Action mechanisms of physiological and pharmacological inotropic interventions on the slow/cardiac striated muscle.

ACTION MECHANISMS OF PHYSIOLOGICAL AND PHARMACOLOGICAL INOTROPIC INTERVENTIONS ON THE SLOW/CARDIAC STRIATED MUSCLE. The mechanical performance of striated muscle is under the control of both thin and thick filament regulation. The start signal is the increase of intracellular Ca2+ concentration, promoted by cell membrane depolarization by the action potential, followed by Ca2+ binding to troponin in the thin filament and structural changes in the troponin–tropomyosin complex that release the actin sites for binding of myosin motors. The second regulatory mechanism, based on thick filament mechano-sensing, recruits myosin motors from their OFF state, in which they lie along the surface of the thick filament folded toward its centre, unable to bind actin and hydrolyze ATP. In cardiac myocytes it has been demonstrated that inotropic interventions potentiate the mechanical output by mechanisms that imply increase in Ca2+ sensitivity of the thin filament but also mobilisation of myosin heads from their OFF state. During my doctorate I investigated the molecular mechanisms responsible for the regulation of the performance of the heart by defining how physiological and pharmacological inotropic interventions modulate the thick filament regulatory mechanisms and its interaction with the thin filament and the mechanokinetic properties of the slow/cardiac myosin motor. In the first part of my PhD research activity , combined mechanical and X-ray diffraction experiments on electrically paced intact trabeculae of rat heart (frequency 0.5 Hz, temperature 27°C) were used to investigate the effect on the thick filament regulatory state of physiological interventions able to potentiate up to twofold the peak of the twitch force in physiological solution with 1 mM Ca2+, such as increase in sarcomere length (SL) from 1.95 to 2.22 μm and addition of the β-adrenergic effector isoprenaline (10-7 M) to the bathing solution. The results show that, in diastole, none of the diffraction signals attributed to the OFF state of the thick filament were significantly affected by either increase in SL or phosphorylation of myofilament proteins, suggesting that the control of thick filament activation is downstream from the Ca2+-dependent thin filament activation, solidifying the idea that in the intact myocyte myosin motors are switched ON only during systole by an energetically well-suited downstream mechanism as thick filament mechano-sensing (Caremani et al., J. Gen. Physiol. 151:53,2019). In the second part of my PhD, the action mechanism of the inotropic agent Omecamtiv Mecarbil (OM) was studied by combining fast-sarcomere level mechanics and ATPase measurements in Ca2+-demembranated fibres from rabbit soleus (SL 2.4 μm, temperature 12°C), which express the β/slow myosin heavy chain isoform. OM is a cardiac myosin activator in phase three clinical trial as a potential treatment for systolic heart failure with reduced ejection fraction. The results show that, at any [Ca2+], OM depresses the force per motor, by preventing the OM-bound motors to complete the force-generating working stroke, but at low [Ca2+] it increases the sarcomere force by increasing the number of attached motors. Increase in the concentration of inorganic phosphate (Pi) in the physiological range (1-10 mM) causes a partial recovery of the force per myosin motor, suggesting that an allosteric competition between OM and Pi allows the no force-generating motors that release OM to re-enter the force-generating cycle (Governali et al., submitted to Nat Commun). This mechanism could underpin an energetically efficient reduction of systolic tension cost in OM-treated patients under exercise, when [Pi] increases with heart-beat frequency.