Unique Role of Intracellular Perinuclear β1-Adrenergic Receptors in Defining Signaling Compartmentation and Pathological Cardiac Remodeling

Abstract

Background: β-Adrenergic receptors (βARs) are prototypical G protein-coupled receptors that regulate contractility in the normal heart and pathological remodeling in disease. Canonical βAR signaling originates at the plasma membrane, but functional βARs have been localized to intracellular membranes such as the endosome, sarcoplasmic reticulum, Golgi, and nuclear envelope. The functional significance of these intracellular receptors remains unclear, including whether they regulate cellular processes distinct from those regulated by plasma membrane receptors and whether they can be independently targeted for therapeutic benefit.

Methods: Live cell imaging of rat and human cardiomyocytes expressing novel compartment-specific modulators of βAR activity and fluorescent biosensors was used to study the compartment-specific βAR regulation of second messengers and to target enzyme activity. Compartmentalized signaling was compared with myocyte gene expression and hypertrophy. Adeno-associated virus gene delivery conferring gain and loss of perinuclear βAR activity was studied in wild-type mice and a mouse model of familial dilated cardiomyopathy.

Results: We demonstrate here that intracellular β₁ARs present on Golgi membrane facing the outer nuclear membrane regulate a perinuclear cAMP compartment containing the A-kinase anchoring protein 6β signalosome, conferring selective regulation of perinuclear cAMP-dependent protein kinase activity independently of βARs at the plasma membrane or endosome. The A-kinase anchoring protein 6β compartment is shown to be of nanometer scale and dependent on local restriction of cAMP diffusion. In addition, perinuclear βARs are shown to be sufficient and necessary for activation of the Ca²⁺-dependent calcineurin-nuclear factor of activated T cells pathway and myocyte hypertrophy in vitro. Accordingly, adeno-associated virus 9-based delivery of an outer nuclear membrane-localized pepducin, which selectively activated perinuclear βARs in vitro, induced dilated cardiomyopathy in wild-type mice. Conversely, in vivo delivery of an outer nuclear membrane-localized nanobody, which selectively inhibited perinuclear βARs in vitro, improved cardiac function and inhibited pathological remodeling in a mouse model of familial dilated cardiomyopathy with established disease.

Conclusions: These results demonstrate that β₁ARs localized to Golgi membranes facing the outer nuclear membrane regulate A-kinase anchoring protein 6β signalosomes required for the induction of pathological cardiac remodeling, defining an intracellular nanocompartment. Proof of concept is provided for a novel therapeutic approach for familial dilated cardiomyopathy, with potential application to other forms of cardiovascular disease.

Keywords: Golgi apparatus; cardiomyopathy, dilated; cell compartmentation; cyclic AMP-dependent protein kinases; receptors, adrenergic, beta; signal transduction.