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1.
Am J Physiol Heart Circ Physiol ; 320(1): H200-H210, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33216625

RESUMO

Sarcolipin (SLN) is an inhibitor of sarco/endoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and expressed at high levels in the ventricles of animal models for and patients with Duchenne muscular dystrophy (DMD). The goal of this study was to determine whether the germline ablation of SLN expression improves cardiac SERCA function and intracellular Ca2+ (Ca2+i) handling and prevents cardiomyopathy in the mdx mouse model of DMD. Wild-type, mdx, SLN-haploinsufficient mdx (mdx:sln+/-), and SLN-deficient mdx (mdx:sln-/-) mice were used for this study. SERCA function and Ca2+i handling were determined by Ca2+ uptake assays and by measuring single-cell Ca2+ transients, respectively. Age-dependent disease progression was determined by histopathological examinations and by echocardiography in 6-, 12-, and 20-mo-old mice. Gene expression changes in the ventricles of mdx:sln+/- mice were determined by RNA-Seq analysis. SERCA function and Ca2+i cycling were improved in the ventricles of mdx:sln+/- mice. Fibrosis and necrosis were significantly decreased, and cardiac function was enhanced in the mdx:sln+/- mice until the study endpoint. The mdx:sln-/- mice also exhibited similar beneficial effects. RNA-Seq analysis identified distinct gene expression changes including the activation of the apelin pathway in the ventricles of mdx:sln+/- mice. Our findings suggest that reducing SLN expression is sufficient to improve cardiac SERCA function and Ca2+i cycling and prevent cardiomyopathy in mdx mice.NEW & NOTEWORTHY First, reducing sarcopolin (SLN) expression improves sarco/endoplasmic reticulum Ca2+ uptake and intracellular Ca2+ handling and prevents cardiomyopathy in mdx mice. Second, reducing SLN expression prevents diastolic dysfunction and improves cardiac contractility in mdx mice Third, reducing SLN expression activates apelin-mediated cardioprotective signaling pathways in mdx heart.


Assuntos
Cardiomiopatias/prevenção & controle , Haploinsuficiência , Proteínas Musculares/deficiência , Distrofia Muscular de Duchenne/complicações , Miocárdio/metabolismo , Proteolipídeos/deficiência , Animais , Apelina/genética , Apelina/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Cardiomiopatias/etiologia , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Modelos Animais de Doenças , Feminino , Fibrose , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Knockout , Proteínas Musculares/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Miocárdio/patologia , Necrose , Proteolipídeos/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Função Ventricular Esquerda
2.
Mol Ther ; 28(3): 845-854, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-31981493

RESUMO

Loss of dystrophin leads to Duchenne muscular dystrophy (DMD). A pathogenic feature of DMD is the significant elevation of cytosolic calcium. Supraphysiological calcium triggers protein degradation, membrane damage, and eventually muscle death and dysfunction. Sarcoplasmic/endoplasmic reticulum (SR) calcium ATPase (SERCA) is a calcium pump that transports cytosolic calcium to the SR during excitation-contraction coupling. We hypothesize that a single systemic delivery of SERCA2a with adeno-associated virus (AAV) may improve calcium recycling and provide long-lasting benefits in DMD. To test this, we injected an AAV9 human SERCA2a vector (6 × 1012 viral genome particles/mouse) intravenously to 3-month-old mdx mice, the most commonly used DMD model. Immunostaining and western blot showed robust human SERCA2a expression in the heart and skeletal muscle for 18 months. Concomitantly, SR calcium uptake was significantly improved in these tissues. SERCA2a therapy significantly enhanced grip force and treadmill performance, completely prevented myocardial fibrosis, and normalized electrocardiograms (ECGs). Cardiac catheterization showed normalization of multiple systolic and diastolic hemodynamic parameters in treated mice. Importantly, chamber dilation was completely prevented, and ejection fraction was restored to the wild-type level. Our results suggest that a single systemic AAV9 SERCA2a therapy has the potential to provide long-lasting benefits for DMD.


Assuntos
Cardiomiopatia Dilatada/etiologia , Cardiomiopatia Dilatada/terapia , Expressão Gênica , Terapia Genética , Distrofia Muscular de Duchenne/complicações , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Administração Intravenosa , Animais , Dependovirus/genética , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Retículo Sarcoplasmático/metabolismo , Fatores de Tempo , Transdução Genética
3.
Am J Physiol Cell Physiol ; 317(4): C813-C824, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31365291

RESUMO

Reduction in the expression of sarcolipin (SLN), an inhibitor of sarco(endo)plasmic reticulum (SR) Ca2+-ATPase (SERCA), ameliorates severe muscular dystrophy in mice. However, the mechanism by which SLN inhibition improves muscle structure remains unclear. Here, we describe the previously unknown function of SLN in muscle differentiation in Duchenne muscular dystrophy (DMD). Overexpression of SLN in C2C12 resulted in decreased SERCA pump activity, reduced SR Ca2+ load, and increased intracellular Ca2+ (Cai2+) concentration. In addition, SLN overexpression resulted in altered expression of myogenic markers and poor myogenic differentiation. In dystrophin-deficient dog myoblasts and myotubes, SLN expression was significantly high and associated with defective Cai2+ cycling. The dystrophic dog myotubes were less branched and associated with decreased autophagy and increased expression of mitochondrial fusion and fission proteins. Reduction in SLN expression restored these changes and enhanced dystrophic dog myoblast fusion during differentiation. In summary, our data suggest that SLN upregulation is an intrinsic secondary change in dystrophin-deficient myoblasts and could account for the Cai2+ mishandling, which subsequently contributes to poor myogenic differentiation. Accordingly, reducing SLN expression can improve the Cai2+ cycling and differentiation of dystrophic myoblasts. These findings provide cellular-level supports for targeting SLN expression as a therapeutic strategy for DMD.


Assuntos
Cálcio/metabolismo , Desenvolvimento Muscular/fisiologia , Proteínas Musculares/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Proteolipídeos/metabolismo , Animais , Diferenciação Celular/fisiologia , Cães , Distrofina/deficiência , Camundongos Knockout , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/fisiopatologia , Mioblastos/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
4.
Cell Signal ; 111: 110886, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37690661

RESUMO

Glucocorticoids through activation of the Glucocorticoid receptor (GR) play an essential role in cellular homeostasis during physiological variations and in response to stress. Our genomic GR binding and transcriptome data from Dexamethasone (Dex) treated cardiomyocytes showed an early differential regulation of mostly transcription factors, followed by sequential change in genes involved in downstream functional pathways. We examined the role of Krüppel-like factor 9 (Klf9), an early direct target of GR in cardiomyocytes. Klf9-ChIPseq identified 2150 genes that showed an increase in Klf9 binding in response to Dex. Transcriptome analysis of Dex treated cardiomyocytes with or without knockdown of Klf9 revealed differential regulation of 1777 genes, of which a reversal in expression is seen in 1640 genes with knockdown of Klf9 compared to Dex. Conversely, only 137 (∼8%) genes show further dysregulation in expression with siKLf9, as seen with Dex treated cardiomyocytes. Functional annotation identified genes of metabolic pathways on the top of differentially expressed genes, including those involved in glycolysis and oxidative phosphorylation. Knockdown of Klf9 in cardiomyocytes inhibited Dex induced increase in glycolytic function and mitochondrial spare respiratory capacity, as measured by glycolysis and mito stress tests, respectively. Thus, we conclude that cyclic, diurnal GR activation, through Klf9 -dependent feedforward signaling plays a central role in maintaining cellular homeostasis through metabolic adaptations in cardiomyocytes.


Assuntos
Miócitos Cardíacos , Receptores de Glucocorticoides , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição/metabolismo , Transdução de Sinais
5.
Cell Signal ; 91: 110245, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35017014

RESUMO

Adaptation of gene expression is one of the most fundamental response of cardiomyocytes to hypertrophic stimuli. G3bp1, an RNA binding protein with site-specific endoribonuclease activity regulates the processing of pre-miR-1 stem-loop, and thus levels of cardiomyocyte -enriched mature miR-1. Here, we examine the role of G3bp1 in regulating gene expression in quiescent cardiomyocytes and those undergoing growth-factor induced hypertrophy. Further, we determine if these changes are facilitated through G3bp1-mediated regulation of miR-1 in these cardiomyocytes. Using isolated cardiomyocytes with knockdown of endogenous G3bp1, we performed high throughput RNA sequencing to determine the change in cardiac transcriptome. Then, using gain and loss of function approach for both, G3bp1 and miR-1, alone or in combination we examine the G3bp1-miR-1 signaling in regulating gene expression and Endothelin (ET-1) -induced cardiomyocyte hypertrophy. We show that knockdown of endogenous G3bp1 results in inhibition of genes involved in calcium handling, cardiac muscle contraction, action potential and sarcomeric structure. In addition, there is inhibition of genes that contribute to hypertrophic and dilated cardiomyopathy development. Conversely, an increase is seen in genes that negatively regulate the Hippo signaling, like Rassf1 and Arrdc3, along with inflammatory genes of TGF-ß and TNF pathways. Knockdown of G3bp1 restricts ET-1 induced cardiomyocyte hypertrophy. Interestingly, concurrent silencing of G3bp1 and miR-1 rescues the change in gene expression and inhibition of hypertrophy seen with knockdown of G3bp1 alone. Similarly, expression of exogenous G3bp1 reverses the miR-1 induced inhibition of gene expression. Intriguingly, expression of Gfp tagged G3bp1 results in perinuclear accumulations of G3bp1-Gfp, resembling Stress Granules. Based on our results, we conclude that G3bp1 through its regulation of mature miR-1 levels plays a critical role in regulating the expression of essential cardiac-enriched genes and those involved in development of cardiomyocyte hypertrophy.


Assuntos
MicroRNAs , Cardiomegalia/genética , Cardiomegalia/metabolismo , Células Cultivadas , DNA Helicases/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo
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