Abstract
Key points Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the thick filament in heart muscle.Omecamtiv mecarbil increases contractility at low levels of activation by stabilizing the ON state of the thick filament.Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto‐myosin ATPase cycle.Blebbistatin reduces contractility by stabilizing the thick filament OFF state and inhibiting acto‐myosin ATPase.Thick filament regulation is a promising target for novel therapeutics in heart disease. Contraction of heart muscle is triggered by a transient rise in intracellular free calcium concentration linked to a change in the structure of the actin‐containing thin filaments that allows the head or motor domains of myosin from the thick filaments to bind to them and induce filament sliding. It is becoming increasingly clear that cardiac contractility is also regulated through structural changes in the thick filaments, although the molecular mechanisms underlying thick filament regulation are still relatively poorly understood. Here we investigated those mechanisms using small molecules – omecamtiv mecarbil (OM) and blebbistatin (BS) – that bind specifically to myosin and respectively activate or inhibit contractility in demembranated cardiac muscle cells. We measured isometric force and ATP utilization at different calcium and small‐molecule concentrations in parallel with in situ structural changes determined using fluorescent probes on the myosin regulatory light chain in the thick filaments and on troponin C in the thin filaments. The results show that BS inhibits contractility and actin‐myosin ATPase by stabilizing the OFF state of the thick filament in which myosin head domains are more parallel to the filament axis. In contrast, OM stabilizes the ON state of the thick filament, but inhibits contractility at high intracellular calcium concentration by disrupting the actin‐myosin ATPase pathway. The effects of BS and OM on the calcium sensitivity of isometric force and filament structural changes suggest that the co‐operativity of calcium activation in physiological conditions is due to positive coupling between the regulatory states of the thin and thick filaments.
Highlights
Contraction of heart muscle is driven by transient interactions between the myosin-containing thick filaments and actin-containing thin filaments, coupled to the hydrolysis of ATP
In this study we have for the first time established detailed structure–function relationships for omecamtiv mecarbil (OM) and BS that integrate functional, biochemical and structural measurements in the native environment of the intact muscle lattice
Our results establish a novel mechanistic basis for the modulation of myosin function via thick filament-based regulation and coupling between the regulatory states of the thick and thin filaments, and suggest how those mechanisms contribute to the normal physiological regulation of contractility in the heart
Summary
Contraction of heart muscle is driven by transient interactions between the myosin-containing thick filaments and actin-containing thin filaments, coupled to the hydrolysis of ATP. In the OFF states of those thick filaments, the myosin head domains are prevented from either binding actin or hydrolysing ATP by a network of intra- and inter-molecular interactions that stabilize a conformation in which they are folded back onto the myosin tails and the thick filament surface. Activation of these myosin-regulated muscles releases the myosin heads from this OFF state, making them available for actin binding and ATP hydrolysis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
