Studies On The Mechanism And Establishment Of Cybrids Cell Lines Of Hypertrophic Cardiomyopathy With M.2336T>C Mutation

Hypertrophic cardiomyopathy (HCM), a common disorder of myocardium with the prevalence of 0.2% in the general population, is usually be recognized as autosomal dominant inherited. While some cases charactered as maternal inheritance noting the mtDNA mutation related mitochondrial dysfunction as one of the mechanisms of hypertrophic cardiomyopathy. m.2336T>C is a novel mutation associated with HCM, which our laboratory reported before. With the breaking of a highly conserved 2336U-A2438 base-pairing of 16S rRNA, m.2336T>C significantly impact the gene and function expression of mitochodria in patient-specific lymphoblastoid cell lines, based on which we established the patient-specific transferring mitochondria cell lines (cybrids) to detect the mitochontrial function and mechanism of HCM futher with a unit nuclear genome.The result show that, after fused with mitochondria-absence p0 cells, the copy number of cybrids returned to a normal level, with all patient-specific cybrids carried m.2336T>C. Detection of the cell doubiling time show the mutant cybrids growed much slower than wild-type cybrids, suggesting that m.2336T>C might weaken the viability of cybrids. Then, we detected the transcription level of mitochondrial 16S rRNA as well as its binding protein MRPL19 and MRPL23, and found a significant drop in mutant cybrids since the m.2336T>C is located on 16S rRNA gene, noting the m.2336T>C might impact assembly and expression of mitochondrial ribinson. Meanwhile, transcription level of oxidation phosphorylation (OXPHOS) complexes subunits of mutant cybrids, no matter mtDNA decided (CⅠ-ND5, CⅢ-CYTB, CⅤ-ATP8) or nDNA decided (CⅠ-DNUFA9, CⅡ-SDHA, CⅢ-UQCRC2, CⅣ-COⅩⅣ, CV-ATP5), showed much lower than wild-type cells. Besides of the transcription level, translation level of mitochondrial OXPHOS complexes subunits in mutant and wild-type cybrids also showed a distinguished differences, which include mtDNA encoded subunits CⅣ-MTCO2, CV-ATP8 and the nDNA encoded subunits CⅠ-DNUFA9, CⅡ-SDHA, CⅢ-UQCRC2, CⅤ-ATP5. Differences between mutant and wild-type cybrids in transcription and translation level of OXPHOS complexes subunits suggested the abnormal of OXPHOS complexes with influence of m.2336T>C, and is probably lead to abnormal of mitochondrial function. So, the functional detction of mitochondria is studied, and a decrease in total and mitochondrial ATP synthesis accompanied with an increase in mitochondrial ROS synthesis of mutant cybrids were found, which confirmed the hypothesis above.Therefore, m.2336T>C decreased the expression level of mitochondrial ribosomal RNA and its binding proteins in mutant cybrids, indicating that the mutation might impact assembly of mitochondrial riboson and then resulted in the abnormal of translation and functional expression of mitochondria. Insufficient energy supply and oxidative damage might be responsible for the occurrence of hypertrophic cardiomyopathy. In conclusion, our study demonstrated the relationship definitly between m.2336T>C and hypertrophic cardiomyopathy, providing a new insight in mechanism study and treatment of hypertrophic cardiomyopathy.