Arachidonic Acid Directly Binds and Activates Beta-Cardiac Myosin in the Regulated Cardiac Actomyosin Complex

Mutation-induced dysfunction or misfolding of heart muscle sarcomere components have been linked to dilated and hypertrophic cardiomyopathies. We have previously shown that the small molecule EMD57033 increases β-cardiac myosin activity and acts as a pharmacological chaperone that stabilizes and refolds the motor domain. Following up on this finding, we investigated whether metabolites such as fatty acids can have a similar effect on β-cardiac myosin. Arachidonic acid (AA) was previously reported as an activator of smooth muscle myosin. We found that among all fatty acids tested, AA induced the largest effect on actin-activated ATPase activity of β-cardiac myosin. Closer characterization revealed a 13-fold increase in β-cardiac myosins affinity for actin in the presence of ATP and a 7-fold increase in the rate of phosphate release. We further aimed to elucidate the effect of AA-induced myosin activation in the context of a reconstituted human cardiac actin-troponin-tropomyosin complex. Both, assays monitoring ATPase and in vitro motility showed a significant increase in activity over a wide range of Ca2+ concentrations. Similar to the effect of EMD57033, the addition of AA increases the stability of myosin and reverses the loss of myosin activity caused by temperature stress. The refolding reaction can be monitored by the recovery of the intrinsic protein fluorescence signal that is associated with ATP binding and active turnover. Alternatively, we followed the clear reduction in the number of immobile filaments following the addition of AA.