Studies On The Molecular Pathogensis And Generation Of HCM-specific Induced Pluripotent Stem Cells With M.2336T>C Mutation

Hypertrophic cardiomyopathy (HCM) is a primary disorder characterized by asymmetric thickening of the septum and left ventricular wall, with a prevalence of 0.2% in the general population. And it is the main cause of sudden cardiac death in adolescents and athletes. Familial HCM is inherited mainly as an autosomal dominant trait and attributed to mutations of nuclear sarcomeric genes. The mutation in mitochondrial DNA (mtDNA) is also one of the molecular bases for this disorder. We previously reported a maternally inherited HCM pedigree with m.2336T>C mutation. This mutation disturbs the 2336U-A2438 base pair in the stem-loop structure of 16S rRNA domain III which is involved in the assembly of mitochondrial ribosome. Functional assays showed that a reduction in mitochondrial oxygen consumption rate and ATP synthesis, and an increase in reactive oxidative species (ROS) production in the mutant cells. However, the molecular pathogenesis of HCM associated with m.2336T>C mutation remains poorly understood due to the lack of cell and animal models. Since no high-performance plasmid systems can be used to make particular mtDNA mutation in the double membrane-bound mitochondria. Recently, patient-specific induced pluripotent stem cells (iPS cells or iPSCs) are attracting increasing attention to establish the disease cell models. Here we reported to generate the HCM-specific iPS cells and differentiated cardiomyocytes, and investigate the molecular pathogenesis of HCM with m.2336T>C mutation. The main results are summarized as following:The urine cells derived from HCM patients with m.2336T>C mutation were reprogrammed to the embryonic stem (ES)-like cells. The biological characterization of these cells were identified such as alkaline phosphatase, immunofluorescence, real-time PCR and semi-quantitative PCR, bisulfite genomic sequencing analyses, and G-banding karyotype analysis. These cells exhibit the morphology and growth properties of ES cells and express ES cell marker genes. The induction of embryoid bodies in vitro and formation of teratoma in vivo showed that HCM-iPS cells can result in tumors containing a variety of tissues from all three germ layers. Mitochondrial genome PCR-sequencing revealed that the cells still maintained the m.2336T>C mutation and no other new mutations arised compared to parental urine cells. These data indicated that we successfully generated HCM-specific iPS cell lines with m.2336T>C mutation.HCM-specific cardiomyocytes (HCM-iPS-CM) differentiated from HCM-iPS cells with m.2336T>C mutation by using single iPS cell differentiation method. These HCM-iPS-CM expressed the cardiomyocyte specific marker proteins. Meanwhile, some aspects of the HCM-specific cellular and electrophysiological properties were identified, which included the increased cellular size and mitochondrial volume, the delayed after depolarizations (DAD) and elongated action potential duration (APD), the beating irregularly. These data indicated that we successfully established a HCM-specific cardiomyocyte cell model.We investigated the role of the m.2336T>C mutation in the pathogenesis of HCM based on mitochondrial functions. The results showed that mtDNA copy number was increased in HCM-iPS-CM. The transcription level of 16S rRNA and binding protein of mitochondrial large ribosomal subunit, including MRPL19 and MRPL23, were decreased significantly. Meanwhile, the transcription and translation level of subunits of oxidative phosphorylation complex encoded by mitochondrial DNA were also decreased. The reduction of the ratio of ADP/ATP which means ATP synthesis efficiency decreased, indicated that mitochondrial function deficiency. Elevated intracellular [Ca2+]i and defect of L-type calcium influxand in HCM-iPS-CM resulted in delayed after depolarizations (DAD) and elongated action potential duration (APD) caused cardiac arrhythmias, which is a serious complication in HCM patients.