Study On Pathogenesis Of Hypertrophic Cardiomyopathy And Cardiac Troponin I Arg145Trp Mutation

Background: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease characterized by ventricular hypertrophy, myofibrillar disarray and myocardial fibrosis. It was the first heritable disorder described in cardiovascular system and the commonest genetic cardiac disease with a prevalence of 0.2% in general population. To date, nearly 300 HCM-causing mutations have been detected, most of which are located in genes encoding sarcomeric proteins, including cardiac troponin I (cTnI) and cardiac troponin T (cTnT) . cTnI, a subunit of cardiac troponin complex combining with actin, plays a pivotal role in the calcium regulation of contractions in cardiac muscle cells. In 1997, Kimura et al reported cTnI mutations were associated with HCM. Since then, 25 mutations in this gene have been detected. cTnT is another component of cardiac troponin complex, which is a Ca²⁺ buffer in cytoplasm and modulates contractions of cardiac myocytes. cTnT mutations first screened in 1994 and more than 20 have been reported now. Although the molecular pathogenesis of HCM has been extensively explored in recent years, key early molecular events still remain elusive and controversial. In the past few years, cardiologists and researchers in China paid more and more attention to hypertophic cardiomyopathy. But most studies focused on its phenotype analysis and mutation screening in one or two genes. No systematic research was executed on the cellular and molecular pathogenesis of HCM in China.Object: To investigate the mutant characterizations of Chinese HCM patients, 71 subjects with a clinical diagnosis of HCM were enrolled in the present study. 7, 8 exons of cTnI and 9, 10, 12 exons of cTnT were screened for mutations using PCR-Sequencing or SSCP analysis. Twomutations of cTnI were determined in two HCM patients: Arg145Trp and Arg162Gln. cTnI Arg145Trp was a novel mutation. To explore the molecular pathogenesis of HCM, we introduced Arg145Trp mutation (Arg146Trp in rat) into rat cardiac troponin I gene with site-directed mutagenesis and constructed recombinant replication-incompetent adenovirus. Adult rat cardiomyocytes, cultured with serum-free medium, were transduced with the recombinant viruses and changes of intracellular Ca²⁺ regulation were determined. Simultaneously, mouse cTnI cDNA were cloned and the same mutation was produced using site-directed mutagenesis technique. With a mouse α-MHC promoter directing, transgenic mice were generated by microinjection.Materials and methods:1. 71 HCM patients were enrolled and genomic DNA was isolated from peripheral blood leukocytes by phenol-chloroform extraction method.2. Whole 7, 8 exons of cTnI were amplified by PCR and the products were sequenced directly.3. PCR products of 9, 10, 12 exons of cTnT were analyzed by SSCP. Sequencing was performed if any abnormal band appeared.4. Arg146Trp mutation was introduced into rat cTnI cDNA by site-directed mutagenesis and Nhe I /BamH I sites were added to its N and C terminals.5. EGFP were ligated to C terminal of cTnI cDNA as a reporter gene. Then recombinant cTnI was subcloned to pShuttle plasmid. After PI-SceI/I-CeuI digestion, the target DNA fragments were cloned to replication-incompetent adenoviral vectors.6. To package and propagate recombinant viruses, HEK293 cells were transfected with Pac I linearized recombinant adenoviral DNA using liposome-based method. Western blotting wasemployed to identify recombinant proteins.7. Adenovirus preparations obtained after lysing infected HEK293 cells were purified by CsCl density gradient.8. Adult rat cardiomyocytes were isolated by Langendoff perfusion and cultured in serum-free medium. After transduced with gene-carrying adenovirus, the cultured cells were observed with fluorescent microscope. In addition, western blotting was used to determine the recombinant proteins.9. Cultured cardiomyocytes transduced with recombinant adenovirus were incubated with Fura-2/AM and intracellular free Ca2+ and caffeine-induced SR Ca2+ release were determined.10. Whole cell patc...