Impact of Para-Nitroblebbistatin on Human Beta-Cardiac Myosin at the Molecular and Tissue Levels

Abstract

Inherited cardiomyopathies are a common form of heart disease caused by mutations in sarcomeric proteins with beta cardiac myosin being one of the most frequently affected genes. Since the discovery of the first cardiomyopathy associated mutation in beta-cardiac myosin, a major goal has been to correlate the in vitro myosin motor properties with the contractile performance of cardiac muscle. Mutations that cause hypertrophic cardiomyopathy are the most common form of the disease and are often associated with increased isometric force and hyper-contractility. Therefore, the development of drugs designed to decrease isometric force by reducing the duty ratio (the proportion of ATPase cycle time myosin spends bound to actin) has been proposed for the treatment of hypertrophic cardiomyopathy. There is still no consensus about whether hypertrophic mutations in beta-cardiac myosin increase myofilament calcium sensitivity as is commonly found in troponin mutations. We examine the impact of a small molecule drug proposed to decrease duty ratio (para-Nitroblebbistatin) using purified human beta cardiac myosin motor assays and studies of permeabilized human cardiac muscle mechanics. We find that this drug reduces actin-activated ATPase and in vitro motility while not changing the ADP release rate constant. Thus, the reduction in the in vitro sliding velocity is likely due to a slowing of attachment rate. In muscle fiber studies we found that para-Nitroblebbistatin reduces steady-state force and calcium sensitivity but does not change maximum contractile velocity and the rate of force development. These results support a model in which the drug reduces the duty ratio without altering the kinetics of attached crossbridges. Thus, small molecule drugs that target the crossbridge attachment rate are an attractive strategy since they are capable of reducing the force generating capacity and calcium sensitivity without altering contractile velocity.