TRPC6 Hyperactivity and Cardiac Dystrophinopathy

 描述(申请人提供)：肌营养不良症涉及胞浆蛋白Dstrophin丢失(Duchenne肌肉营养不良症，或DMD)是一种骨骼和心肌病的破坏性疾病，没有治愈方法，有效的姑息治疗也相对较少。心脏死亡现在越来越普遍，因为更好的护理提高了成年期早期的存活率。在DMD中，肌营养不良蛋白-肌聚糖复合体的溶解导致肌肉对机械负荷的不适应性超敏，涉及增加细胞内钙和氧化应激的拉伸反应机制。我们最近发现，瞬时电位受体阳离子-6(TRPC6)离子通道是DMD心肌(MDX/utroin+/-小鼠)的主要机械感受器，调节缺氧性应激下DMD心肌细胞收缩时的扩张力、钙离子和心律失常。在另一项新的研究中，我们表明，如果蛋白激酶G1a被氧化，它在对抗TRPC6信号方面的效果就会减弱。当DMD启动氧化应激并减少肌肉中的一氧化氮(NO)信号时，PKG1DNA氧化还原的变化可能导致疾病的恶化。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PDE9A针对的是利钠肽而不是NO衍生的cGMP，其抑制作用绕过了钝化NO信号和氧化应激，并改善了心脏对应激的反应。由于在DMD患者和实验模型中钠尿肽水平升高，这种信号级联可能是非常相关的。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，我们测试了TRPC6与DMD中钙处理不当的其他关键决定因素的相互作用，包括肌浆网钙泄漏和钠/钙交换，作为其不利影响的关键因素。然后，我们测试PKG激活是否逆转了这些变化。其次，我们确定TRPC6的激活是否驱动或受到氧化应激的驱动，这些氧化应激特别是由NADPH-氧化酶2(NOX2)产生，它可以与之共定位。在这里，我们使用了新的基因编码的氧化还原传感器，定位于NOX2、小窝和胞浆。我们还利用具有氧化还原死亡的PKG1突变(C42S)的敲入小鼠模型，测试了PKG1TRPC6氧化是否有助于DMD中TRPC6的激活。最后，我们进行了临床前研究，以测试TRPC6的慢性基因缺失或药物抑制PDE9A是否可以改善DMD心脏疾病，改善心肌细胞功能，逆转异常机械刺激和心律失常。总之，这些研究将对DMD心脏疾病形成重大的新见解，重点是TRPC6及其与钙和ROS失调的偶联，并确定涉及PDE9A抑制的新治疗方法是否将有益于这种疾病。

 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.