ラミン遺伝子変異キャリアにおける遺伝子型を用いた心疾患リスクの層別化

京都大学新制・論文博士博士(医学)乙第13568号論医博第2294号京都大学大学院医学研究科医学専攻(主査)教授 森田 智視, 教授 石見 拓, 教授 佐藤 俊哉学位規則第4条第2項該当Doctor of Medical ScienceKyoto UniversityDFA

Recurrent episodes of atrioventricular nodal reentrant tachycardia: Sites of ablation success, ablation endpoint, and primary culprits for recurrence

Abstract Background Atrioventricular nodal reentrant tachycardia (AVNRT) sometimes recurs even after anatomical slow pathway (SP) ablation targeting the rightward inferior extension (RIE). This multicenter study aimed to determine the reasons for AVNRT recurrence. Methods and Results Forty‐six patients were treated successfully for recurrent AVNRT. Initial treatment was for 38 slow‐fast AVNRTs, 3 fast‐slow AVNRTs, 2 slow‐slow AVNRTs, 2 slow‐fast and fast‐slow AVNRTs, and 1 noninducible AVNRT. All initial treatments were of RF application to the RIE; SP elimination was achieved in 11, dual AVN physiology was seen in 29, and AVNRT remained inducible in 5. The recurrent AVNRTs included 34 slow‐fast AVNRTs, 6 fast‐slow AVNRTs, 3 slow‐slow AVNRTs, 2 slow‐fast and fast‐slow AVNRTs, and 1 slow‐fast and slow‐slow AVNRTs. Successful ablation site was within the RIE in 39 and left inferior extension in 7. In 30 of 39, the successful RIE site was in the same area or higher than that of the initial procedure. Conclusion For a high majority (around 85%) of patients in whom AVNRT recurs after initial ablation success, the site of a second successful procedure will be within the RIE even though the RIE was originally targeted. Furthermore, a high majority (around 86%) of sites of successful ablation will be higher than those originally targeted

Patient-Specific Human Induced Pluripotent Stem Cell Model Assessed with Electrical Pacing Validates S107 as a Potential Therapeutic Agent for Catecholaminergic Polymorphic Ventricular Tachycardia.

INTRODUCTION:Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling. However, it is not invariably successful to recapitulate the disease phenotype because of the immaturity of hiPSC-derived cardiomyocytes (hiPSC-CMs). The purpose of this study was to establish and analyze iPSC-based model of catecholaminergic polymorphic ventricular tachycardia (CPVT), which is characterized by adrenergically mediated lethal arrhythmias, more precisely using electrical pacing that could promote the development of new pharmacotherapies. METHOD AND RESULTS:We generated hiPSCs from a 37-year-old CPVT patient and differentiated them into cardiomyocytes. Under spontaneous beating conditions, no significant difference was found in the timing irregularity of spontaneous Ca2+ transients between control- and CPVT-hiPSC-CMs. Using Ca2+ imaging at 1 Hz electrical field stimulation, isoproterenol induced an abnormal diastolic Ca2+ increase more frequently in CPVT- than in control-hiPSC-CMs (control 12% vs. CPVT 43%, p<0.05). Action potential recordings of spontaneous beating hiPSC-CMs revealed no significant difference in the frequency of delayed afterdepolarizations (DADs) between control and CPVT cells. After isoproterenol application with pacing at 1 Hz, 87.5% of CPVT-hiPSC-CMs developed DADs, compared to 30% of control-hiPSC-CMs (p<0.05). Pre-incubation with 10 μM S107, which stabilizes the closed state of the ryanodine receptor 2, significantly decreased the percentage of CPVT-hiPSC-CMs presenting DADs to 25% (p<0.05). CONCLUSIONS:We recapitulated the electrophysiological features of CPVT-derived hiPSC-CMs using electrical pacing. The development of DADs in the presence of isoproterenol was significantly suppressed by S107. Our model provides a promising platform to study disease mechanisms and screen drugs

Gene expression of calcium handling proteins in hiPSC-CMs.

<p>Quantitative real-time PCR of spontaneously contracting embryoid bodies differentiated from control- and CPVT-hiPSCs showed comparable expression levels of the studied calcium handling proteins. All values are relative to the adult human heart and were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). PCR = polymerase chain reaction</p

Characterization of CPVT-hiPSCs.

<p>(A) CPVT-hiPSC colonies derived from the dermal fibroblasts of a patient with CPVT expressed pluripotency markers, shown by immunostaining. Scale bars = 200 μm. (B) CPVT-hiPSCs maintained the normal karyotype. (C) Sequencing analysis of the <i>RYR2</i> gene identified the I4587V heterozygous point mutation in the CPVT-hiPSCs. (D) Hematoxylin-eosin staining of teratomas formed from CPVT-hiPSCs showed differentiation of the cells into various tissues derived from all three germ layers: pigmented epithelium (ectoderm), gut-like structures (endoderm), and cartilage tissue (mesoderm). Scale bar = 100 μm.</p

Ryanodine suppressed diastolic Ca²⁺ waves in CPVT-hiPSC-CMs.

<p>(A) Representative tracings of Ca²⁺ imaging in CPVT-hiPSC-CMs with (lower) and without (upper) ryanodine. Note diastolic Ca²⁺ waves (arrows) without ryanodine (upper, right), however, no diastolic Ca²⁺ waves with ryanodine (lower, right). Vertical bars indicate the time points that the CPVT-hiPSC-CMs were stimulated at 0.5 Hz. (B) Fraction (in %) of CPVT-hiPSC-CMs that showed diastolic Ca²⁺ waves with and without ryanodine. *p < 0.05.</p