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1. Cardiac myosin binding protein-C phosphorylation accelerates β-cardiac myosin detachment rate in mouse myocardium

   https://doi.org/10.1152/ajpheart.00673.2020  

   Tanner, Bertrand C.; Previs, Michael J.; Wang, Yuan; Robbins, Jeffrey; Palmer, Bradley M. (May 2021, American Journal of Physiology-Heart and Circulatory Physiology)

   null (Ed.)

   Cardiac myosin binding protein-C (cMyBP-C) is a thick filament protein that influences sarcomere stiffness and modulates cardiac contraction-relaxation through its phosphorylation. Phosphorylation of cMyBP-C and ablation of cMyBP-C have been shown to increase the rate of MgADP release in the acto-myosin cross-bridge cycle in the intact sarcomere. The influence of cMyBP-C on Pi-dependent myosin kinetics has not yet been examined. We investigated the effect of cMyBP-C, and its phosphorylation, on myosin kinetics in demembranated papillary muscle strips bearing the β-cardiac myosin isoform from nontransgenic and homozygous transgenic mice lacking cMyBP-C. We used quick stretch and stochastic length-perturbation analysis to characterize rates of myosin detachment and force development over 0–12 mM Pi and at maximal (pCa 4.8) and near-half maximal (pCa 5.75) Ca 2+ activation. Protein kinase A (PKA) treatment was applied to half the strips to probe the effect of cMyBP-C phosphorylation on Pi sensitivity of myosin kinetics. Increasing Pi increased myosin cross-bridge detachment rate similarly for muscles with and without cMyBP-C, although these rates were higher in muscle without cMyBP-C. Treating myocardial strips with PKA accelerated detachment rate when cMyBP-C was present over all Pi, but not when cMyBP-C was absent. The rate of force development increased with Pi in all muscles. However, Pi sensitivity of the rate force development was reduced when cMyBP-C was present versus absent, suggesting that cMyBP-C inhibits Pi-dependent reversal of the power stroke or stabilizes cross-bridge attachment to enhance the probability of completing the power stroke. These results support a functional role for cMyBP-C in slowing myosin detachment rate, possibly through a direct interaction with myosin or by altering strain-dependent myosin detachment via cMyBP-C-dependent stiffness of the thick filament and myofilament lattice. PKA treatment reduces the role for cMyBP-C to slow myosin detachment and thus effectively accelerates β-myosin detachment in the intact myofilament lattice. NEW & NOTEWORTHY Length perturbation analysis was used to demonstrate that β-cardiac myosin characteristic rates of detachment and recruitment in the intact myofilament lattice are accelerated by Pi, phosphorylation of cMyBP-C, and the absence of cMyBP-C. The results suggest that cMyBP-C normally slows myosin detachment, including Pi-dependent detachment, and that this inhibition is released with phosphorylation or absence of cMyBP-C. 

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2. Inorganic phosphate accelerates cardiac myofilament relaxation in response to lengthening

   https://doi.org/10.3389/fphys.2022.980662  

   Wakefield, Jane I.; Bell, Stephen P.; Palmer, Bradley M. (September 2022, Frontiers in Physiology)

   Myocardial relaxation in late systole is enhanced by increasing velocities of lengthening. Given that inorganic phosphate (Pi) can rebind to the force-producing myosin enzyme prior to MgADP release and hasten crossbridge detachment, we hypothesized that myocardial relaxation in late systole would be further enhanced by lengthening in the presence of Pi. Wistar rat left ventricular papillary muscles were attached to platinum clips, placed between a force transducer and a length motor at room temperature, and bathed in Krebs solution with 1.8 mM Ca 2+ and varying Pi of 0, 1, 2, and 5 mM. Tension transients were elicited by electrical stimulation at 1 Hz. Peak tension was significantly enhanced by Pi: 0.593 ± 0.088 mN mm −2 at 0 mM Pi and 0.817 ± 0.159 mN mm −2 at 5 mM Pi (mean ± SEM, p < 0.01 by ANCOVA). All temporal characteristics of the force transient were significantly shortened with increasing Pi, e.g., time-to-50% recovery was shortened from 305 ± 14 ms at 0 mM Pi to 256 ± 10 ms at 5 mM Pi ( p < 0.01). A 1% lengthening stretch with varying duration of 10–200 ms was applied at time-to-50% recovery during the descending phase of the force transient. Matching lengthening stretches were also applied when the muscle was not stimulated, thus providing a control for the passive viscoelastic response. After subtracting the passive from the active force response, the resulting myofilament response demonstrated features of faster myofilament relaxation in response to the stretch. For example, time-to-70% relaxation with 100 ms lengthening duration was shortened by 8.8 ± 6.8 ms at 0 Pi, 19.6 ± 4.8* ms at 1 mM Pi, 31.0 ± 5.6* ms at 2 Pi, and 25.6 ± 5.3* ms at 5 mM Pi (* p < 0.01 compared to no change). Using skinned myocardium, half maximally calcium-activated myofilaments underwent a 1% quick stretch, and the tension response was subjected to analysis for sensitivity of myosin detachment rate to stretch, g 1 , at various Pi concentrations. The parameter g 1 was enhanced from 15.39 ± 0.35 at 0 Pi to 22.74 ± 1.31 s −1 /nm at 8 Pi ( p < 0.01). Our findings suggest that increasing Pi at the myofilaments enhances lengthening-induced relaxation by elevating the sensitivity of myosin crossbridge detachment due to lengthening and thus speed the transition from late-systole to early-diastole. 

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3. Regulatory light chain phosphorylation augments length-dependent contraction in PTU-treated rats

   https://doi.org/10.1085/jgp.201812158  

   Breithaupt, Jason J.; Pulcastro, Hannah C.; Awinda, Peter O.; DeWitt, David C.; Tanner, Bertrand C.W. (January 2019, The Journal of General Physiology)

   Force production by actin–myosin cross-bridges in cardiac muscle is regulated by thin-filament proteins and sarcomere length (SL) throughout the heartbeat. Prior work has shown that myosin regulatory light chain (RLC), which binds to the neck of myosin heavy chain, increases cardiac contractility when phosphorylated. We recently showed that cross-bridge kinetics slow with increasing SLs, and that RLC phosphorylation amplifies this effect, using skinned rat myocardial strips predominantly composed of the faster α-cardiac myosin heavy chain isoform. In the present study, to assess how RLC phosphorylation influences length-dependent myosin function as myosin motor speed varies, we used a propylthiouracil (PTU) diet to induce >95% expression of the slower β-myosin heavy chain isoform in rat cardiac ventricles. We measured the effect of RLC phosphorylation on Ca 2+ -activated isometric contraction and myosin cross-bridge kinetics (via stochastic length perturbation analysis) in skinned rat papillary muscle strips at 1.9- and 2.2-µm SL. Maximum tension and Ca 2+ sensitivity increased with SL, and RLC phosphorylation augmented this response at 2.2-µm SL. Subtle increases in viscoelastic myocardial stiffness occurred with RLC phosphorylation at 2.2-µm SL, but not at 1.9-µm SL, thereby suggesting that RLC phosphorylation increases β-myosin heavy chain binding or stiffness at longer SLs. The cross-bridge detachment rate slowed as SL increased, providing a potential mechanism for prolonged cross-bridge attachment to augment length-dependent activation of contraction at longer SLs. Length-dependent slowing of β-myosin heavy chain detachment rate was not affected by RLC phosphorylation. Together with our previous studies, these data suggest that both α- and β-myosin heavy chain isoforms show a length-dependent activation response and prolonged myosin attachment as SL increases in rat myocardial strips, and that RLC phosphorylation augments length-dependent activation at longer SLs. In comparing cardiac isoforms, however, we found that β-myosin heavy chain consistently showed greater length-dependent sensitivity than α-myosin heavy chain. Our work suggests that RLC phosphorylation is a vital contributor to the regulation of myocardial contractility in both cardiac myosin heavy chain isoforms. 

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4. The structural and functional effects of myosin regulatory light chain phosphorylation are amplified by increases in sarcomere length and [Ca ²⁺ ]

   https://doi.org/10.1113/JP286802  

   Turner, Kyrah L; Vander_Top, Blake J; Kooiker, Kristina B; Mohran, Saffie; Mandrycky, Christian; McMillen, Tim; Regnier, Michael; Irving, Thomas C; Ma, Weikang; Tanner, Bertrand CW (October 2024, The Journal of Physiology)

   AbstractPrecise regulation of sarcomeric contraction is essential for normal cardiac function. The heart must generate sufficient force to pump blood throughout the body, but either inadequate or excessive force can lead to dysregulation and disease. Myosin regulatory light chain (RLC) is a thick‐filament protein that binds to the neck of the myosin heavy chain. Post‐translational phosphorylation of RLC (RLC‐P) by myosin light chain kinase is known to influence acto‐myosin interactions, thereby increasing force production and Ca²⁺‐sensitivity of contraction. Here, we investigated the role of RLC‐P on cardiac structure and function as sarcomere length and [Ca²⁺] were altered. We found that at low, non‐activating levels of Ca²⁺, RLC‐P contributed to myosin head disorder, though there were no effects on isometric stress production and viscoelastic stiffness. With increases in sarcomere length and Ca²⁺‐activation, the structural changes due to RLC‐P become greater, which translates into greater force production, greater viscoelastic stiffness, slowed myosin detachment rates and altered nucleotide handling. Altogether, these data suggest that RLC‐P may alter thick‐filament structure by releasing ordered, off‐state myosin. These more disordered myosin heads are available to bind actin, which could result in greater force production as Ca²⁺levels increase. However, prolonged cross‐bridge attachment duration due to slower ADP release could delay relaxation long enough to enable cross‐bridge rebinding. Together, this work further elucidates the effects of RLC‐P in regulating muscle function, thereby promoting a better understanding of thick‐filament regulatory contributions to cardiac function in health and disease.image Key pointsMyosin regulatory light chain (RLC) is a thick‐filament protein in the cardiac sarcomere that can be phosphorylated (RLC‐P), and changes in RLC‐P are associated with cardiac dysfunction and disease.This study assesses how RLC‐P alters cardiac muscle structure and function at different sarcomere lengths and calcium concentrations.At low, non‐activating levels of Ca²⁺, RLC‐P contributed to myofilament disorder, though there were no effects on isometric stress production and viscoelastic stiffness.With increases in sarcomere length and Ca²⁺‐activation, the structural changes due to RLC‐P become greater, which translates into greater force production, greater viscoelastic stiffness, slower myosin detachment rate and altered cross‐bridge nucleotide handling rates.This work elucidates the role of RLC‐P in regulating muscle function and facilitates understanding of thick‐filament regulatory protein contributions to cardiac function in health and disease. 

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5. Strain rate of stretch affects crossbridge detachment during relaxation of intact cardiac trabeculae

   https://doi.org/10.1371/journal.pone.0297212  

   Tanner, Bertrand CW; Palmer, Bradley M; Chung, Charles S (March 2024, PloS one)

   Johnson, Daniel M (Ed.)

   Mechanical Control of Relaxation refers to the dependence of myocardial relaxation on the strain rate just prior to relaxation, but the mechanisms of enhanced relaxation are not well characterized. This study aimed to characterize how crossbridge kinetics varied with strain rate and time-to-stretch as the myocardium relaxed in early diastole. Ramp-stretches of varying rates (amplitude = 1% muscle length) were applied to intact rat cardiac trabeculae following a load-clamp at 50% of the maximal developed twitch force, which provides a first-order estimate of ejection and coupling to an afterload. The resultant stress-response was calculated as the difference between the time-dependent stress profile between load-clamped twitches with and without a ramp-stretch. The stress-response exhibited features of the step-stretch response of activated, permeabilized myocardium, such as distortion-dependent peak stress, rapid force decay related to crossbridge detachment, and stress recovery related to crossbridge recruitment. The peak stress was strain rate dependent, but the minimum stress and the time-to-minimum stress values were not. The initial rapid change in the stress-response indicates enhanced crossbridge detachment at higher strain rates during relaxation in intact cardiac trabeculae. Physiologic considerations, such as time-varying calcium, are discussed as potential limitations to fitting these data with traditional distortion-recruitment models of crossbridge activity. 

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