Abstract 5251: Defective Inter-domain Interaction May Cause Spontaneous Ca 2+ Release Via Reduced Affinity of Calmodulin Binding to RyR2 in Failing Hearts

Abstract

We previously reported that interaction between N-terminal 1–600 and central domains 2000–2500 of ryanodine receptor (RyR2), harboring many mutation sites in CPVT, is defective (i.e. domain unzipping) in failing hearts. Here, we investigated the pathogenic role of calmodulin (CaM), one of the accessory proteins in RyR2, on Ca 2+ release in failing hearts. Sarcoplasmic reticulum (SR) vesicles were isolated from dog LV muscles {normal (N), n=6; 4-weeks rapid RV pacing (HF: n=6). To assess CaM binding to RyR2, SR was mixed with CaM-SANPAH conjugate (16nM-1μM), followed by UV photolysis. Then, the RyR2-bound CaM was detected by Western blotting using anti-CaM antibody. The affinity of CaM binding to RyR2 was lower in failing SR than normal SR (Kd: 47nM in HF: 19nM in N , p<0.01). To assess the possible relationship between domain unzipping and CaM dissociation from RyR2, RyR2 was also fluorescently labeled with methylcoumarin acetamido (MCA) using DP 2460–2495 (DPc10), which harbors a mutation site in CPVT; R2474S, as a site-directing carrier. In failing SR, domain unzipping was already taken place, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. Interestingly, addition of FK506 (10 μM), which was found to dissociate FKBP12.6 from RyR2 and to induce domain unzipping mimicking failing SR, to normal SR indeed reduced the CaM binding affinity to RyR2. In saponin-permeabilized, failing cardiomyocytes ([Ca 2+ ]=buffered at 75 nM), the frequency of Ca 2+ sparks was markedly increased (SpF; s −1 ·100μm −1 : 13.9±0.63 in HF; 7.3±0.6 in N, p<0.01). Addition of CaM (1 μM) in the presence of KN-93 (CaMKII inhibitor) inhibited the increase in SpF (7.9 ±0.41 , p<0.01). This CaM’s effect was, however, markedly inhibited by co-addition of CaM-binding domain peptide within RyR2 (3583–3603) (10.4±0.65, p=ns), strongly suggesting that the re-binding of CaM to RyR2 corrects the abnormal Ca 2+ release in failing cardiomyocytes. In conclusion, the defective inter-domain interaction between N-terminal and central domains within RyR2 seems to increase spontaneous Ca 2+ release events in failing SR, via the reduced CaM-binding to RyR2. Fixing CaM binding to RyR2 may be protective against the diastolic Ca 2+ release in failing hearts.