PhyMedExp, University of Montpellier, CNRS, INSERM, Montpellier , France.
Department of Physiology and Cellular Biophysics, Clyde and Helen Wu Center for Molecular Cardiology, Columbia University Vagelos College of Physicians and Surgeons, New York, NY, USA.
Stem Cell Res Ther. 2023 Sep 23;14(1):266. doi: 10.1186/s13287-023-03502-5.
Polymorphic ventricular tachycardia (PMVT) is a rare genetic disease associated with structurally normal hearts which in 8% of cases can lead to sudden cardiac death, typically exercise-induced. We previously showed a link between the RyR2-H29D mutation and a clinical phenotype of short-coupled PMVT at rest using patient-specific hiPSC-derived cardiomyocytes (hiPSC-CMs). In the present study, we evaluated the effects of clinical and experimental anti-arrhythmic drugs on the intracellular Ca handling, contractile and molecular properties in PMVT hiPSC-CMs in order to model a personalized medicine approach in vitro.
Previously, a blood sample from a patient carrying the RyR2-H29D mutation was collected and reprogrammed into several clones of RyR2-H29D hiPSCs, and in addition we generated an isogenic control by reverting the RyR2-H29D mutation using CRIPSR/Cas9 technology. Here, we tested 4 drugs with anti-arrhythmic properties: propranolol, verapamil, flecainide, and the Rycal S107. We performed fluorescence confocal microscopy, video-image-based analyses and biochemical analyses to investigate the impact of these drugs on the functional and molecular features of the PMVT RyR2-H29D hiPSC-CMs.
The voltage-dependent Ca channel inhibitor verapamil did not prevent the aberrant release of sarcoplasmic reticulum (SR) Ca in the RyR2-H29D hiPSC-CMs, whereas it was prevented by S107, flecainide or propranolol. Cardiac tissue comprised of RyR2-H29D hiPSC-CMs exhibited aberrant contractile properties that were largely prevented by S107, flecainide and propranolol. These 3 drugs also recovered synchronous contraction in RyR2-H29D cardiac tissue, while verapamil did not. At the biochemical level, S107 was the only drug able to restore calstabin2 binding to RyR2 as observed in the isogenic control.
By testing 4 drugs on patient-specific PMVT hiPSC-CMs, we concluded that S107 and flecainide are the most potent molecules in terms of preventing the abnormal SR Ca release and contractile properties in RyR2-H29D hiPSC-CMs, whereas the effect of propranolol is partial, and verapamil appears ineffective. In contrast with the 3 other drugs, S107 was able to prevent a major post-translational modification of RyR2-H29D mutant channels, the loss of calstabin2 binding to RyR2. Using patient-specific hiPSC and CRISPR/Cas9 technologies, we showed that S107 is the most efficient in vitro candidate for treating the short-coupled PMVT at rest.
多形性室性心动过速（PMVT）是一种与结构正常的心脏相关的罕见遗传性疾病，8%的病例可导致心脏性猝死，通常由运动引起。我们之前使用患者特异性诱导多能干细胞衍生的心肌细胞（hiPSC-CMs）证明了 RyR2-H29D 突变与静息时短联 PMVT 的临床表型之间存在关联。在本研究中，我们评估了临床和实验性抗心律失常药物对 PMVT hiPSC-CMs 细胞内 Ca 处理、收缩和分子特性的影响，以便在体外模拟个性化医疗方法。
此前，我们从携带 RyR2-H29D 突变的患者采集了一份血样，并将其重新编程为多个 RyR2-H29D hiPSC 克隆，此外，我们还使用 CRISPR/Cas9 技术逆转 RyR2-H29D 突变生成了一个同基因对照。在这里，我们测试了 4 种具有抗心律失常特性的药物：普萘洛尔、维拉帕米、氟卡尼和 Rycal S107。我们进行了荧光共焦显微镜、基于视频图像的分析和生化分析，以研究这些药物对 PMVT RyR2-H29D hiPSC-CMs 功能和分子特征的影响。
电压依赖性 Ca 通道抑制剂维拉帕米不能防止 RyR2-H29D hiPSC-CMs 中肌浆网（SR）Ca 的异常释放，但 S107、氟卡尼或普萘洛尔可以防止。由 RyR2-H29D hiPSC-CMs 组成的心肌组织表现出异常的收缩特性，而 S107、氟卡尼和普萘洛尔在很大程度上阻止了这种特性。这 3 种药物还恢复了 RyR2-H29D 心肌组织的同步收缩，而维拉帕米则没有。在生化水平上，S107 是唯一能够恢复钙调蛋白 2 与 RyR2 结合的药物，这在同基因对照中可以观察到。
通过在患者特异性 PMVT hiPSC-CMs 上测试 4 种药物，我们得出结论，S107 和氟卡尼在防止 RyR2-H29D hiPSC-CMs 中异常 SR Ca 释放和收缩特性方面是最有效的分子，而普萘洛尔的作用是部分的，维拉帕米似乎无效。与其他 3 种药物不同，S107 能够防止 RyR2-H29D 突变通道的一种主要翻译后修饰，即钙调蛋白 2 与 RyR2 的结合丧失。使用患者特异性 hiPSC 和 CRISPR/Cas9 技术，我们表明 S107 是治疗静息时短联 PMVT 的最有效的体外候选药物。
