Arrhythmogenic NCX Currents in Cardiomyocytes from Transgenic Mice Expressing Mutated RyR2 Receptors

Abstract

We investigated Ca2+ signaling in cardiomyocytes from transgenic mice with a mutation in the calmodulin (CaM) binding domain of RyR2 (W3587A/L3591D/F3603A, RyR2ADA; Yamaguchi et al., Circ Res, 100:293-5, 2007). Ca2+ sparks (Fluo-4) in cardiomyocytes from 11-15 day old homozygous mutant mice were less frequent (0.26±0.17 vs. 3.62±0.54 sparks/sec n=18), but longer lasting than in wild-type. Voltage-clamp depolarization of the mutant cells produced Ca2+ transients of reduced amplitude even though those triggered by caffeine were enhanced and accompanied by disproportionally larger Na+-Ca2+ exchanger (NCX) currents. The distributions of RyR2, NCX and membrane staining in mutant cells indicate a disorganized t-tubular system. During repeated voltage-clamp depolarizations, mutant cardiomyocytes produced alternans or gradually rising Ca2+ transients that in time developed a tendency to generate large delayed Ca2+ releases and NCX currents thereby restarting the sequence. Our findings suggest that the expression RyR2ADA in homozygous mice with cardiac hypertrophy is associated with impaired ICa-induced Ca2+ release resulting, In part, from disruption of the t-tubular system and its associated dyadic junctions. At the same time it is noticeable that the (partially decoupled) SR Ca2+ stores are well maintained and produce a) infrequent, but long lasting Ca2+ sparks, and b) large regenerative Ca2+ transients that are accompanied by prominent NCX currents and can be triggered, not only by caffeine, but also by repeated stimulation. The latter observation suggest a mechanism for delayed after-depolarization, where the arrhythmogenic potential of NCX currents is enhanced by the concurrence of a number of factors: a) impaired Ca2+ signaling by RyR2, b) partial structural decoupling of components of the SR Ca2+ stores, and c) gradual enhancement of the Ca2+ contents of these stores resulting e.g. from rapid beating during adrenergic stimulation. (NIH HL16152; AHA 10SDG3500001).