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

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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.