Mutations in cardiac ryanodine receptor (RyR2) was found to be linked with catecholaminergic polymorphic ventricular tachycardia (CPVT). To study the underlying mechanism of CPVT, we developed knock-in mice harboring the Arg-to-Ser (R2474S) mutation. The RyR2
R2474S/+
knock-in (KI) mice revealed no structural or histological abnormality in hearts. Echocardiography showed no contractile or relaxation dysfunction at rest. In all KI mice (n=6), bidirectional ventricular tachycardia (VT) was observed during or after exercise with treadmill, but never observed in wild-type (WT) mice (n=6). In intact cardiomyocytes, the frequency of Ca
2+
sparks (SpF; s
−1
·100μm
−1
) was significantly increased in KI mice, but not in WT mice (at 2 mM [Ca
2+
]; KI:6.4±0.7, WT:0.9±0.08, p<0.01). To investigate the sensitivity of the RyR2 channel to activation by luminal Ca
2+
{[Ca
2+
] in sarcoplasmic reticulum (SR)}, we measured cytoplasmic [Ca
2+
] ([Ca
2+
]
C
) and luminal [Ca
2+
] ([Ca
2+
]
L
) simultaneously in saponin-permeabilized cardiomyocytes, using Rhod-2 and Fluo-5N AM as Ca
2+
indicators, respectively. When [Ca
2+
]
C
was buffered at 100 nM (by 1 mM EGTA), the spontaneous Ca
2+
sparks were frequently observed both in KI and WT cardiomyocyts (SpF: KI:22.1±0.9, WT:22.0±0.8, p=ns). When we added thapsigargin (1 μM) to the cardiomyocytes under this condition ([Ca
2+
]
C
=100 nM), both SpF and [Ca
2+
]
L
gradually decreased due to a decrease in SR Ca
2+
content caused by an inhibition of SR Ca
2+
ATPase. The relationship curve between SpF and [Ca
2+
]
L
(SpF -[Ca
2+
]
L
) during the addition of thapsigargin was markedly shifted to the left in KI cardiomyocytes compared to WT cardiomyocytes, thereby lowering the threshold of [Ca
2+
]
L
to induce Ca
2+
sparks to approximately one-fifth in KI cardiomyocytes. In conclusion, the enhanced sensitivity of the RyR2 channel to activation by [Ca
2+
]
L
: i.e. decreased threshold [Ca
2+
]
L
to induce spontaneous Ca
2+
release, may be a primary cause of CPVT.
Understanding the Molecular Mechanism of Cation Permeation in the Cardiac Ryanodine Receptor (RyR2) Channel using Computational Electrophysiology
