Andrew R. Marks
The objective of this project is to solve the structures of WT and mutant RyR2 that cause HF, and drug bound channels at a near-atomic level using cryogenic electron microscopy (cryo-EM), and combine these structural studies with in vitro and in vivo analyses to address important unanswered questions concerning the role of RyR2 channels in normal and pathological Ca2+ signaling in the heart. The project will make extensive use of Core A in which human RyR2 mutations linked to chronic HF with reduced ejection fraction (HFrEF) will be identified, and Core B in which murine models of HF will be analyzed. Project 4 is linked to: Project 1 where nanobodies against the PKA site on RYR2 will be generated and tested to determine their ability to specifically block PKA mediated activation of RyR2 and determine whether this is protective against HF progression; Project 2 where mice deficient in adrenergic regulation of either the plasma membrane calcium channel or RyR2 will be crossed to define in unprecedented detail the relative contributions of each channel to cardiac regulation; and to Project 3 where the ability of the Rycal drug ARM210 to fix the presumptive RyR2-mediated leak associated with mutations in the accessory protein junctophilin (Jph2) which links T-tubule and SR will be studied. The central hypothesis of Project 4 is that pathological SR Ca2+ leak can cause both arrhythmias and cardiac dysfunction, the hallmarks of HF, the leading cause of morbidity and mortality in the developed world.
Aim 1: Evaluate the changes in the function and structure of RyR2 variants linked to heart failure.
Aim 2: Evaluate the effect of Rycal molecules in the structure of human RyR2 and its disease related variants.
Aim 3: Examine the mechanism by which adrenergic signaling affects RyR2 function in normal and failing hearts.