Wednesday, August 10, 2022
8/10/2022
PROJECT SUMMARY Cardiomyocytes sense macro scale mechanical cues to adapt their structure and function, and disturbances of these mechanical signals can cause a negative feedback loop that leads to changes in cardiomyocyte structure and ultimately decreased cardiac output. Important to this mechanical sensing are focal adhesions, the mechanosensitive protein complexes that attach the cell cytoskeleton to the underlying extracellular matrix (ECM). Although focal adhesions have been shown to be necessary for myofilament maturation and are sensitive to external substrate characteristics, little is known about the specific forces sensed at these complexes during the physiological contraction cycle or how this adhesive tension is regulated by cardiomyocyte contractility. Furthermore, alterations of cardiomyocyte contractile tension initiate maladaptive cell remodeling, but this mechanism and the involvement of focal adhesions are poorly understood. Given that focal adhesions of other cell types have been shown to distribute focal adhesion tension unevenly within individual cells and microdomains and that there are regional heterogeneities in cardiomyocyte strain, it is important to understand the spatial distribution of mechanosensing in cardiomyocytes as the myofibrils contract against the ECM. This proposal aims to fill the crucial gap in knowledge of the role of focal adhesions and their interactions with both the myofibril structures and the ECM for the mechanical homeostasis of contracting cardiomyocytes. Previous studies of focal adhesion sensation during contraction have been limited by the inability to measure exact force across the focal adhesions in a time and spatially resolved manner. A recently developed tool that has been used to study mechanically driven cell processes is the FRET (Förster Resonant Energy Transfer) tension sensor, which I have engineered to express endogenously in induced pluripotent stem cells within the focal adhesion gene vinculin. Preliminary data from stem cell derived cardiomyocytes that express this sensor show myofibril contraction confers an increase in global focal adhesion tension sensation in static cells. However, the spatial and temporal generation of force in cardiomyocytes during contraction is not yet known and will be examined in this project using cutting edge microscopy and image analysis techniques. Importantly, this model overcomes previous limitations of overexpression artifacts or inconsistent sensor expression. Thus, I propose to first quantify spatial and temporal physiological focal adhesion tension during static and dynamic cardiomyocyte contraction. Second, I will modulate both the inherent contractility of cardiomyocytes and their connection to the ECM to determine the internal and external regulation of focal adhesion tension in cardiomyocytes. These aims will provide a complete understanding of focal adhesion tension generation during CM contraction. This understanding may inform future studies to develop better targeted therapies to maintain mechanical homeostasis in heart disease.
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