Sarcomere Lattice Geometry Influences Cooperative Myosin Binding in Muscle

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

Filament Compliance Influences Cooperative Activation of Thin Filaments and the Dynamics of Force Production in Skeletal Muscle

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

Axial and Radial Forces of Cross-Bridges Depend on Lattice Spacing

Nearly all mechanochemical models of the cross-bridge treat myosin as a simple linear spring arranged parallel to the contractile filaments. These single-spring models cannot account for the radial force that muscle generates (orthogonal to the long axis of the myofilaments) or the effects of changes in filament lattice spacing. We describe a more complex myosin cross-bridge model that uses multiple springs to replicate myosin's force-generating power stroke and account for the effects of lattice spacing and radial force. The four springs which comprise this model (the 4sXB) correspond to the mechanically relevant portions of myosin's structure. As occurs in vivo, the 4sXB's state-transition kinetics and force-production dynamics vary with lattice spacing. Additionally, we describe a simpler two-spring cross-bridge (2sXB) model which produces results similar to those of the 4sXB model. Unlike the 4sXB model, the 2sXB model requires no iterative techniques, making it more computationally efficient. The rate at which both multi-spring cross-bridges bind and generate force decreases as lattice spacing grows. The axial force generated by each cross-bridge as it undergoes a power stroke increases as lattice spacing grows. The radial force that a cross-bridge produces as it undergoes a power stroke varies from expansive to compressive as lattice spacing increases. Importantly, these results mirror those for intact, contracting muscle force production

Molecular and Subcellular-Scale Modeling of Nucleotide Diffusion in the Cardiac Myofilament Lattice

AbstractContractile function of cardiac cells is driven by the sliding displacement of myofilaments powered by the cycling myosin crossbridges. Critical to this process is the availability of ATP, which myosin hydrolyzes during the cross-bridge cycle. The diffusion of adenine nucleotides through the myofilament lattice has been shown to be anisotropic, with slower radial diffusion perpendicular to the filament axis relative to parallel, and is attributed to the periodic hexagonal arrangement of the thin (actin) and thick (myosin) filaments. We investigated whether atomistic-resolution details of myofilament proteins can refine coarse-grain estimates of diffusional anisotropy for adenine nucleotides in the cardiac myofibril, using homogenization theory and atomistic thin filament models from the Protein Data Bank. Our results demonstrate considerable anisotropy in ATP and ADP diffusion constants that is consistent with experimental measurements and dependent on lattice spacing and myofilament overlap. A reaction-diffusion model of the half-sarcomere further suggests that diffusional anisotropy may lead to modest adenine nucleotide gradients in the myoplasm under physiological conditions

Three-dimensional stochastic model of actin–myosin binding in the sarcomere lattice

The effect of molecule tethering in three-dimensional (3-D) space on bimolecular binding kinetics is rarely addressed and only occasionally incorporated into models of cell motility. The simplest system that can quantitatively determine this effect is the 3-D sarcomere lattice of the striated muscle, where tethered myosin in thick filaments can only bind to a relatively small number of available sites on the actin filament, positioned within a limited range of thermal movement of the myosin head. Here we implement spatially explicit actomyosin interactions into the multiscale Monte Carlo platform MUSICO, specifically defining how geometrical constraints on tethered myosins can modulate state transition rates in the actomyosin cycle. The simulations provide the distribution of myosin bound to sites on actin, ensure conservation of the number of interacting myosins and actin monomers, and most importantly, the departure in behavior of tethered myosin molecules from unconstrained myosin interactions with actin. In addition, MUSICO determines the number of cross-bridges in each actomyosin cycle state, the force and number of attached cross-bridges per myosin filament, the range of cross-bridge forces and accounts for energy consumption. At the macroscopic scale, MUSICO simulations show large differences in predicted force-velocity curves and in the response during early force recovery phase after a step change in length comparing to the two simplest mass action kinetic models. The origin of these differences is rooted in the different fluxes of myosin binding and corresponding instantaneous cross-bridge distributions and quantitatively reflects a major flaw of the mathematical description in all mass action kinetic models. Consequently, this new approach shows that accurate recapitulation of experimental data requires significantly different binding rates, number of actomyosin states, and cross-bridge elasticity than typically used in mass action kinetic models to correctly describe the biochemical reactions of tethered molecules and their interaction energetics

Functional Characterization of a Drosophila Transgenic Line Expressing a Chimeric Flightin: Implications on Flight Muscle Structure and Mating Behavior

The asynchronous, indirect flight muscles (IFM) of Drosophila are characterized by their remarkable crystalline myofilament lattice structure that has been proposed to have evolved to power skilled flight for survival, and to produce male courtship song, a sexually selected pre-mating behavior for reproduction. It is not known how physiologically and genetically IFM generates two distinct behaviors under separate evolutionary schemes. Flightin, a 20kDa myofibrillar myosin-binding protein that in Drosophila is exclusively expressed in the IFM, is required for muscle structural integrity and flight. The flightin N-terminal sequence (~65 aa in D. melanogaster) is highly variable among Drosophila species, unlike the rest of the Drosophila protein. Using electron microscopy, fourier image analyses, flight and wing beat frequency tests, I explored the hypothesis that the sequence of amino acids in flightin’s N-terminal region has a strong influence on myofilament lattice spacing and crystallinity. This is investigated by the creation of two independent D.melanogaster transgenic fly lines expressing a D.virilis-D.melanogaster chimeric flightin, both of which exhibit larger myofillament lattice spacing compared to the full length transgenic and D.virilis control fly lines, along with an intermediate wing beat frequency and an equal and/or improved flight ability compared to the control full length transgenic line. These results suggest the N-terminal region is under evolutionary pressures to optimize crystalline lattice structure

The Effect of Muscle Length on Post-Tetanic Potentiation of skMLCK-/- and C57BL/6 Mouse EDL Muscles

Post-tetanic potentiation of force in fast skeletal muscle is inversely related to muscle or sarcomere length, diminishing at longer lengths. This relationship has been mainly attributed to the structural effects of the primary mechanism of potentiation, phosphorylation of the regulatory light chain (RLC) of myosin, which is catalyzed by skeletal myosin light chain kinase (skMLCK). The purpose of this thesis was to compare the relationship between isometric twitch force potentiation and muscle or sarcomere length in fast twitch extensor digitorum longus (EDL) muscles from wildtype and skMLCK-/- mice. It was hypothesized that in addition to reduced potentiation, skMLCK-/- muscles without the ability to phosphorylate the RLC would also display an altered length-dependence of potentiation compared to wildtype muscles with RLC phosphorylation. The main finding was that although twitch potentiation was greater in WT muscles at all lengths, the relationship between potentiation and muscle length was similar in both WT and skMLCK-/- muscles. This indicates that the length-dependence of potentiation cannot necessarily be attributed to RLC phosphorylation. Thus, additional mechanisms, possibly related to Ca2+ handling, thick filament mechanosensing and length-dependent activation may participate in the length-dependence of potentiation displayed by murine fast muscle models

Role of the Drosophila Melanogaster Indirect Flight Muscles in Flight and Male Courtship Song: Studies on Flightin and Mydson Light Chain - 2

Complex behaviors using wings have facilitated the insect evolutionary success and diversification. The Drosophila indirect flight muscles (IFM) have evolved a highly ordered myofilament lattice structure and uses oscillatory contractions by pronounced stretch activation mechanism to drive the wings for high powered flight subject to natural selection. Moreover, the IFM is also utilized during small amplitude wing vibrations for species-specific male courtship song (sine and pulse), an important Drosophila mating behavior subject to sexual selection. Unlike flight, the contractile mechanism and contribution of any muscle gene in courtship song is not known. To gain insight into how separate selection regimes are manifested at the molecular level, we investigated the effect on flight and mating behaviors of mutations in two contractile proteins essential for IFM functions: an IFM-specific protein, flightin (FLN), known to be essential for structural and mechanical integrity of the IFM, and a ubiquitous muscle protein, myosin regulatory light chain (MLC2), known to enhance IFM stretch activation. Comparison of FLN sequences across Drosophila spp., reveal a dual nature with the N-terminal region (63 aa) evolving faster (dN/dS=0.4) than the rest of the protein (dN/dS=0.08). A deletion of the N-terminal region (fln�N62) resulted in reduced IFM fiber stiffness, oscillatory work and power output leading to a decreased flight ability (flight score: 2.8±0.1 vs 4.2±0.4 for fln+ rescued control) despite a normal wing beat frequency. Interestingly, the FLN N-terminal deletion reduced myofilament lattice spacing and order suggesting that this region is required to improve IFM lattice for enhancing power output and flight performance. Moreover, fln�N62 males sing the pulse song abnormally with a longer interpulse interval (IPI, 56±2.5 vs 37±0.7 ms for fln+) and a reduced pulse duty cycle (PDC, 2.6±0.2 vs 7.3±0.2 % for fln+) resulting in a 92% reduction in their courtship success. This suggested that FLN N-terminal region fine-tunes sexually selected song parameters in D. melanogaster, possibly explaining its hypervariability under positive selection. That FLN N-terminal region is not essential but required to optimize IFM functions of both flight and song, indicate that FLN could be an evolutionary innovation for IFM-driven behaviors, possibly through its role in lattice improvement. Mutations of the highly conserved MLC2 [N-terminal 46 aa deletion (Ext), disruption of myosin light chain kinase phosphorylations (Phos), and the two mutations put together (Dual)] are known to impair or abolish flight through severe reductions in acto-myosin contractile kinetics and magnitude of the stretch activation response. Unlike FLN, these MLC2 mutations do not show a pleitropic effect on flight and song. Flight abolished Phos and Dual mutants are capable of singing suggesting that these mutations affect song minimally compared to flight. Moreover, unlike FLN, none of these mutations affect interpulse interval, the most critical sexually selected song parameter in Drosophila. Also, in contrary to the known additive effects of Ext and Phos in the Dual mutant on flight wing beat frequency, a subtractive effect on sine song frequency is found in this study. That mutations in MLC2 are manifested differently for song and flight suggest that stretch activation plays a minimal or no role in song production. The results in this study suggest that the conserved regions of FLN and MLC2 are essential to support underlying IFM contractile structure and function necessary for flight, whereas the fast evolving FLN N-terminal region optimizes IFM\u27s biological performance in flight and species-specific song possibly under positive selection regime

Magnetic susceptibility anisotropy of myocardium imaged by cardiovascular magnetic resonance reflects the anisotropy of myocardial filament α-helix polypeptide bonds.

BackgroundA key component of evaluating myocardial tissue function is the assessment of myofiber organization and structure. Studies suggest that striated muscle fibers are magnetically anisotropic, which, if measurable in the heart, may provide a tool to assess myocardial microstructure and function.MethodsTo determine whether this weak anisotropy is observable and spatially quantifiable with cardiovascular magnetic resonance (CMR), both gradient-echo and diffusion-weighted data were collected from intact mouse heart specimens at 9.4 Tesla. Susceptibility anisotropy was experimentally calculated using a voxelwise analysis of myocardial tissue susceptibility as a function of myofiber angle. A myocardial tissue simulation was developed to evaluate the role of the known diamagnetic anisotropy of the peptide bond in the observed susceptibility contrast.ResultsThe CMR data revealed that myocardial tissue fibers that were parallel and perpendicular to the magnetic field direction appeared relatively paramagnetic and diamagnetic, respectively. A linear relationship was found between the magnetic susceptibility of the myocardial tissue and the squared sine of the myofiber angle with respect to the field direction. The multi-filament model simulation yielded susceptibility anisotropy values that reflected those found in the experimental data, and were consistent that this anisotropy decreased as the echo time increased.ConclusionsThough other sources of susceptibility anisotropy in myocardium may exist, the arrangement of peptide bonds in the myofilaments is a significant, and likely the most dominant source of susceptibility anisotropy. This anisotropy can be further exploited to probe the integrity and organization of myofibers in both healthy and diseased heart tissue