Novel Cardioprotective sGC/cGMP Microdomains: Therapeutic Targets in Medically Treated HF

这一新的早期研究人员R01提案探索了当前指南如何指导药物治疗 (GDMT)已经完全改变了慢性心力衰竭(HF)的疾病状态，特别是在 心肌细胞通过一氧化氮受体、可溶性鸟苷酸环化酶(SGC)传递信号。尽管GDMT是 广泛使用，这种改良的慢性心力衰竭状态实际上被临床前研究忽视了。我们 发现sGC在肥厚、功能不全的心脏中调节失调，而β阻滞剂治疗，一 GDMT的基石，改变了患病心脏中的sGC信号，可能建立 新奇，但潜在的，心脏保护级联反应。压力过载和体积过载都会导致血管再局部化 SGC远离小窝，即分隔信号转导的质膜内陷。 在小窝内，sGC似乎受到保护而不被氧化，并保持对一氧化氮(NO)的反应。在 肥大，心力衰竭，小窝外的sGC被氧化，无反应性 迟钝的。最近，我们发现在面对容量超载压力时，β-BLOCKED阻止了 心肌sGC从小窝分离，恢复小窝外sGC的无反应性 非脂筏膜中β-3肾上腺素能受体(β-3AR)与sGC的诱导功能偶联 微域。有趣的是，这种功能性的β3AR/sGC偶联是非脂筏微域所特有的 在对照组和未经治疗的肥厚心脏中均未发现。目前的提案适用于 PI在心脏sGC信号、分子生理学和临床心衰领域的专业知识 鸟苷一磷酸(CGMP)膜微域信号转导。这个地区有很好的高频治疗。 鉴于膜微域是整合肾上腺素能、钙和钙的关键节点， 环核苷酸信号。这项提议将检验我们的假设，即GDMT改变了NO- 心脏不同膜微区sGC的反应性和功能偶联 心肌细胞，从而导致未开发的心脏保护电位不同于无衰竭或 未经治疗的心脏衰竭。我们的初步研究表明，非脂筏微域可能代表 并元结点。在目标1中，我们将确定质膜凹陷的生理功能。 心肌细胞中二元连接相关的sGC/cGMP信号转导。在目标2中，我们将定义 GDMT对心肌凹陷的影响--与压力下sGC/cGMP二元连接相关信号的比较 心脏超负荷。在目标3中，我们将阐明GDMT如何促进sGC小窝定位和 增强无响应能力。利用我们新的心脏特异性sGC基因敲除，微域靶向sGC 构建和心脏选择性AAV9介导的转基因传递，我们将提供重要的机制 为数百万心力衰竭患者开发创新、协同疗法的证据 已在GDMT上优化。

This new early stage investigator R01 proposal explores how current guideline directed medical therapy (GDMT) has completely modified the chronic heart failure (HF) disease state, specifically with regards to cardiac myocyte signaling via the nitric oxide receptor, soluble guanylyl cyclase (sGC). Although GDMT is widely used, this modified chronic HF disease state is virtually ignored by preclinical studies. We discovered that sGC is dysregulated in the hypertrophied, dysfunctional heart and that β-blocker therapy, a cornerstone of GDMT, alters sGC signaling in the diseased heart in a way that establishes potentially novel, but latent, cardioprotective cascades. Both pressure- and volume-overload induce re-localization of sGC away from caveolae, invaginations of the plasmalemma that compartmentalize signal transduction. Within caveolae, sGC appears protected from oxidation and remains responsive to nitric oxide (NO). In the hypertrophied, failing heart, sGC that is outside of caveolae becomes oxidized and NO-responsiveness is blunted. Recently, we discovered that in the face of volume overload stress, β-blockade prevented myocardial sGC dissociation from caveolae, restored NO-responsiveness of sGC outside of caveolae, and induced functional coupling between β3 adrenoceptor (β3AR) and sGC within a non-lipid raft membrane microdomain. Intriguingly, this functional β3AR/sGC coupling was specific to the non-lipid raft microdomain and was not found in either control nor untreated, hypertrophied hearts. The current proposal applies the PI's expertise in cardiac sGC signaling, molecular physiology, and clinical HF to the field of cyclic guanosine monophosphate (cGMP) membrane microdomain signaling. This area has great HF therapeutic potential given that membrane microdomains act as critical nodes for integrating adrenergic, calcium, and cyclic nucleotide signaling. This proposal will test our hypothesis that GDMT changes the NO- responsiveness and functional coupling of sGC within distinct membrane microdomains of cardiac myocytes, thereby resulting in untapped cardioprotective potential that differs from that in non-failing or untreated failing hearts. Our preliminary studies suggest that the non-lipid raft microdomain may represent dyadic junctions. In Aim 1, we will determine the physiological function of plasmalemmal caveolae- vs. dyadic junction-associated sGC/cGMP signaling in cardiac myocytes. In Aim 2, we will define the effect of GDMT on myocardial caveolae- vs. dyadic junction associated sGC/cGMP signaling in the pressure overloaded heart. In Aim 3, we will elucidate how GDMT promotes sGC caveolae-localization and enhances NO-responsiveness. With our novel cardiac-specific sGC knockout, microdomain-targeted sGC constructs, and cardioselective AAV9-mediated transgene delivery, we will provide vital mechanistic evidence for the development of innovative, synergistic therapies for the millions of HF patients already optimized on GDMT.