Roles of Rad and other CaV1.2 neighboring proteins in regulating cardiac function in health and disease

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The overarching goal of this PPG is to define the molecular mechanisms that regulate local Ca2+ signaling in normal and failing hearts with unprecedented precision. There are three subaims shared by the four projects: 1) explore the precise role of adrenergic signaling in modulating calcium in normal and failing hearts; 2) define novel mechanisms of interactions between t-tubule and SR calcium channels; 3) develop new understandings of genetic based mechanics of inherited forms of CV disease involving calcium. Our long-term goals are to elucidate mechanisms underlying the progressive decompensation towards advanced HF, and to identify novel cellular targets and signaling pathways to prevent and treat the symptoms of HF, and to prevent arrhythmias. Project 2 focuses on the regulation of the Ca2+ channel by the adrenergic nervous system, both under physiological and pathological conditions. Recently, we identified the mechanism by which -adrenergic agonists stimulate voltage-gated Ca2+ channels. We expressed 1C or 2B subunits conjugated to ascorbate- peroxidase in mouse hearts and used multiplexed, quantitative proteomics to track hundreds of proteins in close proximity to CaV1.2. We observed that the Ca2+ channel inhibitor Rad, a monomeric G-protein, is enriched in the CaV1.2 micro-environment but is depleted during -adrenergic stimulation. PKA-catalyzed phosphorylation of specific serine residues on Rad decreases its affinity for auxiliary -subunits and relieves constitutive inhibition of CaV1.2 observed as an increase in channel open probability. We created knock-in mice with alanine substitutions of the four PKA phosphorylation sites. The stimulatory effect of isoproterenol or forskolin on Ca2+ current is completely eliminated when Rad can no longer be phosphorylated in atrial and ventricular cardiomyocytes. Furthermore, loss of PKA phosphorylation of Rad markedly diminished the adrenergic stimulation of the Ca2+ transient and sarcomere shortening. One of our goals is to investigate the relevance of RRAD in humans through analyses of common and rare variants in 20,000 cases with HF, 45,000 cases with MI and equivalent number of controls for each outcome, and to understand the consequences of RRAD deletion and missense mutations through deep phenotyping of carriers and non-carriers of these mutations in consanguineous families in the Pakistan Genome Resource (PGR). Three specific aims are proposed: (1) To determine the impact of -adrenergic stimulation of CaV1.2 in HF. We will use knock-in mice with PKA phosphorylation sites of Rad mutated to alanine, and mice lacking the Rad-subunit interaction in heart. (2) To determine upstream and downstream regulatory pathways modulating adrenergic stimulation of CaV1.2 in physiological and HF states. (3) To explore the role of Rad in regulating cardiac function in humans. We anticipate that this multi-pronged approach will yield novel mechanistic insights into adrenergic signaling in HF and more specifically the adrenergic regulation of Ca2+ channels in HF.