The Mechanisms Of LMNA Mutations Causing Familial Dilated Cardiomyopathy

Mutations in the lamin A/C gene (LMNA) may cause familial dilated cardiomyopathy (DCM) characterized by early onset of atrioventricular block (AVB) before the manifestation of DCM. The molecular mechanisms underlying the disease have not been fully clarified. We suggest a hypothesis that it is due to abnormalities of SCN5A ion channel and gap junctions explored using molecular biology and patch clamping techniques.Part 1 Construction of the Eukaryotic Expression Vector of the LMNA Gene Mutation E82K,R644C,N195KObjective:To construct the eukaryotic expression vector of the HERG gene and express in HEK293 cell. Methods:Amino acid substitution of glutamic for aspartic at position 82,644 and 195(E82K,R644C,N195K)in LMNA was performed by polymerase chain reaction(PCR).Mutagenesis was performed by site-directed mutagenesis PCR. The primers contained the mutant site. The mutant plasmid was confirmed by sequence analysis. Results:The eukaryotic expression vector was constructed correctly. Conclusions:The constrauts was made successfully, it was the foundation for the further functional study. Part 2 Familial dilated cardiomyopathy-related lamin A/C gene mutation E82K reduces sodium current densityObjective:The phenotype of LMNA-related DCM is very similar to the phenotype of SCN5A-related DCM. Therefore, we studied the effects of LMNA mutations on SCN5A channels. Methods and results:The sodium current densities were studied in HEK293 cell lines co-transfected with wild-type (WT) or mutant LMNA (E82K and R644C) and SCN5A cDNA using whole-cell patch-clamp recording techniques. The peak sodium current density was reduced significantly in the cells transfected with LMNA E82K compared to that in cells transfected with WT LMNA (86.19±9.36pA/pF, n=30 vs.155.73±19.09pA/pF, n=47; p=0.001). The reduction of sodium current densities in LMNA E82K mutant cells was due to repressed SCN5A gene translation. The level of sodium channel protein was 0.72±0.14 in LMNA E82K cells relative to control vs.1±0.13 in WT LMNA cells (p<0.05); however, transcription of the SCN5A gene was not altered. Conclusions:Our findings suggest that the LMNA E82K mutation down-regulates sodium channel function by inhibiting SCN5A translation rather than transcription, and provides new insights into the mechanisms of DCM in these patients. The exact mechanism of the effect of the LMNA E82K mutation on SCN5A translation remains to be elucidated.Part 3 Connexin43 Remodeling induced by LMNA gene mutations in family Dilated Cardiomyopathy with atrial ventricular blockObject:Mutations in the lamin A/C gene (LMNA) may cause familial dilated cardiomyopathy (dilated cardiomyopathy) characterized by early onset atrio-ventricular block (A-V block) before the manifestation of dilated cardiomyopathy and high risk of sudden death due to ventricular arrhythmia, which is very similar to the phenotype of gap junction related heart disease. In this part, we studied the effect of LMNA mutations on connexins. Methods:The Cx43 and Cx40 expression in cultured neonatal myocytes transfected with wild-type (WT) or mutant LMNA (E82K and R644C) were studied using confocal imaging and western blot analysis. Results:Cx43 protein expression was reduced by 40% in cells transfected with LMNA E82K than that in cells transfected with WT LMNA cDNA. Confocal imaging showed that the Cx43 located inside of the cells. By contrast, LMNA E82K mutation had no effect on expression and localization of Cx40. LMNA R644C transfection did not show any significant effects on gap junctions at all. Conclusions--Our findings suggest that LMNA E82K significantly reduced the Cx43 expression and altered its localization which may be one of the pathological mechanisms underlie LMNA-related heart disease.