with the reduction of RyR1 protein levels. RESULTS: RYR1 transcripts were reduced in muscle samples of patients suffering from necrotizing myopathy (P = 0.026), inclusion body myopathy (P = 0.003), polymyositis (P < 0.001) and juvenile dermatomyositis (P < 0.001) and in muscle samples of myotonic dystrophy type 2 (P < 0.001), presymptomatic (P < 0.001) and symptomatic (P < 0.001) Duchenne muscular dystrophy, Becker muscular dystrophy (P = 0.004) and limb-girdle muscular dystrophy type 2A (P = 0.004). RyR1 protein content was also significantly decreased in inflammatory myopathy (-75%, P < 0.001) and mitochondrial myopathy (-71%, P < 0.001) muscles. Proteomics data showed that depletion of RyR1 protein in C2C12 myoblasts leads to myotubes recapitulating the common molecular alterations observed in myopathies. Mechanistically, RyR1 protein depletion reduces ER-mitochondria contact length (-26%, P < 0.001), Ca2+ transfer to mitochondria (-48%, P = 0.002) and the mitophagy gene Parkinson protein 2 transcripts (P = 0.037) and induces mitochondrial accumulation (+99%, P = 0.005) and dysfunction (P < 0.001). This was associated to the accumulation of deleterious sphingolipid species. Our data showed increased levels of the ER stress marker chaperone-binding protein/glucose regulated protein 78, GRP78-Bip, in RyR1 knockdown myotubes (+45%, P = 0.046), in mouse RyR1 recessive muscles (+58%, P = 0.001) and in human inflammatory (+96%, P = 0.006) and mitochondrial (+64%, P = 0.049) myopathy muscles. This was accompanied by increased protein levels of the pro-apoptotic protein CCAAT-enhancer-binding protein homologous protein, CHOP-DDIT3, in RyR1 knockdown myotubes (+27%, P < 0.001), mouse RyR1 recessive muscles (+63%, P = 0.009), human inflammatory (+50%, P = 0.038) and mitochondrial (+51%, P = 0.035) myopathy muscles. In publicly available datasets, the decrease in RYR1 content in myopathies was also associated to increased ER stress markers and RYR1 transcript levels are inversely correlated with ER stress markers in the control population. CONCLUSIONS: Decreased RyR1 is commonly observed in myopathies and associated to ER stress in vitro, in mouse muscle and in human myopathy muscles, suggesting a potent role of RyR1 depletion-induced ER stress in the pathogenesis of myopathies.
Assuntos
Doenças Musculares , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Humanos , Camundongos , Estresse do Retículo Endoplasmático , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/patologia , Doenças Musculares/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
Duchenne muscular dystrophy (DMD), the most common muscular dystrophy, is a severe muscle disorder, causing muscle weakness, loss of independence, and premature death. Here, we establish the link between sphingolipids and muscular dystrophy. Transcripts of sphingolipid de novo biosynthesis pathway are up-regulated in skeletal muscle of patients with DMD and other muscular dystrophies, which is accompanied by accumulation of metabolites of the sphingolipid pathway in muscle and plasma. Pharmacological inhibition of sphingolipid synthesis by myriocin in the mdx mouse model of DMD ameliorated the loss in muscle function while reducing inflammation, improving Ca2+ homeostasis, preventing fibrosis of the skeletal muscle, heart, and diaphragm, and restoring the balance between M1 and M2 macrophages. Myriocin alleviated the DMD phenotype more than glucocorticoids. Our study identifies inhibition of sphingolipid synthesis, targeting multiple pathogenetic pathways simultaneously, as a strong candidate for treatment of muscular dystrophies.
Assuntos
Distrofia Muscular de Duchenne , Animais , Modelos Animais de Doenças , Fibrose , Humanos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Esfingolipídeos/metabolismo , Esfingolipídeos/uso terapêuticoRESUMO
Age-related muscle dysfunction and sarcopenia are major causes of physical incapacitation in older adults and currently lack viable treatment strategies. Here we find that sphingolipids accumulate in mouse skeletal muscle upon aging and that both genetic and pharmacological inhibition of sphingolipid synthesis prevent age-related decline in muscle mass while enhancing strength and exercise capacity. Inhibition of sphingolipid synthesis confers increased myogenic potential and promotes protein synthesis. Within the sphingolipid pathway, we show that accumulation of dihydroceramides is the culprit disturbing myofibrillar homeostasis. The relevance of sphingolipid pathways in human aging is demonstrated in two cohorts, the UK Biobank and Helsinki Birth Cohort Study in which gene expression-reducing variants of SPTLC1 and DEGS1 are associated with improved and reduced fitness of older individuals, respectively. These findings identify sphingolipid synthesis inhibition as an attractive therapeutic strategy for age-related sarcopenia and co-occurring pathologies.
Assuntos
Sarcopenia , Animais , Camundongos , Humanos , Idoso , Sarcopenia/prevenção & controle , Músculo Esquelético/metabolismo , Esfingolipídeos/metabolismo , Estudos de Coortes , Envelhecimento/genéticaRESUMO
Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy, and despite advances in genetic and pharmacological disease-modifying treatments, its management remains a major challenge. Mitochondrial dysfunction contributes to DMD, yet the mechanisms by which this occurs remain elusive. Our data in experimental models and patients with DMD show that reduced expression of genes involved in mitochondrial autophagy, or mitophagy, contributes to mitochondrial dysfunction. Mitophagy markers were reduced in skeletal muscle and in muscle stem cells (MuSCs) of a mouse model of DMD. Administration of the mitophagy activator urolithin A (UA) rescued mitophagy in DMD worms and mice and in primary myoblasts from patients with DMD, increased skeletal muscle respiratory capacity, and improved MuSCs' regenerative ability, resulting in the recovery of muscle function and increased survival in DMD mouse models. These data indicate that restoration of mitophagy alleviates symptoms of DMD and suggest that UA may have potential therapeutic applications for muscular dystrophies.
Assuntos
Mitofagia , Distrofia Muscular de Duchenne , Animais , Cumarínicos , Humanos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético , Distrofia Muscular de Duchenne/tratamento farmacológicoRESUMO
Sustained ryanodine receptor (RyR) Ca2+ leak is associated with pathological conditions such as heart failure or skeletal muscle weakness. We report that a single session of sprint interval training (SIT), but not of moderate intensity continuous training (MICT), triggers RyR1 protein oxidation and nitrosylation leading to calstabin1 dissociation in healthy human muscle and in in vitro SIT models (simulated SIT or S-SIT). This is accompanied by decreased sarcoplasmic reticulum Ca2+ content, increased levels of mitochondrial oxidative phosphorylation proteins, supercomplex formation and enhanced NADH-linked mitochondrial respiratory capacity. Mechanistically, (S-)SIT increases mitochondrial Ca2+ uptake in mouse myotubes and muscle fibres, and decreases pyruvate dehydrogenase phosphorylation in human muscle and mouse myotubes. Countering Ca2+ leak or preventing mitochondrial Ca2+ uptake blunts S-SIT-induced adaptations, a result supported by proteomic analyses. Here we show that triggering acute transient Ca2+ leak through RyR1 in healthy muscle may contribute to the multiple health promoting benefits of exercise.