[Reduced Ca2+ current in rat cardiomyocytes transfected with troponin I R145W mutation gene].
OBJECTIVE
To investigate the effects of cardiac troponin I R145W mutation, detected in Chinese patients with hypertrophic cardiomyopathy, on Ca(2+) current modulation.


METHODS
R146W mutation (resemble R145W in human) was introduced into rat cardiac troponin I cDNA by site-directed mutagenesis. With EGFP as a reporter gene, replication-defective adenovirus containing the wild or mutant cTnI gene was constructed. Adult rat cardiomyocytes, were isolated by Langendorff perfusion and cultured with serum-free medium and transduced with the recombinant adenoviruses. Western blot was used to determine the recombinant proteins. Whole cell patch clamp was employed to record L-type Ca(2+) currents on cultured myocytes. Intracellular free Ca(2+) and caffeine-induced sarcoplasmic reticulum (SR) Ca(2+) release were determined after the cells incubated with Fura-2/AM.


RESULTS
DNA sequencing confirmed that R146W mutation was generated in rat cTnI cDNA. Bright green fluorescence was observed in the cultured cardiomyocytes at 48 h after transduction. The recombinant proteins could be identified with cTnI or GFP monoclonal antibody. The peak current of L-type Ca(2+) channel in cells transduced with cTnI R146W was significantly decreased compared to control cells and cells transfected with wild cTnI. Intracellular free Ca(2+) concentrations and caffeine-induced SR Ca(2+) release determined by Fura-2/AM were similar among various cells.


CONCLUSIONS
Reduced peak current of L-type Ca(2+) channel in cells transduced with cTnI R146W might contribute to the disease-causing mechanism of this mutation in patients with hypertrophic cardiomyopathy.
Wu
Heng-Fang
HF
Institute of Cardiovascular Diseases, Clinical Bio Diagnostic & Bio Therapeutic Laboratory, Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
Chen
Xiang-Jian
XJ
Yang
Di
D
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D002478:Cells, Cultured; D005260:Female; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D019210:Troponin I / Q000235:genetics*
D020746:Calcium Channels, L-Type; D019210:Troponin I; D002118:Calcium