Biology subjects

Childers, M. C.

Publications and source records attributed to Childers, M. C..

12 recordsLinked to original sources

bioRxiv2026

The Dilated Cardiomyopathy E525K Mutation Stabilizes the Cardiac Myosin Interacting-Heads Motif While Activating the Isolated Motor Domain

Mutations in {beta}-cardiac myosin are a common cause of inherited cardiomyopathies. The dilated cardiomyopathy E525K mutation alters both thick filament regulation and motor activity, yet the structural basis for these effects has remained unclear. Here, we combined cryo-EM and MD simulations to determine how E525K affects the conformational landscape of cardiac myosin in both its autoinhibited interacting-heads motif (IHM) and isolated myosin head states. E525K-mutated myosin exclusively adopted a single S2 conformation in IHM, in contrast to the conformational heterogeneity observed in wild-type myosin. Structural and electrostatic analyses revealed that the mutation increased positive charge density at the blocked-head (BH) - S2 (subfragment-2) interface, strengthening interactions with negatively charged residues within S2. 3D variability analysis and MD simulations of the E525K IHM demonstrated reduced S2 mobility, consistent with strengthened BH - S2 interactions, and decreased conformational flexibility, providing a structural mechanism for stabilization of the IHM state. To investigate the isolated myosin head, we determined cryo-EM structures of wild-type and E525K subfragment-1 (S1). The mutation induced local conformational rearrangements in the activation-loop and Loop 3 region, the SH3-like domain, and the essential light chain of S1, revealing structural changes that support the enhanced motor activity previously reported for isolated E525K myosin motors. These findings show that E525K stabilizes the autoinhibited IHM through electrostatic stabilization of the BH - S2 interface while inducing structural changes consistent with activation of the isolated motor domain, providing structural insights into how a single DCM mutation differentially regulates cardiac myosin S1 and IHM structure and function.

biophysics↗

bioRxiv2026

The Dilated Cardiomyopathy E525K β-Myosin Mutation Causes Hypocontractility in Cardiomyocytes Without Altering Crossbridge Cycling

The cardiac {beta}-myosin (MYH7) mutation E525K was identified in 2012 in a patient with dilated cardiomyopathy (DCM). Work using engineered constructs has shown that this mutation stabilizes the interacting heads motif (IHM) of myosin and increases the ATPase activity of mutant motor S1 heads. However, no measurements have been made in myofilaments or cardiomyocytes to determine its effect on contractile function. Here, we present force and contractile kinetics measurements from induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) engineered for heterozygous expression of E525K. Single-cell contraction for E525K hiPSC-CMs decreased by 65%, and isometric twitch force in engineered heart tissues (EHTs) decreased by 39%. In contrast, maximal Ca2+ activated isometric force in isolated myofibrils increased by 45% and sub-maximal Ca2+ activated force was similar to WT myofibrils. Structural analysis revealed reduced myofibril content (13.7% decrease) and decreased organization (increased z-disk dispersion angle) in E525K cells. We confirmed that E525K S1 myosin has higher actin affinity than WT S1 and elevated ATPase activity. However, there was no change in S1 ADP release rate. There was also no change in either the rate of force development or relaxation in myofibrils, cells, or EHTs. These findings suggest that E525K myosin crossbridge cycling is not altered during loaded contractions. To understand how twitch force of myocytes was reduced but maximal isometric force was increased, we used a spatially explicit sarcomere model. The results were explained by changing three rates: reduced recruitment from the OFF/IHM state, and increased rates of actin binding and Pi release. Additional force deficits in cells and EHTs likely result from the myofibrillar disorganization. This study demonstrates the value of multi-scale analysis and coupled, computational modeling to understand the molecular mechanisms of sarcomere mutations in cardiomyocytes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/733270v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1a64b4aorg.highwire.dtl.DTLVardef@98f053org.highwire.dtl.DTLVardef@c9f839org.highwire.dtl.DTLVardef@1c795c6_HPS_FORMAT_FIGEXP M_FIG C_FIG A model for how the E525K mutation impacts contracting myofibrils Here, we show that the E525K mutation impacts contraction in multiple ways: (1) Decreased sarcomere organization in cells and tissues drives decreased force generation. (2) Increased binding affinity of E525K myosin for actin and faster Pi release contributes to increased force generation in isolated myofibrils. (3) Increased IHM stability reduces twitch force. (4) The rate-limiting step of loaded crossbridge cycling, ADP release, is unchanged by the E525K mutation, and the rates of loaded contraction and relaxation are unchanged at all scales of contraction.

biophysics↗

bioRxiv2026

The hypertrophic cardiomyopathy mutation G768R makes cardiac myosin a high duty ratio motor

{beta}-cardiac myosin is the primary motor protein in the human heart responsible for force generation by converting chemical energy from ATP hydrolysis to mechanical work. It binds actin, produces force through its powerstroke, and releases actin, and must complete this cycle several times a second within each heartbeat. Like other muscle myosins, cardiac myosin has a low duty ratio of [~]5%, the fraction of time in the actin-bound force-producing state. Here we present a hypertrophic cardiomyopathy (HCM) causing mutation, G768R, that increases cardiac myosins duty ratio to >60%, an unprecedented >10x increase unmatched by any previously studied muscle myosin mutation. In recombinantly expressed human {beta}-cardiac myosin subfragment-1 (sS1), G768R dramatically decreases the load-sensitive actin-detachment rate and step size of single molecules measured by optical tweezers. The [~]15x longer actin-bound time combined with only a modest change in overall ATPase rate predicts a duty ratio of >60%. Motility velocity is also severely slowed, as expected given the long bound time and decreased step size. All-atom molecular dynamic simulations of the pre-powerstroke and post-rigor states predict that the mutation alters lever arm priming and reduces ADP pocket opening, providing a possible structural mechanism to corroborate the experimental observations. Finally, ATPase experiments on 2-headed heavy meromyosin (HMM) constructs suggest that G768R destabilizes the autoinhibited state of myosin. Our findings of high duty ratio and reduced autoinhibition provide molecular mechanisms of cardiac hypercontractility and impaired relaxation. A muscle myosin in the heart that stays bound to actin for over half its period is bound to have significant functional and clinical consequences.

biophysics↗

bioRxiv2026

Single-molecule analysis sheds light on cardiac myosin dysfunction due to hypertrophic cardiomyopathy mutation A57D in ventricular myosin light chain-1 (MLC1v)

Ventricular myosin light chain-1 (MLC1v) is a key structural and function-modulating component of the {beta}-cardiac myosin ({beta}M-II) motor complex. Single-point mutations in MLC1v are linked to severe forms of hypertrophic cardiomyopathy (HCM) and sudden cardiac death (SCD) at a young age. However, the molecular mechanisms underlying the motor dysfunction responsible for HCM phenotype development are not fully understood. Here, we investigated native {beta}M-II motors isolated from septal myectomy sample of an HCM patient, harboring a rare homozygous mutation in MLC1v (A57D). Using a pure population of mutant motors (MUT), and sensitive single-molecule functional analysis approach, we directly assessed the primary functional alterations in {beta}M-II bearing A57D MLC1v mutation. In optical trap single-molecules measurements, the mutant motors displayed increased actomyosin (AM) interaction duration in strongly bound state (ton), corresponding to 3-fold reduced AM detachment rate than wild type myosin (WT). The MUT myosin also generated a shorter powerstroke size ({delta}). Ensemble average analysis of AM interaction events demonstrated that both the first powerstroke ({delta}1) associated with Pi release and the second powerstroke ({delta}2) linked to ADP release were reduced in MUT myosin. Moreover, the increased actomyosin cross-bridge stiffness in the AM.ADP state was observed for MUT compared to WT motors. Consistent with slower AM detachment rate and shorter stroke size, reconstituted human mutant {beta}M-II displayed slower actin filament gliding speed. Alterations in sarcomere-level mechanics included increased Ca2+ sensitivity of force generation and prolonged relaxation time, as predicted by FiberSim modelling. Molecular dynamics simulations indicated that the substitution of alanine by aspartate altered MLC1v interactions with myosin heavy chain (MyHC) and light chain 2 (MLC2v), affecting the curvature and flexibility of the lever arm. Overall, these studies establish the molecular mechanism underlying the primary myosin dysfunction due to A57D MLC1v mutation and further highlight the crucial role of MLC1v-mediated regulation of myosin function. Understanding the precise changes in the mutant myosins biomechanical properties is directly relevant to comprehending the initial triggers for pathological cardiac remodeling in HCM patients and designing tailored therapeutic interventions.

biophysics↗

bioRxiv2025

Inference and validation of a dynamic gene regulatory network influencing cell fate during Duchenne muscular dystrophy initiation

The onset of gene regulatory disturbances leading to developmental disorders remains poorly understood, particularly when symptoms begin after birth. Using induced pluripotent stem cells, single-cell transcriptomics and computational modeling, we determined the gene expression waves guiding early myogenesis and how they are affected by a disease-causing mutation. We uncovered a dynamic gene regulatory network organized in two kernels that function as a toggle-switch controlling stochastic cell fate decisions. In cells derived from patients with Duchenne muscular dystrophy, these waves of regulation are less pronounced and the switch remains in the OFF state with a higher probability, resulting in convergence into a restricted cell fate. Experimental reactivation of the switch through RNA interference led to a rescued transcriptomic state and to a refined network topology. This work uncovers a dynamic regulatory mechanism underlying early cell fate divergence and provides a general framework to infer and validate dynamic gene regulatory networks involved in disease initiation.

bioinformatics↗

bioRxiv2025

The G256E HCM mutation prolongs relaxation via altered nucleotide handling

Mutations in myosin alter its motor functions in diverse ways by affecting different structural and chemo-mechanical events. Multidisciplinary strategies can be used to understand how varying alterations in motor function converge to common phenotypes like hypercontractility and hypertrophic cardiomyopathy (HCM). Here, we combined molecular dynamics (MD) simulations with protein biochemical and myofibril mechanical analyses to study the HCM-causing myosin variant G256E. MD simulations demonstrated that G256E induces structural changes that increase the work required to displace ADP.Mg2+ from actomysoin complex. Stopped-flow biochemical analysis demonstrated increased ADP affinity for actomyosin and single myofibril mechanics analysis demonstrated increased force generation and reduced ADP sensitivity of the early, slow phase of relaxation. Together, these results demonstrate that slower ADP release from myosin during contraction is a significant contributor to pathological contractile nature of the G256E mutation. This study highlights the importance of detailed chemo-mechanical analysis of mutations associated with hereditary cardiac diseases.

biophysics↗

bioRxiv2025

Molecular mechanisms of altered contraction with the β-myosin R403Q mutation in porcine ventricular muscle and a human stem cell-derived cardiomyocyte model.

I.The R403Q mutation in the sarcomere protein beta-myosin heavy chain ({beta}-MHC) is a known genetic cause of hypertrophic cardiomyopathy (HCM), associated with ventricular hypercontractility, impaired relaxation, and cardiac arrhythmias. Despite extensive research, the mutations impact on myosin contractile properties remains unclear, likely due, at least in part, to discrepancies cross different model systems. In this study, we used a multidisciplinary approach to explore mutational effects using two distinct heterozygous R403Q systems: a Yucatan minipig model and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). X-ray diffraction of R403Q minipig ventricular muscle demonstrated reduced order of the thick filament, suggesting destabilization of the inhibited OFF (vs. ON) state of myosin in relaxed muscle, which correlated with elevated force at submaximal calcium. Super-resolution, single-molecule fluorescence microscopy indicated elevated ATPase activity in thick filament zones lacking cMyBP-C. Furthermore, R403Q myofibrils exhibited slower activation and relaxation kinetics, with reduced sensitivity to ADP. Molecular dynamics simulations suggested that altered interactions at the actomyosin interface contribute to these effects, rather than changes at the nucleotide binding pocket, typically associated with ADP release. Human engineered heterozygous R403Q hiPSC-CMs exhibited reduced maximal myofibril force, slowed contractile kinetics, and hypercontraction in engineered heart tissue constructs-consistent with HCM phenotypes observed in the heterozygous porcine model. Our results demonstrate that the R403Q mutation induces early and persistent contractile dysfunction, and that hypercontractility and slower contractile kinetics may result from a combination of an increased population of activated (ON) myosin heads and delayed detachment during cross-bridge cycling, respectively.

biophysics↗

bioRxiv2025

Conformational State of Myosins Disordered Loop 2 Structure Mediates Actomyosin Association During Crossbridge Formation

The binding of myosin to actin to form crossbridges is a critical step for force generation by sarcomeres. A recent cryo-electron microscopy structure has resolved the weakly-bound actomyosin complex (AM.ADP.Pi); however, the structural and dynamic factors that influence actin-myosin association are unclear. The disordered loop 2 of myosin is thought to mediate actomyosin interactions in complex, chemomechanical state-dependent fashion; however, the loop is usually unresolved in structural studies due to its intrinsic disorder. Here, we utilize a combination of molecular dynamics simulations and electrostatic calculations to investigate the dynamics of these actin binding regions of myosin. Our results show that loop 2 experiences disordered dynamics and that specific conformations sampled by loop 2 modulate the strength of the associative electrostatic force between actin and myosin. Variation in the actin-myosin associative force was associated with the presentation and orientation of positively charged residues in loop 2. We provide an in-depth analysis of the conformational state space occupied by loop 2 during nine 500 ns molecular dynamics simulations of pre-powerstroke human {beta}-myosin S1, with three replicates each of wildtype and two different mutant (E525K and V606M) myosin structures. This dataset allowed for exploration of how loop 2 conformational sampling is altered by these two mutations which have been clinically and experimentally associated with cardiomyopathy and altered actin binding affinity. The E525K and V606M mutations altered the conformational ensemble sampled by loop 2 and were associated with associative actin binding strength. These results highlight the importance of the positive charges on loop 2 for actomyosin interactions and demonstrate how disease-causing mutations outside of loop 2 can still affect it.

molecular biology↗

bioRxiv2024

An atomistic model of myosin interacting heads motif dynamics and their modification by 2-deoxy-ADP

The contraction of striated muscle is driven by cycling myosin motor proteins embedded within the thick filaments of sarcomeres. In addition to cross-bridge cycling with actin, these myosin proteins can enter an inactive, sequestered state in which the globular S1 heads rest along the thick filament surface and are unable to perform motor activities. Structurally, this state is called the interacting heads motif (IHM) and is a critical conformational state of myosin that regulates muscle contractility and energy expenditure. Structural perturbation of the sequestered state via missense mutations can pathologically disrupt the mechanical performance of muscle tissue. Thus, the IHM state has become a target for therapeutic intervention. An ATP analogue called 2-deoxy-ATP (dATP) is a potent myosin activator which destabilizes the IHM. Here we use molecular dynamics simulations to study the molecular mechanisms by which dATP modifies the structure and dynamics of myosin in a sequestered state. Simulations with IHM containing ADP.Pi in both nucleotide binding pockets revealed residual dynamics in an otherwise inactive and sequestered state of a motor protein. Replacement of ADP.Pi by dADP.Pi triggered a series of structural changes that modify the protein-protein interface that stabilizes the sequestered state, and changes to this interface were accompanied by allosteric changes in remote regions of the protein complex. A comparative analysis of these dynamics predicted new structural sites that may affect IHM stability.

biophysics↗

bioRxiv2024

Hypertrophic cardiomyopathy mutations Y115H and E497D disrupt the folded-back state of human beta-cardiac myosin allosterically

At the molecular level, clinical hypercontractility associated with many hypertrophic cardiomyopathy (HCM)-causing mutations in {beta}-cardiac myosin appears to be driven by their disruptive effect on the energy-conserving, folded-back, super relaxed (SRX) OFF-state of myosin. A pathological increase in force production results from release of heads from this OFF-state, which results in an increase in the number of heads free to interact with actin and produce force. Pathogenic mutations in myosin can conceivably disrupt the OFF-state by (1) directly affecting the intramolecular interfaces stabilizing the folded-back state, or (2) allosterically destabilizing the folded-back state via disruption of diverse conformational states of the myosin motor along its chemomechanical cycle. However, very little is understood about the mutations that fall in the latter group. Here, using recombinant human {beta}-cardiac myosin, we analysed the biomechanical properties of two such HCM-causing mutations, Y115H (in the transducer) and E497D (in the relay helix), neither of which falls in the regions that interact to stabilize myosins folded-back state. We find these mutations have diverse effects on the contractility parameters of myosin, yet the primary hypercontractile change in both cases is the destabilization of the OFF-state of myosin and increased availability of active myosin heads for actin-binding. Experimental data and molecular dynamics simulations indicate that these mutations likely destabilize the pre-powerstroke state of myosin, the conformation the motor adopts in the inactive folded-back state. We propose that destabilization of the folded-back state of myosin, directly and/or allosterically, is the molecular basis of hypercontractility in HCM in a far greater number of pathogenic mutations than currently thought.

biochemistry↗

bioRxiv2023

Multi-scale models reveal hypertrophic cardiomyopathy MYH7 G256E mutation drives hypercontractility and elevated mitochondrial respiration

RationaleOver 200 mutations in the sarcomeric protein {beta}-myosin heavy chain (MYH7) have been linked to hypertrophic cardiomyopathy (HCM). However, different mutations in MYH7 lead to variable penetrance and clinical severity, and alter myosin function to varying degrees, making it difficult to determine genotype-phenotype relationships, especially when caused by rare gene variants such as the G256E mutation. ObjectiveThis study aims to determine the effects of low penetrant MYH7 G256E mutation on myosin function. We hypothesize that the G256E mutation would alter myosin function, precipitating compensatory responses in cellular functions. MethodsWe developed a collaborative pipeline to characterize myosin function at multiple scales (protein to myofibril to cell to tissue). We also used our previously published data on other mutations to compare the degree to which myosin function was altered. ResultsAt the protein level, the G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 50.9%, suggesting more myosins available for contraction. Myofibrils isolated from hiPSC-CMs CRISPR-edited with G256E (MYH7WT/G256E) generated greater tension, had faster tension development and slower early phase relaxation, suggesting altered myosin-actin crossbridge cycling kinetics. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. Single-cell transcriptomic and metabolic profiling demonstrated upregulation of mitochondrial genes and increased mitochondrial respiration, suggesting altered bioenergetics as an early feature of HCM. ConclusionsMYH7 G256E mutation causes structural instability in the transducer region, leading to hypercontractility across scales, perhaps from increased myosin recruitment and altered crossbridge cycling. Hypercontractile function of the mutant myosin was accompanied by increased mitochondrial respiration, while cellular hypertrophy was modest in the physiological stiffness environment. We believe that this multi-scale platform will be useful to elucidate genotype-phenotype relationships underlying other genetic cardiovascular diseases.

molecular biology↗

bioRxiv2021

Post-translational modification patterns on β-myosin heavy chain are altered in ischemic and non-ischemic human hearts

Phosphorylation and acetylation of sarcomeric proteins are important for fine-tuning myocardial contractility. Here, we used bottom-up proteomics and label-free quantification to identify novel post-translational modifications (PTMs) on beta-myosin heavy chain ({beta}-MHC) in normal and failing human heart tissues. We report six acetylated lysines and two phosphorylated residues: K34-Ac, K58-Ac, S210-P, K213-Ac, T215-P, K429-Ac, K951-Ac, and K1195-Ac. K951-Ac was significantly reduced in both ischemic and non-ischemic failing hearts compared to non-diseased hearts. Molecular dynamics simulations show that K951-Ac may impact stability of thick filament tail interactions and ultimately myosin head positioning. K58-Ac altered the solvent exposed SH3 domain surface - known for protein-protein interactions - but did not appreciably change motor domain conformation or dynamics under conditions studied. Together, K213-Ac/T215-P altered loop 1s structure and dynamics - known to regulate ADP-release, ATPase activity, and sliding velocity. Our study suggests that {beta}-MHC acetylation levels may be influenced more by the PTM location than the type of heart disease since less protected acetylation sites are reduced in both heart failure groups. Additionally, these PTMs have potential to modulate interactions between {beta}-MHC and other regulatory sarcomeric proteins, ADP-release rate of myosin, flexibility of the S2 region, and cardiac myofilament contractility in normal and heart failure hearts.

physiology↗