Abstract 2632: Calmodulin Kinase Inhibition Prevents the Arrhythmogenesis in Ryr2r4496c+/− Knock in Mice
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is caused by mutations in the cardiac ryanodine receptor gene (RyR2) leading to life-threatening arrhythmias elicited by sympathetic activation. We previously demonstrated that Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibition prevents VT in our RyR2R4496C+/− knock-in mice and abolishes triggered activity in isolated RyR2R4496C+/− myocytes. Here we report the effects of CaMKII inhibition on Ca2+handling in RyR2R4496C+/− myocytes. Ca2+ transients were recorded with field stimulation (Fura-2, 1–5 Hz): Isopreterenol (30 nM) exposure did not affect the function of the sodium calcium exchanger and it
induced spontaneous calcium transients,
accelerated the rate of decay of Ca2+ transients
increased SR calcium load (estimated by rapid application of 10 mM caffeine).
Exposure to CAMKII inhibitors (1 μM KN93 or 1 μM CaMKII inhibitory peptide, AIP) significantly inhibited the first two consequences of Iso but failed to attenuate the increase in the SR Ca2+ load (2.1±0.08, 1.9±0.08 and 1.9±0.06 Fratio in Iso treated, KN93 treated and AIP treated cells respectively, p>0.01). Finally we measured Ca2+ sparks in Fluo-4 loaded RyR2R4496C+/− myocytes and showed that CaMKII inhibitors reversed Iso-induced enhancement of Ca2+ sparks frequency (1.3±0.19, 1.1±0.2 and 2.1±0.19 s-1·100 μm-1 in KN93 treated, AIP treated and Iso treated cells respectively, p<0.01), suggesting that inhibition of RyR2 phosphorylation is a critical component of antiarrhythmic action of CMKII inhibitors in CPVT. In agreement with this hypothesis western blot revealed that CaMKII blockers significantly decreased Iso-mediated RyR2 phophorylation (serine 2808 and serine 2814). We conclude that CaMKII inhibition prevents abnormal Ca2+ leak and arrhythmias induced by Iso on RyR2R4496C+/− myocytes through a dual mechanisms
reduction of Iso-induced enhancement of SERCA function and
attenuation of diastolic SR Ca2+ leak from the mutant RyR2 channels.