UC San Diego

Cardiac mechanotransduction is the process by which mechanical signals are transduced into biochemical signals in cardiac myocytes. It is important for moderation of changes in sarcomeric protein assembly, hypertrophic markers, and cell survival. Within a single cardiac myocyte, costameres are complex multi-protein structures which have been shown to be the site where forces generated by cardiac myocytes are transmitted to the external extracellular matrix (ECM) as well as the site where forces externally applied by the ECM are transmitted into the cardiac myocyte. Defects in vinculin and its isoforms, metavinculin, have been implicated in the development of dilated and hypertrophic cardiomyopathy. Due to vinculin's role in contributing to the development of cardiomyopathy and its position in forming a bridge between the ECM and the cytoskeleton, it is hypothesized that vinculin is required for normal mechanotransduction. To study vinculin's role in mechanotransduction, a method using laser tweezers and multifocal multiphoton fluorescence resonance energy transfer (FRET) microscopy was developed. This method allowed the application of calibrated localized forces to cardiomyocytes and the measurement of the cell's response to the force. Results demonstrated that this system can be used to study cardiac mechanotransduction, and that focal adhesion kinase autophosphorylation (FAK-P) as reported by a FRET biosensor changed locally in the cell, but not distally from the site of force application. Additionally, in wildtype cells, FAK-P responses were dependent on cell geometry. These results reveal that types of load depending on orientation of a cell may trigger regulation of hypertrophy. Lastly, using this system, it was determined that anisotropic responses to mechanotransduction as reported by the FAK FRET biosensor is mediated by vinculin. These results reveal the role of the microanatomy of the costamere in determining mechanotransduction events and alterations in hypertrophic signaling. In conclusion, an optically based novel method for studying cardiac mechanotransduction has been developed and it has been used to determine that abnormal vinculin levels alter the biochemical signaling of FAK in response to directionally applied forces. These alterations may contribute to the development of cardiomyopathy and possibly in the development of heart failure