TRPC6 Hyperactivity and Cardiac Dystrophinopathy

TRPC6 过度活跃和心肌肌营养不良症

• 批准号: 9053913
• 负责人: David Alan Kass
• 金额: $ 40.76万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9053913
• 关键词: Acute; Address; Adolescence; Affect; Arrhythmia; Calcium; Cardiac; Cardiac Myocytes; Caring; Cations; Caveolae; Cell membrane; Cells; Chronic; Complex; Contracts; Coupled; Coupling; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cytoplasmic Protein; Cytosol; Data; Defect; Disease; Disease Progression; Duchenne cardiomyopathy; Duchenne muscular dystrophy; Dystrophin; Experimental Models; Feedback; Fibrosis; Figs - dietary; Functional disorder; Gene Deletion; Generations; Genetic; Heart; Heart Diseases; Heart failure; Histopathology; Human; Hyperactive behavior; Image; Ion Channel; Ions; Knock-in Mouse; Link; Mechanics; Membrane; Molecular and Cellular Biology; Mus; Muscle; Muscle Cells; Muscle function; Muscular Dystrophies; Mutation; Myocardium; Myopathy; NADPH Oxidase; Natriuretic Peptides; Nature; Newborn Infant; Nitric Oxide; Oxidation-Reduction; Oxidative Stress; Palliative Care; Pathology; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Phosphorylation; Plasma; Play; Protein Kinase; Proteins; Reactive Oxygen Species; Regulation; Reporting; Role; Sarcoglycans; Sarcoplasmic Reticulum; Signal Transduction; Skeletal Muscle; Sodium; Source; Stress; Stretching; Testing; Therapeutic; Utrophin; aged; base; effective therapy; emerging adult; feeding; forging; improved; improved functioning; in vivo Model; inhibitor/antagonist; insight; male; mortality; mouse model; novel; novel therapeutic intervention; novel therapeutics; oxidant stress; oxidation; phosphoric diester hydrolase; preclinical study; prevent; public health relevance; receptor; response; sensor; skeletal

 DESCRIPTION (provided by applicant):Muscular dystrophy involving loss of the cytoplasmic protein dystrophin (Duchenne Muscular Dystrophy, or DMD) is a devastating disease of both skeletal and cardiac myopathy, with no cure and relatively few effective palliative therapies. Cardiac mortality is now increasingly common, as better care has improved survival into early adulthood. In DMD, dissolution of the dystrophin-sarcoglycan complex results in maladaptive hyper- sensitivity of muscle to mechanical load, involving stretch-responsive mechanisms that increase intracellular calcium and oxidative stress. We recently revealed that transient potential receptor cation-6 (TRPC6) ion channel is a major mechano-sensor in DMD myocardium (mdx/utrophin+/- mice), regulating amplified force, Ca2+ and arrhythmia in contracting DMD cardiac myocytes subjected to auxotonic stress. Acute activation of protein kinase G1a (PKG1) potently blocks this response in a TRPC6-dependent manner, and in another new study, we showed that if PKG1a becomes oxidized, it is less effective in countering TRPC6 signaling. As DMD invokes oxidant stress and reduces nitric oxide (NO) signaling in muscle, the change in PKG1 redox could contribute to worsened disease. In a third study reported in Nature, we revealed a new therapeutic option by showing that PDE9A targets natriuretic peptide not NO derived cGMP - and its inhibition circumvents blunted NO-signaling and oxidant stress and improves cardiac responses to stress. As natriuretic peptide levels are elevated in DMD patients and experimental models, this signaling cascade may be very relevant. The current project synthesizes these exciting new discoveries, and using our sophisticated cell-based mechano-sensing and imaging and in vivo models, will address several novel hypotheses. First, we test that TRPC6 interacts with other critical determinants of calcium mishandling in DMD, including Ca2+ leak from the sarcoplasmic reticulum and sodium/calcium exchange, as key contributors to its adverse impact. We then test if PKG activation reverses these changes. Second, we determine if TRPC6 activation drives or is in driven by oxidant stress particularly generated by NADPH-oxidase 2 (NOX2), with which it can co-localize. Here we employ novel genetically encoded sensors of redox localized to NOX2, caveolae, and cytosol. We also test if PKG1 oxidation contributes to TRPC6 activation in DMD, employing knock-in mouse models with a redox-dead PKG1 mutation (C42S). Lastly, we perform pre-clinical studies to test if either chronic gene deletion of Trpc6 or pharmacological PDE9A inhibition ameliorates DMD cardiac disease, improving myocyte function and reversing abnormal mechano-stimulation and arrhythmia. Together, these studies will forge major new insight into DMD cardiac disease, focusing on TRPC6 and its coupling to calcium and ROS dysregulation, and establish if a new therapeutic approach involving PDE9A inhibition will benefit this disease.

 描述（由申请人提供）：涉及胞质蛋白肌营养不良蛋白缺失的肌营养不良症（杜氏肌营养不良症，或DMD）是骨骼肌和心肌病的毁灭性疾病，无法治愈，有效的姑息疗法相对较少。心脏病死亡率现在越来越普遍，因为更好的护理提高了成年早期的生存率。在DMD中，肌营养不良蛋白-肌聚糖复合物的溶解导致肌肉对机械负荷的适应不良超敏感性，涉及增加细胞内钙和氧化应激的拉伸响应机制。我们最近发现，瞬时电位受体阳离子-6（TRPC-6）离子通道是DMD心肌（mdx/utrophin+/-小鼠）中的主要机械传感器，调节DMD心肌细胞在生长素应激下收缩时的放大力、Ca 2+和心律失常。蛋白激酶G1 a（PKG 1 α）的急性激活以TRPC 6依赖性方式有效地阻断了这种反应，在另一项新的研究中，我们表明，如果PKG 1a被氧化，它在对抗TRPC 6信号传导方面的效果就不那么好。由于DMD引起氧化应激并减少肌肉中的一氧化氮（NO）信号，PKG 1氧化还原酶的变化可能导致疾病恶化。在《自然》杂志上报道的第三项研究中，我们揭示了一种新的治疗选择，表明PDE 9A靶向利钠肽而不是NO衍生的cGMP，它的抑制作用规避了钝化的NO信号传导和氧化应激，并改善了心脏对应激的反应。由于利尿钠肽水平在DMD患者和实验模型中升高，这种信号级联可能非常相关。目前的项目综合了这些令人兴奋的新发现，并使用我们复杂的基于细胞的机械传感和成像以及体内模型，将解决几个新的假设。首先，我们测试TRPC 6与DMD中钙处理不当的其他关键决定因素相互作用，包括肌浆网和钠/钙交换的Ca 2+泄漏，作为其不利影响的关键因素。然后我们测试PKG激活是否逆转这些变化。其次，我们确定TRPC 6激活是否驱动或由氧化应激驱动，特别是由NADPH氧化酶2（NOX 2）产生的，它可以与共定位。在这里，我们采用新的基因编码的传感器的氧化还原定位到NOX 2，小窝，和胞质溶胶。我们还测试了PKG 1 β氧化是否有助于DMD中的TRPC 6活化，采用具有氧化还原死亡PKG 1 β突变（C42 S）的敲入小鼠模型。最后，我们进行临床前研究以测试Trpc 6的慢性基因缺失或药理学PDE 9A抑制是否改善DMD心脏病，改善肌细胞功能并逆转异常机械刺激和心律失常。总之，这些研究将对DMD心脏病形成重大的新见解，重点关注TRPC 6及其与钙和ROS失调的偶联，并确定涉及PDE 9A抑制的新治疗方法是否会使这种疾病受益。