Knockdown of Telethonin Reduces Contractions and Provokes Aberrant Ca2+-waves in Human iPS Cell-induced
Insights
Knocking down the TCAP gene in human iPSC-derived cardiomyocytes impairs cardiac contraction and causes abnormal calcium waves. This TCAP gene knockdown leads to phenotypes consistent with dilated cardiomyopathy (DCM), a leading cause of heart failure.
Area of Science:
- Cardiovascular Biology
- Genetics
- Stem Cell Biology
Background:
- Dilated cardiomyopathy (DCM) is a primary cause of heart failure, with 48 known associated genes.
- Telethonin, encoded by the TCAP gene, is crucial for cardiomyocyte cytoskeletal structure and signaling.
- Six TCAP variants have been identified in DCM patients.
Purpose of the Study:
- To investigate the functional role of the TCAP gene in cardiac function.
- To analyze the impact of TCAP knockdown on cardiomyocyte contractility and calcium handling.
Main Methods:
- CRISPR-Cas9 gene editing was used to knock down the TCAP gene in human induced pluripotent stem cells (iPSCs).
- TCAP expression was validated using RT-qPCR and Western blot.
- Wild-type and TCAP-knockdown iPSC-derived cardiomyocytes (CMs) were differentiated and analyzed for contractility and calcium dynamics.
Main Results:
- TCAP knockdown significantly reduced cardiomyocyte contractility, including contraction velocity, relaxation velocity, and contraction-relaxation duration.
- Aberrant calcium (Ca2+) waves were observed in TCAP-knockdown CMs.
- TCAP-knockdown CMs exhibited triggered activities and abnormal Ca2+ handling.
Conclusions:
- TCAP knockdown in human iPSC-derived CMs impairs cardiac contractility.
- TCAP deficiency leads to abnormal Ca2+ handling and triggered activities, mimicking DCM phenotypes.
- TCAP plays a critical role in maintaining normal cardiac function.
Objectives:
Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure. To date, 48 genes are known to be associated with DCM. Telethonin, encoded by the TCAP gene, is a Z-disk protein that composes cytoskeletal structures and facilitates various signaling pathways in cardiomyocytes. At least, six TCAP variants have been found in patients with DCM. We sought to investigate the role of TCAP in cardiac function using TCAP-knockdown (KD) iPS cell (iPSC)-induced cardiomyocytes (CMs).
Methods & Results:
To investigate the role of TCAP in cardiomyocytes, the TCAP gene was knocked down in human iPS cells established from a healthy subject (201B7) using the CRISPR-Cas9 genome editing. The expressions of TCAP mRNA and telethonin were confirmed by RT-qPCRs and Western blot, respectively. The 201B7 wild type (WT) and the TCAP-knocked down (KD) cells were differentiated into cardiomyocytes (CMs). The contractility measured by a high-resolution block matching-based optical flow technique showed that all contractility parameters, including contraction velocity, relaxation velocity, and contraction-relaxation duration, were decreased in the KD-CMs (n = 54) compared to the WT-CMs (n = 24) (WT vs. KD: 51.28 ± 22.82 vs. 29.11 ± 22.83 µm/s, p < 0.001; 22.05 ± 8.85 vs. 12.48 ± 8.85 µm/s, p < 0.001; 0.82 ± 0.12 vs. 0.58 ± 0.12 s, p < 0.001). Ca2+-imaging studies showed aberrant Ca2+-waves in the KD-CMs.
Conclusions:
We found that TCAP-KD in human iPS cell-induced CMs impairs contraction, induces triggered activities, and abnormal Ca2+-waves, which is consistent with the phenotypes of DCM.
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