Abstract P1057: Implementing A Novel FRET-Based Myofilament Activation Biosensor To Interrogate Dynamic Cardiac Sarcomere Performance With Small Molecule And Genetic Manipulations During A Physiological Twitch

The sarcomere is the functional unit of cardiac muscle contraction. Understanding the precise mechanisms regulating this contraction is key to advancing insights into cardiomyopathies, which affect as many as 1 in 500 adults. Previous work has identified many key sarcomeric components and modulators necessary for the activation of contraction through inter-myofilament signaling. Although these studies have provided valuable information, the majority of this work has been done using in vitro protein recombination or permeabilized muscle. While informative, these systems lack significant physiological features of muscle, including load and intact excitation-contraction coupling mechanisms. Therefore, a large gap exists in the ability to monitor the time-sensitive stepwise physiological mechanism of muscle sarcomere activation in an intact system in real time. In order to further advance the study of sarcomere activation in both physiological and disease states, a cardiac specific myofilament-targeted FRET based biosensor has been designed and validated. The sarcomere activation biosensor reports myofilament dynamics during physiological twitch contractions in live cardiac muscle in real time in the form of simultaneous force and fluorescence measurements. This allows FRET-detection of the global conformational changes in TnC, a nexus point able to integrate many sarcomere-activating ligands, to be observed. From these changes in fluorescence the ensemble of events which lead to sarcomere activation and muscle contraction can be tracked. Data show regulatory ligands of myofilament activation in real time during a physiological twitch contraction. Currently, the biosensor is being utilized to investigate changes in sarcomere activation driven through the use of small molecules, including myosin inhibitor Mavacamten and TnC-TnI inhibitor W7, and those seen in MyBP-C knockout mice. The results of these investigations will be discussed.