DNA-based molecular dissection and targeting of the Gbetagamma-GRK2-interactome for ischemic cardiomyopathy treatment

缺血性心肌病的特征是交感神经动力增强，组织和血浆儿茶酚胺水平上调，试图刺激心肌收缩功能。在这种情况下，G蛋白偶联受体激酶2(GRK2)被上调，并通过释放的Gbetagamma亚基被募集到质膜上；GRK2通过受体脱敏和下调而磷酸化心脏β-肾上腺素能受体(β-AR)，从而抑制心脏β-肾上腺素能受体(β-AR)的变力反应；儿茶酚胺的进一步上调加剧了这一恶性循环，最终促进了心功能障碍。BetaARKct微型蛋白通过结合释放的Gbetagamma亚基来阻断GRK2-Gbetagamma的相互作用，并可以挽救不同的心功能障碍模型。然而，确切的分子模式的有益的贝塔效应在心功能不全仍不清楚。此外，到目前为止，BetaARKct只在啮齿动物模型中进行了测试。由于啮齿动物的生理和分子信号与人类不同，迫切需要在更能反映人类病理生理学的大型动物模型中评估新的治疗方法。在我们的心肌梗死(MI)后缺血性心肌病猪模型中，用腺相关病毒6型(AAV6.betaARKct)基因治疗最近被证明是有效的。然而，关于BetaARKct基因治疗在临床试验中的潜在应用的关键科学问题仍然存在：BetaARKct治疗还是直接抑制/敲除GRK2更可取？与使用β-AR阻滞剂的标准缺血性心肌病治疗相比，BetaARKct基因治疗效果如何？β-ARKct基因治疗或GRK2基因敲除对交感神经系统和心内肌细胞β-AR依赖和Gbetagma依赖的信号通路和基因调控的影响尚不清楚，也从未与β-AR阻滞剂治疗的后果相比较。在这方面，我们研究的中心目标是确定在我们的临床前大动物心肌梗死后心肌病模型中使用BetaARKct基因疗法(AAV6.betaARKct)抑制GRK2的长期效果、治疗概况和基本机制原理，并与标准的β受体阻滞剂治疗进行比较和结合。此外，我们希望建立一种针对GRK2的合成miRNA(AAV6.miGRK2)作为针对Gbetagamma-GRK2-相互作用组的替代治疗方法，并将其与BetaARKct和药理学上的β-AR阻断进行比较。利用我们先进的大型动物平台，这些目标将在我们的缺血后心肌病猪模型中实现。实验被精心安排，以确定对整体和局部心肌功能、交感神经张力的影响，剖析与治疗相关的分子信号通路，并确定安全性方面。

Ischemic cardiomyopathy is characterized by an increased sympathetic drive with upregulation of tissue and plasma catecholamines in an attempt to stimulate myocardial contractile function. G-protein coupled receptor kinase 2 (GRK2) is upregulated under these circumstances and recruited to the plasma membrane via liberated Gbetagamma-subunits; GRK2 phosphorylates cardiac beta-adrenergic receptors (beta-ARs) and thus inhibits cardiac beta-adrenergic receptor (beta-AR) inotropic responsiveness by receptor desensitization and downregulation; further upregulation of catecholamines fuels this vicious cycle finally promoting cardiac dysfunction. The betaARKct miniprotein blocks the GRK2-Gbetagamma interaction by binding of liberated Gbetagamma-subunits and could rescue disparate models of cardiac dysfunction. However, the precise molecular mode of the beneficial betaARKct effects in cardiac dysfunction is still unclear. Furthermore, up to date betaARKct was only tested in rodent models. As rodent physiology and molecular signaling differ from human, it is imminent to evaluate novel therapeutic approaches in large animal models more closely reflecting human pathophysiology. In our post myocardial infarction (MI) ischemic cardiomyopathy pig model betaARKct gene therapy with an adeno-associated virus serotype 6 (AAV6.betaARKct) was recently shown to be effective. However, key scientific issues regarding a potential use of betaARKct gene therapy in clinical trials remain: Is betaARKct therapy or direct GRK2 inhibition/knockdown more desirable? How does betaARKct gene therapy compare to standard ischemic cardiomyopathy treatment with beta-AR blocker therapy? The effects of betaARKct gene therapy or GRK2 knockdown on the sympathetic nervous system and intracardiac myocyte beta-AR dependent and Gbetagamma-dependent signaling pathways and gene regulation are unknown and have never been compared to consequences of beta-AR blocker therapy. In this regard, the central aim of our study is to define longer-term effects, the therapeutic profile and basic mechanistic principles of GRK2 inhibition with betaARKct gene therapy (AAV6.betaARKct) in our preclinical large animal post-MI cardiomyopathy model in comparison to and in combination with standard beta-blocker therapy. Furthermore, we want to establish a synthetic miRNA targeting GRK2 (AAV6.miGRK2) as alternative therapeutic approach targeting the Gbetagamma-GRK2-interactome and compare its effects to betaARKct and pharmacological beta-AR blockade. Taking advantage of our advanced large animal platform these aims will be accomplished in our post-ischemic cardiomyopathy pig model. Experiments are orchestrated to define effects on global and regional myocardial function, sympathetic nervous tone, to dissect therapy-relevant molecular signaling pathways and to determine safety aspects.