Thick-Filament-Based Regulation and the Determinants of Force Generation.

Abstract

Background/Objectives: Thick-filament-based regulation in muscle is generally conceived as processes that modulate the number of myosin heads capable of force generation. It has been generally assumed that biochemical and structural assays of myosin active and inactive states provide equivalent measures of myosin recruitment, but recent studies indicate that this may not always be the case. Here, we studied the steady-state and dynamic mechanical changes in skinned porcine myocardium before and after treatment with omecamtiv mecarbil (OM) or piperine to help decipher how the biochemical and structural states of myosin separately affect contractile force. Methods: Force-Ca²⁺ relationships were obtained from skinned cardiomyocytes isolated from porcine myocardium before and after exposure to 1 μM OM and 7 μM piperine. Crossbridge kinetics were acquired using a step response stretch activation protocol allowing myosin attachment and detachment rates to be calculated. Results: OM augmented calcium-activated force at submaximal calcium levels that can be attributed to increased thick filament recruitment, increases in calcium sensitivity, an increased duty ratio, and from decelerated crossbridge detachment resulting in slowed crossbridge cycling kinetics. Piperine, in contrast, was able to increase activated force at submaximal calcium levels without appreciably affecting crossbridge cycling kinetics. Conclusions: Our study supports the notion that thick filament activation is primarily a process of myosin recruitment that is not necessarily coupled with the chemo-cycling of crossbridges. These new insights into thick filament activation mechanisms will need to be considered in the design of sarcomere-based therapies for treatment of myopathies.