RESUMO
Following a screen for neuromuscular mouse mutants, we identified ostes, a novel N-ethyl N-nitrosourea-induced mouse mutant with muscle atrophy. Genetic and biochemical evidence shows that upregulation of the novel, uncharacterized transient receptor potential polycystic (TRPP) channel PKD1L2 (polycystic kidney disease gene 1-like 2) underlies this disease. Ostes mice suffer from chronic neuromuscular impairments including neuromuscular junction degeneration, polyneuronal innervation and myopathy. Ectopic expression of PKD1L2 in transgenic mice reproduced the ostes myopathic changes and, indeed, caused severe muscle atrophy in Tg(Pkd1l2)/Tg(Pkd1l2) mice. Moreover, double-heterozygous mice (ostes/+, Tg(Pkd1l2)/0) suffer from myopathic changes more profound than each heterozygote, indicating positive correlation between PKD1L2 levels and disease severity. We show that, in vivo, PKD1L2 primarily associates with endogenous fatty acid synthase in normal skeletal muscle, and these proteins co-localize to costameric regions of the muscle fibre. In diseased ostes/ostes muscle, both proteins are upregulated, and ostes/ostes mice show signs of abnormal lipid metabolism. This work shows the first role for a TRPP channel in neuromuscular integrity and disease.
Assuntos
Doenças Neuromusculares/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Regulação para Cima , Animais , Células Cultivadas , Modelos Animais de Doenças , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Feminino , Células HeLa , Humanos , Lactente , Masculino , Camundongos , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Mutação , Doenças Neuromusculares/genética , Ligação Proteica , Receptores Acoplados a Proteínas G/genéticaRESUMO
The KY protein underlies a form of muscular dystrophy in the mouse but its role in muscle remains elusive. Immunodetection of endogenous KY protein in C2C12-derived myotubes and expression of a recombinant form in neonatal cardiomyocytes indicated that KY is a Z-band associated protein. Moreover, characterization of a KY interacting protein fragment led to the identification of Igfn1 (Immunoglobulin-like and fibronectin type 3 domain containing 1). Igfn1 is a transcriptionally complex locus encoding many protein variants. A yeast two-hybrid screen identified the Z-band protein filamin C (FLNC) as an interacting partner. Consistent with this, expression of an IGFN1 recombinant fragment showed that the three N-terminal globular domains, common to at least five IGFN1 variants, are sufficient to provide Z-band targeting. Taken together, the yeast two-hybrid, biochemical and immunofluorescence data support the notion that KY, IGFN1 and FLNC are part of a Z-band associated protein complex likely to provide structural support to the skeletal muscle sarcomere.
Assuntos
Proteínas de Transporte/química , Proteínas Contráteis/química , Proteínas dos Microfilamentos/química , Proteínas Musculares/química , Animais , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Linhagem Celular , Proteínas Contráteis/genética , Proteínas Contráteis/isolamento & purificação , Primers do DNA/genética , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Filaminas , Técnicas In Vitro , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/isolamento & purificação , Complexos Multiproteicos/química , Complexos Multiproteicos/isolamento & purificação , Fibras Musculares Esqueléticas/química , Proteínas Musculares/genética , Proteínas Musculares/isolamento & purificação , Músculo Esquelético/química , Miócitos Cardíacos/metabolismo , Peptídeo Hidrolases , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Sarcômeros/química , Distribuição Tecidual , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: Antiarrhythmic drugs are widely used to treat patients with atrial fibrillation (AF), but the mechanisms conveying their variable effectiveness are not known. Recent data suggested that paired like homeodomain-2 transcription factor (PITX2) might play an important role in regulating gene expression and electrical function of the adult left atrium (LA). OBJECTIVES: After determining LA PITX2 expression in AF patients requiring rhythm control therapy, the authors assessed the effects of Pitx2c on LA electrophysiology and the effect of antiarrhythmic drugs. METHODS: LA PITX2 messenger ribonucleic acid (mRNA) levels were measured in 95 patients undergoing thoracoscopic AF ablation. The effects of flecainide, a sodium (Na+)-channel blocker, and d,l-sotalol, a potassium channel blocker, were studied in littermate mice with normal and reduced Pitx2c mRNA by electrophysiological study, optical mapping, and patch clamp studies. PITX2-dependent mechanisms of antiarrhythmic drug action were studied in human embryonic kidney (HEK) cells expressing human Na channels and by modeling human action potentials. RESULTS: Flecainide 1 µmol/l was more effective in suppressing atrial arrhythmias in atria with reduced Pitx2c mRNA levels (Pitx2c+/-). Resting membrane potential was more depolarized in Pitx2c+/- atria, and TWIK-related acid-sensitive K+ channel 2 (TASK-2) gene and protein expression were decreased. This resulted in enhanced post-repolarization refractoriness and more effective Na-channel inhibition. Defined holding potentials eliminated differences in flecainide's effects between wild-type and Pitx2c+/- atrial cardiomyocytes. More positive holding potentials replicated the increased effectiveness of flecainide in blocking human Nav1.5 channels in HEK293 cells. Computer modeling reproduced an enhanced effectiveness of Na-channel block when resting membrane potential was slightly depolarized. CONCLUSIONS: PITX2 mRNA modulates atrial resting membrane potential and thereby alters the effectiveness of Na-channel blockers. PITX2 and ion channels regulating the resting membrane potential may provide novel targets for antiarrhythmic drug development and companion therapeutics in AF.
Assuntos
Antiarrítmicos/farmacologia , Fibrilação Atrial/tratamento farmacológico , Fibrilação Atrial/fisiopatologia , Flecainida/uso terapêutico , Proteínas de Homeodomínio/fisiologia , Potenciais da Membrana/fisiologia , Fatores de Transcrição/fisiologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêutico , Adulto , Idoso , Animais , Fenômenos Eletrofisiológicos , Feminino , Regulação da Expressão Gênica , Átrios do Coração/fisiopatologia , Proteínas de Homeodomínio/genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Fatores de Transcrição/genética , Proteína Homeobox PITX2RESUMO
Understanding the mechanism of re-entrant arrhythmias in the past 30 years has allowed the development of almost curative therapies for many rhythm disturbances. The complex, polymorphic arrhythmias of atrial fibrillation (AF) and sudden death are, unfortunately, not yet well understood, and hence still in need of adequate therapy. AF contributes markedly to morbidity and mortality in aging Western populations. In the past decade, many genetically altered murine models have been described and characterized. Here, we review genetically altered murine models of AF; powerful tools that will enable a better understanding of the mechanisms of AF and the assessment of novel therapeutic interventions.