Abstract 5289: Abnormally Tight Domain-Domain Interaction at Mutation Site Could be a Primary Cause of Catecholaminergic Polymorphic Ventricular Tachycardia: Insight from RyR2 S2246L/+ Knock-In Mouse Model

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Takeshi Suetomi ◽  
Masafumi Yano ◽  
Xiaojuan Xu ◽  
Makoto Ono ◽  
Hitoshi Uchinoumi ◽  
...  
Keyword(s):  
Ventricular Tachycardia ◽  
Underlying Mechanism ◽  
Catecholaminergic Polymorphic Ventricular Tachycardia ◽  
Polymorphic Ventricular Tachycardia ◽  
Domain Pair ◽  
Mice Model ◽  
Domain Interaction ◽  
Mutation Site ◽  
Lethal Arrhythmia ◽  
Putative Domain

Mutations in cardiac ryanodine receptor 2 (RyR2) have been shown to be associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). To study the underlying mechanism of this lethal arrhythmia, we developed knock-in (KI:RyR2 S2246L/+ ) mice model with the human CPVT-associated RyR2 mutation (S2246L). The KI mice revealed no structural or histological abnormality in hearts, and also had no contractile or relaxation dysfunction at rest. In all KI mice (n=6), however, bi-directional ventricular tachycardia (VT) was observed after exercise with treadmill (Ex:6/6), but not observed in wild-type (WT) mice (Ex:0/7). In isolated cardiomyocytes, line scan images were obtained to measure local Ca 2+ release events using a confocal microscopy with fluo-4 AM as a Ca 2+ indicator. In the KI cardiomyocytes, the frequency of Ca 2+ sparks (SpF: s −1 ·100μm −1 ) was much more increased in response to 100 nM isoproterenol than in WT cardiomyocytes (KI:4.7±0.5 vs WT:1.9±0.2, p<0.01). Using the canine cardiac SR, we fluorescently labeled RyR2 with methylcoumarin acetamido (MCA), using either DP 2232–2266 or DP 2232–2266 mut; Ser is mutated to Leu (S2246L), as a carrier; DP 2232–2266 harbors the same CPVT mutation site as KI mice (S2246L). The binding affinity of DP 2232–2266 mut to RyR2 (Kd=0.08μM) was higher than that of DP 2232–2266 (Kd=0.32μM), suggesting that abnormally tight interaction of the domain pair (between the domain2232–2266 and another putative domain) may be formed by the S2246L mutation. Interestingly, addition of DP 2232–2266 to the SR dose-dependently inhibited the cAMP (30μM)-induced Ca 2+ leak; IC50=0.1μM, although DP 2232–2266 had no effect on the cAMP-induced increase in 2808Ser phosphorylation. In conclusion, some type of RyR2 mutation in CPVT may causatively induce hyper-activated channel gating by forming abnormally tight domain-domain interaction, triggering diastolic Ca 2+ release and hence lethal arrhythmia. Interruption of such abnormal domain-domain interaction (by competing with native domain) may lead to a new therapeutic strategy against CPVT.

Abstract 4625: Dantrolene Inhibits Spontaneous Ca 2+ Release as a Substrate for Catecholaminergic Polymorphic Ventricular Tachycardia (PSVT) by Correcting Defective Inter-Domain Interaction within the Ryanodine Receptor

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Shigeki Kobayashi ◽  
Masafumi Yano ◽  
Takeshi Suetomi ◽  
Makoto Ono ◽  
Xiaojuan Xu ◽  
...  
Keyword(s):  
Ventricular Tachycardia ◽  
Ryanodine Receptor ◽  
Polymorphic Ventricular Tachycardia ◽  
Mice Model ◽  
Domain Interaction ◽  
Mutation Site ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Lethal Arrhythmia ◽  
Fluorescence Quencher ◽  
A Site

We previously reported that dantrolene, a therapeutic agent for malignant hyperthermia (MH), prevented abnormal Ca 2+ leak by correction of the defective inter-domain interaction between N-terminal (1– 600) and central (2000 –2500) domains in MH-type ryanodine receptor (RyR). These N-terminal and central domains of the RyR, harbor many mutations associated with MH in RyR1 and CPVT in RyR2. Here, we examined the effect of dantrolene on the Ca 2+ release in CPVT-associated RyR2 R2474S/ + knock-in (KI) mice model. In KI (but not in wild-type:WT) mice (n=6), ventricular tachycardia was observed during or after exercise with treadmill (6/6), which was prevented by pretreatment of dantrolene (20 mg −1 kg −1 day −1 , for 7 days). Cardiac sarcoplasmic reticulum (SR) vesicles were isolated (n=4), then RyR2 was fluorescently labeled with methylcoumarin acetamido (MCA) using DP 2460 – 2495 (DPc10), harboring the CPVT mutation site; R2474S, as a site-directing carrier. Only in KI (but not WT) SR, cAMP (1 μM) reduced stabilizing interactions between N-terminal and central domains (viz. domain unzipping), as assessed by the quenching of the MCA fluorescence by a large-size fluorescence quencher. Dantrolene (1 μM) inhibited the cAMP-induced domain unzipping (in KI) without effect on the cAMP-induced increase in Ser2808 phosphorylation that showed no difference between WT and KI. Using a quartz crystal microbalance technique (a highly sensitive mass-measuring technique), dantrolene was found to specifically bind to the domain 601– 620 of RyR2. In isolated, saponin-permeabilized cardiomyocytes, local Ca 2+ release events were measured using a confocal microscopy with Rhod-2; [Ca 2+ ] was buffered at 30 nM by 0.5 mM EGTA. In KI, the frequency of Ca 2+ sparks (SpF: s −1 · 100 μm −1 ) was much more increased in response to 1μM cAMP (9.7±0.4 in WT; 14.5±0.4 in KI, p<0.01). In the co-presence of dantrolene (1 μM), the increase in SpF in KI was markedly inhibited. In conclusion, dantrolene, by normally restoring the defective inter-domain interaction, seems to correct the hyper-activated channel gating caused by RyR2 mutation, thereby inhibiting spontaneous Ca 2+ release leading to lethal arrhythmia.

Abstract 5322: Mutation-Linked, Defective Inter-Domain Interaction within the Cardiac Ryanodine Receptor as a Critical Cause of Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT)

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Hitoshi Uchinoumi ◽  
Masafumi Yano ◽  
Takeshi Suetomi ◽  
Xiaojuan Xu ◽  
Makoto Ono ◽  
...  
Keyword(s):  
Ventricular Tachycardia ◽  
Ryanodine Receptor ◽  
Single Point ◽  
Polymorphic Ventricular Tachycardia ◽  
Single Point Mutation ◽  
Mice Model ◽  
Domain Interaction ◽  
Mutation Site ◽  
Fluorescence Quencher ◽  
Cardiac Ryanodine Receptor

We previously reported that interaction of N-terminal (0–600) and central domains (2000–2500) of the cardiac ryanodine receptor (RyR2), harboring many mutations associated with CPVT, is defective (i.e. domain unzipping) in failing hearts. Here, we examined the pathogenic role of the inter-domain interaction within RyR2 in the mutation-linked channel disorder in human CPVT-associated RyR2 R2474S/+ knock-in (KI) mice model. In all KI mice (6/6), bi-directional ventricular tachycardia was observed during or after exercise with treadmill, but not in wild-type (WT) mice (0/7). Sarcoplasmic reticulum (SR) vesicles were isolated (n=4), then RyR2 was fluorescently labeled with methylcoumarin acetamido (MCA) using DP 2460–2495 (DPc10), harboring the same CPVT mutation site as KI mice; R2474S, as a site-directing carrier. Only in KI (but not in WT) mice, partial domain unzipping was taken place at baseline, and further domain unzipping occurred in response to cAMP (1 μM), as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. In the saponin-permiabilized KI cardiomyocytes; [Ca 2+ ] was buffered at 30 nM by 0.5 mM EGTA, the relationship curve between the frequency of Ca 2+ sparks (SpF: s −1 ·100μm −1 ; by Rhod- 2) and SR Ca 2+ content {Ca 2+ con ; by caffeine application}, obtained by incremental addition of cAMP (0.1–1 μM), was markedly shifted to the left (towards lower SR Ca 2+ content) compared to WT cardiomyocytes, although RyR2 2808Ser phosphorylation is similarly increased in WT and KI. For instance, at 1 μM of [cAMP], Ca 2+ con decreased by 43 % from WT, although SpF was more increased in KI than WT (KI: 19.6±0.5; WT: 14.7±0.4, p<0.01). Interestingly, addition of DPc10 (50 μM), which was found to induce similar domain unzipping (competing with native domain) in WT as KI (+cAMP), to WT cardiomyocytes reproduced the leftward shift of the SpF-Ca 2+ con curve seen in KI. In conclusion, single point mutation at specific region may causatively induce defective inter-domain interaction between N-terminal and central domains in response to PKA phosphorylation, and in turn enhance the sensitivity of the channel to activation by luminal [Ca 2+ ]: i.e. decreased threshold [Ca 2+ ] L to induce spontaneous Ca 2+ sparks, leading to CPVT.

Abstract 5314: Domain 2114–2149 Within RyR2 Plays a Key Role for Correcting Channel Disorder in Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT)

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Takeshi Yamamoto ◽  
Masafumi Yano ◽  
Takeshi Suetomi ◽  
Makoto Ono ◽  
Xiaojuan Xu ◽  
...  
Keyword(s):  
Ventricular Tachycardia ◽  
Polymorphic Ventricular Tachycardia ◽  
Mice Model ◽  
Domain Interaction ◽  
Mutation Site ◽  
Large Size ◽  
Central Regions ◽  
Fluorescence Quencher ◽  
A Site

We recently identified the K201(JTV519)-binding site as domain2114–2149 of the ryanodine receptor (RyR2), and the binding of K201 to this domain corrects the defective inter-domain interaction between N-terminal (0–600) and central regions (2000–2500) of RyR2 in pacing-induced failing hearts. Here, we further investigated the role of the domain peptide2114–2149 (DP 2114–2149 ) on the Ca 2+ release in human CPVT-associated RyR2 R2474S/+ knock-in (KI) mice model. The KI mice revealed no structural or histological abnormality in hearts, and also had no contractile or relaxation dysfunction at rest. In all KI mice (n= 6), however, bi-directional ventricular tachycardia (VT) was observed after exercise with treadmill (Ex:6/6), but not observed in wild-type (WT) mice (Ex:0/7 ). In saponin-permeabilized cardiomyocytes from WT and KI mice, line scan images in cardiomyocytes were obtained to measure local Ca 2+ release events using a confocal microscopy with Rhod-2; [Ca 2+ ] was buffered at 30 nM by 0.5 mM EGTA. In KI, the frequency of Ca 2+ sparks (SpF: s −1 ·100μm −1 ) was much more increased in response to cAMP (0.1–1 μM) than in WT (KI:19.2±0.4 vs WT:14.9±0.4; at [cAMP]= 0.3 μM , p<0.01). DP 2114–2149 abolished the cAMP-induced increase in SpF in KI. Sarcoplasmic reticulum (SR) vesicles were isolated from WT and KI hearts (n=4), then RyR2 was fluorescently labeled with methylcoumarin acetamido (MCA) using DP 2460–2495 (DPc10), which harbors the same mutation site in CPVT as KI mice;R2474S, as a site-directing carrier. Addition of cAMP (1 μM) to the SR from KI (but not WT) mice reduced stabilizing interactions between the N-terminal 1–600 and central domains 2000–2500 (i.e. domain unzipping), as assessed by the quenching of the MCA fluorescence by a large-size fluorescence quencher (Kq: 1.16 in KI: 1.10 in WT). Interestingly, DP 2114–2149 corrected this domain unzipping in KI (Kq:1.13), although DP 2114–2149 had no effect on the cAMP-induced increase in Ser2808 phosphorylation. In conclusion, RyR2 mutation seen in human CPVT may causatively induce hyper-activated channel gating in response to PKA phosphorylation, via defective inter-domain interaction. Correction of the defective inter-domain interaction may be a new therapeutic strategy against CPVT.

scholarly journals A Novel Mice Model of Catecholaminergic Polymorphic Ventricular Tachycardia Generated by CRISPR/Cas9

2021 ◽  
Author(s):  
cuilan hou ◽  
Xunwei jiang ◽  
Qingzhu Qiu ◽  
Junmin Zheng ◽  
Shujia Lin ◽  
...  
Keyword(s):  
Ventricular Tachycardia ◽  
Induced Pluripotent Stem Cell ◽  
Catecholaminergic Polymorphic Ventricular Tachycardia ◽  
Calcium Handling ◽  
Polymorphic Ventricular Tachycardia ◽  
Mice Model ◽  
Isolated Cardiomyocytes ◽  
Therapeutic Targeting ◽  
Protein Levels ◽  
Induced Pluripotent

Catecholaminergic polymorphic ventricular tachycardia (CPVT) has been considered as one of the most important causes of children's sudden cardiac death. Mutations in the genes for RyR2 and CASQ2, two mainly subtypes of CPVT, have been identified. However, the mutation in the gene of TECRL was rarely reported, which could be another genetic cause of CPVT. We evaluated myocardial contractility, electrophysiology, calcium handling in Tecrl knockout (Tecrl KO) mice and human induced pluripotent stem cell-derived cardiomyocytes. Immediately after epinephrine plus caffeine injection, Tecrl KO mice showed much more multiple premature ventricular beats and ventricular tachycardia. The Tecrl KO mice demonstrate CPVT phenotypes. Mechanistically, intracellular calcium amplitude was reduced, while time to baseline of 50 was increased in acute isolated cardiomyocytes. RyR2 protein levels decreased significantly upon cycloheximide treatment in TECRL deficiency cardiomyocytes. Overexpression of TECRL and KN93 can partially reverse cardiomyocytes calcium dysfunction, and this is p-CaMKII/CaMKII dependent. Therefore, a new CPVT mouse model was constructed. We propose a previously unrecognized mechanism of TECRL and provide support for the therapeutic targeting of TECRL in treating CPVT

Abstract 795: Enhanced Sensitivity Of The Cardiac Ryanodine Receptor To Activation By Luminal Ca 2+ As A Primary Cause Of Catecholaminergic Polymorphic Ventricular Tachycardia

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Hitoshi Uchinoumi ◽  
Masafumi Yano ◽  
Makoto Ohno ◽  
Xiaojuan Xu ◽  
Hiroki Tateishi ◽  
...  
Keyword(s):  
Ventricular Tachycardia ◽  
Ryanodine Receptor ◽  
Underlying Mechanism ◽  
Catecholaminergic Polymorphic Ventricular Tachycardia ◽  
Polymorphic Ventricular Tachycardia ◽  
Enhanced Sensitivity ◽  
Histological Abnormality ◽  
Ryr2 Channel ◽  
The Relationship ◽  
Cardiac Ryanodine Receptor

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.

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