Danicamtiv Increases Myosin Recruitment and Alters Cross-Bridge Cycling in Cardiac Muscle.

BACKGROUND: Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known that danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. METHODS: Permeabilized porcine cardiac tissue and myofibrils were used for X-ray diffraction and mechanical measurements. A mouse model of genetic dilated cardiomyopathy was used to evaluate the ability of danicamtiv to correct the contractile deficit. RESULTS: Danicamtiv increased force and calcium sensitivity via increasing the number of myosins in the ON state and slowing cross-bridge turnover. Our detailed analysis showed that inhibition of ADP release results in decreased cross-bridge turnover with cross bridges staying attached longer and prolonging myofibril relaxation. Danicamtiv corrected decreased calcium sensitivity in demembranated tissue, abnormal twitch magnitude and kinetics in intact cardiac tissue, and reduced ejection fraction in the whole organ. CONCLUSIONS: As demonstrated by the detailed studies of Danicamtiv, increasing myosin recruitment and altering cross-bridge cycling are 2 mechanisms to increase force and calcium sensitivity in cardiac muscle. Myosin activators such as Danicamtiv can treat the causative hypocontractile phenotype in genetic dilated cardiomyopathy.

Danicamtiv affected isometric force and cross-bridge kinetics similarly in skinned myocardial strips from male and female rats.

Myotropes are pharmaceuticals that have recently been developed or are under investigation for the treatment of heart diseases. Myotropes have had varied success in clinical trials. Initial research into myotropes have widely focused on animal models of cardiac dysfunction in comparison with normal animal cardiac physiology-primarily using males. In this study we examined the effect of danicamtiv, which is one type of myotrope within the class of myosin activators, on contractile function in permeabilized (skinned) myocardial strips from male and female Sprague-Dawley rats. We found that danicamtiv increased steady-state isometric force production at sub-maximal calcium levels, leading to greater Ca2+-sensitivity of contraction for both sexes. Danicamtiv did not affect maximal Ca2+-activated force for either sex. Sinusoidal length-perturbation analysis was used to assess viscoelastic myocardial stiffness and cross-bridge cycling kinetics. Data from these measurements did not vary with sex, and the data suggest that danicamtiv slows cross-bridge cycling kinetics. These findings imply that danicamtiv increases force production via increasing cross-bridge contributions to activation of contraction, especially at sub-maximal Ca2+-activation. The inclusion of both sexes in animal models during the formative stages of drug development could be helpful for understanding the efficacy or limitation of a drug's therapeutic impact on cardiac function.

Cooperative & competitive binding of anti-myosin tail antibodies revealed by super-resolution microscopy.

The MF30 monoclonal antibody, which binds to the myosin subfragment-2 (S2), was found to increase the extent of myofibril shortening. Yet, previous observations found no effect of this antibody on actin sliding over myosin during in vitro motility assays with purified proteins in which myosin binding protein C (MyBPC) was absent. MF30 is hypothesized to enhance the availability of myosin heads (subfragment-1 or S1) to bind actin by destabilizing the myosin S2 coiled-coil and sterically blocking S2 from binding S1. The mechanism of action likely includes MF30's substantial size, thereby inhibiting S1 heads and MyBPC from binding S2. Hypothetically, MF30 should enhance the ON state of myosin, thereby increasing muscle contraction. Our findings indicate that MF30 binds preferentially to the unfolded heavy chains of S2, displaying positive cooperativity. However, the dose-response curve of MF30's enhancement of myofibril shortening did not suggest complex interactions with S2. Single, double, and triple-stained myofibrils with increasing amounts of antibodies against myosin rods indicate a possible competition with MyBPC. Additional assays revealed decreased fluorescence intensity at the C-zone (central zone in the sarcomere, where MyBPC is located), where MyBPC may inhibit MF30 binding. Another monoclonal antibody named MF20, which binds to the light meromyosin (LMM) without affecting myofibril contraction, showed less reduction in fluorescence intensity at the C-zone in expansion microscopy than MF30. Expansion microscopy images of myofibrils labeled with MF20 revealed labeling of the A-band (anisotropic band) and a slight reduction in the labeling at the C-zone. The staining pattern obtained from the expansion microscopy image was consistent with images from photolocalization microscopy which required the synthesis of unique photoactivatable quantum dots, and Zeiss Airyscan imaging as well as alternative expansion microscopy digestion methods. Consistent with the hypothesis that MF30 competes with MyBPC binding to S2, cardiac tissue from MyBPC knockout mice was stained more intensely, especially in the C-zone, by MF30 compared to the wild type.

MgADP Promotes Myosin Head Movement toward Actin at Low [Ca2+] to Increase Force Production and Ca2+-Sensitivity of Contraction in Permeabilized Porcine Myocardial Strips.

Myosin cross-bridges dissociate from actin following Mg2+-adenosine triphosphate (MgATP) binding. Myosin hydrolyses MgATP into inorganic phosphate (Pi) and Mg2+-adenosine diphosphate (ADP), and release of these hydrolysis products drives chemo-mechanical energy transitions within the cross-bridge cycle to power muscle contraction. Some forms of heart disease are associated with metabolic or enzymatic dysregulation of the MgATP-MgADP nucleotide pool, resulting in elevated cytosolic [MgADP] and impaired muscle relaxation. We investigated the mechanical and structural effects of increasing [MgADP] in permeabilized myocardial strips from porcine left ventricle samples. Sarcomere length was set to 2.0 µm at 28 °C, and all solutions contained 3% dextran T-500 to compress myofilament lattice spacing to near-physiological values. Under relaxing low [Ca2+] conditions (pCa 8.0, where pCa = -log10[Ca2+]), tension increased as [MgADP] increased from 0-5 mM. Complementary small-angle X-ray diffraction measurements show that the equatorial intensity ratio, I1,1/I1,0, also increased as [MgADP] increased from 0 to 5 mM, indicating myosin head movement away from the thick-filament backbone towards the thin-filament. Ca2+-activated force-pCa measurements show that Ca2+-sensitivity of contraction increased with 5 mM MgADP, compared to 0 mM MgADP. These data show that MgADP augments tension at low [Ca2+] and Ca2+-sensitivity of contraction, suggesting that MgADP destabilizes the quasi-helically ordered myosin OFF state, thereby shifting the cross-bridge population towards the disordered myosin ON state. Together, these results indicate that MgADP enhances the probability of cross-bridge binding to actin due to enhancement of both thick and thin filament-based activation mechanisms.

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

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 ß-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) Ca2+ 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 ß-myosin detachment in the intact myofilament lattice.NEW & NOTEWORTHY Length perturbation analysis was used to demonstrate that ß-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.

Mavacamten decreases maximal force and Ca2+ sensitivity in the N47K-myosin regulatory light chain mouse model of hypertrophic cardiomyopathy.

Morbidity and mortality associated with heart disease is a growing threat to the global population, and novel therapies are needed. Mavacamten (formerly called MYK-461) is a small molecule that binds to cardiac myosin and inhibits myosin ATPase. Mavacamten is currently in clinical trials for the treatment of obstructive hypertrophic cardiomyopathy (HCM), and it may provide benefits for treating other forms of heart disease. We investigated the effect of mavacamten on cardiac muscle contraction in two transgenic mouse lines expressing the human isoform of cardiac myosin regulatory light chain (RLC) in their hearts. Control mice expressed wild-type RLC (WT-RLC), and HCM mice expressed the N47K RLC mutation. In the absence of mavacamten, skinned papillary muscle strips from WT-RLC mice produced greater isometric force than strips from N47K mice. Adding 0.3 µM mavacamten decreased maximal isometric force and reduced Ca2+ sensitivity of contraction for both genotypes, but this reduction in pCa50 was nearly twice as large for WT-RLC versus N47K. We also used stochastic length-perturbation analysis to characterize cross-bridge kinetics. The cross-bridge detachment rate was measured as a function of [MgATP] to determine the effect of mavacamten on myosin nucleotide handling rates. Mavacamten increased the MgADP release and MgATP binding rates for both genotypes, thereby contributing to faster cross-bridge detachment, which could speed up myocardial relaxation during diastole. Our data suggest that mavacamten reduces isometric tension and Ca2+ sensitivity of contraction via decreased strong cross-bridge binding. Mavacamten may become a useful therapy for patients with heart disease, including some forms of HCM.NEW & NOTEWORTHY Mavacamten is a pharmaceutical that binds to myosin, and it is under investigation as a therapy for some forms of heart disease. We show that mavacamten reduces isometric tension and Ca2+ sensitivity of contraction in skinned myocardial strips from a mouse model of hypertrophic cardiomyopathy that expresses the N47K mutation in cardiac myosin regulatory light chain. Mavacamten reduces contractility by decreasing strong cross-bridge binding, partially due to faster cross-bridge nucleotide handling rates that speed up myosin detachment.

Myosin cross-bridge kinetics slow at longer muscle lengths during isometric contractions in intact soleus from mice.

Muscle contraction results from force-generating cross-bridge interactions between myosin and actin. Cross-bridge cycling kinetics underlie fundamental contractile properties, such as active force production and energy utilization. Factors that influence cross-bridge kinetics at the molecular level propagate through the sarcomeres, cells and tissue to modulate whole-muscle function. Conversely, movement and changes in the muscle length can influence cross-bridge kinetics on the molecular level. Reduced, single-molecule and single-fibre experiments have shown that increasing the strain on cross-bridges may slow their cycling rate and prolong their attachment duration. However, whether these strain-dependent cycling mechanisms persist in the intact muscle tissue, which encompasses more complex organization and passive elements, remains unclear. To investigate this multi-scale relationship, we adapted traditional step-stretch protocols for use with mouse soleus muscle during isometric tetanic contractions, enabling novel estimates of length-dependent cross-bridge kinetics in the intact skeletal muscle. Compared to rates at the optimal muscle length (Lo), we found that cross-bridge detachment rates increased by approximately 20% at 90% of Lo (shorter) and decreased by approximately 20% at 110% of Lo (longer). These data indicate that cross-bridge kinetics vary with whole-muscle length during intact, isometric contraction, which could intrinsically modulate force generation and energetics, and suggests a multi-scale feedback pathway between whole-muscle function and cross-bridge activity.

The spatial distribution of thin filament activation influences force development and myosin activity in computational models of muscle contraction.

Striated muscle contraction is initiated by Ca2+ binding to, and activating, thin filament regulatory units (RU) within the sarcomere, which then allows myosin cross-bridges from the opposing thick filament to bind actin and generate force. The amount of overlap between the filaments dictates how many potential cross-bridges are capable of binding, and thus how force is generated by the sarcomere. Myopathies and atrophy can impair muscle function by limiting cross-bridge interactions between the filaments, which can occur when the length of the thin filament is reduced or when RU function is disrupted. To investigate how variations in thin filament length and RU density affect ensemble cross-bridge behavior and force production, we simulated muscle contraction using a spatially explicit computational model of the half-sarcomere. Thin filament RUs were disabled either uniformly from the pointed end of the filament (to model shorter thin filament length) or randomly throughout the length of the half-sarcomere. Both uniform and random RU 'knockout' schemes decreased overall force generation during maximal and submaximal activation. The random knockout scheme also led to decreased calcium sensitivity and cooperativity of the force-pCa relationship. We also found that the rate of force development slowed with the random RU knockout, compared to the uniform RU knockout or conditions of normal RU activation. These findings imply that the relationship between RU density and force production within the sarcomere involves more complex coordination than simply the raw number of RUs available for myosin cross-bridge binding, and that the spatial pattern in which activatable RU are distributed throughout the sarcomere influences the dynamics of force production.

Omecamtiv Mecarbil Slows Myosin Kinetics in Skinned Rat Myocardium at Physiological Temperature.

Heart failure is a life-threatening condition that occurs when the heart muscle becomes weakened and cannot adequately circulate blood and nutrients around the body. Omecamtiv mecarbil (OM) is a compound that has been developed to treat systolic heart failure via targeting the cardiac myosin heavy chain to increase myocardial contractility. Biophysical and biochemical studies have found that OM increases calcium (Ca2+) sensitivity of contraction by prolonging the myosin working stroke and increasing the actin-myosin cross-bridge duty ratio. Most in vitro studies probing the effects of OM on cross-bridge kinetics and muscle force production have been conducted at subphysiological temperature, even though temperature plays a critical role in enzyme activity and cross-bridge function. Herein, we used skinned, ventricular papillary muscle strips from rats to investigate the effects of [OM] on Ca2+-activated force production, cross-bridge kinetics, and myocardial viscoelasticity at physiological temperature (37°C). We find that OM only increases myocardial contractility at submaximal Ca2+ activation levels and not maximal Ca2+ activation levels. As [OM] increased, the kinetic rate constants for cross-bridge recruitment and detachment slowed for both submaximal and maximal Ca2+-activated conditions. These findings support a mechanism by which OM increases cardiac contractility at physiological temperature via increasing cross-bridge contributions to thin-filament activation as cross-bridge kinetics slow and the duration of cross-bridge attachment increases. Thus, force only increases at submaximal Ca2+ activation due to cooperative recruitment of neighboring cross-bridges, because thin-filament activation is not already saturated. In contrast, OM does not increase myocardial force production for maximal Ca2+-activated conditions at physiological temperature because cooperative activation of thin filaments may already be saturated.

Demembranated skeletal and cardiac fibers produce less force with altered cross-bridge kinetics in a mouse model for limb-girdle muscular dystrophy 2i.

Limb-girdle muscular dystrophy 2i (LGMD2i) is a dystroglycanopathy that compromises myofiber integrity and primarily reduces power output in limb muscles but can influence cardiac muscle as well. Previous studies of LGMD2i made use of a transgenic mouse model in which a proline-to-leucine (P448L) mutation in fukutin-related protein severely reduces glycosylation of α-dystroglycan. Muscle function is compromised in P448L mice in a manner similar to human patients with LGMD2i. In situ studies reported lower maximal twitch force and depressed force-velocity curves in medial gastrocnemius (MG) muscles from male P448L mice. Here, we measured Ca2+-activated force generation and cross-bridge kinetics in both demembranated MG fibers and papillary muscle strips from P448L mice. Maximal activated tension was 37% lower in MG fibers and 18% lower in papillary strips from P448L mice than controls. We also found slightly faster rates of cross-bridge recruitment and detachment in MG fibers from P448L than control mice. These increases in skeletal cross-bridge cycling could reduce the unitary force output from individual cross bridges by lowering the ratio of time spent in a force-bearing state to total cycle time. This suggests that the decreased force production in LGMD2i may be due (at least in part) to altered cross-bridge kinetics. This finding is notable, as the majority of studies germane to muscular dystrophies have focused on sarcolemma or whole muscle properties, whereas our findings suggest that the disease pathology is also influenced by potential downstream effects on cross-bridge behavior.

Flightin maintains myofilament lattice organization required for optimal flight power and courtship song quality in Drosophila.

The indirect flight muscles (IFMs) of Drosophila and other insects with asynchronous flight muscles are characterized by a crystalline myofilament lattice structure. The high-order lattice regularity is considered an adaptation for enhanced power output, but supporting evidence for this claim is lacking. We show that IFMs from transgenic flies expressing flightin with a deletion of its poorly conserved N-terminal domain (flnΔN62 ) have reduced inter-thick filament spacing and a less regular lattice. This resulted in a decrease in flight ability by 33% and in skinned fibre oscillatory power output by 57%, but had no effect on wingbeat frequency or frequency of maximum power output, suggesting that the underlying actomyosin kinetics is not affected and that the flight impairment arises from deficits in force transmission. Moreover, we show that flnΔN62 males produced an abnormal courtship song characterized by a higher sine song frequency and a pulse song with longer pulses and longer inter-pulse intervals (IPIs), the latter implicated in male reproductive success. When presented with a choice, wild-type females chose control males over mutant males in 92% of the competition events. These results demonstrate that flightin N-terminal domain is required for optimal myofilament lattice regularity and IFM activity, enabling powered flight and courtship song production. As the courtship song is subject to female choice, we propose that the low amino acid sequence conservation of the N-terminal domain reflects its role in fine-tuning species-specific courtship songs.

Functional significance of C-terminal mobile domain of cardiac troponin I.

Ca2+-regulation of cardiac contractility is mediated through the troponin complex, which comprises three subunits: cTnC, cTnI, and cTnT. As intracellular [Ca2+] increases, cTnI reduces its binding interactions with actin to primarily interact with cTnC, thereby enabling contraction. A portion of this regulatory switching involves the mobile domain of cTnI (cTnI-MD), the role of which in muscle contractility is still elusive. To study the functional significance of cTnI-MD, we engineered two cTnI constructs in which the MD was truncated to various extents: cTnI(1-167) and cTnI(1-193). These truncations were exchanged for endogenous cTnI in skinned rat papillary muscle fibers, and their influence on Ca2+-activated contraction and cross-bridge cycling kinetics was assessed at short (1.9 µm) and long (2.2 µm) sarcomere lengths (SLs). Our results show that the cTnI(1-167) truncation diminished the SL-induced increase in Ca2+-sensitivity of contraction, but not the SL-dependent increase in maximal tension, suggesting an uncoupling between the thin and thick filament contributions to length dependent activation. Compared to cTnI(WT), both truncations displayed greater Ca2+-sensitivity and faster cross-bridge attachment rates at both SLs. Furthermore, cTnI(1-167) slowed MgADP release rate and enhanced cross-bridge binding. Our findings imply that cTnI-MD truncations affect the blocked-to closed-state transition(s) and destabilize the closed-state position of tropomyosin.

Myosin MgADP Release Rate Decreases as Sarcomere Length Increases in Skinned Rat Soleus Muscle Fibers.

Actin-myosin cross-bridges use chemical energy from MgATP hydrolysis to generate force and shortening in striated muscle. Previous studies show that increases in sarcomere length can reduce thick-to-thin filament spacing in skinned muscle fibers, thereby increasing force production at longer sarcomere lengths. However, it is unclear how changes in sarcomere length and lattice spacing affect cross-bridge kinetics at fundamental steps of the cross-bridge cycle, such as the MgADP release rate. We hypothesize that decreased lattice spacing, achieved through increased sarcomere length or osmotic compression of the fiber via dextran T-500, could slow MgADP release rate and increase cross-bridge attachment duration. To test this, we measured cross-bridge cycling and MgADP release rates in skinned soleus fibers using stochastic length-perturbation analysis at 2.5 and 2.0 µm sarcomere lengths as pCa and [MgATP] varied. In the absence of dextran, the force-pCa relationship showed greater Ca2+ sensitivity for 2.5 vs. 2.0 µm sarcomere length fibers (pCa50 = 5.68 ± 0.01 vs. 5.60 ± 0.01). When fibers were compressed with 4% dextran, the length-dependent increase in Ca2+ sensitivity of force was attenuated, though the Ca2+ sensitivity of the force-pCa relationship at both sarcomere lengths was greater with osmotic compression via 4% dextran compared to no osmotic compression. Without dextran, the cross-bridge detachment rate slowed by ∼15% as sarcomere length increased, due to a slower MgADP release rate (11.2 ± 0.5 vs. 13.5 ± 0.7 s-1). In the presence of dextran, cross-bridge detachment was ∼20% slower at 2.5 vs. 2.0 µm sarcomere length due to a slower MgADP release rate (10.1 ± 0.6 vs. 12.9 ± 0.5 s-1). However, osmotic compression of fibers at either 2.5 or 2.0 µm sarcomere length produced only slight (and statistically insignificant) slowing in the rate of MgADP release. These data suggest that skeletal muscle exhibits sarcomere-length-dependent changes in cross-bridge kinetics and MgADP release that are separate from, or complementary to, changes in lattice spacing.

Effects of myosin light chain phosphorylation on length-dependent myosin kinetics in skinned rat myocardium.

Myosin force production is Ca(2+)-regulated by thin-filament proteins and sarcomere length, which together determine the number of cross-bridge interactions throughout a heartbeat. Ventricular myosin regulatory light chain-2 (RLC) binds to the neck of myosin and modulates contraction via its phosphorylation state. Previous studies reported regional variations in RLC phosphorylation across the left ventricle wall, suggesting that RLC phosphorylation could alter myosin behavior throughout the heart. We found that RLC phosphorylation varied across the left ventricle wall and that RLC phosphorylation was greater in the right vs. left ventricle. We also assessed functional consequences of RLC phosphorylation on Ca(2+)-regulated contractility as sarcomere length varied in skinned rat papillary muscle strips. Increases in RLC phosphorylation and sarcomere length both led to increased Ca(2+)-sensitivity of the force-pCa relationship, and both slowed cross-bridge detachment rate. RLC-phosphorylation slowed cross-bridge rates of MgADP release (∼30%) and MgATP binding (∼50%) at 1.9 µm sarcomere length, whereas RLC phosphorylation only slowed cross-bridge MgATP binding rate (∼55%) at 2.2 µm sarcomere length. These findings suggest that RLC phosphorylation influences cross-bridge kinetics differently as sarcomere length varies and support the idea that RLC phosphorylation could vary throughout the heart to meet different contractile demands between the left and right ventricles.

Myosin MgADP release rate decreases at longer sarcomere length to prolong myosin attachment time in skinned rat myocardium.

Cardiac contractility increases as sarcomere length increases, suggesting that intrinsic molecular mechanisms underlie the Frank-Starling relationship to confer increased cardiac output with greater ventricular filling. The capacity of myosin to bind with actin and generate force in a muscle cell is Ca(2+) regulated by thin-filament proteins and spatially regulated by sarcomere length as thick-to-thin filament overlap varies. One mechanism underlying greater cardiac contractility as sarcomere length increases could involve longer myosin attachment time (ton) due to slowed myosin kinetics at longer sarcomere length. To test this idea, we used stochastic length-perturbation analysis in skinned rat papillary muscle strips to measure ton as [MgATP] varied (0.05-5 mM) at 1.9 and 2.2 µm sarcomere lengths. From this ton-MgATP relationship, we calculated cross-bridge MgADP release rate and MgATP binding rates. As MgATP increased, ton decreased for both sarcomere lengths, but ton was roughly 70% longer for 2.2 vs. 1.9 µm sarcomere length at maximally activated conditions. These ton differences were driven by a slower MgADP release rate at 2.2 µm sarcomere length (41 ± 3 vs. 74 ± 7 s(-1)), since MgATP binding rate was not different between the two sarcomere lengths. At submaximal activation levels near the pCa50 value of the tension-pCa relationship for each sarcomere length, length-dependent increases in ton were roughly 15% longer for 2.2 vs. 1.9 µm sarcomere length. These changes in cross-bridge kinetics could amplify cooperative cross-bridge contributions to force production and thin-filament activation at longer sarcomere length and suggest that length-dependent changes in myosin MgADP release rate may contribute to the Frank-Starling relationship in the heart.

Random myosin loss along thick-filaments increases myosin attachment time and the proportion of bound myosin heads to mitigate force decline in skeletal muscle.

Diminished skeletal muscle performance with aging, disuse, and disease may be partially attributed to the loss of myofilament proteins. Several laboratories have found a disproportionate loss of myosin protein content relative to other myofilament proteins, but due to methodological limitations, the structural manifestation of this protein loss is unknown. To investigate how variations in myosin content affect ensemble cross-bridge behavior and force production we simulated muscle contraction in the half-sarcomere as myosin was removed either (i) uniformly, from the Z-line end of thick-filaments, or (ii) randomly, along the length of thick-filaments. Uniform myosin removal decreased force production, showing a slightly steeper force-to-myosin content relationship than the 1:1 relationship that would be expected from the loss of cross-bridges. Random myosin removal also decreased force production, but this decrease was less than observed with uniform myosin loss, largely due to increased myosin attachment time (ton) and fractional cross-bridge binding with random myosin loss. These findings support our prior observations that prolonged ton may augment force production in single fibers with randomly reduced myosin content from chronic heart failure patients. These simulations also illustrate that the pattern of myosin loss along thick-filaments influences ensemble cross-bridge behavior and maintenance of force throughout the sarcomere.

Kinetics of cardiac myosin isoforms in mouse myocardium are affected differently by presence of myosin binding protein-C.

We tested whether cardiac myosin binding protein-C (cMyBP-C) affects myosin cross-bridge kinetics in the two cardiac myosin heavy chain (MyHC) isoforms. Mice lacking cMyBP-C (t/t) and transgenic controls (WT(t/t)) were fed L-thyroxine (T4) to induce 90/10% expression of α/ß-MyHC. Non-transgenic (NTG) and t/t mice were fed 6-n-propyl-2-thiouracil (PTU) to induce 100% expression of ß-MyHC. Ca(2+)-activated, chemically-skinned myocardium underwent length perturbation analysis with varying [MgATP] to estimate the MgADP release rate (k(-ADP)) and MgATP binding rate (k(+ATP)). Values for (k(-ADP)) were not significantly different between t/t(T4) (102.2 ± 7.0 s(-1)) and WT(t/t)(T4) (91.3 ± 8.9 s(-1)), but k(+ATP)) was lower in t/t(T4) (165.9 ± 12.5 mM(-1) s(-1)) compared to WT(t/t)(T4) (298.6 ± 15.7 mM(-1) s(-1), P < 0.01). In myocardium expressing ß-MyHC, values for k(-ADP) were higher in t/t(PTU) (24.8 ± 1.0 s(-1)) compared to NTG(PTU) (15.6 ± 1.3 s(-1), P < 0.01), and k(+ATP) was not different. At saturating [MgATP], myosin detachment rate approximates k(-ADP), and detachment rate decreased as sarcomere length (SL) was increased in both t/t(T4) and WT(t/t)(T4) with similar sensitivities to SL. In myocardium expressing ß-MyHC, detachment rate decreased more as SL increased in t/t(PTU) (21.5 ± 1.3 s(-1) at 2.2 µm and 13.3 ± 0.9 s(-1) at 3.3 µm) compared to NTGPTU (15.8 ± 0.3 s(-1) at 2.2 µm and 10.9 ± 0.3 s(-1) at 3.3 µm) as detected by repeated-measures ANOVA (P < 0.01). These findings suggest that cMyBP-C reduces MgADP release rate for ß-MyHC, but not for α-MyHC, even as the number of cMyBP-C that overlap with the thin filament is reduced to zero. Therefore, cMyBP-C appears to affect ß-MyHC kinetics independent of its interaction with the thin filament.

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

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.

An inverse power-law distribution of molecular bond lifetimes predicts fractional derivative viscoelasticity in biological tissue.

Viscoelastic characteristics of many materials falling under the category of soft glassy substances, including biological tissue, often exhibit a mechanical complex modulus Y(ω) well described by a fractional derivative model: Y(ω) = E(iω/ϕ)k, where E = a generalized viscoelastic stiffness; i = (-1)1/2; ω = angular frequency; ϕ = scaling factor; and k = an exponent valued between 0 and 1. The term "fractional derivative" refers to the value of k: when k = 0 the viscoelastic response is purely elastic, and when k = 1 the response is purely viscous. We provide an analytical derivation of the fractional derivative complex modulus based on the hypothesis that the viscoelastic response arises from many intermittent molecular crosslinks, whose lifetimes longer than a critical threshold lifetime, tcrit, are distributed with an inverse power law proportional to t-(k+2). We demonstrate that E is proportional to the number and stiffness of crosslinks formed at any moment; the scaling factor ϕ is equivalent to reciprocal of tcrit; and the relative mean lifetime of the attached crosslinks is inversely proportional to the parameter k. To test whether electrostatic molecular bonds could be responsible for the fractional derivative viscoelasticity, we used chemically skinned human skeletal muscle as a one-dimensional model of a soft glassy substance. A reduction in ionic strength from 175 to 110 mEq resulted in a larger E with no change in k, consistent with a higher probability of interfilament molecular interactions. Thick to thin filament spacing was reduced by applying 4% w/v of the osmolyte Dextran T500, which also resulted in a larger E, indicating a greater probability of crosslink formation in proportion to proximity. A 10°C increase in temperature resulted in an increase in k, which corresponded to a decrease in cross-bridge attachment lifetime expected with higher temperatures. These theoretical and experimental results suggest that the fractional derivative viscoelasticity observed in some biological tissue arises as a mechanical consequence of electrostatic interactions, whose longest lifetimes are distributed with an inverse power law.