Journal of Molecular and Cellular Cardiology

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

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.

BackgroundVentricular assist devices (VADs) are used as treatment for end-stage heart failure in children and adults. We previously demonstrated decreased mitochondrial function and changes in cardiolipin, a mitochondrial phospholipid, in explanted pediatric and adult failing hearts. In this study, we tested the hypothesis that VAD unloading of failing hearts leads to positive changes in myocardial cardiolipin in both pediatric and adult hearts. MethodsVentricular tissue was collected from the same patient at time of VAD implantation and at transplant. Ejection fraction (EF), left ventricular internal diameter at end-diastole (LVIDd) and brain natriuretic peptide (BNP) were assessed pre- and post-VAD. Cardiolipin species from paired VAD core and explants were quantified using liquid chromatography mass spectrometry. Mitochondrial respiration was measured in ventricular tissue pre- and post-VAD in paired pediatric samples using the Oroboros Oxygraph-2k. ResultsVAD support led to increased EF and decreased LVIDd and BNP. The predominant cardiolipin species in cardiac mitochondria, tetralinoleoylcardiolipin, was positively remodeled in pediatric post-VAD myocardium, while adult post-VAD myocardium demonstrated significantly increased total cardiolipin and decreased oxidized cardiolipin but did not demonstrate the tetralinoleoylcardiolipin remodeling seen in pediatric hearts. In pediatric patients, VAD support resulted in significant increases in Complex I+II activity, and a trend toward increases in Complex I activity. ConclusionOur data demonstrate age-related differences in VAD-associated cardiolipin remodeling and suggest that improved mitochondrial function in pediatric VAD-supported hearts could be related to increased tetralinoleoylcardiolipin.

BACKGROUNDMono-ADP ribosylation is a post-translational modification that regulates various cellular physiological processes, including cell cycle progression, genomic stability, transcription, and cellular protein turnover. PARP16 is an endoplasmic reticulum (ER)-localized mono-ADP-ribosyltransferase that has been shown to regulate the unfolded protein response and maintain ER homeostasis under stress conditions. Despite its established role in ER stress signaling, the functional significance of PARP16 in cardiac pathophysiology, particularly in cardiac hypertrophy and heart failure, remains poorly understood. In this study, we aim to investigate the role of PARP16 in cardiac hypertrophy and heart failure using in vitro and mouse model systems. METHODSWe analysed PARP16 expression in human heart failure samples as well as in heart failure-based mouse models. We evaluated gene expression by RT-PCR, immunoblotting, and confocal microscopy to understand the role of PARP16 in heart failure under phenylephrine- or isoproterenol-treated conditions. We also investigated the role of PARP16 in regulating cardiac function in genetically engineered mouse models, including whole-body PARP16 knockout, cardiac-specific PARP16 knockout, inducible cardiac-specific PARP16 knockout, and cardiac-specific PARP16 Transgenic mice. We performed echocardiography to assess cardiac function. We also used an in vitro primary cardiomyocyte system to knock down and overexpress PARP16. We performed RNA sequencing and mass spectrometry, followed by molecular docking, molecular dynamics simulation, immunoprecipitation, and luciferase assay to characterise the molecular mechanism by which PARP16 regulates cardiac function. RESULTSHuman heart failure samples showed reduced PARP16 expression. PARP16 expression was also significantly reduced in models of heart failure, including the hearts of isoproterenol-treated C57B/L6 mice and phenylephrine-treated primary cardiomyocytes. PARP16-deficient NRCMs showed signs of pathological remodelling. Whole-body, cardiac-specific, and inducible cardiac-specific PARP16 KO mice exhibited cardiac remodelling and dysfunction. In contrast, cardiac-specific PARP16-overexpressing mice were protected from iso-induced cardiac hypertrophy. Mechanistically, several hypertrophic signalling pathway genes are dysregulated in PARP16 knockout mouse hearts concomitant with upregulated NFAT1 transcriptional activity and nuclear translocation. PARP16 binds to and catalytically downregulates NFAT activity, thereby maintaining cardiac function. Mass spectrometry analysis showed that PARP16 is involved in ADP-ribosylation of NFAT1 at E398 and T533. Pharmacological inhibition of NFAT activation attenuates structural and functional abnormalities associated with PARP16 deficiency. CONCLUSIONSPARP16 binds to and inhibits NFAT1 activity to regulate cardiac function in mice, and its downregulation may activate NFAT1 signalling, leading to hypertrophy. In this manner, PARP16 plays a critical role in cardiac hypertrophy and failure and may serve as a potential therapeutic target for the treatment of heart failure.

BackgroundEndoplasmic reticulum (ER) stress and ER-mitochondria Ca2+ dysregulation contribute to cardiomyocyte injury, yet endogenous regulators at ER-mitochondria interfaces that restrain this cascade remain poorly defined. Pannexin 2 (Panx2), the most structurally divergent pannexin isoform, has been implicated in stress response, but its cardiac localization and function are unclear. MethodsPanx2 localization and function were assessed in human AC16 cardiomyocytes using high-resolution confocal imaging and complementary loss- and gain-of-function approaches during thapsigargin-induced ER stress, with validation in adult mouse ventricular cardiomyocytes. ResultsPanx2 localizes predominantly to the ER and mitochondria-associated membranes, rather than the plasma membrane. Panx2 knockdown reduced ER Ca2+ stores and increased basal cytosolic and mitochondrial Ca2+. During ER stress, Panx2 deficiency markedly amplified Ca2+ dysregulation, mitochondrial dysfunction, unfolded protein response activation, and cytotoxicity, with PERK-dominant signaling and increased IRE1a activation. Notably, PERK inhibition preferentially rescued the Panx2-deficient phenotype, providing the greatest improvement in viability and reduction in cytotoxicity. Panx2 deficiency also enhanced inflammasome/ pyroptotic signaling via the NLRP3-caspase-1-gasdermin D axis. Conversely, Panx2 overexpression suppressed PERK activation and attenuated ER stress-induced injury. Panx2 ablation similarly sensitizes adult ventricular cardiomyocytes to ER stress. ConclusionsPanx2 functions as an organelle-associated checkpoint at ER-mitochondria interfaces that stabilizes Ca2+ homeostasis and limits PERK-dominant ER stress signaling and inflammatory cell death programs in cardiomyocytes, providing a mechanistic framework for cardiomyocyte loss in cardiac disease. Research PerspectiveO_ST_ABS1. What New Question Does This Study Raise?C_ST_ABSDoes Panx2 serve as an endogenous "stress threshold" determinant in cardiomyocytes in vivo, governing when ER stress transitions from adaptive signaling to PERK-driven mitochondrial failure and inflammasome-associated inflammatory cell death during cardiac injury (e.g., ischemia-reperfusion, pressure overload, or cardiometabolic stress)? 2. What Question Should Be Addressed Next?In clinically relevant models of heart disease (ischemia-reperfusion), test whether cardiomyocyte-specific Panx2 loss or augmentation alters infarct size, arrhythmia burden, ventricular remodeling, and functional recovery, and determine whether targeting the Panx2-PERK axis (e.g., selective PERK modulation in the acute reperfusion window or Panx2-directed strategies) reduces cardiomyocyte loss without impairing adaptive stress signaling needed for repair.

BackgroundPost-operative tachycardia is a common and poorly understood complication following the Fontan procedure. Post-operative factors such as surgical scarring and venous hypertension can contribute to tachycardia risk, but the specific molecular signaling cascades triggering acute tachycardia remain uncharacterized, limiting therapeutic innovation and leaving clinicians with only reactive management strategies. Here, we present a retrospective translational study leveraging serum proteomics and machine learning to identify the molecular drivers of post-operative Fontan tachycardia. MethodsWe integrated a clinically relevant ovine animal model of the Fontan circulation with continuous telemetric heart rate monitoring and human patient data. Serum proteomics coupled with machine learning algorithms (LASSO and Boruta) were employed to identify protein panels predictive of post-operative tachycardia. Cross-species validation was performed by comparing proteomic signatures from sheep and pediatric patients undergoing Glenn or Fontan surgery. ResultsOvine Fontan animals demonstrated significant heart rate elevation beginning on post-operative day (POD) 1, peaking at POD 3 (159.4 {+/-} 11.7 bpm vs. pre-operative 105.3 {+/-} 10.5 bpm, p<0.0001), before trending toward baseline by POD 10. This pattern was mirrored in human pediatric patients, though with a more modest magnitude. Surgical controls did not exhibit tachycardia, indicating the response was specific to the Fontan procedure. Proteomic analysis identified distinct separation between pre- and post-operative serum profiles. Principal component analysis revealed that the principal components most correlated with heart rate (PC1: r=0.79, p=6.5x10-; PC8: r=0.40, p=0.04) were significantly enriched for inflammatory and neural pathways. We leveraged the Boruta algorithm to identify a seven-protein panel (ACE, ANGT, ITIH4, SELENOP, W5PHP7, PTX3, and F5) with superior predictive power (AUC=0.926). A cross-species comparison between human and sheep demonstrated that three proteins, angiotensinogen (ANGT), angiotensin-converting enzyme (ACE), and pentraxin 3 (PTX3), were similarly dysregulated in both species post-operatively. ConclusionsThis study provides the first direct molecular evidence implicating a dysregulated neurohormonal-inflammatory axis as a principal driver of acute post-operative Fontan tachycardia. The identified protein signature offers novel mechanistic insights and establishes a foundation for developing targeted diagnostics and therapeutics to predict and mitigate this significant clinical complication.

BackgroundRegulated in development and DNA damage 1 (REDD1) is a highly inducible molecule that plays a role in numerous physiological and pathophysiological processes. It is a well-established negative regulator of mammalian target of rapamycin complex 1 (mTORC1), which is critical for maintaining elevated fatty acid-to-glucose oxidation ratio in the heart. In addition, REDD1 deletion results in hyperglycemia, suggesting that REDD1 is critical for tissue glucose metabolism. The role of REDD1 in regulating cardiac glucose and/or fatty acid metabolism in response to physiologic or pathophysiologic cues, however, remains unexplored. MethodsHerein, we utilize AC16 cardiomyocytes with REDD1 deletion, as well as mice with global or cardiomyocyte-specific deletion of Redd1, and their respective controls. We also subject these mice cardiac pressure overload using transverse aortic constriction (TAC) for 2 weeks or sham operation as a control. To examine the molecular regulators of glucose oxidation, we utilized qPCR and western blotting to evaluate pyruvate dehydrogenase (PDH) kinase (PDK) and phospho-PDH (pPDH) levels, respectively. We also directly measured PDH activity and glucose-driven cellular respiration. To investigate the complete REDD1-dependent transcriptome and metabolome, we performed RNA-sequencing (RNA-Seq) and untargeted metabolomics, respectively. To determine if the observed gene expression changes were dependent upon transcription factor peroxisome proliferator-activated receptor alpha (PPAR), we utilized an established pharmacologic PPAR inhibitor, GW6471. Here, we measured PPAR activity directly, as well as the expression of its target genes. In order to determine if our observed effects were mTORC1-dependent, we utilized mTORC1-specific inhibitor, everolimus. Finally, we measured cardiac hypertrophy using gravimetric analyses (heart weight (HW)-to-body weight (BW) or HW-to-tibia length (TL) ratios) and histological analyses of cardiomyocyte cross sectional area (CSA). We also measured mRNA and protein levels of pathological hypertrophic markers Natriuretic Peptide B (Nppb) and Cardiac Ankyrin Repeat Protein (CARP), respectively. ResultsOur data demonstrate that physiological levels of glucose induce REDD1 expression in cardiomyocytes. Further, we show that in cardiomyocytes or the hearts of mice with REDD1 deletion, there is elevated PDK4 expression, as well as increased levels of pPDH (S300 and/or S293) and reduced PDH activity. Interestingly, everolimus treatment has no effect on these alterations. In vitro, we also observe elevated glycolysis and glycolytic capacity, and reduced maximal respiratory capacity (MRC) in the presence of glucose. Interestingly, our RNA-Seq data reveals the upregulation of genes involved in fatty acid catabolism. Further, we demonstrate that PPAR activity is enhanced, and everolimus treatment also has no effect on this parameter. Additionally, we show that treatment of cardiomyocytes with GW6471 normalizes the expression of its target genes (PDK4, ACSL1) and levels of pPDH (S300), that are elevated in cells with REDD1 deletion. Finally, we observe elevated REDD1 in the hearts of mice following TAC. Moreover, we show reduced HW/BW, HW/TL, cardiomyocyte CSA, and levels of cardiac Nppb and CARP in mice with cardiomyocyte Redd1 deletion subjected to TAC versus controls also subjected to TAC. Importantly, TAC-induced reductions in cardiac Pdk4 and pPDH (S293 and S300), are normalized to control levels in mice with Redd1 deletion subjected to TAC. ConclusionsTogether, our findings suggest that physiological glucose-induced and pathological pressure overload-induced REDD1 is required for enhancing glucose oxidation and suppressing fatty acid oxidation in cardiomyocytes. In this way, REDD1 supports cardiac hypertrophic growth. We also outline a mechanism whereby REDD1 inhibits PPAR activity, thereby inhibiting the expression of its target genes, including PDK4 and those involved in fatty acid oxidation. Finally, we demonstrate that these effects are independent of REDD1s ability to inhibit mTORC1.

BackgroundThe cardiac isoform of phosphofructokinase-2/fructose 2,6-bisphosphatase (PFKFB2) is the hearts strongest glycolytic regulator but is degraded in the absence of insulin signaling. This makes PFKFB2 loss critical to understand in metabolic heart disease, of which impaired insulin signaling is a hallmark. Prolongation of the QT interval, risk of arrhythmia, and sudden cardiac death are also augmented in metabolic heart disease, raising a question as to whether potential crosstalk between glycolytic dysregulation and electrophysiological dysfunction exists. MethodsWe therefore assessed the impact of PFKFB2 loss on cardiac electrophysiology using a cardiomyocyte-specific PFKFB2 knockout mouse model (cKO) and litter-matched controls (CON). To do so, we employed electrocardiography in the fed state and following 12 hours of fasting, examining physiology both at baseline and in the presence of an acute stimulant stress. To further investigate the arrhythmia mechanism, we used patch-clamp electrophysiology and IonOptix Ca2+ transient measurements in ventricular cardiomyocytes isolated from CON and cKO hearts. ResultsThe hearts of cKO mice exhibited prolonged repolarization, marked by QT and action potential duration prolongations. This occurred with impaired Ca2+ reuptake and increased spontaneous Ca2+ release events in ventricular cardiomyocytes. Ultimately, these changes culminated in ventricular tachyarrhythmia in cKO mice, which was enhanced in the fed relative to the fasted state. ConclusionThese data suggest that in the presence of sufficient glucose availability, cardiac glycolytic dysregulation at the phosphofructokinase nexus is sufficient to promote cardiac electrophysiological instability. Clinical PerspectiveO_ST_ABSWhat is KnownC_ST_ABSO_LIMetabolic heart diseases, such as heart failure with preserved ejection fraction and diabetic cardiomyopathy, are associated with heightened risks of arrhythmogenesis and sudden cardiac death. C_LI What the Study AddsO_LIHere, we show for the first time that PFKFB2 is decreased in human hearts with heart failure with preserved ejection fraction. C_LIO_LIFurthermore, we show that loss of cardiac PFKFB2 is sufficient to promote impaired ventricular repolarization at baseline and ventricular tachyarrhythmia upon stress test. C_LIO_LIThis identifies PFKFB2 stabilization and activation as key potential targets in conferring electrophysiological stability in metabolic heart disease. C_LI

BackgroundHeart failure, arrhythmia, and sudden cardiac death are common outcomes of cardiomyopathies, which are molecularly diverse heart muscle disorders marked by structural and functional myocardial dysfunction. The lack of sensitive molecular biomarkers that precede overt physiological deterioration makes early diagnosis difficult despite advancements in imaging and clinical classification. The immune transcriptional landscape across cardiomyopathy subtypes is still poorly understood, despite growing evidence linking both innate and adaptive immune dysregulation, such as macrophage activation and T-cell and inflammatory cytokine networks, as active contributors to myocardial remodelling and disease progression. MethodsWe performed a multi-cohort integrative transcriptomic analysis of 1,068 cardiac tissue samples from five publicly available GEO datasets (GSE57338, GSE5406, GSE36961, GSE141910, GSE47495) spanning dilated, ischemic, hypertrophic, and peripartum cardiomyopathy. Using a fully scripted R and Python pipeline, we conducted differential expression analysis (limma), immune cell deconvolution (xCell), pathway enrichment (clusterProfiler), weighted gene co-expression network analysis (WGCNA), and regularised machine learning classification (LASSO, Random Forest). Cross-dataset validation was performed in two independent cohorts on different microarray platforms. ResultsDifferential expression analysis identified 43 primary DEGs (FDR < 0.05, |log2FC| > 1.0), revealing a coherent immune-fibrotic program characterized by loss of anti-inflammatory macrophage markers (CD163, VSIG4), complement dysregulation (FCN3), innate interferon activation (IFI44L, IFIT2), and ECM remodelling (ASPN, SFRP4, LUM). xCell deconvolution identified coordinated depletion of adaptive immune populations in failing myocardium. WGCNA defined a fibrosis hub module (brown; CTSK, SULF1, SFRP4) and an immune collapse module (turquoise; MYD88, TNFRSF1A, LAPTM5). A nine-gene LASSO classifier achieved a cross-validated AUC of 0.986, with HMOX2 as the top-discriminating feature, implicating ferroptosis in cardiomyocyte death. Cross-platform validation in an independent HCM cohort (GSE36961) demonstrated a directional concordance of 34.9%. ConclusionsThis study defines a reproducible immune-fibrotic transcriptional signature of human cardiomyopathy, nominates HMOX2 and ferroptosis as central pathomechanisms, and provides a validated nine-gene biomarker panel for future translational investigation.

BackgroundMyocardial Infarction (MI) remains a leading cause of mortality worldwide, despite advancements in clinical therapies and interventions. MI results from prolonged ischemia, leading to hypoxia-induced damage to cardiac tissue and reperfusion-injury (R/I) that aggravates cardiomyocyte (CM) loss. One key cellular event during this process is accumulation of dysfunctional mitochondria, resulting from environmental hypoxia and subsequent oxidative stress upon reperfusion. Post-MI cardiac-remodeling involves changes in both cellular and extracellular matrix (ECM). Ubiquitin-dependent and independent autophagy are crucial for cardio protection during this phase. The ECM provides structural integrity and functions as a reservoir for signaling molecules. Asporin (ASPN), a small leucine-rich proteoglycan, plays a role in modulating cardiac-remodeling by limiting excessive fibrosis and protecting CMs from cell death. MethodsWe investigated the therapeutic potential of ASPN by using an exogenous recombinant peptide of ASPN (rASPN), testing its effects using an in-vitro ischemia-reperfusion (I/R) model simulating MI conditions. Two I/R models were developed using an immortalized human embryonic cardiac cell line to reflect the hypoxia-reperfusion (H/R) phases of MI. In the No-Reoxygenation (No-ReOx) model, cells were subjected to hypoxia for 18 hours, with or without exogenous rASPN. In the Reoxygenation (ReOx) model, cells underwent 18 hours of hypoxia, then 12 hours of reoxygenation (simulating reperfusion), with or without rASPN. ResultsProteomics revealed that ASPN modulates key pathways involved in apoptosis, non-canonical autophagy, and metabolic reprogramming. Additionally, ASPN influenced immune response pathways and significantly affected TGF-{beta} signaling, a central mediator of cardiac fibrosis and remodeling post-MI. These findings indicate that ASPN plays a multifaceted role in regulating cellular responses to hypoxia and R/I. ConclusionsOur H/R model simulates key aspects of MI and R/I. The protective role of ASPN observed in this model suggests it as a promising candidate for developing cardioprotective therapies to minimize R/I and adverse cardiac-remodeling following MI.

BackgroundCardiac myosin inhibitors (CMIs) demonstrate advantages over other guideline-directed therapy for patients with obstructive hypertrophic cardiomyopathy (oHCM). By reducing hypercontractility, CMIs abrogate excessive systolic function and improve diastolic function; diminish hypertrophy of the left ventricle (LV); and improve exercise capacity, functional class, and symptoms. Whether CMIs are therapeutic in heart failure with preserved ejection fraction (HFpEF) is of interest because a significant subset of these patients demonstrate supranormal ejection fractions and abnormal LV structure, characteristics in common with HCM, where CMIs have proved effective. ObjectivesOur goal was to characterize the mechanism of myosin inhibition for ulacamten and determine its efficacy in a rodent model of HFpEF. MethodsUlacamten was characterized using biophysical and biochemical approaches, cardiomyocytes from humans and the ZSF1 obese rat model of HFpEF, hypercontractile human-engineered heart tissues, and echocardiography in the ZSF1 rat model. ResultsUnlike the other CMIs, aficamten and mavacamten, ulacamten binds outside the S1 domain of myosin and requires the regulatory light chain domain to bind and inhibit the activity of 2-headed myosin. Ulacamten only partially inhibits the myosin ATPase activity in both myofibrillar and protein systems, but inhibition of contractility was nearly complete in cardiomyocytes. Improvement in relaxation was demonstrated in hypercontractile-engineered heart tissues, and chronic treatment of ZSF1 obese rats showed benefits in both cardiac structure and function. ConclusionsUlacamten inhibits myosin in a manner distinct from aficamten and mavacamten, potentially broadening the mechanistic properties of CMIs available for treatment of hypercontractile cardiac dysfunction. CONDENSED ABSTRACTCardiac myosin inhibitors (CMIs) abrogate excessive systolic function and improve diastolic function, diminish cardiac hypertrophy, and improve exercise capacity in humans with obstructive hypertrophic cardiomyopathy (oHCM). Supranormal ejection fraction underlies heart failure with preserved ejection fraction (HFpEF) in some patients. We describe a new CMI, ulacamten, with binding and inhibitory properties distinct from two other FDA-approved CMIs, aficamten and mavacamten. Specifically, ulacamten requires 2-headed myosin to inhibit activity, whereas aficamten and mavacamten inhibit single-headed myosin. Ulacamten inhibits contractility in primary myocytes isolated from control human and hypercontractile ZSF1 obese rat hearts, as well as engineered heart tissues created with induced pluripotent stem cell cardiomyocytes bearing an HCM mutation. Chronic treatment of ZSF1 obese rats as a preclinical model of HFpEF improves diastolic function and reduces hypertrophy and fibrosis, broadening the potential mechanistic landscape of CMIs. Visual abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/701387v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@11f9cecorg.highwire.dtl.DTLVardef@776847org.highwire.dtl.DTLVardef@15f19ddorg.highwire.dtl.DTLVardef@9b20c6_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundDepending upon the type of pathological stress, the heart undergoes concentric or eccentric remodeling. This structural change is associated with diastolic and/or systolic ventricular dysfunction reflecting differentially altered cardiomyocyte morphology, ultrastructure, metabolism, contractility, and survival, as well as interstitial myocardial fibrosis. Despite an association of both concentric and eccentric remodeling with heart failure and sudden death, the molecular mechanisms resulting in abnormal cardiac geometry remain poorly understood. A better understanding of the basic mechanisms conferring these contrasting forms of remodeling should inform novel approaches to preserve normal cardiac structure and function in cardiovascular disease. The protein phosphatase Cell Division Cycle 14A (CDC14A) and its substrate the lysine methyltransferase KMT5A are identified herein as key regulators of the balance between concentric and eccentric pathological cardiac remodeling. MethodsThe regulation of adult rat ventricular myocyte morphology by CDC14A and KMT5A was studied in vitro following gain and loss of function by expression of wild-type and mutant proteins and RNA interference (RNAi). Epigenomic regulation by KMT5A was studied by mapping histone 4 lysine 20 mono-methylation (H4K20me1) modified chromatin sites and correlating them with gene transcription. Regulation of pathological cardiac remodeling in vivo was demonstrated by CDC14A and KMT5A RNAi using adeno-associated virus (AAV) mediated cardiomyocyte-specific small hairpin RNA (shRNA) expression in mice. ResultsCDC14A inhibited the growth in width of cultured adult myocytes stimulated by -adrenergic receptor activation or by serum response factor. KMT5A was downregulated by CDC14A in cardiomyocytes and was required for myocyte growth in width. -adrenergic stimulation of KMT5A-dependent H4K20 mono-methylation across transcription units correlated with regulation of gene transcription. Accordingly, AAV-expressed KMT5A shRNA induced eccentric remodeling and cardiac dysfunction in wild-type mice. Conversely, expression of Cdc14A shRNA improved systolic function and cardiac structure and inhibited pathological gene expression in the Tpm1 E54K mouse with Dilated Cardiomyopathy. ConclusionsCDC14A-KMT5A-dependent epigenomic regulation of gene transcription constitutes a molecular switch that determines concentric versus eccentric cardiac remodeling. These findings identify CDC14A as a potential therapeutic target for the treatment of dilated cardiomyopathy and other forms of heart failure with reduced ejection fraction. Clinical PerspectiveO_ST_ABSWhat is newC_ST_ABSO_LIA function is identified for the first time for the protein phosphatase CDC14A in the heart, regulation of cardiomyocyte morphology and overall cardiac geometry in pathological cardiac remodeling. C_LIO_LIThe lysine methyltransferase KMT5A is shown to mediate the effects of CDC14A in the adult cardiomyocyte by regulating H4K20 mono-methylation, such that reduced KMT5A expression promotes a phenotype resembling Dilated Cardiomyopathy. C_LIO_LIH4K20me1 epigenomic modification is identified as a regulator of cardiac structure and function. C_LI Clinical implicationsO_LICDC14A loss of function experimentation in vivo, resulting in improved cardiac structure and function in a mouse model of Dilated Cardiomyopathy, suggests that CDC14A is a novel therapeutic target for heart failure with reduced ejection fraction. C_LI

Glucagon-like peptide-1 receptor (GLP1R) agonists improve glycaemic control, induce weight loss, and consistently reduce major adverse cardiovascular events. However, the mechanistic basis of their cardioprotective effects remains incompletely understood, particularly whether benefits arise solely from systemic actions or also involve direct cardiac GLP1R signalling. To address this, we performed integrated single-cell and single-nucleus transcriptomics to map GLP1R gene expression across human and murine organs, cardiac cell types, disease states, and hiPSC-derived cardiac organoids. In humans, GLP1R expression was predominantly pancreatic, with low cardiac expression largely restricted to cardiomyocytes and consistently upregulated across ischaemic, dilated, and hypertrophic cardiomyopathy. In contrast, murine cardiac Glp1r expression was confined to endocardial cells and remained unchanged in heart disease. Other cardiac cell types, including fibroblasts, endothelial cells, and mural cells, showed minimal GLP1R expression in both species. Human cardiac organoids recapitulated ventricular GLP1R patterns closer to adult human myocardium than murine tissue. Together, these findings indicate that GLP1R is primarily extracardiac but selectively induced in failing human myocardium, supporting a model in which myocardial GLP1R signalling augments systemic mechanisms to confer GLP1R agonist-mediated cardioprotection.

Iron overload cardiomyopathy (IOC) is a serious heart condition that is caused by elevated levels of systemic iron. IOC is characterized by both systolic and diastolic dysfunction as well as arrhythmias. It has been challenging to isolate the cardiac-specific cellular and molecular mechanisms driving IOC because the disease affects multiple interconnected organ systems. Here, we leverage stem cell technologies, cardiac tissue engineering, and protein reconstitution assays to model key aspects of human IOC in vitro and to probe the cellular and molecular mechanisms driving cardiac dysfunction. We demonstrate that human engineered heart tissues consisting of both cardiomyocytes and cardiac fibroblasts faithfully recapitulate key aspects of the human disease, including reduced systolic function, impaired diastolic function, and increased prevalence of arrhythmogenic events. We demonstrate that while both cardiomyocytes and cardiac fibroblasts show increased intracellular iron levels, leading to reduced viability, cardiomyocytes show higher levels of iron accumulation and higher levels of reactive oxygen species production. Moreover, we show that in a tissue, iron overload has little effect on the action potential kinetics; however, it directly impacts the amplitude and kinetics of the calcium transient, potentially driving arrhythmogenesis. Finally, we demonstrate that iron overload decreases force production, in part, through oxidative damage of sarcomeric proteins and direct iron-based inhibition of myosin. In summary, our results reveal new insights into the cellular and molecular mechanisms of human IOC pathogenesis, and they establish new in vitro models that can be harnessed to faithfully recapitulate key aspects of the human disease phenotype. HighlightsO_LIContractile aspects of iron overload cardiomyopathy have been difficult to study in vitro. C_LIO_LIWe developed engineered heart tissues to model key aspects of the human disease. C_LIO_LIIn vitro iron overload reduces contractility and induces arrhythmogenesis. C_LIO_LIIron differentially affects cardiomyocytes and cardiac fibroblasts. C_LIO_LIIron overload directly impacts the actomyosin contractile apparatus. C_LI

BackgroundHeart failure with preserved ejection fraction (HFpEF) remains a major therapeutic challenge due to its complex pathophysiology and pronounced heterogeneity. Regenerative approaches using neonatal mesenchymal stromal cells (nMSCs) and their secretome (SEC) have shown promise in other heart failure contexts. ObjectivesHowever, the effect of these therapies in HFpEF, and the underlying molecular mechanisms and causal pathways remain poorly understood. MethodsHFpEF was established in two distinct murine models, followed by treatment with either nMSCs or SEC. Functional and histological endpoints were assessed. We developed a novel machine learning framework, VIPcell, which integrates data augmentation, Partial Least Squares (PLS) regression, and causal structure inference to identify genes causally linked to cardiac function using single-nucleus RNA sequencing (snRNA-seq) data. VIPcell was applied to heart tissues from treated HFpEF animals to uncover key regulators of cardiac remodeling. ResultsBoth nMSC and SEC therapies significantly improved diastolic function in two independent rodent HFpEF models. These improvements were associated with reduced inflammation, attenuated myocardial fibrosis, and improved exercise capacity. Intercellular communication analysis revealed widespread, system-level signaling in nMSC-treated hearts, compared to more localized endothelial-cardiomyocyte crosstalk in SEC-treated hearts. Causal inference via VIPcell suggested overlapping upstream regulators in both treatment groups, particularly genes involved in regulatory T cell (Treg) biology and immunomodulatory signaling pathways, including FOXO signaling, NLRP3 inflammasome inhibition, and Tie2 activation. In vivo validation confirmed selective expansion of Tregs following nMSC and SEC therapy. In vitro, nMSCs induced significantly greater Treg expansion compared to multiple adult stem cell types. Critically, chemical depletion of Tregs abrogated the therapeutic effects of both treatments, establishing Tregs as central mediators of diastolic function recovery in the HFpEF preclinical model. ConclusionsnMSC and SEC therapies improve diastolic function in HFpEF through distinct remodeling mechanisms converging on Treg-mediated immune modulation. VIPcell supported identification of causal regulators, highlighting Treg-related signaling as a key driver of myocardial recovery in HFpEF. These findings offer mechanistic insight into cellular therapies for HFpEF and support the development of targeted, Treg-focused interventions.

Heart as one high ATP consuming organ accounts for 5% of the total oxygen demands. The central question of heart health is how mitochondria fit its needs. Impaired mitochondrial dynamics (fission and fusion) have been observed in failing heart, but whether and how phosphorylation events involved in mitochondrial quality control are still imperceptive. The phosphatase 2A catalytic subunit (PP2A c) cardiac-specific knockout mouse (KO), which exhibited a hypertrophic cardiomyopathy phenotype, was studied. We profiled the pattern of morphological and functional alteration of cardiac mitochondria that appeared during postnatal development. Increased heterogeneity of mitochondria and a decreased ATP yield was displayed. Notably, a fission procedure escalated. To illustrate the protagonist of the mitochondrial dynamics, we applied a high-throughput spectrometry-based phosphoproteomic screening following by GO and KEGG pathway annotations for 788 phosphosites, accounting for 90 proteins. Results suggested that the MAPK signaling may be a predominant factor associated with those mitochondrial alternations in KO hearts. Furthermore, we identified hyperphosphorylated ERK2 accumulated into the nucleus regarding PP2Ac depletion. Consequently, Fis1 expression was accelerated at the transcriptional level which facilitated recruitment of Drp1 onto the outer mitochondrial membrane. The mitochondrial fission towards shifting led to excessed mitophagy and is considered the culprit in early mortality. These findings are indicative of the fundamental role of PP2A in mitochondrial dynamics regulation and cardiomyopathy progression. During the progression of heart failure, the phospho-regulation of ERK2 could be a novel therapeutic approach to prevent or attenuate adverse hypertrophic cardiomyopathy.

Ischemic injury and adverse post-infarction myocardial remodeling are major causes of heart failure. We previously reported that microRNA (miR)-200c inhibition in murine embryos increased cardiogenic transcription factors (TFs) Tbx5, Gata4, and Mef2c to activate an immature cardiomyocyte cell state, suggesting miR-200 inhibition as a therapy for cardiac repair. We performed permanent ligation of the left anterior descending artery (a severe myocardial infarct, MI) on PMIS-miR-200c (inhibition of miR-200c; PMIS-C), PMIS-A (inhibition of miR-200a) and wildtype adult mice. Echocardiographic left ventricular (LV) ejection fraction (EF) at 3 WPI (weeks post-injury) was 22% {+/-} 4.31% (WT) but increased to 56% {+/-} 4.25% (PMIS-C) (p [&le;] 0.0001). Post-infarction LV chamber dilation was reversed in PMIS-C mice compared to WT, and trichrome staining showed a decrease in fibrosis 3 WPI. By 9 WPI, PMIS-C heart function was like that of WT mice before injury. Tbx5, Gata4, Mef2c, and Isl-1 were increased after MI in PMIS-C hearts. PMIS-C mice recover cardiac function and reverse ischemic pathology of acute cardiac injury in adult mice. Inhibition of miR-200c activates several important pathways in heart development and repair mechanisms after an MI in adult hearts. The PMIS-miR-200c transgenic mice demonstrate an important role for miR-200c in regulating heart repair after ischemic injury. Novelty and SignificanceO_ST_ABSWhat is known?C_ST_ABS*The microRNA-200 (miR-200) family targets several heart factors in vitro. *miR-200c inhibition was shown to protect cardiomyocytes in a myocardial ischemia-reperfusion injury, myocardial cellular model. *miR-200 may play a role in cardiovascular fibrosis, however there are no in vivo reports of the role miR-200 plays in heart repair. What New Information Does This Article Contribute?*PMIS-miR-200c transgenic mice reveal a role for miR-200c inhibition in rapid repair of the heart after a myocardial infarct (MI). After an MI, miR-200c expression increases, to levels observed during early heart development. *Inhibition of miR-200c allows for expression of Tbx5, Gata4, Pitx2, Mef2c, Yap, Nppa and Sox5 factors to repair the heart after ischemic injury. *PMIS-miR-200c mice have increased cardiomyocyte proliferation and reduced cardiac fibroblasts resulting in decreased fibrosis. *Heart function in PMIS-miR-200c mice is significantly restored 3-weeks post-MI. While microRNAs have been extensively studied in heart development and ischemic injury, little is known about the miR-200 family in the cardiovascular system. In other cell types and systems, miR-200 is upregulated under oxidative stress and hypoxia. miR-200c targets Zeb1, eNOS, Sirt1 and Fox01 to regulate cell growth and arrest, apoptosis and senescence in other tissues. miR-200 members are increased in response to ischemia, but this has not been evaluated in the heart. We show a direct effect of miR-200c inhibition and decreased fibrosis in the MI heart. The miR-200 family targets stem cell factors such as Sox2, Klf4, and Bmi1 and our recent sn-RNA multiomics analyses of PMIS-miR-200c mice revealed de-differentiated or immature cardiomyocytes. Thus, inhibition of miR-200c reactivates transcription factors after an MI, important for cardiomyocyte renewal. This research demonstrates how inhibition of miR-200 regulates cardiac function after an MI.

BackgroundHeart failure with preserved ejection fraction (HfpEF) is increasingly recognized as a multisystem disorder linked to the cardiovascular-kidney-metabolic (CKM) syndrome. While the falling heart undergoes metabolic reprogramming, the interorgan crosstalk regulating myocardial substrate preference in HFpEF remains elusive. We aimed to clarify the role of systemic and local ketogenesis in the pathogenesis of cardiac hypertrophy and HFpEF. MethodsA mouse model of HFpEF was employed using a high-fat diet combined with NG-Nitro-L-arginine methyl ester hydrochloride (L-NAME). Cardiac hypertrophy and systemic metabolic profiling including ketogenesis were evaluated. To dissect the role of site-specific ketogenesis, we generated inducible cardiomyocyte-specific (Hmgcs2{Delta}iCM) and hepatocyte-specific (Hmgcs2{Delta}Hep) knockout mice of HMG-CoA synthase 2 (Hmgcs2), deficient in the rate-limiting enzyme for ketogenesis. Cardiomyocyte -specific nuclei were isolated for transcriptomic (RNA-seq) and in vitro assays in H9C2 cells were used to elucidate molecular mechanisms. ResultsThe HFpEF model successfully exhibited diastolic dysfunction, impaired exercise capacity and cardiac hypertrophy with elevated circulating ketone body concentration. Myocardial metabolomics and snRNA-seq identified a profound metabolic shift characterized by the accumulation of long-chain fatty acids and Krebs cycle intermediates, coupled with the transcriptional downregulation of insulin signaling and fatty acid degradation pathways. Although circulating ketone body level was upregulated, Hmgcs2{Delta}iCM mice showed no exacerbation of the HFpEF phenotype. In contrast, Hmgcs2{Delta}Hep mice exhibited significantly aggravated cardiac hypertrophy (HW/TL; Hmgcs2flox: 7.41 {+/-} 0.87: Hmgcs2{Delta}Hep: 8.29 {+/-} 0.73; p = 0.0154). Mechanistically, hepatic ketogenesis was required to maintain circulating beta-hydroxybutyrate (BHB) levels, which directly modulated cardiomyocyte metabolism. BHB acted as a metabolic signal to dampen fatty acid overload and facilitate glucose utilization. ConclusionsOur study identifies a critical "liver-heart axis" where hepatic ketogenesis serves as an essential regulator of myocardial metabolic resilience. Impaired hepatic ketogenesis creates a metabolic mismatch that drives pathological cardiac remodeling. These findings highlight the liver as a therapeutic target within the CKM syndrome framework, suggesting that restoring the hepato-cardiac metabolic bridge may ameliorate HFpEF progression. What is New?O_LIThis study identifies a novel liver-adipose-heart axis that governs myocardial metabolic resilience during the development of heart failure with preserved ejection fraction (HFpEF). C_LIO_LIWe demonstrate that while both the liver and heart upregulate ketogenesis under metabolic stress, only hepatic ketogenesis--and not cardiac-intrinsic ketogenesis--is essential for mitigating pathological cardiac remodeling. C_LIO_LIMechanistically, liver-derived {beta} -hydroxybutyrate acts as a critical C_LIO_LIendocrine signal that dampens fatty acid oxidation and facilitates myocardial glucose utilization. C_LI What Are the Clinical Implications?O_LIOur findings highlight the liver as a central therapeutic target within the cardiovascular-kidney-metabolic (CKM) syndrome framework, where hepatic metabolic failure directly drives cardiac dysfunction. C_LIO_LIRestoring the hepato-cardiac metabolic bridge, through either hepatic-targeted therapies or ketone body supplementation, represents a promising strategy to enhance myocardial metabolic flexibility and ameliorate HfpEF in patients with multi-organ metabolic disorders. C_LI

Pathologic cardiac hypertrophy requires increased protein synthesis, but the mechanosensors that link membrane stretch to translational control remain poorly understood. Polycystin-1 (PC1), encoded by PKD1, has been proposed as a cardiac mechanosensor, with its C-terminal tail (PC1-CT) promoting hypertrophy in rodent cardiomyocytes. However, its subcellular localization and downstream signaling remain incompletely defined, especially in human cardiomyocytes. Here, we examined endogenous PC1 C-terminus localization and the effects of adenoviral PC1-CT overexpression in human iPSC-derived ventricular cardiomyocytes (hiPSC-CMs) and adult mouse ventricular myocytes. Immunofluorescence revealed a striking striated pattern for both endogenous PC1 C-terminus (detected with a PC1-CT antibody) and the overexpressed PC1-CT fragment. In hiPSC-CMs, the PC1 C-terminus localized between the -actinin bands. In contrast, in adult cardiomyocytes, the overexpressed protein colocalized with -actinin and desmin, suggesting that PC1-CT sarcomeric distribution depends on cardiomyocyte maturation. We performed RNA-seq to assess transcriptional responses downstream of PC1-CT overexpression in hiPSC-CMs relative to LacZ controls. Gene Set Enrichment Analysis (GSEA) revealed enrichment of gene sets related to ribosome biogenesis, RNA processing, and protein synthesis, while classical hypertrophic markers remained unchanged. Pathway analysis suggested increased PI3K activity. PC1-CT overexpression increased phosphorylation of Akt, ERK, S6K1, and ribosomal protein S6 without altering 4EBP1 phosphorylation, suggesting preferential activation of the mTOR-S6K1-S6 branch. Pharmacological studies showed that pan-PI3K inhibition abolished S6 phosphorylation, whereas MEK blockade did not affect it; pertussis toxin and PI3K{gamma}-selective inhibitors also did not affect S6, suggesting a Gi/o-independent PI3K/Akt signaling driving mTOR-S6K1-S6 activation. Collectively, these data identify a sarcomere-associated pool of PC1-CT that engages PI3K-Akt-mTOR-S6K1-S6 signaling to enhance transcriptional programs related to ribosome biogenesis and protein synthesis, without activating a canonical hypertrophic gene program. These findings reveal a mechanistic link between PC1-CT and cardiomyocyte growth.

BackgroundPediatric dilated cardiomyopathy (DCM) is a rare, progressive heart disease with variable outcomes that range from recovery to heart transplantation. To date, there are no prognostic biomarkers for children with DCM. Identifying circulating biomarkers that are associated with clinical outcomes is critical for personalized management. MethodsmiRNAs were identified by RNA-seq, whereas proteins were identified by SomaScan(R). Machine learning methodologies were used to explore the predictive ability of circulating factors identified from serum samples collected at the time of presentation with acute heart failure. ResultsThirty patients experienced poor outcomes (cardiac transplantation, mechanical circulatory support, or death) and 19 patients recovered left ventricular function. Distinct miRNA and protein signatures differentiated outcomes groups. Top candidate proteins (COL2A1, CXCL12, and ADGRF5) and miRNAs (miR-874-3p, miR-335-3p, miR-323a-3p) demonstrated strong discriminatory performance within the study cohort (recovered vs poor outcomes; Area Under the Curve of 0.92). Ingenuity Pathway Analysis implicates cardiac remodeling, fibrosis, and inflammatory signaling as central pathways differentiating patient outcomes. ConclusionsCirculating miRNA and protein signatures at presentation identify a circulating molecular signature associated with divergent clinical trajectories in pediatric DCM. These findings support the potential utility of multi-omic biomarkers for early risk stratification and provide insight into mechanisms underlying divergent outcomes. CLINICAL PERSPECTIVEWhat Is New? O_LICirculating miRNA and protein profiles measured at presentation distinguish children with pediatric DCM who recover from those who progress to advanced heart failure. C_LIO_LIA combined multi-omic biomarker demonstrated strong discriminatory performance in this cohort (AUC 0.92). C_LIO_LIPathway analysis implicates extracellular matrix remodeling, fibrosis, and inflammatory signaling in children with adverse clinical trajectories. C_LI What Are the Clinical Implications? O_LISerum-based molecular biomarkers may enable earlier risk stratification in children presenting with dilated cardiomyopathy. C_LIO_LIMulti-omic integration may improve identification of pediatric patients at risk for transplantation, mechanical circulatory support, or death. C_LIO_LIThese findings support further validation of circulating biomarker panels to guide personalized management in this rare disease. C_LI RESEARCH PERSPECTIVEWhat New Question Does This Study Raise? O_LICan integrated circulating miRNA-protein signatures identify biologically distinct trajectories of recovery versus progression in children with dilated cardiomyopathy? C_LIO_LIDo circulating molecular profiles reflect underlying disease mechanisms that determine divergent clinical outcomes in pediatric DCM? C_LI What Question Should Be Addressed Next? O_LIDo the pathways identified by integrated miRNA-protein analysis (fibrosis, remodeling, and inflammation) play causal roles in determining recovery versus progression? C_LIO_LICan multi-omic biomarkers be incorporated into prospective studies to improve early risk stratification and guide clinical management? C_LI