Tail length and E525K dilated cardiomyopathy mutant alter human ß-cardiac myosin super-relaxed state.

Dilated cardiomyopathy (DCM) is a condition characterized by impaired cardiac function, due to myocardial hypo-contractility, and is associated with point mutations in ß-cardiac myosin, the molecular motor that powers cardiac contraction. Myocardial function can be modulated through sequestration of myosin motors into an auto-inhibited "super-relaxed" state (SRX), which may be further stabilized by a structural state known as the "interacting heads motif" (IHM). Here, we sought to determine whether hypo-contractility of DCM myocardium results from reduced function of individual myosin molecules or from decreased myosin availability to interact with actin due to increased IHM/SRX stabilization. We used an established DCM myosin mutation, E525K, and characterized the biochemical and mechanical activity of wild-type and mutant human ß-cardiac myosin constructs that differed in the length of their coiled-coil tail, which dictates their ability to form the IHM/SRX state. We found that short-tailed myosin constructs exhibited low IHM/SRX content, elevated actin-activated ATPase activity, and fast velocities in unloaded motility assays. Conversely, longer-tailed constructs exhibited higher IHM/SRX content and reduced actomyosin ATPase and velocity. Our modeling suggests that reduced velocities may be attributed to IHM/SRX-dependent sequestration of myosin heads. Interestingly, longer-tailed E525K mutants showed no apparent impact on velocity or actomyosin ATPase at low ionic strength but stabilized IHM/SRX state at higher ionic strength. Therefore, the hypo-contractility observed in DCM may be attributable to reduced myosin head availability caused by enhanced IHM/SRX stability in E525K mutants.

N-Terminal Fragment of Cardiac Myosin Binding Protein C Modulates Cooperative Mechanisms of Thin Filament Activation in Atria and Ventricles.

Cardiac myosin binding protein C (cMyBP-C) is one of the essential control components of the myosin cross-bridge cycle. The C-terminal part of cMyBP-C is located on the surface of the thick filament, and its N-terminal part interacts with actin, myosin, and tropomyosin, affecting both kinetics of the ATP hydrolysis cycle and lifetime of the cross-bridge, as well as calcium regulation of the actin-myosin interaction, thereby modulating contractile function of myocardium. The role of cMyBP-C in atrial contraction has not been practically studied. We examined effect of the N-terminal C0-C1-m-C2 (C0-C2) fragment of cMyBP-C on actin-myosin interaction using ventricular and atrial myosin in an in vitro motility assay. The C0-C2 fragment of cMyBP-C significantly reduced the maximum sliding velocity of thin filaments on both myosin isoforms and increased the calcium sensitivity of the actin-myosin interaction. The C0-C2 fragment had different effects on the kinetics of ATP and ADP exchange, increasing the affinity of ventricular myosin for ADP and decreasing the affinity of atrial myosin. The effect of the C0-C2 fragment on the activation of the thin filament depended on the myosin isoforms. Atrial myosin activates the thin filament less than ventricular myosin, and the C0-C2 fragment makes these differences in the myosin isoforms more pronounced.

Mechanical and signaling responses of unloaded rat soleus muscle to chronically elevated ß-myosin activity.

It has been reported that muscle functional unloading is accompanied by an increase in motoneuronal excitability despite the elimination of afferent input. Thus, we hypothesized that pharmacological potentiation of spontaneous contractile soleus muscle activity during hindlimb unloading could activate anabolic signaling pathways and prevent the loss of muscle mass and strength. To investigate these aspects and underlying molecular mechanisms, we used ß-myosin allosteric effector Omecamtiv Mekarbil (OM). We found that OM partially prevented the loss of isometric strength and intrinsic stiffness of the soleus muscle after two weeks of disuse. Notably, OM was able to attenuate the unloading-induced decrease in the rate of muscle protein synthesis (MPS). At the same time, the use of drug neither prevented the reduction in the markers of translational capacity (18S and 28S rRNA) nor activation of the ubiquitin-proteosomal system, which is evidenced by a decrease in the cross-sectional area of fast and slow muscle fibers. These results suggest that chemically-induced increase in low-intensity spontaneous contractions of the soleus muscle during functional unloading creates prerequisites for protein synthesis. At the same time, it should be assumed that the use of OM is advisable with pharmacological drugs that inhibit the expression of ubiquitin ligases.

Inducing positive inotropy in human iPSC-derived cardiac muscle by gene editing-based activation of the cardiac α-myosin heavy chain.

Human induced pluripotent stem cells and their differentiation into cardiac myocytes (hiPSC-CMs) provides a unique and valuable platform for studies of cardiac muscle structure-function. This includes studies centered on disease etiology, drug development, and for potential clinical applications in heart regeneration/repair. Ultimately, for these applications to achieve success, a thorough assessment and physiological advancement of the structure and function of hiPSC-CMs is required. HiPSC-CMs are well noted for their immature and sub-physiological cardiac muscle state, and this represents a major hurdle for the field. To address this roadblock, we have developed a hiPSC-CMs (ß-MHC dominant) experimental platform focused on directed physiological enhancement of the sarcomere, the functional unit of cardiac muscle. We focus here on the myosin heavy chain (MyHC) protein isoform profile, the molecular motor of the heart, which is essential to cardiac physiological performance. We hypothesized that inducing increased expression of α-MyHC in ß-MyHC dominant hiPSC-CMs would enhance contractile performance of hiPSC-CMs. To test this hypothesis, we used gene editing with an inducible α-MyHC expression cassette into isogeneic hiPSC-CMs, and separately by gene transfer, and then investigated the direct effects of increased α-MyHC expression on hiPSC-CMs contractility and relaxation function. Data show improved cardiac functional parameters in hiPSC-CMs induced with α-MyHC. Positive inotropy and relaxation was evident in comparison to ß-MyHC dominant isogenic controls both at baseline and during pacing induced stress. This approach should facilitate studies of hiPSC-CMs disease modeling and drug screening, as well as advancing fundamental aspects of cardiac function parameters for the optimization of future cardiac regeneration, repair and re-muscularization applications.

Binding pocket dynamics along the recovery stroke of human ß-cardiac myosin.

The druggability of small-molecule binding sites can be significantly affected by protein motions and conformational changes. Ligand binding, protein dynamics and protein function have been shown to be closely interconnected in myosins. The breakthrough discovery of omecamtiv mecarbil (OM) has led to an increased interest in small molecules that can target myosin and modulate its function for therapeutic purposes (myosin modulators). In this work, we use a combination of computational methods, including steered molecular dynamics, umbrella sampling and binding pocket tracking tools, to follow the evolution of the OM binding site during the recovery stroke transition of human ß-cardiac myosin. We found that steering two internal coordinates of the motor domain can recapture the main features of the transition and in particular the rearrangements of the binding site, which shows significant changes in size, shape and composition. Possible intermediate conformations were also identified, in remarkable agreement with experimental findings. The differences in the binding site properties observed along the transition can be exploited for the future development of conformation-selective myosin modulators.

Hypertrophic cardiomyopathy mutations in the pliant and light chain-binding regions of the lever arm of human ß-cardiac myosin have divergent effects on myosin function.

Mutations in the lever arm of ß-cardiac myosin are a frequent cause of hypertrophic cardiomyopathy, a disease characterized by hypercontractility and eventual hypertrophy of the left ventricle. Here, we studied five such mutations: three in the pliant region of the lever arm (D778V, L781P, and S782N) and two in the light chain-binding region (A797T and F834L). We investigated their effects on both motor function and myosin subfragment 2 (S2) tail-based autoinhibition. The pliant region mutations had varying effects on the motor function of a myosin construct lacking the S2 tail: overall, D778V increased power output, L781P reduced power output, and S782N had little effect on power output, while all three reduced the external force sensitivity of the actin detachment rate. With a myosin containing the motor domain and the proximal S2 tail, the pliant region mutations also attenuated autoinhibition in the presence of filamentous actin but had no impact in the absence of actin. By contrast, the light chain-binding region mutations had little effect on motor activity but produced marked reductions in autoinhibition in both the presence and absence of actin. Thus, mutations in the lever arm of ß-cardiac myosin have divergent allosteric effects on myosin function, depending on whether they are in the pliant or light chain-binding regions.

Nanosurfer assay dissects ß-cardiac myosin and cardiac myosin-binding protein C interactions.

Cardiac myosin-binding protein C (cMyBP-C) modulates cardiac contractility through putative interactions with the myosin S2 tail and/or the thin filament. The relative contribution of these binding-partner interactions to cMyBP-C modulatory function remains unclear. Hence, we developed a "nanosurfer" assay as a model system to interrogate these cMyBP-C binding-partner interactions. Synthetic thick filaments were generated using recombinant human ß-cardiac myosin subfragments (HMM or S1) attached to DNA nanotubes, with 14- or 28-nm spacing, corresponding to the 14.3-nm myosin spacing in native thick filaments. The nanosurfer assay consists of DNA nanotubes added to the in vitro motility assay so that myosins on the motility surface effectively deliver thin filaments to the DNA nanotubes, enhancing thin filament gliding probability on the DNA nanotubes. Thin filament velocities on nanotubes with either 14- or 28-nm myosin spacing were no different. We then characterized the effects of cMyBP-C on thin filament motility by alternating HMM and cMyBP-C N-terminal fragments (C0-C2 or C1-C2) on nanotubes every 14 nm. Both C0-C2 and C1-C2 reduced thin filament velocity four- to sixfold relative to HMM alone. Similar inhibition occurred using the myosin S1 construct, which lacks the myosin S2 region proposed to interact with cMyBP-C, suggesting that the cMyBP-C N terminus must interact with other myosin head domains and/or actin to slow thin filament velocity. Thin filament velocity was unaffected by the C0-C1f fragment, which lacks the majority of the M-domain, supporting the importance of this domain for inhibitory interaction(s). A C0-C2 fragment with phospho-mimetic replacement in the M-domain showed markedly less inhibition of thin filament velocity compared with its phospho-null counterpart, highlighting the modulatory role of M-domain phosphorylation on cMyBP-C function. Therefore, the nanosurfer assay provides a platform to precisely manipulate spatially dependent cMyBP-C binding-partner interactions, shedding light on the molecular regulation of ß-cardiac myosin contractility.

Alpha and beta myosin isoforms and human atrial and ventricular contraction.

Human atrial and ventricular contractions have distinct mechanical characteristics including speed of contraction, volume of blood delivered and the range of pressure generated. Notably, the ventricle expresses predominantly ß-cardiac myosin while the atrium expresses mostly the α-isoform. In recent years exploration of the properties of pure α- & ß-myosin isoforms have been possible in solution, in isolated myocytes and myofibrils. This allows us to consider the extent to which the atrial vs ventricular mechanical characteristics are defined by the myosin isoform expressed, and how the isoform properties are matched to their physiological roles. To do this we Outline the essential feature of atrial and ventricular contraction; Explore the molecular structural and functional characteristics of the two myosin isoforms; Describe the contractile behaviour of myocytes and myofibrils expressing a single myosin isoform; Finally we outline the outstanding problems in defining the differences between the atria and ventricles. This allowed us consider what features of contraction can and cannot be ascribed to the myosin isoforms present in the atria and ventricles.

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 preserves length-dependent contractility and improves diastolic function in human engineered heart tissue.

Comprehensive functional characterization of cardiac tissue includes investigation of length and load dependence. Such measurements have been slow to develop in engineered heart tissues (EHTs), whose mechanical characterizations have been limited primarily to isometric and near-isometric behaviors. A more realistic assessment of myocardial function would include force-velocity curves to characterize power output and force-length loops mimicking the cardiac cycle to characterize work output. We developed a system that produces force-velocity curves and work loops in human EHTs using an adaptive iterative control scheme. We used human EHTs in this system to perform a detailed characterization of the cardiac ß-myosin specific inhibitor, mavacamten. Consistent with the clinically proposed application of this drug to treat hypertrophic cardiomyopathy, our data support the premise that mavacamten improves diastolic function through reduction of diastolic stiffness and isometric relaxation time. Meanwhile, the effects of mavacamten on length- and load-dependent muscle performance were mixed. The drug attenuated the length-dependent response at small stretch values but showed normal length dependency at longer lengths. Peak power output of mavacamten-treated EHTs showed reduced power output as expected but also shifted peak power output to a lower load. Here, we demonstrate a robust method for the generation of isotonic contraction series and work loops in engineered heart tissues using an adaptive-iterative method. This approach reveals new features of mavacamten pharmacology, including previously unappreciated effects on intrinsic myosin dynamics and preservation of Frank-Starling behavior at longer muscle lengths.NEW & NOTEWORTHY We applied innovative methods to comprehensively characterize the length and load-dependent behaviors of engineered human cardiac muscle when treated with the cardiac ß-myosin specific inhibitor mavacamten, a drug on the verge of clinical implementation for hypertrophic cardiomyopathy. We find mechanistic support for the role of mavacamten in improving diastolic function of cardiac tissue and note novel effects on work and power.

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.

Mavacamten: a novel small molecule modulator of ß-cardiac myosin for treatment of hypertrophic cardiomyopathy.

INTRODUCTION: Hypertrophic cardiomyopathy (HCM) is a common known monogenetic cardiovascular disorder which frequently leads to symptoms such as dyspnea and exercise intolerance. Current guideline-recommended pharmacotherapies have variable therapeutic responses. Mavacamten, a small molecule modulator of ß-cardiac myosin, reduces hypercontractility, a central mechanism in the pathogenesis of HCM. Mavacamten has recently been evaluated in Phase 2 and 3 clinic trials for obstructive and nonobstructive symptomatic HCM. AREAS COVERED: This article reviews available preclinical and clinical trials assessing the efficacy and safety of Mavacamten for the treatment of symptomatic obstructive and nonobstructive HCM. EXPERT OPINION: Findings from Phase 2 and 3 trials suggest that Mavacamten represents a very promising new therapy for the treatment of symptomatic patients with HCM. Treatment leads to an improvement in symptomatic and physiologic metrics for symptomatic patients with HCM with minimal adverse events. Patients with obstructive HCM demonstrated a significant improvement in LVOT gradient, NYHA functional class, Kansas City Cardiomyopathy Questionnaire (KCCQ), Overall Summary Score (OSS), and numerical rating scale (NRS) dyspnea scores; and patients with both obstructive and nonobstructive HCM had significant improvement in serum N-terminal pro-B-type natriuretic peptide (NT-proBNP) concentrations.

Ca2+-Dependent NOX5 (NADPH Oxidase 5) Exaggerates Cardiac Hypertrophy Through Reactive Oxygen Species Production.

NOX5 (NADPH oxidase 5) is a homolog of the gp91phox subunit of the phagocyte NOX, which generates reactive oxygen species. NOX5 is involved in sperm motility and vascular contraction and has been implicated in diabetic nephropathy, atherosclerosis, and stroke. The function of NOX5 in the cardiac hypertrophy is unknown. Because NOX5 is a Ca2+-sensitive, procontractile NOX isoform, we questioned whether it plays a role in cardiac hypertrophy. Studies were performed in (1) cardiac tissue from patients undergoing heart transplant for cardiomyopathy and heart failure, (2) NOX5-expressing rat cardiomyocytes, and (3) mice expressing human NOX5 in a cardiomyocyte-specific manner. Cardiac hypertrophy was induced in mice by transverse aorta coarctation and Ang II (angiotensin II) infusion. NOX5 expression was increased in human failing hearts. Rat cardiomyocytes infected with adenoviral vector encoding human NOX5 cDNA exhibited elevated reactive oxygen species levels with significant enlargement and associated increased expression of ANP (atrial natriuretic peptides) and ß-MHC (ß-myosin heavy chain) and prohypertrophic genes (Nppa, Nppb, and Myh7) under Ang II stimulation. These effects were reduced by N-acetylcysteine and diltiazem. Pressure overload and Ang II infusion induced left ventricular hypertrophy, interstitial fibrosis, and contractile dysfunction, responses that were exaggerated in cardiac-specific NOX5 trangenic mice. These phenomena were associated with increased reactive oxygen species levels and activation of redox-sensitive MAPK (mitogen-activated protein kinase). N-acetylcysteine treatment reduced cardiac oxidative stress and attenuated cardiac hypertrophy in NOX5 trangenic. Our study defines Ca2+-regulated NOX5 as an important NOX isoform involved in oxidative stress- and MAPK-mediated cardiac hypertrophy and contractile dysfunction.

Taurine attenuates isoproterenol-induced H9c2 cardiomyocytes hypertrophy by improving antioxidative ability and inhibiting calpain-1-mediated apoptosis.

Pathological cardiac hypertrophy is ultimately accompanied by cardiomyocyte apoptosis. Apoptosis mainly related to calpain-1-mediated apoptotic pathways. Studies had proved that taurine can maintain heart health through antioxidation and antiapoptotic functions, but the effect of taurine on cardiac hypertrophy is still unclear. This study aimed to determine whether taurine could inhibit calpain-1-mediated mitochondria-dependent apoptotic pathways in isoproterenol (ISO)-induced hypertrophic cardiomyocytes. We found that taurine could inhibit the increase in cell surface area and reduce the protein expression levels of the hypertrophic markers atrial natriuretic peptide, brain natriuretic polypeptide, and ß-myosin heavy chain. Taurine also reduced ROS, intracellular Ca2+ overload and mitochondrial membrane potential. Moreover, taurine inhibited cardiomyocyte apoptosis by decreasing the protein expression of calpain-1, Bax, t-Bid, cytosolic cytochrome c, Apaf-1, cleaved caspase-9 and cleaved caspase-3 and by enhancing calpastatin and Bcl-2 protein expression. Calpain-1 small interfering RNA transfection results showed similar antiapoptotic effects as the taurine prevention group. However, compared with the two treatments, taurine inhibited the expression of cleaved caspase-9 more significantly. Therefore, we believe that taurine prevents ISO-induced H9c2 cardiomyocyte hypertrophy by inhibiting oxidative stress, intracellular Ca2+ overload, the calpain-1-mediated mitochondria-dependent apoptotic pathway and cleaved caspase-9 levels.

Hypertrophic Cardiomyopathy: Diverse Pathophysiology Revealed by Genetic Research, Toward Future Therapy.

Hypertrophic cardiomyopathy (HCM) is an intractable disease that causes heart failure mainly due to unexplained severe cardiac hypertrophy and diastolic dysfunction. HCM, which occurs in 0.2% of the general population, is the most common cause of sudden cardiac death in young people. HCM has been studied extensively using molecular genetic approaches. Genes encoding cardiac ß-myosin heavy chain, cardiac myosin-binding protein C, and troponin complex, which were originally identified as causative genes, were subsequently reported to be frequently implicated in HCM. Indeed, HCM has been considered a disease of sarcomere gene mutations. However, fewer than half of patients with HCM have mutations in sarcomere genes. The others have been documented to have mutations in cardiac proteins in various other locations, including the Z disc, sarcoplasmic reticulum, plasma membrane, nucleus, and mitochondria. Next-generation sequencing makes it possible to detect mutations at high throughput, and it has become increasingly common to identify multiple cardiomyopathy-causing gene mutations in a single HCM patient. Elucidating how mutations in different genes contribute to the disease pathophysiology will be a challenge. In studies using animal models, sarcomere mutations generally tend to increase myocardial Ca2+ sensitivity, and some mutations increase the activity of myosin ATPase. Clinical trials of drugs to treat HCM are ongoing, and further new therapies based on pathophysiological analyses of the causative genes are eagerly anticipated.

Musa balbisiana Fruit Rich in Polyphenols Attenuates Isoproterenol-Induced Cardiac Hypertrophy in Rats via Inhibition of Inflammation and Oxidative Stress.

Musa balbisiana Colla (Family: Musaceae), commonly known as banana and native to India and other parts of Asia, is very rich in nutritional value and has strong antioxidant potential. In the present study, we have developed Musa balbisiana (MB) fruit pulp powder and evaluated its cardioprotective effect in cardiac hypertrophy, which is often associated with inflammation and oxidative stress. An ultra-high-pressure liquid chromatography-mass spectrometer (UPLC-MS/MS) has been used for the detection and systematic characterization of the phenolic compounds present in Musa balbisiana fruit pulp. The cardioprotective effect of MB was evaluated in a rat model of isoproterenol- (ISO-) induced cardiac hypertrophy by subcutaneous administration of isoproterenol (5 mg/kg-1/day-1), delivered through an alzet minipump for 14 days. Oral administration of MB fruit pulp powder (200 mg/kg/day) significantly (p < 0.001) decreased heart weight/tail length ratio and cardiac hypertrophy markers like ANP, BNP, ß-MHC, and collagen-1 gene expression. MB also attenuated ISO-induced cardiac inflammation and oxidative stress. The in vivo data were further confirmed in vitro in H9c2 cells where the antihypertrophic and anti-inflammatory effect of the aqueous extract of MB was observed in the presence of ISO and lipopolysaccharide (LPS), respectively. This study strongly suggests that supplementation of dried Musa balbisiana fruit powder can be useful for the prevention of cardiac hypertrophy via the inhibition of inflammation and oxidative stress.

Dilated cardiomyopathy mutation in the converter domain of human cardiac myosin alters motor activity and response to omecamtiv mecarbil.

We investigated a dilated cardiomyopathy (DCM) mutation (F764L) in human ß-cardiac myosin by determining its motor properties in the presence and absence of the heart failure drug omecamtive mecarbil (OM). The mutation is located in the converter domain, a key region of communication between the catalytic motor and lever arm in myosins, and is nearby but not directly in the OM-binding site. We expressed and purified human ß-cardiac myosin subfragment 1 (M2ß-S1) containing the F764L mutation, and compared it to WT with in vitro motility as well as steady-state and transient kinetics measurements. In the absence of OM we demonstrate that the F764L mutation does not significantly change maximum actin-activated ATPase activity but slows actin sliding velocity (15%) and the actomyosin ADP release rate constant (25%). The transient kinetic analysis without OM demonstrates that F764L has a similar duty ratio as WT in unloaded conditions. OM is known to enhance force generation in cardiac muscle while it inhibits the myosin power stroke and enhances actin-attachment duration. We found that OM has a reduced impact on F764L ATPase and sliding velocity compared with WT. Specifically, the EC50 for OM induced inhibition of in vitro motility was 3-fold weaker in F764L. Also, OM reduces maximum actin-activated ATPase 2-fold in F764L, compared with 4-fold with WT. Overall, our results suggest that F764L attenuates the impact of OM on actin-attachment duration and/or the power stroke. Our work highlights the importance of mutation-specific considerations when pursuing small molecule therapies for cardiomyopathies.

Taxifolin ameliorates DEHP-induced cardiomyocyte hypertrophy via attenuating mitochondrial dysfunction and glycometabolism disorder in chicken.

Di-(2-ethylhexyl) phthalate (DEHP) is a prevalent environmental contaminant that severely impacts the health of human and animals. Taxifolin (TAX), a plant flavonoid isolated from yew, exerts protective effects on cardiac diseases. Nevertheless, whether DEHP could induce cardiomyocyte hypertrophy and its mechanism remains unclear. This study aimed to highlight the specific molecular mechanisms of DEHP-induced cardiomyocyte hypertrophy and the protective potential of TAX against it. Chicken primary cardiomyocytes were treated with DEHP (500 µM) and/or TAX (0.5 µM) for 24 h. The levels of glucose and adenosine triphosphate (ATP) were detected, and cardiac hypertrophy-related genes were validated by real-time quantitative PCR (qRT-PCR) and Western blot (WB) in vitro. The results showed that DEHP-induced cardiac hypertrophy was ameliorated by TAX, as indicated by the increased cardiomyocyte area and expression of atrial natriuretic peptide (ANP), natriuretic peptides A-like (BNP) and ß-myosin heavy cardiac muscle (ß-MHC). Furthermore, DEHP induced cardiac hypertrophy via the interleukin 6 (IL-6)/Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway in vitro. In addition, DEHP disrupted mitochondrial function and glycometabolism by activating the insulin-like growth factor 1 (IGF1)/phosphatidylinositol 3-kinase (PI3K) pathway and the peroxisome proliferator activated receptors (PPARs)/PPARG coactivator 1 alpha (PGC-1α) pathway to induce cardiac hypertrophy in vitro. Intriguingly, those DEHP-induced changes were obviously alleviated by TAX treatment. Taken together, cardiac hypertrophy was induced by DEHP via activating the IL-6/JAK/STAT3 signaling pathway, triggering glycometabolism disorder and mitochondrial dysfunction in vitro, can be ameliorated by TAX. Our findings may provide a feasible molecular mechanism for the treatment of cardiomyocyte hypertrophy induced by DEHP.

Apela Promotes Cardiomyocyte Differentiation from Transgenic Human Embryonic Stem Cell Lines.

Although embryonic stem (ES) cells (ESCs) may be a promising donor source for the repair of infarcted or ischemic heart tissues, their successful application in regenerative medicine has been hampered by difficulties in enriching, identifying, and selecting cardiomyocytes from the differentiating cells. We established transgenic human ES cell lines by transcriptional control of the α-cardiac myosin heavy chain (α-MHC) promoter driving green fluorescent protein (GFP) expression. Differentiated GFP-expressing cells display the characteristics of cardiomyocytes (CMs). Apela, a recently identified short peptide, up-regulated the expression of the cardiac-restricted transcription factors Tbx5 and GATA4 as well as differentiated the cardiomyocyte markers α-MHC and ß-MHC. Flow cytometric analysis showed that apela increased the percentage of GFP-expressing cells in the beating foci of the embryoid bodies. The percentage of cardiac troponin T (TNT)-positive cells and the protein expression of TNT were increased in the ES cell-derived CMs with apela treatment. Functionally, the contractile frequency of the ES-derived CMs responded appropriately to the vasoactive drugs isoprenaline and carbachol. Our work presented a protocol for specially labelling and enriching CMs by combining transgenic human ES cell lines and exogenous growth factor treatment.

Three perspectives on the molecular basis of hypercontractility caused by hypertrophic cardiomyopathy mutations.

Several lines of evidence suggest that the primary effect of hypertrophic cardiomyopathy mutations in human ß-cardiac myosin is hypercontractility of the heart, which leads to subsequent hypertrophy, fibrosis, and myofilament disarray. Here, I describe three perspectives on the molecular basis of this hypercontractility. The first is that hypercontractility results from changes in the fundamental parameters of the actin-activated ß-cardiac myosin chemo-mechanical ATPase cycle. The second considers that hypercontractility results from an increase in the number of functionally accessible heads in the sarcomere for interaction with actin. The final and third perspective is that load dependence of contractility is affected by cardiomyopathy mutations and small-molecule effectors in a manner that changes the power output of cardiac contraction. Experimental approaches associated with each perspective are described along with concepts of therapeutic approaches that could prove valuable in treating hypertrophic cardiomyopathy.