Biology subjects

Moussavi-Harami, F.

Publications and source records attributed to Moussavi-Harami, F..

9 recordsLinked to original sources

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↗

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

Myosin modulator Aficamten inhibits force in cardiac muscle by altering myosin's biochemical activity without changing thick filament structure

BackgroundInhibiting contractility by targeting cardiac myosin is an effective treatment for patients with hypertrophic cardiomyopathy (HCM). Aficamten is a second in class myosin inhibitor with promising clinical data showing improvements in hemodynamics and symptoms in patients with HCM. While it is known that Aficamten inhibits force and cardiomyocyte contractility by stabilizing the weak pre-powerstroke conformation, effects on myosin structure and kinetics during loaded contraction are lacking. MethodsPermeabilized porcine cardiac tissue and myofibrils were used for single-molecule imaging of ATP turn over, X-ray diffraction, and mechanical measurements. Engineered heart tissues from human induced pluripotent stem cell cardiomyocytes were used to evaluate effects on force and contraction kinetics. ResultsIn contrast to Mavacamten, Aficamten does not structurally sequester myosin heads along the thick filament. Aficamten inhibits ATPase activity by shifting myosin heads from higher to slower ATPase state, with the emergence of a super slow biochemical nucleotide turnover state. This results in decreased force and calcium sensitivity without altering cross-bridge cycling. These contractile mechanical changes are comparable to Mavacamten. Our myofibril mechanical assay showed inhibition of force with accelerated relaxation. In EHTs, while Mavacamten and Aficamten inhibit cardiac twitch forces, Mavacamten reduces the activation kinetics while both result in faster relaxation. ConclusionsWe used a combination of biochemical and biomechanical assays to show that Aficamten inhibits myosin ATPase without appreciably altering myosin structure. This is different from Mavacamten that strongly affects both. While both compounds inhibit contractility, differences in mechanisms of action and kinetics of force activation and relaxation could allow use in different patient populations.

biophysics↗

bioRxiv2024

Identifying mechanisms and therapeutic targets in muscle using Bayesian parameter estimation with conditional variational autoencoders

Cardiomyopathies, often caused by mutations in genes encoding muscle proteins, are traditionally treated by phenotyping hearts and addressing symptoms post irreversible damage. With advancements in genotyping, early diagnosis is now possible, potentially introducing earlier treatment. However, the intricate structure of muscle and its myriad proteins make treatment predictions challenging. Here we approach the problem of estimating therapeutic targets for a mutation in mouse muscle using a spatially explicit half sarcomere muscle model. We selected 9 rate parameters in our model linked to both small molecules and cardiomyopathy-causing mutations. We then randomly varied these rate parameters and simulated an isometric twitch for each combination to generate a large training dataset. We used this dataset to train a Conditional Variational Autoencoder (CVAE), a technique used in Bayesian parameter estimation. Given simulated or experimental isometric twitches, this machine learning model is able to then predict the set of rate parameters which are most likely to yield that result. We then predict the set of rate parameters associated with twitches from control mice with the cardiac Troponin C (cTnC) I61Q variant and control twitches treated with the myosin activator Danicamtiv, as well as model parameters that recover the abnormal I61Q cTnC twitches. SIGNIFICANCEMachine learning techniques have potential to accelerate discoveries in biologically complex systems. However, they require large data sets and can be challenging in high dimensional systems such as cardiac muscle. In this study, we combined experimental measures of cardiac muscle twitch forces with mechanistic simulations and a newly developed mixture of Bayesian inference with neural networks (in autoencoders) to solve the inverse problem of determining the underlying kinetics for observed force generation by cardiac muscle. The autoencoders are trained on millions of simulations spanning parameter spaces that correspond to the mechanochemistry of cardiac sarcomeres. We apply the trained model to experimental data in order to infer parameters that can explain a diseased twitch and ways to recover it.

biophysics↗

bioRxiv2023

Late-life Rapamycin Treatment Enhances Cardiomyocyte Relaxation Kinetics and Reduces Myocardial Stiffness

Diastolic dysfunction is a key feature of the aging heart. We have shown that late-life treatment with mTOR inhibitor, rapamycin, reverses age-related diastolic dysfunction in mice but the molecular mechanisms of the reversal remain unclear. To dissect the mechanisms by which rapamycin improves diastolic function in old mice, we examined the effects of rapamycin treatment at the levels of single cardiomyocyte, myofibril and multicellular cardiac muscle. Compared to young cardiomyocytes, isolated cardiomyocytes from old control mice exhibited prolonged time to 90% relaxation (RT90) and time to 90% Ca2+ transient decay (DT90), indicating slower relaxation kinetics and calcium reuptake with age. Late-life rapamycin treatment for 10 weeks completely normalized RT90 and partially normalized DT90, suggesting improved Ca2+ handling contributes partially to the rapamycin-induced improved cardiomyocyte relaxation. In addition, rapamycin treatment in old mice enhanced the kinetics of sarcomere shortening and Ca2+ transient increase in old control cardiomyocytes. Myofibrils from old rapamycin-treated mice displayed increased rate of the fast, exponential decay phase of relaxation compared to old controls. The improved myofibrillar kinetics were accompanied by an increase in MyBP-C phosphorylation at S282 following rapamycin treatment. We also showed that late-life rapamycin treatment normalized the age-related increase in passive stiffness of demembranated cardiac trabeculae through a mechanism independent of titin isoform shift. In summary, our results showed that rapamycin treatment normalizes the age-related impairments in cardiomyocyte relaxation, which works conjointly with reduced myocardial stiffness to reverse age-related diastolic dysfunction.

physiology↗

bioRxiv2023

Cell based dATP delivery as a therapy for chronic heart failure.

Withdrawal StatementThe authors have withdrawn this manuscript because the senior authors have identified inconsistencies in the data presented in this manuscript, which affect the reliability of the main conclusions. In keeping with our commitment to scientific rigor and transparency, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding authors.

pathology↗

bioRxiv2023

Danicamtiv increases myosin recruitment and alters the chemomechanical cross bridge cycle in cardiac muscle

Modulating myosin function is a novel therapeutic approach in patients with cardiomyopathy. Detailed mechanism of action of these agents can help predict potential unwanted affects and identify patient populations that can benefit most from them. Danicamtiv is a novel myosin activator with promising preclinical data that is currently in clinical trials. While it is known danicamtiv increases force and cardiomyocyte contractility without affecting calcium levels, detailed mechanistic studies regarding its mode of action are lacking. Using porcine cardiac tissue and myofibrils we demonstrate that Danicamtiv increases force and calcium sensitivity via increasing the number of myosin in the "on" state and slowing cross bridge turnover. Our detailed analysis shows that inhibition of ADP release results in decreased cross bridge turnover with cross bridges staying on longer and prolonging myofibril relaxation. Using a mouse model of genetic dilated cardiomyopathy, we demonstrated that Danicamtiv corrected calcium sensitivity in demembranated and abnormal twitch magnitude and kinetics in intact cardiac tissue. Significance StatementDirectly augmenting sarcomere function has potential to overcome limitations of currently used inotropic agents to improve cardiac contractility. Myosin modulation is a novel mechanism for increased contraction in cardiomyopathies. Danicamtiv is a myosin activator that is currently under investigation for use in cardiomyopathy patients. Our study is the first detailed mechanism of how Danicamtiv increases force and alters kinetics of cardiac activation and relaxation. This new understanding of the mechanism of action of Danicamtiv can be used to help identify patients that could benefit most from this treatment.

physiology↗

bioRxiv2023

Correcting dilated cardiomyopathy with fibroblast-targeted p38 deficiency

Inherited mutations in contractile and structural genes, which decrease cardiomyocyte tension generation, are principal drivers of dilated cardiomyopathy (DCM)- the leading cause of heart failure1,2. Progress towards developing precision therapeutics for and defining the underlying determinants of DCM has been cardiomyocyte centric with negligible attention directed towards fibroblasts despite their role in regulating the best predictor of DCM severity, cardiac fibrosis3,4. Given that failure to reverse fibrosis is a major limitation of both standard of care and first in class precision therapeutics for DCM, this study examined whether cardiac fibroblast-mediated regulation of the hearts material properties is essential for the DCM phenotype. Here we report in a mouse model of inherited DCM that prior to the onset of fibrosis and dilated myocardial remodeling both the myocardium and extracellular matrix (ECM) stiffen from switches in titin isoform expression, enhanced collagen fiber alignment, and expansion of the cardiac fibroblast population, which we blocked by genetically suppressing p38 in cardiac fibroblasts. This fibroblast-targeted intervention unexpectedly improved the primary cardiomyocyte defect in contractile function and reversed ECM and dilated myocardial remodeling. Together these findings challenge the long-standing paradigm that ECM remodeling is a secondary complication to inherited defects in cardiomyocyte contractile function and instead demonstrate cardiac fibroblasts are essential contributors to the DCM phenotype, thus suggesting DCM-specific therapeutics will require fibroblast-specific strategies.

cell biology↗

bioRxiv2022

dATP Elevation Induces Myocardial Metabolic Remodeling to Support Improved Cardiac Function

Hallmark features of systolic heart failure are reduced contractility and impaired metabolic flexibility of the myocardium. Cardiomyocytes (CMs) with elevated deoxy ATP (dATP) via overexpression of ribonucleotide reductase (RNR) enzyme robustly improve contractility. However, the effect of dATP elevation on cardiac metabolism is unknown. Here, we developed proteolysis-resistant versions of RNR and demonstrate that elevation of dATP/ATP to ~1% in CMs in a transgenic mouse (TgRRB) resulted in robust improvement of cardiac function. Pharmacological approaches showed that CMs with elevated dATP have greater basal respiratory rates by shifting myosin states to more active forms, independent of its isoform, in relaxed CMs. Targeted metabolomic profiling revealed a significant reprogramming towards oxidative phosphorylation in TgRRB-CMs. Higher cristae density and activity in the mitochondria of TgRRB-CMs improved respiratory capacity. Our results revealed a critical property of dATP to modulate myosin states to enhance contractility and induce metabolic flexibility to support improved function in CMs. HighlightsO_LIUbiquitylation-resistant variant RRB in a transgenic mice model (TgRRB) elevates dATP level up to 1% (of the total ATP pool) in the heart and improves function. C_LIO_LITgRRB-CMs show greater basal oxygen consumption due to changes in myosin state by dATP. C_LIO_LITgRRB-CMs respond to elevated function with a metabolic shift, such that there are higher pools of oxidative metabolites, with elevated OXPHOS, FAO, and energy reserve. C_LIO_LILong-term mitochondrial remodeling may occur to accommodate for the higher energy demands of the high functioning TgRRB-CMs. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/515235v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@11fa00borg.highwire.dtl.DTLVardef@14460e9org.highwire.dtl.DTLVardef@369f31org.highwire.dtl.DTLVardef@d879f_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗