Dissecting Roles of Cav1.2 (α1C) Intracellular Loops in Cardiac Excitation-Contraction Coupling

Abstract

Cardiac excitation-contraction (EC) coupling relies on Ca2+-induced Ca2+ release (CICR) enabled by an intimate relationship between L-type (CaV1.2) channels and ryanodine receptors (RyRs) at dyadic junctions. The determinants underlying CaV1.2/RyR functional proximity responsible for effective CICR are unknown, but likely entail protein interactions involving one or more intracellular loops of CaV1.2 pore-forming α1C subunit. We hypothesized that over-expressing α1C intracellular loops that play a critical role in CaV1.2/RyR communication would disrupt CICR in cardiomyocytes. We used adenoviruses to overexpress CFP-tagged α1C intracellular loops and termini (NT, I-II, II-III, III-IV, CT) in cardiomyocytes and determined their impact on field-stimulation-evoked rhod-2-reported Ca2+ transients. Over-expressed NT, II-III, and III-IV loops had minimal effect on CICR. By contrast, over-expressed I-II and CT produced dramatic disruption of EC coupling characterized by two distinct signatures: a sharp augmentation in CICR failure, and an increased propensity for arrhythmic Ca2+ transients. The defective EC coupling was not due to a gross misalignment of CaV1.2 and RyR as reported by co-immunolocalization assays. Surprisingly, overexpressed I-II had no impact on CaV1.2 current (ICa,L), whereas CT strongly suppressed ICa,L. The results identify a dominant role for I-II and CT in establishing CaV1.2/RyR functional interrelationship, and suggest a new approach to dissect molecular components and interactions critical for CICR in the heart.