Cardiomyocytes generated from CPVTD307H patients are arrhythmogenic in response to β-adrenergic stimulation

Sudden cardiac death caused by ventricular arrhythmias is a disastrous event, especially when it occurs in young individuals. Among the five major arrhythmogenic disorders occurring in the absence of a structural heart disease is catecholaminergic polymorphic ventricular tachycardia (CPVT), which is a highly lethal form of inherited arrhythmias. Our study focuses on the autosomal recessive form of the disease caused by the missense mutation D307H in the cardiac calsequestrin gene, CASQ2. Because CASQ2 is a key player in excitation contraction coupling, the derangements in intracellular Ca(2+) handling may cause delayed afterdepolarizations (DADs), which constitute the mechanism underlying CPVT. To investigate catecholamine-induced arrhythmias in the CASQ2 mutated cells, we generated for the first time CPVT-derived induced pluripotent stem cells (iPSCs) by reprogramming fibroblasts from skin biopsies of two patients, and demonstrated that the iPSCs carry the CASQ2 mutation. Next, iPSCs were differentiated to cardiomyocytes (iPSCs-CMs), which expressed the mutant CASQ2 protein. The major findings were that the β-adrenergic agonist isoproterenol caused in CPVT iPSCs-CMs (but not in the control cardiomyocytes) DADs, oscillatory arrhythmic prepotentials, after-contractions and diastolic [Ca(2+) ](i) rise. Electron microscopy analysis revealed that compared with control iPSCs-CMs, CPVT iPSCs-CMs displayed a more immature phenotype with less organized myofibrils, enlarged sarcoplasmic reticulum cisternae and reduced number of caveolae. In summary, our results demonstrate that the patient-specific mutated cardiomyocytes can be used to study the electrophysiological mechanisms underlying CPVT.     

CaMKII inhibition rectifies arrhythmic phenotype in a patient-specific model of catecholaminergic polymorphic ventricular tachycardia

Induced pluripotent stem cells (iPSC) offer a unique opportunity for developmental studies, disease modeling and regenerative medicine approaches in humans. The aim of our study was to create an in vitro ‘patient-specific cell-based system'' that could facilitate the screening of new therapeutic molecules for the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited form of fatal arrhythmia. Here, we report the development of a cardiac model of CPVT through the generation of iPSC from a CPVT patient carrying a heterozygous mutation in the cardiac ryanodine receptor gene (RyR2) and their subsequent differentiation into cardiomyocytes (CMs). Whole-cell patch-clamp and intracellular electrical recordings of spontaneously beating cells revealed the presence of delayed afterdepolarizations (DADs) in CPVT-CMs, both in resting conditions and after β-adrenergic stimulation, resembling the cardiac phenotype of the patients. Furthermore, treatment with KN-93 (2-[N-(2-hydroxyethyl)]-N-(4methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), an antiarrhythmic drug that inhibits Ca²⁺/calmodulin-dependent serine–threonine protein kinase II (CaMKII), drastically reduced the presence of DADs in CVPT-CMs, rescuing the arrhythmic phenotype induced by catecholaminergic stress. In addition, intracellular calcium transient measurements on 3D beating clusters by fast resolution optical mapping showed that CPVT clusters developed multiple calcium transients, whereas in the wild-type clusters, only single initiations were detected. Such instability is aggravated in the presence of isoproterenol and is attenuated by KN-93. As seen in our RyR2 knock-in CPVT mice, the antiarrhythmic effect of KN-93 is confirmed in these human iPSC-derived cardiac cells, supporting the role of this in vitro system for drug screening and optimization of clinical treatment strategies.     

Depressed β-adrenergic inotropic responsiveness and intracellular calcium handling abnormalities in Duchenne Muscular Dystrophy patients’ induced pluripotent stem cell–derived cardiomyocytes

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an X-linked disease affecting male and rarely adult heterozygous females, resulting in death by the late 20s to early 30s. Previous studies reported depressed left ventricular function in DMD patients which may result from deranged intracellular Ca²⁺-handling. To decipher the mechanism(s) underlying the depressed LV function, we tested the hypothesis that iPSC-CMs generated from DMD patients feature blunted positive inotropic response to β-adrenergic stimulation. To test the hypothesis, [Ca²⁺]_i transients and contractions were recorded from healthy and DMD-CMs. While in healthy CMs (HC) isoproterenol caused a prominent positive inotropic effect, DMD-CMs displayed a blunted inotropic response. Next, we tested the functionality of the sarcoplasmic reticulum (SR) by measuring caffeine-induced Ca²⁺ release. In contrast to HC, DMD-CMs exhibited reduced caffeine-induced Ca²⁺ signal amplitude and recovery time. In support of the depleted SR Ca²⁺ stores hypothesis, in DMD-CMs the negative inotropic effects of ryanodine and cyclopiazonic acid were smaller than in HC. RNA-seq analyses demonstrated that in DMD CMs the RNA-expression levels of specific subunits of the L-type calcium channel, the β1-adrenergic receptor (ADRβ1) and adenylate cyclase were down-regulated by 3.5-, 2.8- and 3-fold, respectively, which collectively contribute to the depressed β-adrenergic responsiveness.     

胚胎干细胞及诱导多能干细胞向心肌细胞分化和调控的研究进展

胚胎干细胞（embryonic stem cells,ESCs）具有自我更新、无限增殖和多向分化的特性,包括分化成心脏组织的多种类型细胞。经体细胞重编程产生的诱导多能干细胞（induced pluripotent stem cells,iPS）也被证明有类似胚胎干细胞的特性。但这些多能干细胞向心肌细胞自发分化的效率非常低,因此,如何有效地诱导这些多能干细胞向心肌细胞的定向分化对深入认识心肌发生发育的关键调控机制和实现其在药物发现和再生医学,如心肌梗塞、心力衰竭的细胞治疗以及心肌组织工程中的应用均具有非常重要的意义。该文重点综述了近年来胚胎干细胞及诱导多能干细胞向心肌细胞分化和调控的研究进展,并探讨了这一研究领域亟待解决的关键问题和这些多能干细胞的应用前景。     

猪多能干细胞的研究进展

多能干细胞,如胚胎干细胞（embryonic stem cells,ESCs）、诱导多能干细胞（induced pluripotent stem cells,iPSCs）和成体干细胞（adultstemcells,ASCs）,是一类具有巨大潜能的独特细胞。猪作为试验材料,在遗传、代谢、生理生化及基因序列等方面较小鼠更接近于人类,正逐渐成为人类异种移植和再生医学研究的理想生物学模型。然而,目前对猪多能干细胞种类、来源、特征及机制的有限认识直接阻碍了其相关应用。该文将分别对猪ASCs的研究现状、猪类ESCs的分离培养、猪iPSCs的研究进展、多能干细胞间的联系和展望进行论述,以期为从事该领域研究的科研人员提供参考。     

诱导多能干细胞研究进展

人类诱导多能干细胞（induced pluripotent stem cells,iPS细胞）的建立被公认为目前最重要的科技进展之一。iPS细胞在动物疾病模型上的成功治疗,病患特异性iPS细胞的研究及iPS细胞的定向分化研究将有可能使人们避开治疗性克隆的伦理和技术障碍,给人类疾病的干细胞治疗带来光明的前景。本文从iPS细胞的诱导策略和方法,来源细胞及筛选、重编程机制的研究现状、应用前景以及研究中存在的问题等方面对其作一综述和讨论。     

Stem cell therapy: an exercise in patience and prudence

In recent times, the epigenetic study of pluripotency based on cellular reprogramming techniques led to the creation of induced pluripotent stem cells. It has come to represent the forefront of a new wave of alternative therapeutic approaches in the field of stem cell therapy. Progress in drug development has saved countless lives, but there are numerous intractable diseases where curative treatment cannot be achieved through pharmacological intervention alone. Consequently, there has been an unfortunate rise in incidences of organ failures, degenerative disorders and cancers, hence novel therapeutic interventions are required. Stem cells have unique self-renewal and multilineage differentiation capabilities that could be harnessed for therapeutic purposes. Although a number of mature differentiated cells have been characterized in vitro, few have been demonstrated to function in a physiologically relevant context. Despite fervent levels of enthusiasm in the field, the reality is that other than the employment of haematopoietic stem cells, many other therapies have yet to be thoroughly proven for their therapeutic benefit and safety in application. This review shall focus on a discussion regarding the current status of stem cell therapy, the issues surrounding it and its future prospects with a general background on the regulatory networks underlying pluripotency.     

诱导型细胞的研究进展

胚胎干细胞(ES细胞)和诱导型多能干细胞(iPS细胞)的研究进展为生物学基础研究注入了新的活力,然而免疫排斥、致瘤性以及诱导效率低等缺陷制约其进一步快速发展和临床应用．最近,科学家借鉴iPS细胞诱导技术和传统的诱导体系,将终末分化细胞直接诱导为功能性细胞,如心肌细胞、神经细胞和肝脏细胞,称为诱导型细胞．这些研究进展极大地促进了细胞分化、重编程和表观遗传学的研究,也为人类再生医学的研究提供了新的途径．     

Expression of membrane-bound carbonic anhydrases IV, IX, and XIV in the mouse heart.

Expression of membrane-bound carbonic anhydrases (CAs) of CA IV, CA IX, CA XII, and CA XIV has been investigated in the mouse heart. Western blots using microsomal membranes of wild-type hearts demonstrate a 39-, 43-, and 54-kDa band representing CA IV, CA IX, and CA XIV, respectively, but CA XII could not be detected. Expression of CA IX in the CA IV/CA XIV knockout animals was further confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Cardiac cells were immunostained using anti-CA/FITC and anti-alpha-actinin/TRITC, as well as anti-CA/FITC and anti-SERCA2/TRITC. Subcellular CA localization was investigated by confocal laser scanning microscopy. CA localization in the sarcolemmal (SL) membrane was examined by double immunostaining using anti-CA/FITC and anti-MCT-1/TRITC. CAs showed a distinct distribution pattern in the sarcoplasmic reticulum (SR) membrane. CA XIV is predominantly localized in the longitudinal SR, whereas CA IX is mainly expressed in the terminal SR/t-tubular region. CA IV is present in both SR regions, whereas CA XII is not found in the SR. In the SL membrane, only CA IV and CA XIV are present. We conclude that CA IV and CA XIV are associated with the SR as well as with the SL membrane, CA IX is located in the terminal SR/t-tubular region, and CA XII is not present in the mouse heart. Therefore, the unique subcellular localization of CA IX and CA XIV in cardiac myocytes suggests different functions of both enzymes in excitation-contraction coupling.     

人胚胎干细胞和诱导型人多能干细胞分化的心肌细胞在药物心脏毒性筛选中的应用

心脏毒性是药物研发失败的主要原因之一,也是临床前安全评价研究的难题之一。人胚胎干细胞和诱导型人多能干细胞均具有无限增殖、自我更新和多向分化的特性,为体外心脏毒性筛选实验提供了细胞资源。人胚胎干细胞和诱导型人多能干细胞诱导分化的心肌细胞相似,具有相同的形态结构,且随着培养时间的推移,功能性心、Na^＋、Ca^2＋通道密度逐渐增加、心肌特异性基因ANF、α—MHC、MLC-2α的表达量增加,具有相似的动作电位时程和收缩性等特点,相当于幼稚型心肌细胞。将它们应用于已知作用药物的心脏毒性筛选,检测心肌细胞离子通道、动作电位、心脏损伤标志物、收缩功能的变化,获得与临床相似的结果。因此,建立人胚胎干细胞和诱导型人多能干细胞诱导分化心肌细胞的体外评价模型,大大减少了药物研发的时间和成本,克服了种属间的差异,推动了心脏毒性体外评价方法的发展。