Project Summary The overarching goal of this PPG is to define the molecular mechanisms that regulate local calcium (Ca2+) signaling in normal and failing hearts with unprecedented precision. There are three goals 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 sarcoplasmic reticulum (SR) calcium channels; 3) develop new understandings of genetic based mechanisms of inherited forms of CV disease involving calcium. Project four provides the essential link to SR calcium release by studying the structure-function relationships of the type 2 ryanodine receptor (RyR2)/calcium release channel present on the sarcoplasmic and endoplasmic reticula (SR/ER) of many cell types in the context of heart failure (chronic HF with reduced ejection fraction, HFrEF). RyR2 channels are required for Ca2+ release from intracellular stores that triggers excitation-contraction (EC) coupling in the cardiac muscle. Inherited RyR2 mutations can cause arrhythmias including exercise-induced sudden death or catecholaminergic polymorphic ventricular tachycardia (CPVT), and stress-induced post- translational modifications of RyR2 contribute to heart failure (HF) progression. In both cases RyR2 channels are leaky either due to inherited mutations (CPVT) or acquired post-translational modifications (HF). Unanswered questions include: 1) can (and if so how) RyR2 mutations can cause HF; 2) what is the precise mechanism by which PKA phosphorylation activates RyR2 and plays a role in HF? Preliminary data using cryo-EM to solve the structure of human RyR2 at ~2.4 Å show that a human CPVT mutation RyR2-R2474S puts the channel into a “primed state” from which it can be readily and pathologically activated at low, normally non-activating [Ca2+]cyt explaining why these channels are leaky and cause fatal ventricular arrhythmias during exercise or stress. The Rycal drug ARM210 binds to RyR2 and restores the mutant channel back to a stable closed state preventing leak and arrhythmias. The applicant hypothesizes that RyR2 missense mutations, 14 of which are found in patients with a ClinVar prediction of cardiomyopathy in patients from the Pakistan Genome Resource (PGR), a unique cohort of individuals with extensive phenotype data and high rates of consanguinity, (Core A), may be gain of function (GoF) mutations that also put the channel into a primed state which is even more sensitive to activation compared to the CPVT mutations. Thus, these patients have leaky RyR2 channels and develop HF due to depletion of SR Ca2+ resulting in impaired cardiac contractility. These questions will be addressed using functional and structural assays and using a novel drug, Rycal (ARM210), that fixes the RyR2 mediated SR Ca2+ leak via a well-defined mechanism. Three aims are proposed: 1) Evaluate changes in the function and structure of RyR2 GoF missense variants li...