Abstract 1874: Scavenging Free Radicals by Low-Dose Carvedilol Prevents Redox-Dependent Ca2+ Leak via Stabilization of Ryanodine Receptor in Heart Failure
We have reported that the interaction between the N-terminal and central domains of the ryanodine receptor-2 (RyR2), the domains where many mutations have been found in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), is defective in heart failure (HF). Here, we investigated whether the defectiveness is corrected by carvedilol (CV), which has a potent antioxidant activity.
Methods and Results. Sarcoplasmic reticulum (SR) vesicles were isolated from canine LV muscle (normal:n=6, or rapid RV pacing for four weeks:n=6). The RyR2 was labeled with fluorescent conformational probe methylcoumarin acetate (MCA) using DPc10 (a synthetic peptide corresponding to Gly2460-Pro2495 of RyR2; one of the mutable domains in CPVT) as a site-direction carrier. The content of free thiols in RyR2 was determined using the monobromobimane fluorescence technique. In failing SR vesicles, oxidative stress of RyR2 (% decrease in the number of free thiols from maximally reduced state) was severe (-45 %, p<0.01). The RyR2 in failing SR revealed a destabilized inter-domain interaction (viz. domain unzipped state), as confirmed by a marked quenching of MCA fluorescence by a large-size fluorescence quencher. In failing cardiomyocytes, diastolic Ca2+ spark was frequently seen compared to normal cardiomyocytes (failure:3.7±1.3 s-1 100μm-1; normal:1.5±0.3 s-1 100μm-1, p<0.01). Incubation of failing myocytes with carvedilol (30 nM), at which cell shortening of normal cardiomyocytes was unchanged, indeed inhibited Ca2+ spark (2.0±0.6 s-1 100μm-1), concurrently with a marked reduction of intracellular reactive oxygen species (ROS) assessed by 2′,7′-dichlorofluorescin diacetate (DCFH-DA) fluorescence. The nitric oxide donor, 3-morpholinosydnonimine (SIN-1) reproduced, in normal SR, several abnormal features seen in failing SR, such as defective inter-domain interaction and Ca2+ spark, which were reversed by addition of carvedilol.
Conclusions. CV, at a concentration which is sufficient to produce antioxidant effect, but is not sufficiently high to produce negative inotropic effect, improves intracellular Ca2+ handling and contractile dysfunction, by correcting defective inter-domain interaction within RyR2 in the failing hearts.