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Updated: Jul 7, 2026
Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes
Published on: May 25, 2022
[Reduced Ca2+ current in rat cardiomyocytes transfected with troponin I R145W mutation gene]
Heng-Fang Wu1, Xiang-Jian Chen, Di Yang
1Institute of Cardiovascular Diseases, Clinical Bio Diagnostic & Bio Therapeutic Laboratory, Department of Cardiology, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
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.
Conclusion:
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.
Insights
The cardiac troponin I R145W mutation, found in hypertrophic cardiomyopathy patients, significantly reduces L-type calcium channel current in heart cells. This finding suggests a potential disease mechanism for this genetic variant.
Area of Science:
- Cardiovascular Research
- Molecular Cardiology
- Genetic Mutations
Context:
- Hypertrophic cardiomyopathy (HCM) is a significant cause of heart failure.
- Genetic mutations in cardiac troponin I (cTnI) are linked to HCM.
- The R145W mutation in cTnI has been identified in Chinese HCM patients.
Purpose:
- To investigate the functional impact of the cTnI R145W mutation on cardiac function.
- To determine the effect of the R145W mutation on calcium (Ca2+) current modulation in cardiomyocytes.
Summary:
- The R146W mutation (human R145W equivalent) was introduced into rat cTnI cDNA using site-directed mutagenesis and delivered to adult rat cardiomyocytes via adenovirus.
- Whole-cell patch clamp recordings revealed a significant decrease in peak L-type Ca2+ channel current in cardiomyocytes expressing the cTnI R146W mutant compared to wild-type.
- Intracellular Ca2+ levels and sarcoplasmic reticulum Ca2+ release remained similar across experimental groups.
Impact:
- The reduced L-type Ca2+ current associated with the cTnI R145W mutation may be a key factor in the pathogenesis of hypertrophic cardiomyopathy.
- This study provides mechanistic insights into how specific cTnI mutations contribute to cardiac dysfunction.
- Findings may inform future diagnostic and therapeutic strategies for HCM patients with this genetic variant.
