Abstract 334: Cardiac Myosin Binding Protein C Phosphorylation Regulates Calcium Homeostasis

Abstract

Rationale: Cardiac myosin binding protein-C (cMyBP-C) is heavily phosphorylated to regulate normal cardiac function under basal conditions. However, its phosphorylation level is significantly decreased in patients with heart failure and atrial fibrillation. Furthermore, decreased cMyBP-C phosphorylation causes reduced myofilament contractility and decreased calcium sensitivity. The impact of such decreases in cMyBP-C phosphorylation and myofilament calcium sensitivity on the overall calcium handling and potential induction of arrhythmogenesis is not known. Objective: To determine the necessity and sufficiency of cMyBP-C phosphorylation to regulate calcium cycling and contractility at the isolated cardiomyocyte level. Methods and Results: Contractile properties and calcium kinetics were measured in intact cardiomyocytes isolated from 3-month-old, mixed sex, mice expressing phospho-ablated (S273A/S282A/S302A) cMyBP-C (AAA) or phospho-mimetic (S273D/S282D/S302D) cMyBP-C (DDD) and nontransgenic (NTG) control mice. AAA cells displayed a significant decrease in fractional shortening compared to NTG and DDD cells (9.4% vs 12.8% in NTG, and 12.3% in DDD, p<0.0001). Similarly, AAA myocytes demonstrated abnormal calcium kinetics with increased diastolic calcium levels (20%, p<0.001 vs . NTG and DDD) and prolonged decay time of the calcium transient (26%, p<0.01 vs NTG and DDD) when compared with NTG and DDD myocytes. Caffeine-induced calcium release in AAA myocytes indicated no change in SR calcium content, while sodium-calcium exchanger function, assessed as the time constant (τau) of calcium decline, was increased (89%, p<0.01 vs NTG). However, these depressive effects in AAA myocytes were relieved by isoproterenol (100 nmol/L) stimulation. Furthermore, stress conditions (2 Hz + ISO) increased after-contractions in AAA cardiomyocytes (60% in AAA vs 13% in NTG, p<0.001 and 15% in DDD, p<0.001). In addition, preliminary data indicate increased susceptibility of the AAA mice to arrhythmias under stress conditions in vivo. Conclusion: Dephosphorylation of cMyBP-C is sufficient to reduce sarcomere contractility and impair calcium cycling resulting in spontaneous after-contractions and arrhythmias under stress conditions.