The Synergic Role of Actomyosin Architecture and Biased Detachment in Muscle Energetics: Insights in Cross Bridge Mechanism Beyond the Lever-Arm Swing.

Muscle energetics reflects the ability of myosin motors to convert chemical energy into mechanical energy. How this process takes place remains one of the most elusive questions in the field. Here, we combined experimental measurements of in vitro sliding velocity based on DNA-origami built filaments carrying myosins with different lever arm length and Monte Carlo simulations based on a model which accounts for three basic components: (i) the geometrical hindrance, (ii) the mechano-sensing mechanism, and (iii) the biased kinetics for stretched or compressed motors. The model simulations showed that the geometrical hindrance due to acto-myosin spatial mismatching and the preferential detachment of compressed motors are synergic in generating the rapid increase in the ATP-ase rate from isometric to moderate velocities of contraction, thus acting as an energy-conservation strategy in muscle contraction. The velocity measurements on a DNA-origami filament that preserves the motors' distribution showed that geometrical hindrance and biased detachment generate a non-zero sliding velocity even without rotation of the myosin lever-arm, which is widely recognized as the basic event in muscle contraction. Because biased detachment is a mechanism for the rectification of thermal fluctuations, in the Brownian-ratchet framework, we predict that it requires a non-negligible amount of energy to preserve the second law of thermodynamics. Taken together, our theoretical and experimental results elucidate less considered components in the chemo-mechanical energy transduction in muscle.

Excessive Accumulation of Ca2 + in Mitochondria of Y522S-RYR1 Knock-in Mice: A Link Between Leak From the Sarcoplasmic Reticulum and Altered Redox State.

Mice (Y522S or YS), carrying a mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of skeletal muscle fibers (ryanodine receptor type-1, RyR1) which causes Ca2+ leak, are a widely accepted and intensively studied model for human malignant hyperthermia (MH) susceptibility. Since the involvement of reactive oxygen species (ROS) and of mitochondria in MH crisis has been previously debated, here we sought to determine Ca2+ uptake in mitochondria and its possible link with ROS production in single fibers isolated from flexor digitorum brevis (FDB) of YS mice. We found that Ca2+ concentration in the mitochondrial matrix, as detected with the ratiometric FRET-based 4mtD3cpv probe, was higher in YS than in wild-type (WT) fibers at rest and after Ca2+ release from SR during repetitive electrical stimulation or caffeine administration. Also mitochondrial ROS production associated with contractile activity (detected with Mitosox probe) was much higher in YS fibers than in WT. Importantly, the inhibition of mitochondrial Ca2+ uptake achieved by silencing MCU reduced ROS accumulation in the matrix and Ca2+ release from SR. Finally, inhibition of mitochondrial ROS accumulation using Mitotempo reduced SR Ca2+ release in YS fibers exposed to caffeine. The present results support the view that mitochondria take up larger amounts of Ca2+ in YS than in WT fibers and that mitochondrial ROS production substantially contributes to the increased caffeine-sensitivity and to the enhanced Ca2+ release from SR in YS fibers.

Proposed mechanism for the length dependence of the force developed in maximally activated muscles.

The molecular bases of the Frank-Starling law of the heart and of its cellular counterpart, the length dependent activation (LDA), are largely unknown. However, the recent discovery of the thick filament activation, a second pathway beside the well-known calcium mediated thin filament activation, is promising for elucidating these mechanisms. The thick filament activation is mediated by the tension acting on it through the mechano-sensing (MS) mechanism and can be related to the LDA via the titin passive tension. Here, we propose a mechanism to explain the higher maximum tension at longer sarcomere lengths generated by a maximally activated muscle and test it in-silico with a single fiber and a ventricle model. The active tension distribution along the thick filament generates a reservoir of inactive motors at its free-end that can be activated by passive tension on a beat-to-beat timescale. The proposed mechanism is able to quantitatively account for the observed increment in tension at the fiber level, however, the ventricle model suggests that this component of the LDA is not crucial in physiological conditions.