RESUMO
BACKGROUND: Although walking is the most common exercise modality in daily life for most humans, running and cycling are the most applied exercise modalities during maximal oxygen uptake (VO
Assuntos
Exercício Físico , Consumo de Oxigênio , Humanos , Reprodutibilidade dos Testes , Caminhada , Teste de Esforço/métodosRESUMO
Calcineurin (Cn) signaling has been implicated in nerve activity-dependent fiber type specification in skeletal muscle, but the downstream effector pathway has not been established. We have investigated the role of the transcription factor nuclear factor of activated T cells (NFAT), a major target of Cn, by using an in vivo transfection approach in regenerating and adult rat muscles. NFAT transcriptional activity was monitored with two different NFAT-dependent reporters and was found to be higher in slow compared to fast muscles. NFAT activity is decreased by denervation in slow muscles and is increased by electrostimulation of denervated muscles with a tonic low-frequency impulse pattern, mimicking the firing pattern of slow motor neurons, but not with a phasic high-frequency pattern typical of fast motor neurons. To determine the role of NFAT, we transfected regenerating and adult rat muscles with a plasmid coding for VIVIT, a specific peptide inhibitor of Cn-mediated NFAT activation. VIVIT was found to block the expression of slow myosin heavy chain (MyHC-slow) induced by slow motor neuron activity in regenerating slow soleus muscle and to inhibit the expression of MyHC-slow transcripts and the activity of a MyHC-slow promoter in adult soleus. The role of NFAT was confirmed by the finding that a constitutively active NFATc1 mutant stimulates the MyHC-slow, inhibits the fast MyHC-2B promoter in adult fast muscles, and induces MyHC-slow expression in regenerating muscles. These results support the notion that Cn-NFAT signaling acts as a nerve activity sensor in skeletal muscle in vivo and controls nerve activity-dependent myosin switching.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Neurônios Motores/metabolismo , Músculo Esquelético/fisiologia , Miosinas/metabolismo , Proteínas Nucleares , Fatores de Transcrição/metabolismo , Animais , Calcineurina/genética , Calcineurina/metabolismo , Proteínas de Ligação a DNA/genética , Estimulação Elétrica , Genes Reporter , Técnicas In Vitro , Masculino , Fibras Musculares de Contração Rápida/citologia , Fibras Musculares de Contração Rápida/fisiologia , Fibras Musculares de Contração Lenta/citologia , Fibras Musculares de Contração Lenta/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/inervação , Miosinas/genética , Fatores de Transcrição NFATC , Oligopeptídeos/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/genéticaRESUMO
Nerve activity controls fiber size and fiber type in skeletal muscle, but the underlying molecular mechanisms remain largely unknown. We have previously shown that Ras-mitogen-activated protein kinase and calcineurin control fiber type but not fiber size in regenerating rat skeletal muscle. Here we report that constitutively active protein kinase B (PKB), also known as Akt, increases fiber size and prevents denervation atrophy in regenerating and adult rat muscles but does not affect fiber type profile. The coexistence of hypertrophic muscle fibers overexpressing activated PKB with normal-size untransfected fibers within the same muscle points to a cell-autonomous control of muscle growth by PKB. The physiological role of this pathway is confirmed by the finding that PKB kinase activity and phosphorylation status are significantly increased in innervated compared with denervated regenerating muscles in parallel with muscle growth. Muscle fiber hypertrophy induced by activated PKB and by a Ras double mutant (RasV12C40) that activates selectively the phosphoinositide 3-kinase-PKB pathway is completely blocked by rapamycin, showing that the mammalian target of rapamycin kinase is the major downstream effector of this pathway in the control of muscle fiber size. On the other hand, nerve activity-dependent growth of regenerating muscle is only partially inhibited by dominant negative PKB and rapamycin, suggesting that other nerve-dependent signaling pathways are involved in muscle growth. The present results support the notion that fiber size and fiber type are regulated by nerve activity through different mechanisms.
Assuntos
Músculo Esquelético/enzimologia , Músculo Esquelético/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Animais , Estimulação Elétrica , Hipertrofia , Masculino , Denervação Muscular , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/enzimologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/inervação , Atrofia Muscular/prevenção & controle , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Wistar , Regeneração/efeitos dos fármacos , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TORRESUMO
We have examined protein kinase A (PKA) subunit expression in adult rat skeletal muscles. Northern blots identified PKA catalytic alpha and regulatory (R) I alpha and RII alpha subunits as the major subunits expressed in slowly contracting soleus (SOL) and rapidly contracting extensor digitorum longus (EDL) muscles. In addition, the steady-state RNA levels of PKA subunit mRNAs and activities of RI alpha and RII alpha promoters are similar in SOL and EDL. These data indicate that posttranscriptional mechanisms account for the twofold differences in PKA subunit protein levels reported earlier. Electrical stimulation of denervated SOL with an EDL-like activity pattern (fast pattern) transformed SOL into an EDL-like muscle with regard to PKA protein levels. These experiments suggest that the posttranscriptional regulation is activity pattern-dependent. Denervation specifically increased RI alpha promoter activity and RI alpha mRNA levels in SOL and EDL. Further experiments indicated that the RI alpha 1a upstream sequences were activated following denervation. Direct electrical stimulation prevented the rise in RI alpha mRNA levels following denervation, demonstrating that electrical muscle activity regulates transcription.