Journal of Molecular and Cellular Cardiology

BackgroundLeft ventricular hypertrabeculation (LVHT) is a heterogenous cardiac condition with a complex and poorly understood aetiology. We comprehensively characterised the effect of a novel P1891A mutation in the SCN5A gene, which encodes the voltage-gated sodium channel Nav1.5, identified in a Finnish family diagnosed with LVHT. MethodsWe generated SCN5A-P1891A mutation-carrying human induced pluripotent stem cell-derived cardiomyocytes (P1891A-hiPSC-CMs) and performed electrophysiological assessments, including patch-clamp studies, and fluorescent calcium imaging, to determine the mutations effect on hiPSC-CM electrophysiology. We also evaluated the impact of the mutation on the proliferative capacity in response to mitogenic stimuli and on the hypertrophic response following cyclic mechanical stretch or endothelin-1 treatment. Further, we assessed the effect on contractile parameters in three-dimensional (3D) contractile hydrogels (engineered heart tissues, EHTs) and conducted advanced proteomics to understand the consequences of the mutation on Nav1.5 protein-protein interactions. ResultsThe SCN5A-P1891A mutation reduced the sodium current densities and increased both the sodium window current and arrhythmogenicity; however, action potential parameters were unaffected. Advanced proteomics characterised, for the first time, the complete Nav1.5 interactome and revealed that the SCN5A-P1891A mutation negated interactions with fibroblast growth factor 12 (FGF12) and FGF13, that are known to modulate sodium channel activity. Baseline proliferation was unchanged, although aged P1891A-hiPSC-CMs demonstrated enhanced proliferative capacity following mitogenic stimulation. Further, P1891A-hiPSC-CMs exhibited a heightened stress response upon mechanical stretch, resulting in the upregulation of heart failure-associated genes. Strikingly, EHTs derived from P1891A-hiPSC-CMs yielded disparate phenotypes. Whilst the majority condensed only partially and failed to beat synchronously, a small subset condensed fully yet exhibited weak contractile properties, alongside age-associated functional decline. In contrast, EHTs derived from healthy control hiPSC-CMs consistently condensed fully and demonstrated a positive correlation between post-fabrication age and contractile properties ConclusionsOur study presents a unique aetiology of LVHT and reveals a novel association between SCN5A mutations and enhanced human cardiomyocyte proliferation. Further, the inability of P1891A-hiPSC-CMs to consistently form fully condensed 3D cardiac tissues may be linked to their abnormal response to mechanical stretch and provides a powerful 3D model for future mechanistic research and drug development studies to better understand and treat LVHT.

BackgroundMaladaptive changes in the function, expression and localization of proteins involved in calcium-handling worsen the impaired contractility of systolic heart failure (HF). Standard proteomics techniques require cell lysis and so are unable to characterize changes specific to the critical sub-cellular domain bounded by the T-tubule and the sarcoplasmic reticulum, known as the cardiac dyad. Traditional approaches are also less likely to capture low-affinity protein-protein interactions on lipid membranes. To improve our understanding of heart failure pathophysiology, we applied proximity proteomics to the cardiac dyad of mice with ischemic cardiomyopathy. MethodsUsing two lines of transgenic mice expressing fusion proteins of the engineered ascorbate peroxidase APEX2 with subunits of the cardiac voltage gated calcium channel, CaV1.2, we labeled the dyad proteome of live, intact myocytes from healthy mice (N=6) and mice with coronary artery ligation HF (N=5) by peroxidase-catalyzed biotinylation. Quantitative mass spectrometry with isobaric tandem mass tags (TMT) was used to assess alterations in the local dyad proteome in myocytes from mice with chronic, remodeled HF. We subsequently generated a mouse with inducible cardiac overexpression of Galectin-1 to examine the effects of this protein on cardiac function and more specifically on the calcium-handling properties of mature myocytes, using cellular electrophysiology, calcium imaging, and echocardiography. ResultsFrom mice with HF, we found significant enrichment of 43 proteins defined by their abundance and proximity to transgenic CaV1.2 1C channels, and a significant reduction in 22 proteins, out a of a total of 2326 proteins quantified. We also significantly enriched 286 proteins, and saw a reduction in 13 proteins, defined by proximity and abundance to CaV1.2 {beta}2B-subunits, out of a total 2236 proteins identified. Pathway analysis revealed HF is associated with increased abundance of components of the 26s proteasome and microtubules in the dyad, as well as the dimerizing, carbohydrate binding protein Galectin-1. Cardiac specific overexpression of Galectin-1 in healthy mice increases activation of CaV1.2 and the Ryanodine receptor and accelerates myocyte relaxation through phosphorylation of Phospholamban. ConclusionsUsing proximity proteomics to examine the effects of HF in vivo, we find increased localization of Galectin-1 to the cardiac dyad, and that overexpression of Galectin-1 accelerates calcium kinetics in the heart.

Three-dimensional engineered cardiac tissue (ECT) using purified human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has emerged as an appealing model system for the study of human cardiac biology and disease. A recent study reported widely-used metabolic (lactate) purification of monolayer hiPSC-CM cultures results in an ischemic cardiomyopathy-like phenotype compared to magnetic antibody-based cell sorting (MACS) purification, complicating the interpretation of studies using lactate-purified hiPSC-CMs. Herein, our objective was to determine if use of lactate relative to MACs-purified hiPSC-CMs impacts the properties of resulting hiPSC-ECTs. Therefore, hiPSC-CMs were differentiated and purified using either lactate-based media or MACS. After purification, hiPSC-CMs were combined with hiPSC-cardiac fibroblasts to create 3D hiPSC-ECT constructs maintained in culture for four weeks. There were no structural differences observed, and there was no significant difference in sarcomere length between lactate and MACS hiPSC-ECTs. Assessment of isometric twitch force, Ca2+ transients, and {beta}-adrenergic response revealed similar functional performance between purification methods. High-resolution mass spectrometry (MS)-based quantitative proteomics showed no significant difference in any protein pathway expression or myofilament proteoforms. Taken together, this study demonstrates lactate- and MACS-purified hiPSC-CMs generate ECTs with comparable molecular and functional properties, and suggests lactate purification does not result in an irreversible change in hiPSC-CM phenotype. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/539642v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@54b370org.highwire.dtl.DTLVardef@d0bbc8org.highwire.dtl.DTLVardef@1d847e3org.highwire.dtl.DTLVardef@578c1b_HPS_FORMAT_FIGEXP M_FIG C_FIG

AimsJunctophilin-2 is required for the development, maturation and integrity of the t-tubule system and the gating stability of RyR2 in cardiomyocytes. This study investigated whether and how junctophilin-2 maintained junctin, a scaffold protein stabilizing RyR2, to prevent cardiomyocyte death under stress. MethodsCardiomyocytes were exposed to conditions of stress including palmitate, doxorubicin, or hypoxia/re-oxygenation. Adenoviral vectors were employed to manipulate expression of junctophilin-2 and junctin in cardiomyocytes. Molecular/cellular/biochemical analyses were conducted. ResultsDifferent conditions of stress decreased junctophilin-2 expression through aberrant autophagy and concomitantly induced a reduction of junctin protein in cardiomyocytes. Over-expression of junctophilin-2 preserved the protein levels of junctin and attenuated cytosolic Ca2+ and apoptosis in cardiomyocytes under stress. Knockdown of junctophilin-2 reproduced the detrimental phenotypes of stress in cardiomyocytes. Notably, over-expression of junctin prevented cardiomyocyte death under stress whereas knockdown of junctin offset the protective effects conferred by junctophilin-2 over-expression. Mechanistically, junctophilin-2 blocked MURF1-junctin interaction thereby preventing junctin ubiquitination and proteasome-dependent degradation. Mass spectrometry analysis identified multiple ubiquitination sites on the junctin protein and the non-ubiquitinated junctin mutant (K8A/K102A/K107A/K140A) was resistant to degradation. ConclusionsThis study uncovers an unrecognized role of junctophilin-2 in preventing junctin ubiquitination and degradation in maintaining cytosolic Ca2+ homeostasis. Both junctophilin-2 and junctin represent two new survival factors of cardiomyocytes and thus, may be new therapeutic targets for cardiac protection.

BackgroundHypertrophic cardiomyopathy (HCM) is a heritable pathological condition resulting from mutations in sarcomere-related proteins, leading to severe structural abnormalities without effective treatment options. Although reduced fibroblast growth factor 12(FGF12) expression is observed in HCM patients, its functional role remains unclear. MethodsEmploying immunoprecipitation (IP)-mass spectrometry (MS) and CUT&Tag sequencing, we investigated FGF12-interacting proteins in myocardial samples from healthy volunteers and HCM patients. CRISPR-Cas9 was utilized to explore the function and interaction partners of FGF12 in cardiomyocytes induced from human pluripotent stem cells (hiPSCs-CMs), other cell lines, and mouse models (MYH7R403Q and MYBPC3c.790G>A, transverse aortic constriction (TAC)). During hypertrophy, FGF12 localizes intranuclearly, prompting investigations into its binding to gene promoter regions through CUT&Tag sequencing and dual-luciferase experiments using myocardial tissues from patients. The beating frequency of hiPS-CMs was assessed using the CardioExcyte 96 real-time label-free cardiomyocyte functional analysis system. ResultsFGF12 was found to associate with proteins involved in energy metabolism, predominantly localizing to the perinuclear space under physiological conditions but shifting into the nucleus of hypertrophic cardiomyocytes. Co-IP-MS revealed significant interactions between FGF12 and metabolism-associated proteins, particularly GATA binding protein 4 (GATA4) and mitogen-activated protein kinase 1/3 (MAPK1/3) in the perinuclear space. In a hypertrophic state, FGF12 bound to the GATA4 promoter region, increasing its expression upon nuclear translocation. Both in vitro and in vivo models demonstrated that FGF12 interaction with GATA4 inhibited GATA4 and MAPK1/3 phosphorylation, inducing the expression of hypertrophy-associated genes. Overexpression of FGF12-NLS-del (nuclear localization signal deletion) resulted in decreased GATA4 phosphorylation, suggesting inhibition in the perinuclear region. ConclusionsThis study elucidates a pathological mechanism of HCM involving FGF12, where its nuclear localization enhances phosphorylation, GATA4 expression, and activation of the ERK1/2-pGATA4 pathway genes associated with hypertrophy. Beyond advancing our understanding of HCM, these findings propose FGF12 as a potential therapeutic target for HCM, warranting further exploration to potentially alleviate this condition affecting millions of individuals.

Inefficiency of regeneration underlies many of the pathologies associated with heart injury and disease. Ventricular diploid cardiomyocytes (CMs) are a candidate population that may have enhanced proliferative and regenerative properties [1-3], but subpopulations of diploid CMs and their regenerative capacities are not yet known. Here, using the expression marker Cntn2-GFP and the lineage marker Etv1CreERT2, we demonstrate that peripheral ventricular conduction CMs (Purkinje CMs) are disproportionately diploid (35%, vs. 4% of bulk ventricular CMs). However, this lineage had no enhanced competence to support regeneration after adult infarction. Furthermore, the CM-specific kinase Tnni3k, which strongly influences bulk ventricular CM ploidy [3] and is also associated with conduction system defects [4], had no influence on the ploidy or organization of the ventricular conduction system. Unlike the bulk diploid CM population, a significant fraction of conduction CMs remain diploid by avoiding neonatal cell cycle activity, likely contributing to these properties.

BackgroundThe heart undergoes growth in response to both pathological and physiological stimuli. Pathological hypertrophy often leads to cardiomyocyte loss and heart failure (HF), whereas physiological hypertrophy paradoxically protects the heart and enhances cardiomyogenesis. The molecular mechanisms that distinguish these two forms of hypertrophy remain unclear. MethodsIn this study, we utilized single-cell transcriptomics from transverse aortic constriction (TAC) models at 2, 5, 8, and 11 weeks (GSE120064), along with bulk RNA sequencing from mice subjected to 12 months of exercise-induced physiological hypertrophy and cardiomyogenesis (CRA007207), to investigate the molecular differences between pathological and physiological hypertrophy. ResultsOur results reveal the following. Mitochondrial-related pathways are the primary drivers of the pathological changes that occur following TAC. The mitochondrial fission and fusion pathways exhibited increased activity at 2 weeks but decreased activity at 5, 8, and 11 weeks post TAC. The expression pattern of exercise-induced physiological hypertrophy was similar to that of 2-week TAC-induced changes, indicating that the early stage of TAC represents an adaptive physiological response or physiological hypertrophy. Notably, during HF, the fission genes Fis1 and Dnm1l increase, in contrast to the expected decrease in fusion genes. These findings were experimentally validated, indicating that the mitochondrial fission genes Fis1 and Dnm1l are key promoters of HF. ConclusionsOur data indicate that the balance between mitochondrial fission and fusion plays a critical role in the transition from physiological to pathological hypertrophy. The fission-related genes Fis1 and Dnm1l have emerged as key drivers of pathological hypertrophy and heart failure. These findings suggest that targeting fission genes, particularly Fis1 and Dnm1l, may represent promising therapeutic strategies for managing heart failure.

BACKGROUNDMYBPC3, 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). METHODSThe hiPSC-derived cardiomyocytes (hiPSC-CMs) and hCOs were generated from human subjects to define the molecular, cellular, and functional changes caused by the MYBPC3D389V 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. RESULTSConfocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of the MYBPC3D389V 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 MYBPC3D389V than NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation in MYBPC3D389V hCOs was observed, but without changes in total protein levels, in addition to higher oxidative stress and lower mitochondrial membrane potential ({Delta}{Psi}m). 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(R)) in MYBPC3D389V hCOs. Lastly, various in vitro binding assays revealed a significant loss of affinity in the presence of MYBPC3D389V with myosin S2 region as a likely mechanism for hypercontraction. CONCLUSIONSConceptually, 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 MYBPC3D389V hypercontractile phenotype, which was rescued by administration of a myosin inhibitor. Novelty and SignificanceO_ST_ABSWhat Is Known?C_ST_ABSO_LIMYBPC3 mutations have been implicated in hypertrophic cardiomyopathy. C_LIO_LID389V 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. C_LIO_LIThe molecular mechanism by which the D389V variant develops pathological cardiac dysfunction remains to be conclusively determined. C_LI What New Information Does This Article Contribute?O_LIThe 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. C_LIO_LIThe MYBPC3D389V-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. C_LIO_LIOur 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(R)), a myosin inhibitor. C_LIO_LIIn addition, hypercontractility causes secondary mitochondrial defects such as higher oxidative stress and lower mitochondrial membrane potential ({Delta}{Psi}m), highlighting a possible early adaptive response to primary sarcomeric changes. C_LIO_LIEarly treatment of MYBPC3D389V carriers with mavacamten may prevent or reduce early HCM-related pathology. C_LI GRAPHICAL ABSTRACTA graphical abstract is available for this article. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/596463v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1489301org.highwire.dtl.DTLVardef@1ab60c1org.highwire.dtl.DTLVardef@5d49d1org.highwire.dtl.DTLVardef@9944d6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background and aimsIncreased levels of -tubulin and its post-translational modifications (PTMs) are found in human heart failure and could initiate diastolic dysfunction by modulating cardiomyocyte stiffness. How these modifications occur and how they may underlie cardiac dysfunction remains unknown. Upstream kinases may play a critical role, but this has not been explored. Methods and resultsHere we address this question by, for the first time ever, determining levels of the enzymes involved in microtubule (MT) detyrosination and acetylation (TAT1, HDAC6) in a well-characterized cohort of patients with hypertrophic cardiomyopathy (HCM). In HCM patients (N=10-11), protein levels of detyrosination enzymes remain unaltered, whilst levels of TAT1 and HDAC6 were decreased and increased, respectively. Phosphoproteomics in HCM (N=24) and control (N=8) myocardium identified significant differences in over 1900 serine/threonine and 160 tyrosine phosphosites, in addition to increased EGFR/IGF1R-MAPK signaling in HCM. We subsequently showed that MT repolymerization was increased in HCM MYBPC3Arg943X hiPSC-CMs. Isoprenaline-mediated PKA activation decreased MT repolymerization in hiPSC-CMs and revealed CLASP1, MAST4 and MAP1A as potential MT modifiers in HCM. ConclusionsWe show that the altered HCM MT code cannot be attributed to levels of key MT-modifying enzymes. By combining kinome analyses in human HCM hearts with hiPSC-CM studies on MT dynamics, PTMs and contractility we unveiled a regulatory role for MTs in the cardiomyocyte response to beta-adrenergic receptor stimulation. Disease-mediated changes in the MT code thereby exert both a direct, and indirect effect on cardiac function via mediating the response to adrenergic activation. Graphical Abstract created with BioRender.com O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/706710v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1c58dc4org.highwire.dtl.DTLVardef@de502eorg.highwire.dtl.DTLVardef@1621512org.highwire.dtl.DTLVardef@557b82_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundProtein quality control (PQC) is critical for maintaining sarcomere structure and function in cardiac myocytes, and mutations in PQC pathway proteins, such as CRYAB (arginine to glycine at position 120, R120G) and BAG3 (proline to lysine at position 209, P209L) induce protein aggregate pathology with cardiomyopathy in humans. Novel observations in yeast and mammalian cells demonstrate mitochondrial uptake of cytosolic protein aggregates. We hypothesized that mitochondrial uptake of cytosolic protein aggregates and their removal by mitophagy, a lysosomal degradative pathway essential for myocardial homeostasis, facilitates cytosolic protein quality control in cardiac myocytes. MethodsMice with inducible cardiac myocyte specific ablation of TRAF2 (TRAF2icKO), which impairs mitophagy, were assessed for protein aggregates with biochemical fractionation and super-resolution imaging in comparison to floxed controls. Induced pluripotent stem cell (iPSC)-derived cardiac myocytes with R120G knock-in to the CRYAB locus were assessed for localization of the CRYAB protein. Transgenic mice expressing R120G CRYAB protein (R120G-TG) were subjected to both TRAF2 gain-of-function (with AAV9-cardiac Troponin T promoter-driven TRAF2 transduction) and TRAF2 loss-of-function (with tamoxifen-inducible ablation of one Traf2 allele) in cardiac myocytes to determine the effect of mitophagy modulation on cardiac structure, function, and protein aggregate pathology. ResultsCardiomyocyte-specific ablation of TRAF2 results accumulation of mitochondrial and cytosolic protein aggregates and DESMIN mis-localization to protein aggregates. Isolated mitochondria take up cardiomyopathy-associated aggregate-prone cytosolic chaperone proteins, namely arginine to glycine (R120G) CRYAB mutant and proline to lysine (P209L) BAG3 mutant. R120G-CRYAB mutant protein increasingly localizes to mitochondria in human and mouse cardiomyocytes. R120G-TG mice demonstrate upregulation of TRAF2 in the mitochondrial fraction with increased mitophagy as compared with wild type. Adult-onset inducible haplo-insufficiency of TRAF2 resulted in accelerated mortality, impaired left ventricular systolic function and increased protein aggregates in R120G-TG mice as compared with controls. Conversely, AAV9-mediated TRAF2 transduction in R120G-TG mice reduced mortality and attenuated left ventricular systolic dysfunction, with reduced protein aggregates and restoration of normal localization of DESMIN, a cytosolic scaffolding protein chaperoned by CRYAB, as compared with control AAV9-GFP group. ConclusionsTRAF2-mediated mitophagy in cardiac myocytes facilitates removal of cytosolic protein aggregates and can be stimulated to ameliorate proteotoxic cardiomyopathy.

BackgroundWhile operative and perioperative care continues to improve for single ventricle congenital heart disease (SV), long-term morbidities and mortality remain high. Importantly, phosphodiesterase-5 inhibitor therapies (PDE5i) are increasingly used, however, little is known regarding the direct myocardial effects of PDE5i therapy in the SV population. ObjectivesOur group has previously demonstrated that the failing SV myocardium is characterized by increased PDE5 activity and impaired mitochondrial bioenergetics. Here we sought to determine whether serum circulating factors contribute to pathological metabolic remodeling in SV, and whether PDE5i therapy abrogates these changes. MethodsUsing an established in vitro model whereby primary cardiomyocytes are treated with patient sera +/- PDE5i, we assessed the impact of circulating factors on cardiomyocyte metabolism. Mass spectrometry-based lipidomics and metabolomics were performed to identify phospholipid and metabolite changes. Mitochondrial bioenergetics were assessed using the Seahorse Bioanalyzer and a stable isotope based mitochondrial enzyme activity assay. Relative mitochondrial copy number was quantified using RT-qPCR. ResultsOur data suggest that serum circulating factors contribute to fundamental changes in cardiomyocyte bioenergetics, including impaired mitochondrial function associated with decreased cardiolipin and other phospholipid species, increased reactive oxygen species (ROS) generation, and altered metabolite milieu. Treatment with PDE5i therapy was sufficient to abrogate a number of these metabolic changes, including a rescue of phosphatidylglycerol levels, a reduction in ROS, improved energy production, and normalization of several key metabolic intermediates. ConclusionsTogether, these data suggest PDE5i therapy has direct cardiomyocyte effects and contributes to beneficial cardiomyocyte metabolic remodeling in SV failure.

RationaleMitochondrial fission and fusion are relatively infrequent in adult cardiomyocytes compared to another cell types1-3. This is surprising considering that proteins involved in mitochondrial dynamics are highly expressed in the heart. It has been previously reported that dynamin-related protein 1 (DRP1) has a critical role in mitochondrial fitness and cardiac protection1, 4. Cardiac DRP1 ablation in the adult heart evokes a progressive dilated cardiac myopathy and lethal heart failure1. Nevertheless, the conditional cardiac-specific DRP1 knock-out animals present a significantly longer survival rate compared with global DRP1 KO models1, 4, 5. We have described before the great importance for cardiac physiology of the strategic positioning of mitochondrial proteins in the cardiac tissue6, 7. Therefore, we hypothesize that DRP1 plays a regulatory role in cardiac physiology and mitochondrial fitness by preferentially accumulating at mitochondria and junctional sarcoplasmic reticulum (jSR) contact sites, where the high Ca2+ microdomain is formed during excitation-contraction (EC) coupling. ObjectiveThis study aims to determine whether mitochondria-associated DRP1 is preferentially accumulated in the mitochondria and jSR contact sites, the mechanism responsible for such a biased distribution, and its functional implication. Methods and ResultsUsing high-resolution imaging approaches, we found that mitochondria-associated DRP1 in cardiomyocytes was localized in the discrete regions where T-tubule, jSR, and mitochondria are adjacent to each other. Western blot results showed that mitochondria-bound DRP1 was restricted to the mitochondria-associated membranes (MAM), with undetectable levels in purified mitochondria. Furthermore, in comparison to the cytosolic DRP1, the membrane-bound DRP1 in SR and MAM fractions formed high molecular weight oligomers demosntratd by 2D blue native technique. In both electrically paced adult cardiomyocytes and Langendorff-perfused beating hearts, the oscillatory Ca2+ pulses preserved MAM-associated DRP1 accumulation. Interestingly, similar to DRP1, all mitochondria-bound {beta}-ACTIN only exists in MAM and not in the purified mitochondria. Additionally, co-immunoprecipitation pulls down both DRP1 and {beta}-ACTIN together. Inhibition of {beta}-ACTIN polymerization with Cytochalasin D disrupts the tight association between DRP1 and {beta}-ACTIN. In cardiac-specific DRP1 knock-out mouse after 6 weeks of tamoxifen induction (DRP1icKo), the cardiomyocytes show disarray of sarcomere, a decrease of cardiac contraction, loss of mitochondrial membrane potential, significantly decreased spare respiratory capacity, and frequent occurrence of early after contraction (EAC), suggesting the heart is susceptible to arrhythmias and heart failure. Despite of this phenotype, DRP1icKo animals have longer life span than other DRP1 KO models. Strikingly, DRP1 levels are only modestly decreased in the MAM when compared with the rest of the cellular fractions. These preserved levels were accompanied by the preservation of the mitochondrial pool in the MAM fraction obtained from the DRP1icKO hearts. ConclusionsThe results show that in adult cardiomyocytes, mitochondria bound DRP1 clusters in high molecular weight protein complexes at MAM. This clustering is fortified by EC coupling mediated Ca2+ transients and requires its interaction with {beta}-ACTIN. Together with the better preserved DRP1 levels in the DRP1icKO model in the MAM, we conclude that DRP1 is anchored at the mitochondria-SR interface through {beta}-ACTIN and positions itself to play a fundamental role in regulating mitochondrial quality control in the working heart.

BackgroundMyocarditis leads to dilated cardiomyopathy (DCM) with one-third failing to recover normal ejection fraction (EF50%), and there is a critical need for prognostic biomarkers to assess risk of nonrecovery. Cardiac myosin (CM) autoantibodies (AAbs) cross-reactive with the {beta}-adrenergic receptor ({beta}AR) are associated with myocarditis/DCM, but their potential for prognosis and functional relevance is not fully understood. MethodsCM AAbs and myocarditis-derived human monoclonal antibodies (mAbs) were investigated to define pathogenic mechanisms and CM epitopes of nonrecovery. Myocarditis patients who do not recover ejection fraction (EF<50%) by one year were studied in a longitudinal (n=41) cohort. Sera IgG and human mAbs were investigated for autoreactivity with CM and CM peptides by ELISA, protein kinase A (PKA) activation, and transcriptomic analysis in H9c2 heart cell line. ResultsCM AAbs were significantly elevated in nonrecovered compared to recovered patients and correlated with reduced EF (<50%). CM epitopes specific to nonrecovery were identified. Transcriptomic analysis revealed serum IgG and mAb 2C.4 induced fibrosis/apoptosis pathways in vitro similar to isoproterenol treated cells. Sera IgG and 2C.4 activated PKA in an IgG and {beta}AR-dependent manner. Endomyocardial biopsies from myocarditis/DCM revealed IgG+ trichrome+ tissues. ConclusionsCM AAbs were significantly elevated in nonrecovered patients, suggesting novel prognostic relevance. CM AAbs correlated with lower EF, and Ab-induced fibrosis/apoptosis pathways suggested a role for CM AAbs in patients who do not recover and develop irreversible heart failure. Homology between CM and {beta}ARs supports mechanisms related to cross-reactivity of CM AAbs with the {beta}AR, a potential AAb target in nonrecovery.

ObjectiveTo investigate the effects of carnosine on heart failure and to examine whether this is associated with reduced immunogenicity of oxidatively-generated aldehyde modified proteins. BackgroundHeart failure is associated with the accumulation of lipid derived aldehydes that form immunogenic protein adducts. However, the pathological impact of these aldehydes and aldehyde-modified proteins in heart failure has not been assessed. Histidyl dipeptides, such as carnosine found in the heart, bind to aldehydes, and their protein adducts. However, the effects of carnosine on heart failure or the antigenicity of aldehyde modified proteins have not been studied. MethodsMale, wild type C57BL/6J mice were subjected to either sham or transverse aortic constriction (TAC) surgery. To increase carnosine levels, they were placed on drinking water with or without {beta}-alanine prior to surgery, and for the remainder of the study. Cardiac function was evaluated by echocardiography, and the levels of histidyl dipeptides, immune cell populations, and CD4+ T cell activation were assessed via LC-MS/MS and flow cytometry, respectively. ResultsMyocardial levels of histidyl dipeptides decreased at both 3- and 8-weeks post-TAC. Supplementation with {beta}-alanine increased myocardial histidyl dipeptide levels, attenuated adverse cardiac remodeling, and reduced aldehyde stress. Carnosine formed covalent bond with protein-bound aldehydes in the failing heart, reducing their antigenic potential and decreasing activation of dendritic cells and CD4+ T cells in vitro. {beta}-alanine supplementation decreased the population of CD11b+CD64-Ly6G+ neutrophils and CD4+ CD44+ effector T cells in the failing heart. ConclusionsIncreasing myocardial carnosine levels reduces aldehyde stress, dampens maladaptive immune responses, and preserves cardiac function during heart failure. HIGHLIGHTSO_LILevels of endogenous dipeptide carnosine are depleted in failing hearts, while supplementation of the carnosine precurson {beta}-alanine increases myocardial carnosine and preserves cardiac function during heart failure. C_LIO_LIHeart failure is associated with increased activation and infiltration of CD4+ T cells and generation of aldehyde modified protein adducts in failing hearts. C_LIO_LIThe free aldehyde moiety of aldehyde modified protein adducts activates CD4+ T cells through dendritic cell presentation and capping this moiety with carnosine diminishes their antigencity. C_LIO_LIIncreasing myocardial carnosine levels diminishes aldehyde stress and activation of CD4+ T cells during heart failure. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/671840v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@28fc7corg.highwire.dtl.DTLVardef@d851ccorg.highwire.dtl.DTLVardef@1e24a5dorg.highwire.dtl.DTLVardef@18023b6_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundImpaired left ventricular relaxation, high filling pressures, and dysregulation of Ca2+ homeostasis are common findings contributing to diastolic dysfunction in hypertrophic cardiomyopathy (HCM). Studies have shown that impaired relaxation is an early observation in the sarcomere-gene-positive preclinical HCM cohort which suggests potential involvement of myofilament regulators of relaxation. Yet, a molecular level understanding of mechanism(s) at the level of the myofilament is lacking. We hypothesized that mutation-specific, allosterically mediated, changes to the cardiac troponin C-cardiac troponin I (cTnC-cTnI) interface can account for the development of early-onset diastolic dysfunction via decreased PKA accessibility to cTnI. MethodsHCM mutations R92L-cTnT (Arg92Leu) and {Delta}160E-cTnT (Glu160 deletion) were studied in vivo, in vitro, and in silico via 2D echocardiography, western blotting, ex vivo hemodynamics, stopped-flow kinetics, time resolved fluorescence resonance energy transfer (TR-FRET), and molecular dynamics simulations. ResultsThe HCM-causative mutations R92L-cTnT and {Delta}160E-cTnT result in different time-of-onset of diastolic dysfunction. R92L-cTnT demonstrated early-onset diastolic dysfunction accompanied by a localized decrease in phosphorylation of cTnI. Constitutive phosphorylation of cTnI (cTnI-D23D24) was sufficient to recover diastolic function to Non-Tg levels only for R92L-cTnT. Mutation-specific changes in Ca2+ dissociation rates associated with R92L-cTnT reconstituted with cTnI-D23D24 led us to investigate potential involvement of structural changes in the cTnC-cTnI interface as an explanation for these observations. We probed the interface via TR-FRET revealing a repositioning of the N-terminus of cTnI, closer to cTnC, and concomitant decreases in distance distributions at sites flanking the PKA consensus sequence. Implementing TR-FRET distances as constraints into our atomistic model identified additional electrostatic interactions at the consensus sequence. ConclusionThese data indicate that the early diastolic dysfunction observed in a subset of HCM is likely attributable to structural changes at the cTnC-cTnI interface that impair accessibility of PKA thereby blunting {beta}-adrenergic responsiveness and identifying a potential molecular target for therapeutic intervention.

BackgroundPhosphoglucomutase-1 (PGM1) plays a pivotal role in glycolysis, glycogen metabolism, and glycosylation. Pathogenic variants in PGM1 cause PGM1-congenital disorder of glycosylation (PGM1-CDG), a multisystem disorder with cardiac involvement. While glycosylation abnormalities in PGM1-CDG are treatable with galactose, cardiomyopathy does not improve suggesting a glycosylation-independent pathomechanism. Recently, mitochondrial abnormalities have been shown in a heart of a PGM1-deficicient patient and PGM1-mouse model. In addition, PGM1 has been associated with LDB3 (ZASP/Cypher), a sarcomeric Z-disk protein also associated with cardiomyopathy. However, the cardiac-specific role of PGM1 remains poorly understood, and targeted therapies for PGM1-related cardiomyopathy are currently lacking. MethodsInduced pluripotent stem cell-derived cardiomyocytes (iCMs) were generated from PGM1-deficient patient fibroblasts. Multielectrode array (MEA) recordings, untargeted (glyco)proteomics, and pathway analysis were performed to assess functional and molecular changes. Key findings were validated using tracer metabolomics and mitochondrial respiration assays. ResultsPGM1-deficient iCMs exhibited reduced beating frequency, impaired contractility, and prolonged contraction kinetics. Proteomic analyses revealed depletion of Z-disk components, including LDB3. AlphaFold3 structural modeling predicted a direct interaction between PGM1 and LDB3, implicating PGM1 in Z-disk integrity, which was confirmed in vitro. In addition, mitochondrial proteins were severely depleted, prompting us to investigate mitochondrial function. Functional validation confirmed extensive metabolic rewiring, energy depletion, and severely impaired mitochondrial respiration. Finally, the in silico drug repurposing identified possible therapeutic options that could target PGM1-deficient cardiomyopathy. ConclusionPGM1 is a key regulator of cardiomyocyte function, linking sarcomeric Z-disk integrity with mitochondrial metabolism. These mechanistic insights offer a foundation for developing targeted therapies for PGM1-CDG and potentially other cardiomyopathies involving Z-disk dysfunction. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=175 HEIGHT=200 SRC="FIGDIR/small/662580v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@17da449org.highwire.dtl.DTLVardef@1acb669org.highwire.dtl.DTLVardef@1fbbeecorg.highwire.dtl.DTLVardef@b38b6f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Microtubule detyrosination and re-tyrosination on the C-terminus of -tubulin are mediated by the vasohibin (VASH)-small vasohibin-binding protein (SVBP) complex and tubulin tyrosine ligase (TTL), respectively. Elevated levels of detyrosinated -tubulin (dTyr-tub) are observed in heart failure, and reducing this modification improves cardiac function, suggesting that clinically used heart failure therapies may modulate microtubule detyrosination. We investigated whether sacubitrilat and valsartan, the active components of the angiotensin receptor-neprilysin inhibitor LCZ696, influence dTyr-tub levels in endothelin-1 (ET1)-induced hypertrophy in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). While both sacubitrilat and valsartan prevented hypertrophy, only sacubitrilat prevented ET1-induced dTyr-tub accumulation. RNA sequencing revealed that sacubitrilat normalized several ET1-induced dysregulated pathways. Sacubitrilat slightly increased cyclic guanosine 3,5-monophosphate (cGMP) levels and lowered dTyr-tub, whereas inhibition or knockdown of the cGMP-dependent protein kinase 1 (PRKG1) increased dTyr-tub level. Mechanistically, PRKG1 alpha phosphorylated native VASH1. Incubation of microtubules with the VASH1-SVBP complex containing wild-type VASH1 increased detyrosination, while incubation of the complex containing a VASH1 phosphomimic, in which seven C-terminal serine residues were mutated to glutamate (VASH1-7E) did not. Consistently, overexpression of VASH1-7E gave rise to lower dTyr-tub level than overexpression of a non-phosphorylatable form of VASH1 (VASH1-7A) in hiPSC-CMs deficient in VASH1. In conclusion, these findings identify a cGMP-PRKG1-VASH1 signaling axis that reduces microtubule detyrosination in cardiomyocytes. Our work provides mechanistic insight into how neprilysin inhibition may contribute to therapeutic benefit in heart failure. One Sentence SummaryWe establish a neprilysin-cGMP-PRKG1-VASH1 signaling axis that reduces microtubule detyrosination in cardiomyocytes.

IntroductionMyocardial accumulation of the protein transthyretin (TTR) can result in amyloid TTR cardiomyopathy (ATTR-CM), a form of restrictive heart disease with limited therapies and still generally poor clinical outcomes. The mechanisms by which TTR fibril accumulation elicits cardiac toxicity at the protein level remain largely unknown. Accordingly, we performed untargeted proteomics of ventricular myocardium from patients with ATTR-CM versus controls. MethodsMyocardial tissue from non-failing (NF) controls (n=7) and ATTR-CM (n=4) were assayed by mass spectrometry. HFrEF, HCM, and HFpEF proteomics were acquired from published databases. ResultsA total of 539/7093 (7.6% of total) proteins were found to be differentially expressed in ATTR-CM, 227/359 (42%) upregulated and 312/539 (58%) downregulated. Gene ontology pathway analysis found that downregulated proteins were enriched for oxidative phosphorylation and mitochondrial protein translation pathways, while upregulated proteins were enriched for enhanced endocytosis and intracellular vesicle mediated transport. The latter is not observed in other forms of heart failure. We further identify a profound downregulation of sarcomere protein content, which is also not seen in other cardiomyopathies. ConclusionThe ATTR-CM myocardial proteome identifies endocytosis and intracellular transport as uniquely upregulated processes, whereas sarcomere protein content is uniquely downregulated. Both maybe potential therapeutic targets.

BackgroundCT1 is a 25 amino acid therapeutic peptide incorporating the Zonula Occludens-1 (ZO-1)-binding domain of connexin43 (Cx43) that is currently in Phase III clinical testing for healing chronic skin wounds. In preclinical studies in mice, we reported that CT1 reduces arrhythmias and improves ventricular function following cardiac injury, effects that were accompanied by increases in PKC{varepsilon} phosphorylation of Cx43 at serine 368 (pS368). In this study, we undertake a systematic characterization of the molecular mode-of-action of CT1 in mitigating the effects of ischemia reperfusion injury on ventricular contractile function.\n\nMethods and ResultsTo determine the basis of CT1-mediated increases in pS368 we undertook tandem mass spectrometry of reactants in an in vitro assay of PKC{varepsilon} phosphorylation, identifying an interaction between negatively charged amino acids in the CT1 Asp-Asp-Leu-Glu-Iso sequence and positively charged lysines (Lys345, Lys346) in a short -helical sequence (H2) within the Cx43 CT domain. In silico modeling provided further support of the specificity of this interaction, leading us to conclude that CT1 has potential to directly interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift and phosphorylation assays, we characterized a series of CT1 variant peptides, identifying sequences competent to interact with either ZO-1 PDZ2 or the Cx43 CT, but with limited or no ability to bind both polypeptides. Based on this analysis, it was found that only those peptides competent to interact with Cx43, but not ZO-1 alone, resulted in increased pS368 phosphorylation in vitro and in vivo. Moreover, in a mouse model of global ischemia reperfusion injury we determined that pre-ischemic infusion only with those peptides competent to bind Cx43 preserved left ventricular (LV) contractile function following injury. Interestingly, a short 9 amino acid (MW=1110) Cx43-binding variant of the original 25 amino acid CT1 sequence demonstrated potent LV-protecting effects when infused either before or after ischemic injury.\n\nConclusionsInteraction of CT1 with the Cx43 CT, but not ZO-1 PDZ2, explains cardioprotection mediated by this therapeutic peptide. Pharmacophores targeting the Cx43 carboxyl terminus could provide a novel translational approach to preservation of ventricular function following ischemic injury.

Cardiac myosin binding protein-C (cMyBP-C) is an essential regulator of cardiac contractility through its interactions with the thick and thin filament. cMyBP-C is heavily influenced by post-translational modifications, including phosphorylation which improves cardiac inotropy and lusitropy, and S-glutathionylation, which impairs phosphorylation and is increased in heart failure. Palmitoylation is an essential cysteine modification that regulates the activity of cardiac ion channels and soluble proteins, however, its relevance to myofilament proteins has not been investigated. In the present study, we purified palmitoylated proteins from ventricular cardiomyocytes and identified that cardiac actin, myosin and cMyBP-C are palmitoylated. The palmitoylated form of cMyBP-C was more resistant to salt extraction from the myofilament lattice than the non-palmitoylated form. Isometric tension measurements suggest c-MyBP-C palmitoylation reduces myofilament Ca2+ sensitivity, with no change to maximum force or passive tension. Importantly, cMyBP-C palmitoylation levels are reduced at the site of injury in a rabbit model of heart failure but increased in samples from patients with ischaemic heart failure. Identification of cMyBP-C palmitoylation site revealed S-glutathionylated cysteines C635 and C651 are required for cMyBP-C palmitoylation, suggesting an interplay between the modifications at these sites. We conclude that structural and contractile proteins within the myofilament lattice are palmitoylated, with important functional consequences for cardiac contractile performance.