Mechanotransduction in Single Cardiac Myocyte Studied Using Laser Tweezers and FRET

Deletion or mutation of a variety of proteins localized at cell-matrix and cell-cell junctions, such as vinculin and its splice-variant metavinculin, can lead to dilated cardiomyopathy in mice and humans, leading some to hypothesize that these molecules are involved in mechanotransmission or mechanotransduction in the heart.To investigate cardiac mechanotransduction mechanisms in single cells, we have combined laser tweezers with a fluorescence resonance energy transfer (FRET) biosensor to apply localized forces and probe localized signaling events in isolated mouse ventricular myocytes.Isolated murine ventricular cardiac myocytes were transfected with a focal adhesion kinase (FAK) FRET reporter to monitor integrin-mediated activation events. An integrin ligand-coated microsphere was adhered to the cell surface. Then laser tweezers were used to apply localized piconewton forces parallel or normal to the image plane either cyclically or statically. To ensure a constant force application on the microsphere, 10υM of blebbistatin was added to the imaging media. In conjunction with force application, a pulsed Ti:sapphire infrared laser was used for two-photon excitation of the FRET reporter.Phase contrast and fluorescent images were captured simultaneously, allowing quantification of applied forces and FRET ratio changes. Results indicate that piconewton level forces can be applied to the microsphere and FRET ratio changes validate that integrin-mediated events are being activated by the locally applied forces. This data suggests that the use of laser tweezers combined with FRET provides a means to study integrin-mediated events in cardiac mechanotransduction. Future studies include varying the type of integrin ligand-coated microsphere and FRET biosensors, as well as studying genetically manipulated murine lines in isolated adult cardiac myocytes.