BPS2026 – Resolving calmodulin-RyR2 conformational dynamics by real-time FRET in permeabilized and intact cardiac myocytes

Calmodulin (CaM) stabilizes the cardiac ryanodine receptor (RyR2) and suppresses arrhythmogenic sarcoplasmic reticulum (SR) Ca 2+ leak. In heart failure (HF), the CaM affinity for RyR2 is reduced, but the conformational dynamics of CaM binding with RyR2 during the cardiac cycle remain poorly defined. Using FRET between fluorescently labeled FKBP12.6 and CaM in permeabilized ventricular myocytes, we found that CaM remains bound during Ca 2+ waves at 0.2–1 Hz, with no evidence of dissociation between beats even in HF myocytes. Fluorescence-lifetime imaging microscopy (FLIM) further revealed Ca 2+ -dependent repositioning of the CaM N terminus relative to FKBP. Notably, this conformational movement was blunted in myocytes from HF mice (cardiac-specific CaMKII overexpression), suggesting that pathological SR Ca 2+ leak may arise from altered CaM positioning on RyR2 rather than acute dissociation. As a control for Ca 2+ -dependent effects, we are employing a Ca 2+ -insensitive CaM 1234 mutant combined with thapsigargin treatment to test whether Ca 2+ -induced FRET changes are due to altered CaM-RyR2 interactions vs. Ca 2+ -CaM binding. To extend these findings toward physiological conditions, we developed a patch-dialysis approach to introduce fluorescent CaM and FKBP into intact myocytes while monitoring Ca transients and FRET in real time confocal line-scan imaging. Preliminary results demonstrate robust incorporation of fluorescent proteins in striated binding patterns consistent with RyR2 localization. Together, these studies provide new mechanistic insight into CaM regulation of RyR2 in health and disease and establish a framework for directly testing whether conformational signatures of CaM observed in vitro translate into the intact cellular environment.