Browsing by Subject "cyclic mechanical stretching"

Congenital heart diseases develop during heart development and encompass structural abnormalities in the heart present at birth, with hypoplastic left heart syndrome (HLHS) representing a rare but life-threatening subtype. HLHS is characterised by the underdevelopment of left-sided heart structures, resulting in a major blood flow obstruction of the heart, impairing systemic circulation. Current knowledge of HLHS aetiology is scarce, which makes the development of effective treatments challenging. Therefore, identifying the disease mechanisms causing HLHS is essential. Notably, HLHS is linked with mutations in the NKX2-5 gene, which encodes for a cardiac transcription factor and has a pivotal role in heart development together with the transcription factor GATA4. This makes these genes intriguing research targets in HLHS. This study aims to enlighten how HLHS patient-derived human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) differ from those derived from healthy donors in terms of stress response by subjecting hiPSC-CMs to pro-hypertrophic stimuli, namely endothelin-1 (ET-1) and cyclic mechanical stretching. Additionally, the effects of GATA4-targeted compounds on these hypertrophy models were also studied, which included two inhibitors (3i-1262 and 3i-1000) and one activator (3i-0777) of GATA4-NKX2-5 interaction. Differentiation of CMs was performed using a small-molecule induction protocol based on sequential Wnt pathway activation and inhibition. The effects of ET-1 and cyclic mechanical stretching were analysed by High-content analysis for pro-B-type natriuretic peptide (proBNP) expression, and quantitative PCR for hypertrophic gene expression, respectively. Both ET-1 and cyclic mechanical stretching effectively induced hypertrophy in their respective models. This was observed in all cell lines as a higher hypertrophic response of proBNP in ET-1 exposed hiPSC-CMs and upregulation of hypertrophic genes NPPA and NPPB in stretched hiPSC-CMs. GATA4-targeted compounds did not show statistically significant effects on ET-1-induced hypertrophy or stretching-induced hypertrophic gene expression in any cell line, but various trends could be distinguished. As expected, both inhibitor compounds, 3i-1262 and 3i-1000, showed a tendency for antihypertrophic effects since they decreased the percentage of proBNP+ cells in all cell lines. Unexpectedly, the activator compound 3i-0777 also decreased the percentage of proBNP+ cells. We also observed that HLHS-disease cell line HEL 149 seemed to differ from the three other cell lines showing a phenotype that exhibits similar gene expression patterns as seen in heart failure patients. This was mainly observed as a statistically significantly lower basal MYH6 gene expression. However, the limited experimental setup of this study requires further experiments to detect significant differences and draw definitive conclusions regarding the effects of GATA4-targeted compounds on hypertrophic stimuli.