Dilated Cardiomyopathy Disease Genes Lmna Mutation Function And Cardiac Specific The Gene Tnni3k Regulation Mechanism

Dilated cardiomyopathy (DCM) is a form of heart muscle disease characterized by impaired systolic function and ventricular dilation of the left, or both ventricles, representing the most common heart-failure eventually requiring heart transplantation. This condition is also associated with a high rate of sudden death due to ventricular arrhythmias and a high mortality rate of 15% to 50% at 5 years. The prevalence of DCM in the US population, using diagnostic criteria for advanced disease, was estimated to be 36.5 per 100,000 individuals. The aetiology and the pathogenetic mechanisms are still largely unknown in approximately half of the patients. Several prospective studies have clearly demonstrated the existence of genetic transmission of the disease detectable in at least 25% of DCM patients.Familial DCM is suggested by different patterns of inheritance, with autosomal trait prevailing, and variable clinical features. Clinical and molecular genetic studies have resulted in the identification of 26 candidate disease loci, and 22 genes responsible for familial DCM. One of genes is the lamin A/C gene, that encodes for major structural components of the lamina network that underlies and supports the nuclear envelope. Mutations in lamin A/C have been associated with 10 different inherited diseases including DCM, Emery-Dreifuss muscular dystrophy (EDMD) and Hutchinson-Gilford progeria syndrome (HGPS). A new term, laminopathies has been coined to describe the growing number of disorders caused by mutations in nuclear lamins and lamin-binding proteins.We developed a study to screen for genetic mutations in a large Chinese DCM family including 76 members using PCR to amplify the 12 exons of the lamin A/C gene and products sequencing analysis. We found a Glu82Lys substitution mutation located in the rod domain of the lamin A/C protein in 8 family members, of these, 3 members have been diagnosed as DCM definitely, 1 member presented with dilated heart and their age were older than 30 year-old, the rest 4 members no clinical phenotype carried this mutation were younger than 30 year-old. To date, the pathogenic mechanism of lamin A/C gene defect is poorly understood. We expressed Glu82Lys mutated lamin A/C protein in HEK293 cells and found the mutated protein couldn't localized normally at the inner nuclear membrane as well as led the emerin protein who interacted with lamin A/C protein aberrantly distributed. We also observed the nuclear membrane structure was disrupted and heterochromatin aggregated aberrantly in the nucleus of the HEK293 cells stable transfected mutated lamin A/C gene by transmission electron microscope. Hochest33342 staining indicated that Glu82Lys mutation could dramatically increase apoptosis of HEK293 cell after being treated with H₂O₂. The heart is the first organ to form and function in the embryo. Many reports indicate that cardiac specific genes play pivotal roles in maintaining functions of embryo and adult heart.The cardiac ankyrin repeat kinase (CARK) gene, also named TNNI3K for its interaction with cardiac troponin I, is a both unique expression and heart-enriched gene. Previous reports indicate that CARK protein is a functional kinase and belongs to the MAPKKKs superfamily. To understand the mechanisms of CARK gene expression and regulation, we first cloned the full-length mRNA sequence and mapped the transcription start site of the mouse CARK gene and characterized its promoter regions. Two transcriptional isoforms of the CARK were identified in mouse heart tissue, both are highly identical to human and rat CARK genes. Truncation analysis of the CARK promoter identified a minimal 151bp region that has strong basal promoter activity.To map cis-acting elements that were required for transcriptional activity of the CARK minimal promoter region (from -151 to -1), we performed a multiple alignment of this sequence of mouse with human, chimpanzee, dog and rat. By searching all the conserved sequences in this region for cardiac transcription factor binding sites in the CardioSignal database, five conserved cardiac TFBSs including Tbx5, SRE, M-CAT box, MEF2 and GATA4 were identified.Mutational and loss-of-functional analysis and co-transfection studies indicated that MEF2 is the most critical cis-acting element that regulates CARK promoter activity. Binding to the MEF2 sites by mef2c protein was confirmed by electrophoretic mobility shift assay and competition and supershift electrophoretic mobility shift assays.