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

这项新的早期研究者R01提案探讨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）疾病状态，特别是关于 心肌细胞信号通过一氧化氮受体，可溶性鸟苷酸环化酶（sGC）。虽然GDMT是 广泛使用，这种改良的慢性HF疾病状态实际上被临床前研究忽略。我们 发现sGC在肥大、功能障碍的心脏中失调，β受体阻滞剂治疗， GDMT的基石，改变了患病心脏中的sGC信号传导， 新颖但潜在的心脏保护级联反应。压力和体积超负荷均诱导 sGC远离小窝，质膜的内陷，分隔信号转导。 在小窝内，sGC似乎受到保护而不受氧化，并保持对一氧化氮（NO）的反应。在 肥厚、心力衰竭时，小窝外的sGC被氧化，NO反应性降低。 钝的最近，我们发现，面对容量超负荷应激，β-受体阻滞剂阻止了 心肌sGC与小窝分离，恢复小窝外sGC的NO反应性，以及 在非脂筏膜内诱导β 3肾上腺素受体（β 3 AR）和sGC之间的功能偶联 微域有趣的是，这种功能性β 3AR/sGC偶联特异于非脂筏微结构域， 在对照组和未治疗的肥大心脏中均未发现。目前的建议适用于 PI在心脏sGC信号传导、分子生理学和临床HF领域的专业知识， 鸟苷一磷酸（cGMP）膜微区信号传导。这个地区有很好的HF治疗 考虑到膜微区作为整合肾上腺素能、钙和 环核苷酸信号传导。这一提议将检验我们的假设，即GDMT改变了NO- sGC在心脏不同膜微区内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潜力，不同于非衰竭或 未经治疗的衰竭心脏我们的初步研究表明，非脂筏微区可能代表 二元结在目标1中，我们将确定质膜小窝的生理功能-与。 心肌细胞中的二元连接相关sGC/cGMP信号传导。在目标2中，我们将定义 GDMT对压力下心肌小窝与二进连接相关sGC/cGMP信号传导的影响 超负荷的心脏在目标3中，我们将阐明GDMT如何促进sGC小窝定位， 增强NO反应性。通过我们的新型心脏特异性sGC敲除，微结构域靶向sGC 构建体和心脏选择性AAV9介导的转基因递送，我们将提供重要的机制， 为数百万HF患者开发创新的协同疗法的证据已经 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.