Myosin Binding protein-C Research Articles

Molecular regulation of stretch activation.

Stretch activation is defined as a delayed increase in force after rapid stretches. Although there is considerable evidence for stretch activation in isolated cardiac myofibrillar preparations, few studies have measured mechanisms of stretch activation in mammalian skeletal muscle fibers. We measured stretch activation following rapid step stretches [∼1%-4% sarcomere length (SL)] during submaximal Ca2+ activations of rat permeabilized slow-twitch skeletal muscle fibers before and after protein kinase A (PKA), which phosphorylates slow myosin binding protein-C. PKA significantly increased stretch activation during low (∼25%) Ca2+ activation and accelerated rates of delayed force development (kef) during both low and half-maximal Ca2+ activation. Following the step stretches and subsequent force development, fibers were rapidly shortened to original sarcomere length, which often elicited a shortening-induced transient force overshoot. After PKA, step shortening-induced transient force overshoot increased ∼10-fold following an ∼4% SL shortening during low Ca2+ activation levels. kdf following step shortening also increased after PKA during low and half-maximal Ca2+ activations. We next investigated thin filament regulation of stretch activation. We tested the interplay between cardiac troponin I (cTnI) phosphorylation at the canonical PKA and novel tyrosine kinase sites on stretch activation. Native slow-skeletal Tn complexes were exchanged with recombinant human cTn complex with different human cTnI N-terminal pseudo-phosphorylation molecules: 1) nonphosphorylated wild type (WT), 2) the canonical S22/23D PKA sites, 3) the tyrosine kinase Y26E site, and 4) the combinatorial S22/23D + Y26E cTnI. All three pseudo-phosphorylated cTnIs elicited greater stretch activation than WT. Following stretch activation, a new, elevated stretch-induced steady-state force was reached with pseudo-phosphorylated cTnI. Combinatorial S22/23D + Y26E pseudo-phosphorylated cTnI increased kdf. These results suggest that slow-skeletal myosin binding protein-C (sMyBP-C) phosphorylation modulates stretch activation by a combination of cross-bridge recruitment and faster cycling kinetics, whereas cTnI phosphorylation regulates stretch activation by both redundant and synergistic mechanisms; and, taken together, these sarcomere phosphoproteins offer precision targets for enhanced contractility.

MYBPC1 is a key regulator for laryngeal carcinoma formation.

Laryngeal carcinoma represents one of the most common types of tumor of the respiratory tract. The aim of the present study was to evaluate the functions of myosin-binding protein C1 (MYBPC1) in the progression of laryngeal carcinoma and to unravel the potential underlying molecular mechanism(s). Significantly differentially expressed mRNAs and miRNAs were analyzed, and potential genes were verified using clinically recruited patients with laryngeal carcinoma. The human laryngeal carcinoma cell lines TU686, TU212 and AMC-HN-8, as well as the control nasopharyngeal epithelial cell line NP69, were selected for the functional analysis of MYBPC1. The interaction between MYBPC1 and miR-451a was also explored in depth. The functions of MYBPC1 in the laryngeal carcinoma cell lines were examined using colony formation assay, cell proliferation and invasion assays, and via measuring the extent of apoptosis. The intracellular function of MYBPC1 was subsequently confirmed by constructing an in vivo xenograft model through the subcutaneous injection of laryngeal carcinoma cells into 4-week-old male nude mice. Compared with normal tissue, MYBPC1 was found to be the most significantly downregulated gene, whereas activating transcription factor-2 (ATF-2) was the most significantly upregulated one. At the same time, miR-451a was found to be the most significantly downregulated miRNA in laryngeal squamous cell carcinoma tissues. According to the WHO classification system, we found that the level of MYBPC1 was significantly decreased in grade IV tissues compared with grade II and grade III tissues, a finding that was consistent with the observed activity of miR-451a. MiR-451a was found to cause a marked enhancement of the activity of MYBPC1 in TU212 cells, which in turn was attenuated by ATF overexpression, suggesting that miR-451a could indirectly modulate the function of MYBPC1 through the ATF2-dependent signaling axis. MYBPC1 suppressed the invasion of cells induced by ATF2 in laryngeal carcinoma cells. Moreover, subcutaneous injection of MYBPC1 to construct an in vivo xenograft mouse model enabled rescue of the mice from laryngeal carcinoma formation. Taken together, the results of the present study have shown that MYBPC1 fulfills a pivotal role in laryngeal carcinoma formation, and these findings may provide both a new avenue for research planning and a potential therapeutic target for laryngeal carcinoma.

Abstract P3022: Multiscale Characterization Of Left Ventricular Diastolic Dysfunction In Diabetic And Cardiac Myosin Binding Protein-C Phospho-Ablated Murine Models

Introduction: Left ventricular (LV) diastolic dysfunction (LVDD) is often characterized by organ-level (global) metrics whose quantitative connections to mechanical remodeling events at the tissue and fiber levels remain poorly studied. Methods: We performed measurements in the LV at three length scales, namely, organ, tissue, and fiber, in two murine models of LVDD: (i) diabetic (db/db) (n=10) versus wild-type (WT; n=10) mice, and (ii) novel knock-in cardiac myosin binding protein-C (cMyBP-C) phospho-ablated (KI-3A; n=8) versus cMyBP-C phospho-mimetic (KI-3D, n=6) and WT (n=8) mice. Echocardiography was done in vivo. Left ventricular free wall (LVFW) specimens were harvested and passively stretched along with circumferential (C) and longitudinal (L) directions (schematically identified in Fig. 1c) in select mice. Passive tests were followed by activation-relaxation tests of the LVFW specimens at resting biaxial stretches. Active force-pCa measurements at resting stretch were carried out on the skinned papillary muscle fibers. Results: Both db/db and KI-3A mice demonstrated slow relaxation at the organ level indicated by increased E/e' (Figs. 1a-b). db/db LVFW specimens showed increased circumferential stiffness at the tissue level under biaxial passive stretching (p<0.05 for strains >0.25). Biaxial activation-relaxation tests of LVFW specimens in knock-in mice (Fig. 1c shows the results for KI-3A mice) revealed that KI-3A mice relax significantly slower than KI-3D mice along the longitudinal direction (Fig. 1d). Fiber-level tests demonstrated that active force development was significantly enhanced in db/db mice (Fig. 1e) while it was significantly suppressed in KI-3A mice relative to KI-3D and WT mice (Fig. 1f). Conclusions: Although both db/db and KI-3A mice showed LVDD at the organ level, they demonstrated different mechanical alterations at the fiber and tissue fiber levels, suggesting that global metrics should be complemented with metrics characterizing the LV properties at the fiber and tissue levels to fully characterize LVDD.

Abstract EC101: Premature Stop Mutations In Mybphl Share The Common Pathomechanism Of Preventing Protein Incorporation Into The Cardiac Myofilament

Cardiomyopathy and arrhythmia are diseases with significant genetic linkage. Cardiac myosin binding protein-C (cMyBP-C) is a myofilament associated protein with a C-terminal composed of repeating immunoglobulin (Ig) and fibronectin III (FnIII) domains that bind to the myosin thick filaments and titin. Truncating mutations in the C-terminal of MyBP-C result in improper myofilament incorporation and degradation of the truncated protein and is a major cause of hypertrophic cardiomyopathy. Myosin-binding protein-H like ( MyBP-HL ) is a protein that was recently identified that is structurally related to MyBP-C. Both cMyBP-C and MyBP-HL contain repeated C-terminal Ig and FnIII domains that bind to myosin. MyBP-HL is different in that it is primarily expressed in the atria where it interacts with the myofilament. MyBP-HL premature stop mutations are associated with improper myofilament incorporation, and has been linked to ventricular conduction system abnormalities, atria enlargement, and dilated cardiomyopathy (DCM) in humans. The gnomAD database reports 9 stop-gain mutations for MYBPHL in humans including: Gln29, Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250, Arg255 and Tyr307. Prior investigation modeled the MyBP-HL Arg255X mutation and showed no MyBP-HL localization into the myofilament. A null mouse model showed that loss of MyBP-HL resulted in arrhythmia and cardiomyopathy, similar to that observed in humans. We hypothesize that all MyBP-HL truncation mutations will share a common mechanism by failing to produce MyBP-HL that integrates into the sarcomere. We test this hypothesis by transfecting neonatal rat cardiomyocytes with MyBP-HL constructs containing these mutations. We tested Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250 and Arg255 MyBP-HL mutations. We measured co-localization events between MyBP-HL mutants and MyBP-C in atrial and ventricular overexpressed cells and observed that stop gained mutations disrupt normal localization as we expected. Moreover, we tested wild-type MyBP-HL overexpression, and both showed to be able to incorporate in a similar pattern to cMyBP-C C-zone doublets. These truncating mutations in MYBP-HL may increase risk for developing arrhythmias, atria enlargement and DCM.

Abstract GS101: Myosin Binding Protein H-like Competes With Cardiac Myosin Binding Protein-c To Prolong Atrial Contraction

Myosin binding protein-H like (MyBP-HL) has recently been identified as a novel component of myofilaments in the cardiac atria. Loss of MyBP-HL is associated with dilated cardiomyopathy and arrhythmia in humans and mice. MyBP-HL is highly homologous to cardiac myosin binding protein-C (cMyBP-C), which has been studied in the context of genetic cardiomyopathy. Prior work using mouse atrial tissue lysates established MyBP-HL and cMyBP-C bind competitively and maintain a stoichiometric ratio compared to myosin heavy chain. We now confirm this competitive binding using neonatal rat ventricular cardiomyocytes (NRVMs) transfected with mouse Mybphl . Using confocal microscopy with internal controls, we found a linear, inverse relationshib between myosin binding protein levels with NRVMs with high levels of MyBP-HL expression showing a reduction in cMyBP-C levels compared to untransfected NRVMs that only express cMyBP-C. While we show that these two myosin binding proteins bind comparatively with each other, the functional consequences are unclear. We performed force-calcium measurements of permeabilized single atrial cardiomyocytes and found that loss of MyBP-HL does not significantly alter the calcium sensitivity of force development, or the specific force generated by isometric contraction. Because cMyBP-C is a known regulator of crossbridge sliding velocity, we evaluated contraction and relaxation kinetics of Mybphl single-myofibrils from WT and Mybphl homozygous null mice. Atrial myofibrils lacking Mybphl showed a significantly faster rate and shorter duration of their linear phase of relaxation, which is governed by the off-rate of myosin from actin. This suggests that extra cMyBP-C in the atria can hasten myofibril relaxation. In healthy atria with MyBP-HL replacing approximately half the cMyBP-C, the slow phase of relaxation is prolonged. While the physiological importance of this is not explored, it provides a mechanism by which loss of function mutations in MYBPHL cause contractile dysfunction.

Heterogeneous Distribution of Genetic Mutations in Myosin Binding Protein-C Paralogs.

Myosin binding protein-C (MyBP-C) is a sarcomeric protein which regulates the force of contraction in striated muscles. Mutations in the MYBPC family of genes, including slow skeletal (MYBPC1), fast skeletal (MYBPC2) and cardiac (MYBPC3), can result in cardiac and skeletal myopathies. Nonetheless, their evolutionary pattern, pathogenicity and impact on MyBP-C protein structure remain to be elucidated. Therefore, the present study aimed to systematically assess the evolutionarily conserved and epigenetic patterns of MYBPC family mutations. Leveraging a machine learning (ML) approach, the Genome Aggregation Database (gnomAD) provided variants in MYBPC1, MYBPC2, and MYBPC3 genes. This was followed by an analysis with Ensembl’s variant effect predictor (VEP), resulting in the identification of 8,618, 3,871, and 3,071 variants in MYBPC1, MYBPC2, and MYBPC3, respectively. Missense variants comprised 61%–66% of total variants in which the third nucleotide positions in the codons were highly altered. Arginine was the most mutated amino acid, important because most disease-causing mutations in MyBP-C proteins are arginine in origin. Domains C5 and C6 of MyBP-C were found to be hotspots for most mutations in the MyBP-C family of proteins. A high percentage of truncated mutations in cMyBP-C cause cardiomyopathies. Arginine and glutamate were the top hits in fMyBP-C and cMyBP-C, respectively, and tryptophan and tyrosine were the most common among the three paralogs changing to premature stop codons and causing protein truncations at the carboxyl terminus. A heterogeneous epigenetic pattern was identified among the three MYBP-C paralogs. Overall, it was shown that databases using computational approaches can facilitate diagnosis and drug discovery to treat muscle disorders caused by MYBPC mutations.

Myosin Heavy Chain Converter Domain Mutations Drive Early-Stage Changes in Extracellular Matrix Dynamics in Hypertrophic Cardiomyopathy.

More than 60% of hypertrophic cardiomyopathy (HCM)-causing mutations are found in the gene loci encoding cardiac myosin-associated proteins including myosin heavy chain (MHC) and myosin binding protein C (MyBP-C). Moreover, patients with more than one independent HCM mutation may be at increased risk for more severe disease expression and adverse outcomes. However detailed mechanistic understanding, especially at early stages of disease progression, is limited. To identify early-stage HCM triggers, we generated single (MYH7 c.2167C > T [R723C] with a known pathogenic significance in the MHC converter domain) and double (MYH7 c.2167C > T [R723C]; MYH6 c.2173C > T [R725C] with unknown significance) myosin gene mutations in human induced pluripotent stem cells (hiPSCs) using a base-editing strategy. Cardiomyocytes (CMs) derived from hiPSCs with either single or double mutation exhibited phenotypic characteristics consistent with later-stage HCM including hypertrophy, multinucleation, altered calcium handling, metabolism, and arrhythmia. We then probed mutant CMs at time points prior to the detection of known HCM characteristics. We found MYH7/MYH6 dual mutation dysregulated extracellular matrix (ECM) remodeling, altered integrin expression, and interrupted cell-ECM adhesion by limiting the formation of focal adhesions. These results point to a new phenotypic feature of early-stage HCM and reveal novel therapeutic avenues aimed to delay or prohibit disease onset.

NOS isoforms facilitate Ca2+‐cycling‐dependent and ‐independent cardiac contractility

Increasing biased G‐protein coupled receptor (GPCR) drugs are now under clinical investigation. More understanding of the biased β‐adrenergic receptor (β‐AR) signaling is urgently required for a better cardiovascular drug. Here we found that two nitric oxide synthase (NOS) isotypes, NOS1, and NOS3 coupled with β1ARs and transduce distinct β1AR‐cGMP signaling at sarcoplasmic reticulum and myofilament microdomain, respectively. Intriguingly, activation of NOS1 enhanced contractility and Ca2+ cycling, whereas NOS3 promoted contraction without affecting intracellular Ca2+. Quantitative proteomic revealed that NOS3 activation increased phosphorylation of myosin proteins including myosin binding protein C (MyBP‐C), myosin light chain kinase (MYLK) and myosin phosphatase target subunit 1 (MYPT1), to enhance contractility by Ca2+sensitization. Moreover, in heart failure, excessive adrenergic stimulation dissociated β1AR from NOS1 and impaired the NOS1‐cGMP signaling. Accordingly, stimulating NOS3 but NOS1 enhances systolic cardiac contraction in failing mice without rousing intracellular Ca2+. We propose NOS facilitate biased β1AR‐cGMP signaling via Ca2+‐dependent or independent mechanisms and define NOS3‐cGMP as specific therapeutic strategy targeting myofilament for heart failure.

Single-cell transcriptomics provides insights into hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a genetic heart disease that is characterized by unexplained segmental hypertrophy that is usually most pronounced in the septum. While sarcomeric gene mutations are often the genetic basis for HCM, the mechanistic origin for the heterogeneous remodeling remains largely unknown. A better understanding of the gene networks driving the cardiomyocyte (CM) hypertrophy is required to improve therapeutic strategies. Patients suffering from HCM often receive a septal myectomy surgery to relieve outflow tract obstruction due to hypertrophy. Using single-cell RNA sequencing (scRNA-seq) on septal myectomy samples from patients with HCM, we identify functional links between genes, transcription factors, and cell size relevant for HCM. The data show the utility of using scRNA-seq on the human hypertrophic heart, highlight CM heterogeneity, and provide a wealth of insights into molecular events involved in HCM that can eventually contribute to the development of enhanced therapies.

Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G).

Human wild type (wt) cardiac α-actin and its mutants p.A295S or p.R312H and p.E361G correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by using the baculovirus/Sf21 insect cell system. The c-actin variants inhibited DNase I, indicating maintenance of their native state. Electron microscopy showed the formation of normal appearing actin filaments though they showed mutant specific differences in length and straightness correlating with their polymerization rates. TRITC-phalloidin staining showed that p.A295S and p.R312H exhibited reduced and the p.E361G mutant increased lengths of their formed filaments. Decoration of c-actins with cardiac tropomyosin (cTm) and troponin (cTn) conveyed Ca2+-sensitivity of the myosin-S1 ATPase stimulation, which was higher for the HCM p.A295S mutant and lower for the DCM p.R312H and p.E361G mutants than for wt c-actin. The lower Ca2+-sensitivity of myosin-S1 stimulation by both DCM actin mutants was corrected by the addition of levosimendan. Ca2+-dependency of the movement of pyrene-labeled cTm along polymerized c-actin variants decorated with cTn corresponded to the relations observed for the myosin-S1 ATPase stimulation though shifted to lower Ca2+-concentrations. The N-terminal C0C2 domain of cardiac myosin-binding protein-C increased the Ca2+-sensitivity of the pyrene-cTM movement of bovine, recombinant wt, p.A295S, and p.E361G c-actins, but not of the p.R312H mutant, suggesting decreased affinity to cTm.

Modulation of myosin by cardiac myosin binding protein-C peptides improves cardiac contractility in ex-vivo experimental heart failure models

Cardiac myosin binding protein-C (cMyBP-C) is an important regulator of sarcomeric function. Reduced phosphorylation of cMyBP-C has been linked to compromised contractility in heart failure patients. Here, we used previously published cMyBP-C peptides 302A and 302S, surrogates of the regulatory phosphorylation site serine 302, as a tool to determine the effects of modulating the dephosphorylation state of cMyBP-C on cardiac contraction and relaxation in experimental heart failure (HF) models in vitro. Both peptides increased the contractility of papillary muscle fibers isolated from a mouse model expressing cMyBP-C phospho-ablation (cMyBP-CAAA) constitutively. Peptide 302A, in particular, could also improve the force redevelopment rate (ktr) in papillary muscle fibers from cMyBP-CAAA (nonphosphorylated alanines) mice. Consistent with the above findings, both peptides increased ATPase rates in myofibrils isolated from rats with myocardial infarction (MI), but not from sham rats. Furthermore, in the cMyBP-CAAA mouse model, both peptides improved ATPase hydrolysis rates. These changes were not observed in non-transgenic (NTG) mice or sham rats, indicating the specific effects of these peptides in regulating the dephosphorylation state of cMyBP-C under the pathological conditions of HF. Taken together, these studies demonstrate that modulation of cMyBP-C dephosphorylation state can be a therapeutic approach to improve myosin function, sarcomere contractility and relaxation after an adverse cardiac event. Therefore, targeting cMyBP-C could potentially improve overall cardiac performance as a complement to standard-care drugs in HF patients.

Mapping the binding interaction of MyBP-C and myosin in hypertrophic cardiomyopathy

Cardiac myosin binding protein-C (MyBP-C) accounts for about half of hypertrophic cardiomyopathy (HCM) mutations, and in the events where another sarcomeric protein is mutated, MyBP-C phosphorylation is frequently altered. MyBP-C is thought to regulate normal cardiac contractility through its interactions with actin and myosin, and this regulation is dependent on phosphorylation. Efforts in elucidating the details of MyBP-C interactions with myosin and actin have been limited due to low-throughput and labor-intensive assays.

A homozygous missense mutation in the M-domain of cardiac myosin binding protein-C is lethal in a CRISPR-gene edited mouse model

Myosin binding protein-C (MyBP-C) is a regulatory protein that modulates striated muscle contraction. Mutations in MYBPC3, the gene encoding cardiac MyBP-C, are a leading cause of hypertrophic cardiomyopathy (HCM). The L348P substitution (L352P in humans) is an HCM-associated missense mutation that increases binding of cardiac MyBP-C's M-domain to actin (Bezold et al., J Biol Chem, 2013). We previously developed a transgenic mouse model where transgenic expression of the L348P mutant protein was 39 ± 4% of total cardiac MyBP-C (van Dijk et al., J Mol Cell Cardiol, 2018).

Mechanisms of dysfunction in patient-derived cardiomyocytes with the hypertrophic cardiomyopathy-myosin binding protein-C e258K mutation

Mutations in cardiac myosin binding protein-C (cMyBP-C) are the most common cause of hypertrophic cardiomyopathy (HCM). We recently described the highly penetrant MYBPC3-c.772G>A (p.E258K) mutation with founder effect in Tuscany, Italy. Isolated myofibrils from patient myectomies indicated accelerated cross-bridge kinetics and higher energy cost of tension. Concurrently, prolonged action potentials and calcium transient duration preserves twitch contraction amplitude and duration in intact cells and trabeculae (Pioner et al., Biophys J, 2020;118:35a).

Understanding the molecular basis of HCM-causing mutations in cardiac myosin and cardiac myosin binding protein-C

Hypertrophic cardiomyopathy (HCM), a heritable cardiovascular disease marked by hypercontractility and abnormal thickening of heart muscle, affects up to 1 in 200 individuals. Mutations in sarcomere proteins result in HCM, but the mechanism by which these mutant proteins lead to hypercontractility and heart failure remains poorly understood, limiting clinical interventions for the same. We are currently focusing on two sarcomere proteins that together comprise ∼70% of all known HCM mutations: β-cardiac myosin and cardiac myosin binding protein-C, cMyBPC (a regulatory protein in the thick filament).

Interaction of the M-domain of cardiac myosin binding protein-C with the native cardiac thin filament

Cardiac muscle contraction depends on precise interactions between thin (actin) and thick (myosin) filaments (TFs). TFs are regulated by intracellular Ca2+ levels as well as other sarcomeric proteins, such as cardiac myosin binding protein C (cMyBP-C) and myosin. Recent studies have shown the first four N-terminal domains (NTDs) of cMyBP-C (i.e. C0, C1, M and C2), are effective modulators of the TF activity. Their collective activation of the TF is regulated by the phosphorylation of the M domain but the mechanism is not fully understood.

In situ replacement of slow skeletal myosin binding protein-c

Myosin binding protein (MyBP-C) is a modular protein that interacts with both thin and thick filaments and regulates striated muscle contraction. The functional characteristics of MyBP-C have yet to be fully uncovered in all 3 isoforms (cardiac, fast skeletal, and slow skeletal). To manipulate cardiac (c) MyBP-C at its precise position in the sarcomere in situ, we created gene-edited ‘SpyC’ mice that allow us to use a novel ‘cut and paste’ method for cMyBP-C recombination in cardio-myocytes. This ‘cut and paste’ method allows for the in situ removal of endogenous N’-terminal domains from MyBP-C via TEV proteolysis, followed by its replacement with any desired recombinant N’-terminal MyBP-C.

Functional and Molecular Characterisation of Heart Failure Progression in Mice and the Role of Myosin Regulatory Light Chains in the Recovery of Cardiac Muscle Function

Heart failure (HF) as a result of myocardial infarction (MI) is a major cause of fatality worldwide. However, the cause of cardiac dysfunction succeeding MI has not been elucidated at a sarcomeric level. Thus, studying the alterations within the sarcomere is necessary to gain insights on the fundamental mechansims leading to HF and potentially uncover appropriate therapeutic targets. Since existing research portrays regulatory light chains (RLC) to be mediators of cardiac muscle contraction in both human and animal models, its role was further explored In this study, a detailed characterisation of the physiological changes (i.e., isometric force, calcium sensitivity and sarcomeric protein phosphorylation) was assessed in an MI mouse model, between 2D (2 days) and 28D post-MI, and the changes were related to the phosphorylation status of RLCs. MI mouse models were created via complete ligation of left anterior descending (LAD) coronary artery. Left ventricular (LV) papillary muscles were isolated and permeabilised for isometric force and Ca2+ sensitivity measurement, while the LV myocardium was used to assay sarcomeric proteins’ (RLC, troponin I (TnI) and myosin binding protein-C (MyBP-C)) phosphorylation levels and enzyme (myosin light chain kinase (MLCK), zipper interacting protein kinase (ZIPK) and myosin phosphatase target subunit 2 (MYPT2)) expression levels. Finally, the potential for improving the contractility of diseased cardiac papillary fibres via the enhancement of RLC phosphorylation levels was investigated by employing RLC exchange methods, in vitro. RLC phosphorylation and isometric force potentiation were enhanced in the compensatory phase and decreased in the decompensatory phase of HF failure progression, respectively. There was no significant time-lag between the changes in RLC phosphorylation and isometric force during HF progression, suggesting that changes in RLC phosphorylation immediately affect force generation. Additionally, the in vitro increase in RLC phosphorylation levels in 14D post-MI muscle segments (decompensatory stage) enhanced its force of isometric contraction, substantiating its potential in HF treatment. Longitudinal observation unveils potential mechanisms involving MyBP-C and key enzymes regulating RLC phosphorylation, such as MLCK and MYPT2 (subunit of MLCP), during HF progression. This study primarily demonstrates that RLC phosphorylation is a key sarcomeric protein modification modulating cardiac function. This substantiates the possibility of using RLCs and their associated enzymes to treat HF.

Left Ventricular Hypertrophy and Hypertrophic Cardiomyopathy in Adult Solid Organ Transplant Recipients.

Background.Hypertrophic cardiomyopathy (HCM) in pediatric solid organ transplant recipients has been reported in association with use of calcineurin inhibitors. However, data on the incidence and prevalence of HCM in adult posttransplant patients are limited. We sought to describe the clinical characteristics of solid organ transplant recipients who were diagnosed with HCM from 2011 to 2021 at a single center.Methods.Patients who had undergone solid organ transplant and exhibited left ventricular hypertrophy with left ventricular wall thickness ≥13 mm on transthoracic echocardiography were included. Clinical history, pedigree analysis, clinical genetic testing, transthoracic echocardiography, cardiac magnetic resonance imaging, treatment, and follow-up testing results were collected. Categorical variables were described as n (%). Continuous variables were described with medians and interquartile ranges and compared using the Wilcoxon rank-sum and Kruskal-Wallis tests. A 2-sided P < 0.05 was considered statistically significant.Results.Three lung, 5 kidney, and 4 liver transplant recipients from 12 different families were included. Seven patients (58%) did not carry a preexisting diagnosis of hypertension, and none had a history of aortic or subaortic stenosis. A majority of patients exhibited asymmetric septal hypertrophy (67%; medial septal thickness versus left ventricular posterior wall thickness 17 versus 13 mm; P < 0.001) and dynamic left ventricular outflow tract (LVOT) obstruction (58%). All patients were managed long term with calcineurin inhibitors. Clinical genetic testing in 6 patients identified 2 with disease-causing variants in 2 sarcomere genes, myosin binding protein-C and myosin heavy chain 7. Four patients (33%) underwent successful septal reduction therapy for treatment of symptomatic LVOT obstruction.Conclusions.Symptomatic HCM with dynamic LVOT obstruction can develop in solid organ transplant recipients, and genetic testing can identify individuals with sarcomeric HCM. Medical management and septal reduction therapies are treatment options for severe symptomatic disease.

Abstract 14317: Left Ventricular Free Wall Adaptations in Heart Failure With Preserved Ejection Fraction: Insights From a Murine Model

Introduction: Heart failure with preserved ejection fraction has heterogeneous causes. Understanding the cause of diastolic dysfunction can help with pairing correct treatment. Hypothesis: We hypothesize that one can elucidate the underlying cause using echocardiography. Methods: We performed echocardiography on hearts of cardiac myosin binding protein-C phosphorylation deficient mouse t3SA vs. its control tWT at 3 months of age with both sexes (n=6 for each group). Left ventricular free wall (LVFW) specimens were harvested and biaxially stretched along circumferential and longitudinal directions, followed by picrosirius red staining. Fiber architecture was quantified by analyzing histology images. Results: t3SA hearts demonstrated hypertrophy (see Fig. 1a-b for representative mice) and diastolic dysfunction (slowed e’ and increased E/e’ ratio; see Table 1). t3SA LVFW specimens showed increased longitudinal stiffness (p&lt;0.05 for strains &gt;0.25; Fig. 1c-d). Consistent with this stiffening, t3SA specimens exhibited pronounced fiber orientation towards the longitudinal direction (see Fig. 1e-f for representative mice). The smaller e’+S showed that t3SA mice had slowed cross-bridge cycling rates. Biomechanical analyses indicated that LVFW myocardial adaptations contributed to diastolic dysfunction. Conclusions: Echocardiography indices can be used to noninvasively infer underlying remodeling events contributing to diastolic dysfunction facilitating remodeling-targeted therapy selection.