Danon病(DD)是由编码溶酶体相关膜蛋白2型(LAMP2)的基因突变引起的,导致自噬受损,糖原积累,和心脏肥大.然而,目前尚不清楚为什么大部分DD患者会出现心律失常和心源性猝死.在目前的研究中,我们产生了LAMP2敲除(KO)人iPSC衍生的心肌细胞(CM),模拟DD心脏的LAMP2功能障碍。形态学分析证实了LAMP2KOCMs的肌节排列。在功能研究中,LAMP2KOCM在基础水平上显示出接近正常的钙处理。然而,前成熟培养基(MM)的治疗夸大了KO细胞的疾病表型,因为它们表现出受损的钙再循环和增加的不规则搏动事件,概括了DD患者的心律失常表型。进一步的机制研究证实,MM治疗显着增强了LAMP2KOCMs的自噬应激,伴随着细胞和线粒体活性氧(ROS)水平的增加。LAMP2KOCM中过量的ROS积累导致钙/钙调蛋白依赖性蛋白激酶IIδ(CaMKIIδ)的过度激活和心律失常发生,通过ROS清除剂的治疗部分挽救了。总之,我们的研究揭示了ROS诱导的CaMKIIδ过度激活是促进DD患者心律失常的关键机制。
Danon disease (DD) is caused by mutations of the gene encoding lysosomal-associated membrane protein type 2 (LAMP2), which lead to impaired autophagy, glycogen accumulation, and cardiac hypertrophy. However, it is not well understood why a large portion of DD patients develop arrhythmia and sudden cardiac death. In the current study, we generated LAMP2 knockout (KO) human iPSC-derived cardiomyocytes (CM), which mimic the LAMP2 dysfunction in DD heart. Morphologic analysis demonstrated the sarcomere disarrangement in LAMP2 KO CMs. In functional studies, LAMP2 KO CMs showed near-normal calcium handling at base level. However, treatment of pro-maturation medium (MM) exaggerated the disease phenotype in the KO cells as they exhibited impaired calcium recycling and increased irregular beating events, which recapitulates the pro-arrhythmia phenotypes of DD patients. Further mechanistic study confirmed that MM treatment significantly enhanced the autophagic stress in the LAMP2 KO CMs, which was accompanied by an increase of both cellular and mitochondrial reactive oxygen species (ROS) levels. Excess ROS accumulation in LAMP2 KO CMs resulted in the over-activation of calcium/calmodulin dependent protein kinase IIδ (CaMKIIδ) and arrhythmogenesis, which was partially rescued by the treatment of ROS scavenger. In summary, our study has revealed ROS induced CaMKIIδ overactivation as a key mechanism that promotes cardiac arrhythmia in DD patients.