Roles of cMyBP-C phosphorylation on cardiac contractile dysfunction in db/db mice.

Type 2 diabetes mellitus (T2DM) is a metabolic disease and comorbidity associated with several conditions, including cardiac dysfunction leading to heart failure with preserved ejection fraction (HFpEF), in turn resulting in T2DM-induced cardiomyopathy (T2DM-CM). However, the molecular mechanisms underlying the development of T2DM-CM are poorly understood. It is hypothesized that molecular alterations in myopathic genes induced by diabetes promote the development of HFpEF, whereas cardiac myosin inhibitors can rescue the resultant T2DM-mediated cardiomyopathy. To test this hypothesis, a Leptin receptor-deficient db/db homozygous (Lepr db/db) mouse model was used to define the pathogenesis of T2DM-CM. Echocardiographic studies at 4 and 6 months revealed that Lepr db/db hearts started developing cardiac dysfunction by four months, and left ventricular hypertrophy with diastolic dysfunction was evident at 6 months. RNA-seq data analysis, followed by functional enrichment, revealed the differential regulation of genes related to cardiac dysfunction in Lepr db/db heart tissues. Strikingly, the level of cardiac myosin binding protein-C phosphorylation was significantly increased in Lepr db/db mouse hearts. Finally, using isolated skinned papillary muscles and freshly isolated cardiomyocytes, CAMZYOS ® (mavacamten, MYK-461), a prescription heart medicine used for symptomatic obstructive hypertrophic cardiomyopathy treatment, was tested for its ability to rescue T2DM-CM. Compared with controls, MYK-461 significantly reduced force generation in papillary muscle fibers and cardiomyocyte contractility in the db/db group. This line of evidence shows that 1) T2DM-CM is associated with hyperphosphorylation of cardiac myosin binding protein-C and 2) MYK-461 significantly lessened disease progression in vitro, suggesting its promise as a treatment for HFpEF.

MYBPC3 D389V Variant Induces Hypercontractility in Cardiac Organoids.

BACKGROUND: MYBPC3 , encoding cardiac myosin binding protein-C (cMyBP-C), is the most mutated gene known to cause hypertrophic cardiomyopathy (HCM). However, since little is known about the underlying etiology, additional in vitro studies are crucial to defining the underlying molecular mechanisms. Accordingly, this study aimed to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with a polymorphic variant (D389V) in MYBPC3 by using human-induced pluripotent stem cell (hiPSC)-derived cardiac organoids (hCOs). METHODS: The hiPSC-derived cardiomyocytes (hiPSC-CMs) and hCOs were generated from human subjects to define the molecular, cellular, and functional changes caused by the MYBPC3 D389V variant. This variant is associated with increased fractional shortening and is highly prevalent in South Asian descendants. Recombinant C0-C2, N'-region of cMyBP-C (wildtype and D389V), and myosin S2 proteins were also utilized to perform binding and motility assays in vitro . RESULTS: Confocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of the MYBPC3 D389V variant revealed the presence of highly organized sarcomeres. Furthermore, functional experiments showed hypercontractility with increased contraction velocity, faster calcium cycling, and faster contractile kinetics in hCOs expressing MYBPC3 D389V than NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation in MYBPC3 D389V hCOs was observed, but without changes in total protein levels, in addition to higher oxidative stress and lower mitochondrial membrane potential (ΔΨm). Next, spatial mapping revealed the presence of endothelial cells, fibroblasts, macrophages, immune cells, and cardiomyocytes in the hCOs. The hypercontractile function was significantly improved after treatment with the myosin inhibitor mavacamten (CAMZYOS®) in MYBPC3 D389V hCOs. Lastly, various in vitro binding assays revealed a significant loss of affinity in the presence of MYBPC3 D389V with myosin S2 region as a likely mechanism for hypercontraction. CONCLUSIONS: Conceptually, we showed the feasibility of assessing the functional and molecular mechanisms of HCM using highly translatable hCOs through pragmatic experiments that led to determining the MYBPC3 D389V hypercontractile phenotype, which was rescued by administration of a myosin inhibitor. Novelty and Significance: What Is Known?: MYBPC3 mutations have been implicated in hypertrophic cardiomyopathy. D389V is a polymorphic variant of MYBPC3 predicted to be present in 53000 US South Asians owing to the founder effect. D389V carriers have shown evidence of hyperdynamic heart, and human-induced pluripotent stem cells (hiPSC)-derived cardiomyocytes with D389V show cellular hypertrophy and irregular calcium transients. The molecular mechanism by which the D389V variant develops pathological cardiac dysfunction remains to be conclusively determined.What New Information Does This Article Contribute ?: The authors leveraged a highly translational cardiac organoid model to explore the role of altered cardiac calcium handling and cardiac contractility as a common pathway leading to pathophysiological phenotypes in patients with early HCM. The MYBPC3 D389V -mediated pathological pathway is first studied here by comparing functional properties using three-dimensional cardiac organoids differentiated from hiPSC and determining the presence of hypercontraction. Our data demonstrate that faster sarcomere kinetics resulting from lower binding affinity between D389V-mutated cMyBP-C protein and myosin S2, as evidenced by in vitro studies, could cause hypercontractility which was rescued by administration of mavacamten (CAMZYOS®), a myosin inhibitor. In addition, hypercontractility causes secondary mitochondrial defects such as higher oxidative stress and lower mitochondrial membrane potential (ΔΨm), highlighting a possible early adaptive response to primary sarcomeric changes. Early treatment of MYBPC3 D389V carriers with mavacamten may prevent or reduce early HCM-related pathology. GRAPHICAL ABSTRACT: A graphical abstract is available for this article.

Fast skeletal myosin binding protein-C expression exacerbates dysfunction in heart failure.

During heart failure, gene and protein expression profiles undergo extensive compensatory and pathological remodeling. We previously observed that fast skeletal myosin binding protein-C (fMyBP-C) is upregulated in diseased mouse hearts. While fMyBP-C shares significant homology with its cardiac paralog, cardiac myosin binding protein-C (cMyBP-C), there are key differences that may affect cardiac function. However, it is unknown if the expression of fMyBP-C expression in the heart is a pathological or compensatory response. We aim to elucidate the cardiac consequence of either increased or knockout of fMyBP-C expression. To determine the sufficiency of fMyBP-C to cause cardiac dysfunction, we generated cardiac-specific fMyBP-C over-expression mice. These mice were further crossed into a cMyBP-C null model to assess the effect of fMyBP-C in the heart in the complete absence of cMyBP-C. Finally, fMyBP-C null mice underwent transverse aortic constriction (TAC) to define the requirement of fMyBP-C during heart failure development. We confirmed the upregulation of fMyBP-C in several models of cardiac disease, including the use of lineage tracing. Low levels of fMyBP-C caused mild cardiac remodeling and sarcomere dysfunction. Exclusive expression of fMyBP-C in a heart failure model further exacerbated cardiac pathology. Following 8 weeks of TAC, fMyBP-C null mice demonstrated greater protection against heart failure development. Mechanistically, this may be due to the differential regulation of the myosin super-relaxed state. These findings suggest that the elevated expression of fMyBP-C in diseased hearts is a pathological response. Targeted therapies to prevent upregulation of fMyBP-C may prove beneficial in the treatment of heart failure. Significance Statement: Recently, the sarcomere - the machinery that controls heart and muscle contraction - has emerged as a central target for development of cardiac therapeutics. However, there remains much to understand about how the sarcomere is modified in response to disease. We recently discovered that a protein normally expressed in skeletal muscle, is present in the heart in certain settings of heart disease. How this skeletal muscle protein affects the function of the heart remained unknown. Using genetically engineered mouse models to modulate expression of this skeletal muscle protein, we determined that expression of this skeletal muscle protein in the heart negatively affects cardiac performance. Importantly, deletion of this protein from the heart could improve heart function suggesting a possible therapeutic avenue.

Distinct Mechanisms for Increased Cardiac Contraction Through Selective Alteration of Either Myosin or Troponin Activity.

Modulation of sarcomere contractility represents a new therapeutic opportunity for the treatment of heart failure by directly targeting the thick and thin filament proteins of the sarcomere to increase cardiac muscle contraction. This study compared the effect of 2 small molecules (M and T) that selectively alter myosin thick filament (M) or troponin thin filament (T) activity on overall cardiac muscle mechanics. This study revealed key differences related to the mechanism utilized by M and T to increase contractile force generation and suggests that targeting different proteins within the sarcomere may result in differentiating therapeutic profiles.

Mutations in myosin S2 alter cardiac myosin-binding protein-C interaction in hypertrophic cardiomyopathy in a phosphorylation-dependent manner.

Hypertrophic cardiomyopathy (HCM) is an inherited cardiovascular disorder primarily caused by mutations in the ß-myosin heavy-chain gene. The proximal subfragment 2 region (S2), 126 amino acids of myosin, binds with the C0-C2 region of cardiac myosin-binding protein-C to regulate cardiac muscle contractility in a manner dependent on PKA-mediated phosphorylation. However, it is unknown if HCM-associated mutations within S2 dysregulate actomyosin dynamics by disrupting its interaction with C0-C2, ultimately leading to HCM. Herein, we study three S2 mutations known to cause HCM: R870H, E924K, and E930Δ. First, experiments using recombinant proteins, solid-phase binding, and isothermal titrating calorimetry assays independently revealed that mutant S2 proteins displayed significantly reduced binding with C0-C2. In addition, CD revealed greater instability of the coiled-coil structure in mutant S2 proteins compared with S2Wt proteins. Second, mutant S2 exhibited 5-fold greater affinity for PKA-treated C0-C2 proteins. Third, skinned papillary muscle fibers treated with mutant S2 proteins showed no change in the rate of force redevelopment as a measure of actin-myosin cross-bridge kinetics, whereas S2Wt showed increased the rate of force redevelopment. In summary, S2 and C0-C2 interaction mediated by phosphorylation is altered by mutations in S2, which augment the speed and force of contraction observed in HCM. Modulating this interaction could be a potential strategy to treat HCM in the future.

Amino terminus of cardiac myosin binding protein-C regulates cardiac contractility.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N')-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N'-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N'-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.

South Asian-Specific MYBPC3 Δ25bp Deletion Carriers Display Hypercontraction and Impaired Diastolic Function Under Exercise Stress.

Background: A 25-base pair (25bp) intronic deletion in the MYBPC3 gene enriched in South Asians (SAs) is a risk allele for late-onset left ventricular (LV) dysfunction, hypertrophy, and heart failure (HF) with several forms of cardiomyopathy. However, the effect of this variant on exercise parameters has not been evaluated. Methods: As a pilot study, 10 asymptomatic SA carriers of the MYBPC3 Δ25bp variant (52.9 ± 2.14 years) and 10 age- and gender-matched non-carriers (NCs) (50.1 ± 2.7 years) were evaluated at baseline and under exercise stress conditions using bicycle exercise echocardiography and continuous cardiac monitoring. Results: Baseline echocardiography parameters were not different between the two groups. However, in response to exercise stress, the carriers of Δ25bp had significantly higher LV ejection fraction (%) (CI: 4.57 ± 1.93; p < 0.0001), LV outflow tract peak velocity (m/s) (CI: 0.19 ± 0.07; p < 0.0001), and higher aortic valve (AV) peak velocity (m/s) (CI: 0.103 ± 0.08; p = 0.01) in comparison to NCs, and E/A ratio, a marker of diastolic compliance, was significantly lower in Δ25bp carriers (CI: 0.107 ± 0.102; p = 0.038). Interestingly, LV end-diastolic diameter (LVIDdia) was augmented in NCs in response to stress, while it did not increase in Δ25bp carriers (CI: 0.239 ± 0.125; p = 0.0002). Further, stress-induced right ventricular systolic excursion velocity s' (m/s), as a marker of right ventricle function, increased similarly in both groups, but tricuspid annular plane systolic excursion increased more in carriers (slope: 0.008; p = 0.0001), suggesting right ventricle functional differences between the two groups. Conclusions: These data support that MYBPC3 Δ25bp is associated with LV hypercontraction under stress conditions with evidence of diastolic impairment.

Structure, Rheology, and Electrokinetics of Soft Colloidal Suspension Electrolytes.

When ion transport in a binary liquid electrolyte is driven at potentials above the thermal voltage, an extended space charge region forms at the electrolyte/electrode interface and triggers the hydrodynamic instability termed electroconvection. We experimentally show that this instability can be completely arrested in soft colloidal suspension electrolytes composed of low concentrations of polymer-grafted nanoparticles in a liquid host. The mechanism is revealed by means of X-ray scattering, Brownian dynamics calculations, and linear stability analysis to involve overlap of the soft particles at low particle fractions to create a jammed, nanoporous medium that resists convective flow by a Darcy-Brinkman like drag on the electrolyte solvent.

Cardiac myosin binding protein-C phosphorylation regulates the super-relaxed state of myosin.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) accelerates cardiac contractility. However, the mechanisms by which cMyBP-C phosphorylation increases contractile kinetics have not been fully elucidated. In this study, we tested the hypothesis that phosphorylation of cMyBP-C releases myosin heads from the inhibited super-relaxed state (SRX), thereby determining the fraction of myosin available for contraction. Mice with various alanine (A) or aspartic acid (D) substitutions of the three main phosphorylatable serines of cMyBP-C (serines 273, 282, and 302) were used to address the association between cMyBP-C phosphorylation and SRX. Single-nucleotide turnover in skinned ventricular preparations demonstrated that phosphomimetic cMyBP-C destabilized SRX, whereas phospho-ablated cMyBP-C had a stabilizing effect on SRX. Strikingly, phosphorylation at serine 282 site was found to play a critical role in regulating the SRX. Treatment of WT preparations with protein kinase A (PKA) reduced the SRX, whereas, in nonphosphorylatable cMyBP-C preparations, PKA had no detectable effect. Mice with stable SRX exhibited reduced force production. Phosphomimetic cMyBP-C with reduced SRX exhibited increased rates of tension redevelopment and reduced binding to myosin. We also used recombinant myosin subfragment-2 to disrupt the endogenous interaction between cMyBP-C and myosin and observed a significant reduction in the population of SRX myosin. This peptide also increased force generation and rate of tension redevelopment in skinned fibers. Taken together, this study demonstrates that the phosphorylation-dependent interaction between cMyBP-C and myosin is a determinant of the fraction of myosin available for contraction. Furthermore, the binding between cMyBP-C and myosin may be targeted to improve contractile function.

Data on whole length myosin binding protein C stabilizes myosin S2 as measured by gravitational force spectroscopy.

Data presented in this article relates to the research article entitled "Whole length myosin binding protein C stabilizes myosin subfragment-2 (S2) flexibility as measured by gravitational force spectroscopy." (Singh et al., 2018) [1]. The data exhibits the purified skeletal myosin binding protein C (MyBPC) from rabbit back muscle was of slow skeletal type confirmed by chromatography and in unphosphorylated state based on its isoelectric point (pI) by chromatofocussing. The competitive enzyme linked immunosorbent assay (cELISA) data displayed the site specificity of polyclonal anti-S2 antibody to myosin S2. This polyclonal antibody binding site corresponds to a familial hypertrophic cardiomyopathy (FHC) point mutation hotspot on myosin S2 illustrated in a figure of compiled data.

Whole length myosin binding protein C stabilizes myosin S2 as measured by gravitational force spectroscopy.

The mechanical stability of the myosin subfragment-2 (S2) was tested with simulated force spectroscopy (SFS) and gravitational force spectroscopy (GFS). Experiments examined unzipping S2, since it required less force than stretching parallel to the coiled coil. Both GFS and SFS demonstrated that the force required to destabilize the light meromyosin (LMM) was greater than the force required to destabilize the coiled coil at each of three different locations along S2. GFS data also conveyed that the mechanical stability of the S2 region is independent from its association with the myosin thick filament using cofilaments of myosin tail and a single intact myosin. The C-terminal end of myosin binding protein C (MyBPC) binds to LMM and the N-terminal end can bind either S2 or actin. The force required to destabilize the myosin coiled coil molecule was 3 times greater in the presence of MyBPC than in its absence. Furthermore, the in vitro motility assay with full length slow skeletal MyBPC slowed down the actin filament sliding over myosin thick filaments. This study demonstrates that skeletal MyBPC both enhanced the mechanical stability of the S2 coiled coil and reduced the sliding velocity of actin filaments over polymerized myosin filaments.

A Molecularly Complete Planar Bacterial Outer Membrane Platform.

The bacterial outer membrane (OM) is a barrier containing membrane proteins and liposaccharides that fulfill crucial functions for Gram-negative bacteria. With the advent of drug-resistant bacteria, it is necessary to understand the functional role of this membrane and its constituents to enable novel drug designs. Here we report a simple method to form an OM-like supported bilayer (OM-SB), which incorporates native lipids and membrane proteins of gram-negative bacteria from outer membrane vesicles (OMVs). We characterize the formation of OM-SBs using quartz crystal microbalance with dissipation (QCM-D) and fluorescence microscopy. We show that the orientation of proteins in the OM-SB matches the native bacterial membrane, preserving the characteristic asymmetry of these membranes. As a demonstration of the utility of the OM-SB platform, we quantitatively measure antibiotic interactions between OM-SBs and polymyxin B, a cationic peptide used to treat Gram-negative infections. This data enriches understanding of the antibacterial mechanism of polymyxin B, including disruption kinetics and changes in membrane mechanical properties. Combining OM-SBs with microfluidics will enable higher throughput screening of antibiotics. With a broader view, we envision that a molecularly complete membrane-scaffold could be useful for cell-free applications employing engineered membrane proteins in bacterial membranes for myriad technological purposes.

Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions on protein activity and the roles of membrane proteins in disease pathways.

Use of Infant Feeding Tube for Staged Flexor Tendon Reconstruction.

A two-stage flexor tendon reconstruction using a silicone rod in the first stage and a free tendon graft through the pseudo sheath formed around the silicone in the second stage was described by Hunter and Salisbury for a neglected and failed flexor tendon reconstruction. We are describing a technique where we have used an infant feeding tube as a substitute for silicone rods, which substantially reduces the cost of procedure but delivers the same results.

Posterior scrotal artery flap to cover a groin defect: a new technique.

Commonly, a groin defect is reconstructed with flaps from ipsilateral thigh or lower abdomen. Here we present a case report of use of a pedicled flap from the posterior scrotum based on posterior scrotal artery to cover a groin defect exposing femoral vessels. Posterior scrotal artery, to best of our knowledge, has not been described in the literature to cover a groin defect.

Simple and effective method of heat therapy in lymphoedema.

Heat therapy is a well known conservative management for lymphoedema. We are describing here a heat therapy apparatus which is easy to make, cheap, transportable, easily reproducible and maintenance free and found to be very effective.