Fluorescently Labelled Myosin Regulatory Light Chains As Biosensors For Thick Filament Activation In Heart Muscle

Calcium binding to the actin-containing thin filaments has long been known to be the trigger for heart muscle contraction. The resulting change in the thin filament conformation allows myosin head domains from the neighboring thick filaments to attach to actin and drive force generation and filament sliding. There is growing interest, however, in the involvement of proteins of the thick filament in the regulation of cardiac muscle contractility. The myosin-associated regulatory light chains (RLC) form an integral part of the myosin head or motor domain and can control the availability of the heads for actin interaction. To develop an effective and targeted therapeutic strategy for heart failure, we have employed fluorescent probes on the RLC to detect structural changes in intact thick filaments in isolated cardiac myofibrils. We generated a series of single cysteine mutants of RLC by site directed mutagenesis and labelled these proteins with solvato-chromic fluorescent dyes. These labelled RLCs were exchanged into rat and bovine cardiac myofibrils, replacing ∼40% of the native RLCs. We recorded emission spectra from these myofibrils in rigor and relaxing conditions and observed significant spectral changes for some probes, with the peak intensity of fluorescence shifting towards shorter wavelength in the relaxed state. We conclude that biosensor-RLCs can be used to screen potential small molecule modulators of the regulatory state of the myosin-containing thick filaments. The robustness of this technique makes it suitable for scaling up for high throughput screens. These results also provide further insight into the role of RLC in thick filament-based regulation and cardiac muscle contractility.