Reduced Ca2+ current in rat cardiomyocytes transfected with troponin I R145W mutation gene / 中华心血管病杂志
Chinese Journal of Cardiology
;
(12): 1000-1004, 2007.
Article
in Chinese
| WPRIM
| ID: wpr-299540
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of cardiac troponin I R145W mutation, detected in Chinese patients with hypertrophic cardiomyopathy, on Ca(2+) current modulation.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Cardiomyopathy, Hypertrophic
/
Transfection
/
Cells, Cultured
/
Calcium
/
Mutagenesis, Site-Directed
/
Rats, Sprague-Dawley
/
Patch-Clamp Techniques
/
Troponin I
/
Calcium Channels, L-Type
/
Myocytes, Cardiac
Type of study:
Prognostic study
Limits:
Animals
Language:
Chinese
Journal:
Chinese Journal of Cardiology
Year:
2007
Type:
Article
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