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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.

Mutations in sarcomeric actins have been linked to various skeletal and cardiac myopathies. In contrast, only a small number of mutations in the cytoplasmic isoforms β-actin and γ-actin have been identified and linked to disease processes. Here, we describe the characterization of disease-related human β-actin mutants R183W and E364K. Mutation R183W is associated with developmental malformations, deafness and dystonia. The residue is located in subdomain 4 in the nucleotide binding cleft and participates in forming a gate-like structure for the nucleotide. Mutation E364K was found in a patient with neutrophil dysfunction. E364 is a surface residue in subdomain 1. We produced both mutants in Sf9 cells and characterized their properties in vitro. The midpoints of the thermal transition Tm, which correspond to the temperatures at which half of all protein molecules are in the native state and the remaining half is in the denatured state, are unaffected by the mutations suggesting proper folding and normal protein stability. Both mutants display increased affinity for DNase-I indicating conformational changes in subdomain 2 that appear to alter the binding interface between monomers. Activated neutrophils are highly motile phagocytic cells and they are thus highly sensitive to changes that affect actin polymerization and F-actin stability. We observed reduced rates of nucleotide exchange and elongation velocities during polymerization for R183W and E364K β-actin. Mutation R183W accelerates ATP hydrolysis by F-actin. R183W β-actin shows impaired ability to activate non-muscle myosin 2A ATPase.