In muscle, force emerges from myosin binding with actin (forming a cross-bridge). This actomyosin binding depends upon myofilament geometry, kinetics of thin-filament Ca2+ activation, and kinetics of cross-bridge cycling. Binding occurs within a compliant network of protein filaments where there is mechanical coupling between myosins along the thick-filament backbone and between actin monomers along the thin filament. Such mechanical coupling precludes using ordinary differential equation models when examining the effects of lattice geometry, kinetics, or compliance on force production. This study uses two stochastically driven, spatially explicit models to predict levels of cross-bridge binding, force, thin-filament Ca2+ activation, and ATP utilization. One model incorporates the 2-to-1 ratio of thin to thick filaments of vertebrate striated muscle (multi-filament model), while the other comprises only one thick and one thin filament (two-filament model). Simulations comparing these models show that the multi-filament predictions of force, fractional cross-bridge binding, and cross-bridge turnover are more consistent with published experimental values. Furthermore, the values predicted by the multi-filament model are greater than those values predicted by the two-filament model. These increases are larger than the relative increase of potential inter-filament interactions in the multi-filament model versus the two-filament model. This amplification of coordinated cross-bridge binding and cycling indicates a mechanism of cooperativity that depends on sarcomere lattice geometry, specifically the ratio and arrangement of myofilaments.

Striated muscle contraction is a highly cooperative process initiated by Ca2+ binding to the troponin complex, which leads to tropomyosin movement and myosin cross-bridge (XB) formation along thin filaments. Experimental and computational studies suggest skeletal muscle fiber activation is greatly augmented by cooperative interactions between neighboring thin filament regulatory units (RU-RU cooperativity; 1 RU = 7 actin monomers+1 troponin complex+1 tropomyosin molecule). XB binding can also amplify thin filament activation through interactions with RUs (XB-RU cooperativity). Because these interactions occur with a temporal order, they can be considered kinetic forms of cooperativity. Our previous spatially-explicit models illustrated that mechanical forms of cooperativity also exist, arising from XB-induced XB binding (XB-XB cooperativity). These mechanical and kinetic forms of cooperativity are likely coordinated during muscle contraction, but the relative contribution from each of these mechanisms is difficult to separate experimentally. To investigate these contributions we built a multi-filament model of the half sarcomere, allowing RU activation kinetics to vary with the state of neighboring RUs or XBs. Simulations suggest Ca2+ binding to troponin activates a thin filament distance spanning 9 to 11 actins and coupled RU-RU interactions dominate the cooperative force response in skeletal muscle, consistent with measurements from rabbit psoas fibers. XB binding was critical for stabilizing thin filament activation, particularly at submaximal Ca2+ levels, even though XB-RU cooperativity amplified force less than RU-RU cooperativity. Similar to previous studies, XB-XB cooperativity scaled inversely with lattice stiffness, leading to slower rates of force development as stiffness decreased. Including RU-RU and XB-RU cooperativity in this model resulted in the novel prediction that the force-[Ca2+] relationship can vary due to filament and XB compliance. Simulations also suggest kinetic forms of cooperativity occur rapidly and dominate early to get activation, while mechanical forms of cooperativity act more slowly, augmenting XB binding as force continues to develop.

We measured myosin crossbridge detachment rate and the rates of MgADP release and MgATP binding in mouse and rat myocardial strips bearing one of the two cardiac myosin heavy chain (MyHC) isoforms. Mice and rats were fed an iodine-deficient, propylthiouracil diet resulting in ~100% expression of β-MyHC in the ventricles. Ventricles of control animals expressed ~100% α-MyHC. Chemically-skinned myocardial strips prepared from papillary muscle were subjected to sinusoidal length perturbation analysis at maximum calcium activation pCa 4.8 and 17°C. Frequency characteristics of myocardial viscoelasticity were used to calculate crossbridge detachment rate over 0.01 to 5 mM [MgATP]. The rate of MgADP release, equivalent to the asymptotic value of crossbridge detachment rate at high MgATP, was highest in mouse α-MyHC (111.4±6.2 s−1) followed by rat α-MyHC (65.0±7.3 s−1), mouse β-MyHC (24.3±1.8 s−1) and rat β-MyHC (15.5±0.8 s−1). The rate of MgATP binding was highest in mouse α-MyHC (325±32 mM−1.s−1) then mouse β-MyHC (152±23 mM−1.s−1), rat α-MyHC (108±10 mM−1.s−1) and rat β-MyHC (55±6 mM−1.s−1). Because the events of MgADP release and MgATP binding occur in a post power-stroke state of the myosin crossbridge, we infer that MgATP release and MgATP binding must be regulated by isoform- and species-specific structural differences located outside the nucleotide binding pocket, which is identical in sequence for these four myosins. We postulate that differences in the stiffness profile of the entire myosin molecule, including the thick filament and the myosin-actin interface, are primarily responsible for determining the strain on the nucleotide binding pocket and the subsequent differences in the rates of nucleotide release and binding observed among the four myosins examined here.

Background and Purpose: Heart failure can reflect impaired contractile function at the myofilament level. In healthy hearts, myofilaments become more sensitive to Ca 2+ as cells are stretched. This represents a fundamental property of the myocardium that contributes to the Frank-Starling response, although the molecular mechanisms underlying the effect remain unclear. Mavacamten, which binds to myosin, is under investigation as a potential therapy for heart disease. We investigated how mavacamten affects the sarcomere-length dependence of Ca 2+-sensitive isometric contraction to determine how mavacamten might modulate the Frank-Starling mechanism. Experimental Approach: Multicellular preparations from the left ventricular-free wall of hearts from organ donors were chemically permeabilized and Ca 2+ activated in the presence or absence of 0.5-μM mavacamten at 1.9 or 2.3-μm sarcomere length (37 C). Isometric force and frequency-dependent viscoelastic myocardial stiffness measurements were made. Key Results: At both sarcomere lengths, mavacamten reduced maximal force and Ca 2+ sensitivity of contraction. In the presence and absence of mavacamten, Ca 2+ sensitivity of force increased as sarcomere length increased. This suggests that the length-dependent activation response was maintained in human myocardium, even though mavacamten reduced Ca 2+ sensitivity. There were subtle effects of mavacamten reducing force values under relaxed conditions (pCa 8.0), as well as slowing myosin cross-bridge recruitment and speeding cross-bridge detachment under maximally activated conditions (pCa 4.5). Conclusion and Implications: Mavacamten did not eliminate sarcomere lengthdependent increases in the Ca 2+ sensitivity of contraction in myocardial strips from organ donors at physiological temperature. Drugs that modulate myofilament function may be useful therapies for cardiomyopathies. K E Y W O R D S cardiac muscle mechanics, human myosin, mavacamten, sarcomere length Abbreviations: (Pi), inorganic phosphate; (pCa), −log 10 [Ca 2+ ]; (F pas), passive stress under relaxed conditions; (F act), maximal Ca 2+-activated stress; (pCa 50), free Ca 2+ concentration required to develop half the maximum Ca 2+-activated stress; (nH), Hill coefficient.

The purpose of our study was to evaluate the cytotoxicity of incubation solutions used in heart surgery to endothelial cells. The endothelial layer of human saphenous veins (HSV) and bovine internal mammary arteries (BMA) and veins (BMV) were studied after a two-hour storage interval and compared with control vessel segments prepared immediately after harvesting. To visualize the endothelial cell damage, specimens were stained with a silver nitrate technique. The surface covered by light-microscopically intact endothelial cells was computed in percent. In the control HSV segments 70.8 +/- 4.6% of the endothelium were found to be morphologically intact. The results for stored HSV segments were 50.0 +/- 4.2% (Bretschneider's solution), 14.8 +/- 4.5% (physiological saline), 0.45 +/- 0.1% (physiological saline with heparin), 16.7 +/- 4.7% (Ringer's lactate) and 37.2 +/- 5.3% (heparinized blood). Comparable values obtained with BMA specimens were 98.3 +/- 0.7% (controls), 78.1 +/- 4.7% (Bretschneider's solution), 39.2 +/- 3.3% (physiological saline), 8.4 +/- 2.0% (physiological saline with heparin), 11.3 +/- 1.7% (Ringer's lactate) and 67.8 +/- 6.2% (heparinized blood). A similar trend was found with BMV segments: 85.2 +/- 4.7% (controls), 75.6 +/- 6.0% (Bretschneider's solution), 49.5 +/- 8.9% (physiological saline), 5.95 +/- 0.7% (physiological saline with heparin), 6.2 +/- 0.7% (Ringer's lactate) and 54.3 +/- 5.1% (heparinized blood). In conclusion, Bretschneider's solution proved to be superior for storage of bypass grafts in comparison to all other tested solutions in this series.