Cross-bridge Binding Research Articles

Cooperative mechanisms underlie differences in myocardial contractile dynamics between large and small mammals.

Ca2+ binding to troponin C (TnC) and myosin cross-bridge binding to actin act in a synergistic cooperative manner to modulate myocardial contraction and relaxation. The responsiveness of the myocardial thin filament to the activating effects of Ca2+ and myosin cross-bridge binding has been well-characterized in small mammals (e.g., mice). Given the nearly 10-fold difference in resting heart rates and twitch kinetics between small and large mammals, it is unlikely that the cooperative mechanisms underlying thin filament activation are identical in these two species. To test this idea, we measured the Ca2+ dependencies of steady-state force and the rate constant of force redevelopment (ktr) in murine and porcine permeabilized ventricular myocardium. While murine myocardium exhibited a steep activation-dependence of ktr, the activation-dependent profile of ktr was significantly reduced in porcine ventricular myocardium. Further insight was attained by examining force-pCa and ktr-pCa relationships. In the murine myocardium, the pCa50 for ktr was right-shifted compared with the pCa50 for force, meaning that increases in steady-state force occurred well before increases in the rate of force redevelopment were observed. In the porcine myocardium, we observed a tighter coupling of the force-pCa and ktr-pCa relationships, as evidenced by near-maximal rates of force redevelopment at low levels of Ca2+ activation. These results demonstrate that the molecular mechanisms underlying the cooperative activation of force are a dynamic property of the mammalian heart, involving, at least in part, the species- and tissue-specific expression of cardiac myosin heavy chain isoforms.

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.

Sensitivity of muscle force response of a two-state cross-bridge model to variations in model parameters.

Muscle models based on the cross-bridge theory (Huxley-type models) are frequently used to calculate muscle forces for different contractile conditions. Dynamic and nonlinear characteristics of muscle forces produced during isometric, concentric, and eccentric contractions can be represented to a limited extent by using cross-bridge models. Cross-bridge models use various parameters to simulate force responses. However, there remains uncertainty as to the effect of changes in model parameters on force responses in Huxley-type models. In this study, we aimed to analyze the sensitivity of force response to changes in model parameters in Huxley-type models. A two-state Huxley model was used to determine the cross-bridge attachment distributions and forces for shortening and lengthening contractions. Sensitivity of muscle force to changes in attachment rate, detachment rate, and cross-bridge binding distance was examined within a range of ±20% of the nominal value using Monte Carlo simulations. Changes in the detachment rate influenced the predicted muscle forces the most for lengthening contractions, while changes in attachment rate and binding distance affected forces the most for shortening contractions. These results show once more the asymmetry between shortening and lengthening contractions and the difficulty in using a single cross-bridge model to predict forces during shortening and elongation accurately.

2-Deoxy-ATP improves ventricular function via combined effects on myosin recruitment from the super-relaxed state, crossbridge cycling and calcium dynamics in healthy and failing conditions

2-deoxy-ATP (dATP) has been shown to enhance cardiac contractility by acting on myosin to increase the rate of crossbridge binding and cycling. An increased rate of Ca2+ transient decay has also been observed with elevated dATP. However, it is unclear how dATP affects feedback between sarcomere force and Ca2+ dynamics, and to what degree these cellular level effects can explain experimentally observed improvements in ventricular function with elevated dATP. Further, low concentrations of dATP have been shown to significantly improve cardiomyocyte and ventricular function, but the mechanisms behind this are not well understood.

2'-Deoxy-ATP release myosin heads from the super relaxed state in permeabilized porcine myocardium

The nucleotide 2-deoxy-ATP (dATP) is a naturally occurring energy substrate for myosin that enhances cross-bridge binding and cycling kinetics. In relaxed rodent cardiac and skeletal muscle, dATP repositions myosin heads away from the thick filament backbone towards thin filaments, but the effects of dATP on the myocardium from large mammals has been much less explored. Here we titrated permeabilized porcine myocardium in relaxing solution with a series of dATP concentrations ranging from 0 to 100% of the total nucleotide pool and examined myosin and sarcomere structure using small-angle X-ray diffraction.

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.

CMyBP-C phosphorylation modulates the time-dependent slowing of unloaded shortening in murine skinned myocardium.

In myocardium, phosphorylation of cardiac myosin-binding protein-C (cMyBP-C) is thought to modulate the cooperative activation of the thin filament by binding to myosin and/or actin, thereby regulating the probability of cross-bridge binding to actin. At low levels of Ca2+ activation, unloaded shortening velocity (Vo) in permeabilized cardiac muscle is comprised of an initial high-velocity phase and a subsequent low-velocity phase. The velocities in these phases scale with the level of activation, culminating in a single high-velocity phase (Vmax) at saturating Ca2+. To test the idea that cMyBP-C phosphorylation contributes to the activation dependence of Vo, we measured Vo before and following treatment with protein kinase A (PKA) in skinned trabecula isolated from mice expressing either wild-type cMyBP-C (tWT), nonphosphorylatable cMyBP-C (t3SA), or phosphomimetic cMyBP-C (t3SD). During maximal Ca2+ activation, Vmax was monophasic and not significantly different between the three groups. Although biphasic shortening was observed in all three groups at half-maximal activation under control conditions, the high- and low-velocity phases were faster in the t3SD myocardium compared with values obtained in either tWT or t3SA myocardium. Treatment with PKA significantly accelerated both the high- and low-velocity phases in tWT myocardium but had no effect on Vo in either the t3SD or t3SA myocardium. These results can be explained in terms of a model in which the level of cMyBP-C phosphorylation modulates the extent and rate of cooperative spread of myosin binding to actin.

Fibersim, an Open-Source Spatially-Explicit Model of the Half-Sarcomere

FiberSim (https://campbell-muscle-lab.github.io/FiberSim/) is an open source spatially explicit model of a half-sarcomere, that represents a hexagonal lattice of compliant thin and thick filaments. Similar to prior frameworks developed by the Daniel, Tanner, and Mijailovich groups, FiberSim incorporates a variety of molecular mechanisms taking place during striated muscle contraction, such as thin filament activation and cross-bridge binding. Like any mathematical model of a biological system, FiberSim provides a convenient way to test hypotheses about the role of different molecular mechanisms and is a useful adjunct to experimental techniques.

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.

The Positively Charged C-Terminal Region of Human Skeletal Troponin T Retards Activation and Decreases Calcium Sensitivity

Calcium binding to troponin C (TnC) activates striated muscle contraction by removing TnI (troponin I) from its inhibitory site on actin. Troponin T (TnT) links TnI with tropomyosin, causing tropomyosin to move from an inhibitory position on actin to an activating position. Positive charges within the C-terminal region of human cardiac TnT limit Ca2+ activation. We now show that the positively charged region of TnT has an even larger impact on skeletal muscle regulation. We prepared one variant of human skeletal TnT that had the C-terminal 16 residues truncated (Δ16) and another with an added C-terminal Cys residue and Ala substituted for the last 6 basic residues (251C-HAHA). Both mutants reduced (based on S1 binding kinetics) or eliminated (based on acrylodan-tropomyosin fluorescence) the first inactive state of actin at <10 nM free Ca2+. 251C-HAHA-TnT and Δ16-TnT mutants greatly increased ATPase activation at 0.2 mM Ca2+, even without high-affinity cross-bridge binding. They also shifted the force-pCa curve of muscle fibers to lower Ca2+ by 0.8-1.2 pCa units (the larger shift for 251C-HAHA-TnT). Shifts in force-pCa were maintained in the presence of para-aminoblebbistatin. The effects of modification of the C-terminal region of TnT on the kinetics of S1 binding to actin were somewhat different from those observed earlier with the cardiac analogue. In general, the C-terminal region of human skeletal TnT is critical to regulation, just as it is in the cardiac system, and is a potential target for modulating activity.

Myosin dynamics during relaxation in mouse soleus muscle and modulation by 2'-deoxy-ATP.

Skeletal muscle relaxation has been primarily studied by assessing the kinetics of force decay. Little is known about the resultant dynamics of structural changes in myosin heads during relaxation. The naturally occurring nucleotide 2-deoxy-ATP (dATP) is a myosin activator that enhances cross-bridge binding and kinetics. X-ray diffraction data indicate that with elevated dATP, myosin heads were extended closer to actin in relaxed muscle and myosin heads return to an ordered, resting state after contraction more quickly. Molecular dynamics simulations of post-powerstroke myosin suggest that dATP induces structural changes in myosin heads that increase the surface area of the actin-binding regions promoting myosin interaction with actin, which could explain the observed delays in the onset of relaxation. This study of the dATP-induced changes in myosin may be instructive for determining the structural changes desired for other potential myosin-targeted molecular compounds to treat muscle diseases. Here we used time-resolved small-angle X-ray diffraction coupled with force measurements to study the structural changes in FVB mouse skeletal muscle sarcomeres during relaxation after tetanus contraction. To estimate the rate of myosin deactivation, we followed the rate of the intensity recovery of the first-order myosin layer line (MLL1) and restoration of the resting spacing of the third and sixth order of meridional reflection (SM3 and SM6 ) following tetanic contraction. A transgenic mouse model with elevated skeletal muscle 2-deoxy-ATP (dATP) was used to study how myosin activators may affect soleus muscle relaxation. X-ray diffraction evidence indicates that with elevated dATP, myosin heads were extended closer to actin in resting muscle. Following contraction, there is a slight but significant delay in the decay of force relative to WT muscle while the return of myosin heads to an ordered resting state was initially slower, then became more rapid than in WT muscle. Molecular dynamics simulations of post-powerstroke myosin suggest that dATP induces structural changes in myosin that increase the surface area of the actin-binding regions, promoting myosin interaction with actin. With dATP, myosin heads may remain in an activated state near the thin filaments following relaxation, accounting for the delay in force decay and the initial delay in recovery of resting head configuration, and this could facilitate subsequent contractions.

Cycling Cross-Bridges Contribute to Thin Filament Activation in Human Slow-Twitch Fibers.

It has been shown that not only calcium but also strong binding myosin heads contribute to thin filament activation in isometrically contracting animal fast-twitch and cardiac muscle preparations. This behavior has not been studied in human muscle fibers or animal slow-twitch fibers. Human slow-twitch fibers are interesting since they contain the same myosin heavy chain isoform as the human heart. To explore myosin-induced activation of the thin filament in isometrically contracting human slow-twitch fibers, the endogenous troponin complex was exchanged for a well-characterized fast-twitch skeletal troponin complex labeled with the fluorescent dye N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (fsTn-IANBD). The exchange was ≈70% complete (n = 8). The relative contributions of calcium and strong binding cross-bridges to thin filament activation were dissected by increasing the concentration of calcium from relaxing (pCa 7.5) to saturating levels (pCa 4.5) before and after incubating the exchanged fibers in the myosin inhibitor para-aminoblebbistatin (AmBleb). At pCa 4.5, the relative contributions of calcium and strong binding cross-bridges to thin filament activation were ≈69 and ≈31%, respectively. Additionally, switching from isometric to isotonic contraction at pCa 4.5 revealed that strong binding cross-bridges contributed ≈29% to thin filament activation (i.e., virtually the same magnitude obtained with AmBleb). Thus, we showed through two different approaches that lowering the number of strong binding cross-bridges, at saturating calcium, significantly reduced the activation of the thin filament in human slow-twitch fibers. The contribution of myosin to activation resembled that which was previously reported in rat cardiac and rabbit fast-twitch muscle preparations. This method could be applied to slow-twitch human fibers obtained from the soleus muscle of cardiomyopathy patients. Such studies could lead to a better understanding of the effect of point mutations of the cardiac myosin head on the regulation of muscle contraction and could lead to better management by pharmacological approaches.

Passive force enhancement in striated muscle.

Passive force enhancement is defined as the increase in passive, steady-state, isometric force of an actively stretched muscle compared with the same muscle stretched passively to that same length. Passive force enhancement is long lasting, increases with increasing muscle length and increasing stretch magnitudes, contributes to the residual force enhancement in skeletal and cardiac muscle, and is typically only observed at muscle lengths at which passive forces occur naturally. Passive force enhancement is typically equal to or smaller than the total residual force enhancement, it persists when a muscle is deactivated and reactivated, but can be abolished instantaneously when a muscle is shortened quickly from its stretched length. There is strong evidence that the passive force enhancement is caused by the filamentous sarcomeric protein titin, although the detailed molecular mechanisms underlying passive force enhancement remain unknown. Here I propose a tentative mechanism based on experimental evidence that associates passive force enhancement with the shortening of titin's free spring length in the I-band region of sarcomeres. I suggest that this shortening is accomplished by titin binding to actin and that the trigger for titin-actin interactions is associated with the formation of strongly bound cross bridges between actin and myosin that exposes actin attachment sites for titin through movement of the regulatory proteins troponin and tropomyosin.

The actin 'A-triad's' role in contractile regulation in health and disease.

Striated muscle contraction is regulated by Ca2+ -dependent modulation of myosin cross-bridge binding to F-actin by the thin filament troponin (Tn)-tropomyosin (Tm) complex. In the absence of Ca2+ , Tn binds to actin and constrains Tm to an azimuthal location where it sterically occludes myosin binding sites along the thin filament surface. This limits force production and promotes muscle relaxation. In addition to Tn-actin interactions, inhibitory Tm positioning requires associations between other thin filament constituents. For example, the actin 'A-triad', composed of residues K326, K328 and R147, forms numerous, highly favourable electrostatic contacts with Tm that are critical for establishing its inhibitory azimuthal binding position. Here, we review recent findings, including the identification and interrogation of modifications within and proximal to the A-triad that are associated with disease and/or altered muscle behaviour, which highlight the surface feature's role in F-actin-Tm interactions and contractile regulation.

Changes in the fraction of strongly attached cross bridges in mouse atrophic and hypertrophic muscles as revealed by continuous wave electron paramagnetic resonance.

Electron paramagnetic resonance (EPR), coupled with site-directed spin labeling, has been proven to be a particularly suitable technique to extract information on the fraction of myosin heads strongly bound to actin upon muscle contraction. The approach can be used to investigate possible structural changes occurring in myosin of fiber s altered by diseases and aging. In this work, we labeled myosin at position Cys707, located in the SH1-SH2 helix in the myosin head cleft, with iodoacetamide spin label, a spin label that is sensitive to the reorientational motion of this protein during the ATPase cycle and characterized the biochemical states of the labeled myosin head by means of continuous wave EPR. After checking the sensitivity and the power of the technique on different muscles and species, we investigated whether changes in the fraction of strongly bound myosin heads might explain the contractile alterations observed in atrophic and hypertrophic murine muscles. In both conditions, the difference in contractile force could not be justified simply by the difference in muscle mass. Our results showed that in atrophic muscles the decrease in force generation was attributable to a lower fraction of strongly bound cross bridges during maximal activation. In contrast in hypertrophic muscles, the increase in force generation was likely due to several factors, as pointed out by the comparison of the EPR experiments with the tension measurements on single skinned fibers.

X-Ray Diffraction Resolves How Actin-Myosin Spacing Explains the Differences of Two Muscles with Identical Steady State Properties

The actin-myosin lattice underlying muscle is dynamic. Radial lattice spacing changes during activation and during periodic axial strain like muscle experiences during locomotion. Actin-myosin spacing in muscle affects crossbridge binding rates and the steady-state length-tension properties of muscle. The interaction of macroscopic strain, lattice spacing and binding kinetics raises the possibility that small changes in lattice spacing could have profound consequences for the dynamic behavior of muscle. To study the functional implications of lattice spacing, we examined two muscles in Blaberus discoidalis. These muscles have nearly identical length-tension and force-velocity relationships and share innervation. However, when periodically activated under identical cyclical length changes, one of them does net positive mechanical work while the other is dissipative. Using the BioCAT x-ray beamline at the Advanced Photon Source at Argonne National Lab, we measured lattice spacing during dynamic oscillations and isometric twitches. We found passive lattice spacing in the two muscles to be 50.01+/−1.54 nm and 50.51+/−.6 nm (95% CI) with the second muscle's spacing consistently higher (p≈10−5). We found that in the isometric twitch case, the first muscle's lattice spacing increased to 51.15+/−1.7 while the other did not change significantly (51.3+/−.65), meaning that the spacing difference vanishes under steady state, activated conditions (p≈.19). A difference in spacing under passive conditions, but not active, means that during cyclic length changes the lattice spacing changes were larger in one muscle. In the muscle with the larger dynamic range, lattice spacing correlates with the timing of increased stress and positive mechanical work. A 1 nm transient difference in actin-myosin spacing mediates the dynamic difference between the two muscles (motor vs. brake) but also predicts their identical quasi-static properties, allowing nanoscale structure to influence macroscopic muscle function.

Enhanced Crossbridge Binding with 2-Deoxy-ATP Results from Increased Electrostatic Interactions between Myosin and Actin in Cardiac Muscle

Enhanced Crossbridge Binding with 2-Deoxy-ATP Results from Increased Electrostatic Interactions between Myosin and Actin in Cardiac Muscle

Molecular Dynamics Studies of Myosin Structure with 2-Deoxy-ADP

Myosin uses ATP catalysis to drive muscle contraction by facilitating cross-bridge cycling. During the cross-bridge powerstroke, coordinated release of the byproducts of nucleotide catalysis (Pi, ADP) triggers a series of conformational changes in myosin that alter its interactions with actin and ultimately generate force. In addition to ATP, myosins can utilize other nucleotides, such as the naturally occurring 2-deoxy-ATP (dATP), to catalyze the powerstroke. We have reported that dATP enhances cross-bridge binding and cycling dynamics, resulting in stronger, faster contraction especially in cardiac muscle. In a recent study (Nowakowski 2017, Protein Sci 26:749-62) our simulations of myosin in the pre-powerstroke state showed that dADP induces conformational changes that expose polar residues and enhance its affinity for actin. In a current study using x-ray structural analysis, we found that dATP increases the rate of myosin detachment following tetanic contraction. To understand the molecular basis for the enhanced rate of detachment in the presence of elevated dATP, we performed molecular dynamics simulations of the myosin motor domain modeled in the post-powerstroke conformation in the presence of Mg2+ and either ADP or dADP. We found that dADP was more flexible than ADP, more mobile within the nucleotide binding pocket and had different interactions with myosin relative to ADP. Furthermore, changes in the interactions between dADP and myosin were transmitted to the actin binding surface. Additionally, we observed partial release of the nucleotides from the binding pocket and have delineated the molecular mechanisms by which regulatory loops in myosin control nucleotide release. Our results have revealed molecular mechanisms underlying dATP-enhanced cross-bridge cycling dynamics. More generally they have yielded insights for the development of novel myosin-targeted therapies for cardiomyopathies based the structural advantages produced by dATP in resting and contracting muscle.

Prolonged cross-bridge binding triggers muscle dysfunction in a Drosophila model of myosin-based hypertrophic cardiomyopathy.

K146N is a dominant mutation in human β-cardiac myosin heavy chain, which causes hypertrophic cardiomyopathy. We examined how Drosophila muscle responds to this mutation and integratively analyzed the biochemical, physiological and mechanical foundations of the disease. ATPase assays, actin motility, and indirect flight muscle mechanics suggest at least two rate constants of the cross-bridge cycle are altered by the mutation: increased myosin attachment to actin and decreased detachment, yielding prolonged binding. This increases isometric force generation, but also resistive force and work absorption during cyclical contractions, resulting in decreased work, power output, flight ability and degeneration of flight muscle sarcomere morphology. Consistent with prolonged cross-bridge binding serving as the mechanistic basis of the disease and with human phenotypes, 146N/+ hearts are hypercontractile with increased tension generation periods, decreased diastolic/systolic diameters and myofibrillar disarray. This suggests that screening mutated Drosophila hearts could rapidly identify hypertrophic cardiomyopathy alleles and treatments.

Abstract 439: Elevating 2-deoxy Adenosine Triphosphate Improves Contraction in a Mouse Model of Genetic Dilated Cardiomyopathy

Several studies have identified reduced tension and Ca 2+ sensitivity as key regulators that precipitate dilated cardiomyopathy (DCM). We are developing a novel therapy to increase tension using adeno-associated viral (AAV) vectors that increase intracellular 2 deoxy-ATP (dATP) via overexpression of the enzyme ribonucleotide reductase (RNR). We have reported that dATP substitution for ATP increases the magnitude and rate of contraction and that RNR overexpression increases intracellular dATP and enhances cardiac function. Here we use a multiscale approach to test the effect of dATP in a genetic model of dilated cardiomyopathy (DCM) with a missense mutation (D230N) in alpha-tropomyosin (Tm). Using recombinant proteins in the in vitro motility assay, we found that filament velocity was lower with D230N compared to WT Tm (2.03±0.04 vs. 2.44±0.04 μm/s). This deficit was corrected when dATP was substituted for ATP (2.90±0.04 μm/s). Next, we used transgenic D230N mice that show progressive ventricular dilation and systolic dysfunction. Intact and demembranated trabeculae from these mice have decreased tension and calcium sensitivity of force, and loss of length-dependent activation (LDA) compared to WT mice. Substitution of dATP for ATP, increased Ca 2+ sensitivity in D230N Tm myocardium (pCa 50 =5.48±0.02 vs. 5.57±0.02) and restored LDA. Complete replacement of ATP with dATP resulted in an ~70% increase in force at submaximal [Ca 2+ ] and force at submaximal Ca 2+ was significantly increased by replacement of only 5% of the nucleotide pool with dATP. For isolated myofibrils from D230N Tm and WT mice, tension at submaximal Ca 2+ (pCa=5.6) was lower in D230N mice compared to WT mice (51±4 vs. 75±8 mN/mm 2 ) and this deficit was improved with dATP (68±5 mN/mm 2 ). Similarly, the rate of activation in D230N myofibrils was slower than in WT mice (2.1±0.2 vs.3.3±0.2 s -1 ) and partially corrected with dATP (2.7±0.2 s -1 ). These results demonstrate that enhanced cross bridge binding, via increasing dATP, can recover lost contractile function in a thin filament mutation model of DCM. In ongoing studies, we are testing the ability of elevated dATP, via AAV- RNR, to protect against reduced systolic function and progressive ventricular dilation in 2-3 week old D230N mice .