Substrate and mechanotransduction influence SERCA2a localization in human pluripotent stem cell-derived...

Area of Science:

• Cellular and Molecular Biology
• Biomedical Engineering
• Stem Cell Research

Background:

• Physical cues significantly influence cellular phenotype, impacting cell structure and function.
• In vitro cellular models, including human pluripotent stem cell (hPSC)-derivatives, are crucial for disease modeling and drug discovery.
• Optimizing culture systems is essential for developing accurate cellular models and understanding cellular organization.

Purpose of the Study:

• To investigate the role of mechanotransduction pathways in the structural reorganization of hPSC-derived cardiomyocytes.
• To understand how substrate mechanics affect cardiomyocyte structure, calcium handling, and SERCA2 pump localization.
• To identify specific signaling pathways involved in substrate-mediated cellular changes in hPSC-cardiomyocytes.

Main Methods:

• Studied hPSC-derived cardiomyocytes cultured on hydrogels of varying stiffness (4kPa and 16kPa) and glass.
• Investigated the involvement of the RhoA/ROCK pathway using pharmacological inhibitors.
• Analyzed structural reorganization, sarcomeric alignment, and SERCA2 pump localization via microscopy and functional assays.

Main Results:

• Cell adhesion to the culture substrate triggers structural reorganization in hPSC-derived cardiomyocytes, mediated by the RhoA/ROCK pathway.
• Physiological substrate stiffness (16kPa) promotes sarcomeric alignment and SERCA2a pump relocalization, enhancing calcium cycling.
• Inhibition of Rho-associated protein kinase (ROCK) affects SERCA2 localization and calcium handling, confirming its critical role.

Conclusions:

• The mechanotransduction pathway RhoA/ROCK is essential for substrate-induced structural reorganization and functional improvements in hPSC-derived cardiomyocytes.
• Substrate mechanical properties directly influence cardiomyocyte structure, SERCA2 localization, and calcium handling capabilities in vitro.
• This research provides insights for optimizing hPSC-based cellular models for disease research and drug screening.