Journal of Molecular and Cellular Cardiology

Background: Cardiometabolic diseases frequently involve concurrent cardiovascular and hepatic dysfunction, yet the conserved molecular mechanisms underlying these systemic responses remain poorly defined. Objectives: To identify conserved molecular responses across complementary manifestations of cardiometabolic stress and determine whether integrated multi-organ analyses reveal therapeutically actionable targets for heart failure. Methods: Cardiac functional phenotyping, hepatic injury profiling, and bulk RNA sequencing were performed across three complementary mouse models representing distinct manifestations of cardiometabolic stress: high-fat diet plus L-NAME (HFD+LN)-induced heart failure with preserved ejection fraction (HFpEF; cardiovascular disease), Western diet (WD)-induced obesity (systemic metabolic stress), and choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-induced steatotic liver disease (hepatic metabolic stress). Comparative transcriptomic analyses distinguished organ-specific responses from conserved molecular signatures. Results: Each model produced distinct systemic, hepatic, and cardiac phenotypes accompanied by divergent transcriptional responses within individual organs. Cross-model and cross-organ integration identified a limited set of conserved molecular responses to cardiometabolic stress, with Serpine1, encoding plasminogen activator inhibitor-1 (PAI-1), emerging as a highly conserved candidate that exhibited preferential induction in the heart. Pharmacologic inhibition of PAI-1 significantly improved cardiac function and attenuated adverse remodeling in established HFpEF, whereas hepatic pathology was comparatively less affected, indicating differential organ-specific dependence on this pathway. Conclusions: Integrated analyses across complementary manifestations of cardiometabolic stress identified conserved molecular signatures that transcend individual disease models and organs. These findings establish a comparative framework for discovering cardiovascular therapeutic targets and identify PAI-1 as a promising mediator of cardiac remodeling in cardiometabolic disease.

Precise quantification of myofilament protein phosphorylation is essential for understanding the regulation of cardiac contractility in health and disease. Although Phos-tag SDS-PAGE is widely used to resolve phosphorylated protein isoforms, its reproducibility and quantitative reliability are often limited by variability in the key experimental factors, including gel composition, electrophoretic conditions, protein loading, and sample preparation. Here, we present a standardized manganese (Mn2+)-Phos-tag SDS-PAGE workflow optimized for cardiac myofilament proteins, using myosin regulatory light chain 2 (MLC2) and cardiac troponin I (cTnI) as model targets. We systematically evaluated critical parameters - including Mn2+ and Phos-tag concentrations, acrylamide composition, electrophoretic regime, buffer chemistry, protein loading, and EDTA-mediated transfer - to define conditions that maximize phospho-species resolution while preserving quantitative fidelity. We further demonstrate that electrophoresis rate, sample loading, and extraction strategy significantly influence band morphology, signal intensity, and the apparent distribution of phospho-species. As a use case scenario, we compared Trichloroacetic acid (TCA) extracted mouse left ventricular homogenates with myofibrils prepared using a widely adopted Triton-X-100 tissue-demembranization protocol. Myofibril preparation was associated with profound MLC2 dephosphorylation at the earliest stages of preparation, whereas cTnI exhibited a marked reduction in higher-order, low-stoichiometry phosphoforms. Further evaluation of Myosin-binding protein C (MyBP-C) showed progressive loss of phosphorylation over the course of 24 hours. We submit that TCA-extracted heart standards in combination with Phos-tag gels can provide valuable quality control for the phosphorylation status of myofibril preparations, and that inclusion of a high-affinity PP2A and PP1 phosphatase inhibitor like okadaic acid may benefit future myofibril mechanics studies.

BackgroundDuchenne muscular dystrophy (DMD) cardiomyopathy is characterized by progressive microtubule remodeling, connexin-43 (Cx43) dysregulation, and ventricular arrhythmias. We previously demonstrated phospho-mimic knock-in of {beta}III-tubulin S172E preserves microtubule organization and attenuates cardiac pathology in mdx mice. However, whether these protective effects can be reproduced using a clinically relevant gene-delivery strategy remains unknown. Methods and ResultsWe generated a cardiomyocyte-specific adeno-associated virus serotype 9 (AAV9) vector expressing phospho-mimic {beta}III-tubulin (Tubb3-S172E) under the cardiac troponin T promoter and delivered it to 4-5-month-old wild-type and mdx mice. Cardiac Tubb3-S172E expression was confirmed by quantitative qPCR and immunoblotting. In mdx mice, AAV9-mediated Tubb3-S172E expression significantly reduced mononuclear inflammatory infiltration, restored Cx43 localization at intercalated discs, and attenuated isoproterenol-induced arrhythmia susceptibility. In contrast, cardiac fibrosis, Nav1.5 protein expression, and peak sodium current density were not significantly improved. Overexpression of wild-type {beta}III-tubulin in healthy hearts increased Cx43 lateralization and arrhythmia susceptibility, indicating that {beta}III-tubulin phosphorylation state rather than protein abundance determines its protective function. ConclusionsCardiomyocyte-targeted delivery of phospho-mimic {beta}III-tubulin partially recapitulates the protective effects observed in the genetic S172E knock-in model. These findings identify {beta}III-tubulin Ser172 phosphorylation as a critical regulator of microtubule-dependent electrical remodeling and support therapeutic modulation of this pathway in Duchenne muscular dystrophy cardiomyopathy. Research PerspectiveO_LICardiomyocyte-targeted AAV9 delivery of phospho-mimic aIII-tubulin improves Cx43 organization, inflammatory remodeling, and arrhythmia susceptibility in dystrophic hearts, demonstrating that therapeutic modulation of {beta}III-tubulin Ser172 phosphorylation partially recapitulates the protective effects observed in the genetic S172E model. C_LIO_LIThe dissociation between improved electrical remodeling and persistent Nav1.5 and fibrotic abnormalities suggests that {beta}III-tubulin Ser172 phosphorylation selectively regulates specific microtubule-dependent pathological pathways in dystrophic cardiomyopathy. C_LIO_LIFuture studies should define the molecular mechanisms linking {beta}III-tubulin Ser172 phosphorylation to cardiomyocyte-immune cell communication and determine how this pathway coordinates electrical and inflammatory remodeling in dystrophic hearts. C_LI

Background and AimsCardiac fibrosis is a major contributor to heart failure (HF), yet mechanisms limiting pathological fibroblast activation remain incompletely understood. We identified dysferlin (DYSF), a membrane repair protein, as highly induced in HF fibroblasts and investigated its role in regulating profibrotic responses. MethodsCardiac fibroblasts from patients with end-stage HF and unaffected donor hearts were analysed by liquid chromatography-tandem mass spectrometry and bulk RNA sequencing. Dysferlin expression was validated in independent cohorts. Selected gene/protein expression was validated using single-cell/single-nucleus RNA sequencing and multiplex immunofluorescence of human myocardium from dilated cardiomyopathy (DCM), ischaemic cardiomyopathy (ICM), acute myocardial infarction (AMI), and unaffected hearts. Functional studies were performed in human and mouse cardiac fibroblasts using siRNA-mediated silencing and TGF-{beta} stimulation, and in engineered human 3D cardiac microtissues. Fibrotic remodelling, autophagy, apoptosis, and contractile function were assessed by molecular, histological, biochemical and functional analyses. ResultsDysferlin abundance was markedly increased in HF fibroblasts. Across HF myocardium, DYSF was enriched in activated fibroblasts but largely excluded from COMP-enriched fibrotic regions, consistent with a role in restraining fibroblast state transitions. Although induced by TGF-{beta}, DYSF silencing enhanced extracellular matrix production, increased FOSL2 expression, and promoted differentiation into COMP-positive matrifibrocytes. In engineered human cardiac microtissues, DYSF silencing exacerbated fibrosis, increased apoptosis, and impaired contractility. Mechanistically, dysferlin restrained the TGF-{beta}-FOSL2-autophagy signalling axis, whereas FOSL2 suppressed DYSF expression, defining a reciprocal regulatory circuit. Silencing FOSL2 or MXRA5 increased dysferlin levels, while mRNA-protein discordance implicated S-acylation as a potential regulator of dysferlin protein abundance. ConclusionsDysferlin is a stress-inducible antifibrotic regulator that limits maladaptive fibroblast differentiation and myocardial fibrosis, thereby representing a potential therapeutic target to attenuate adverse cardiac remodelling in HF. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/745492v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@b52f7dorg.highwire.dtl.DTLVardef@140f781org.highwire.dtl.DTLVardef@3964f5org.highwire.dtl.DTLVardef@131404_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: HFpEF accounts for roughly half of heart failure admissions and lacks disease-modifying therapy. Autotaxin (ENPP2) generates lysophosphatidic acid (LPA), a profibrotic and pro-inflammatory bioactive lipid. Whether circulating lysophospholipid metabolism is altered in HFpEF, and whether autotaxin inhibition modifies an established experimental HFpEF phenotype, is untested. Methods: Plasma from patients with HFpEF (n=210) and non-heart-failure comparators (n=27) underwent untargeted and LPA-targeted mass spectrometry and a nine-analyte multiplex immunoassay. Male C57BL/6J mice received a high-fat diet plus L-NAME (0.85 g/L) or chow for 5 weeks; after phenotype confirmation, they received oral PF-8380 (30 mg/kg/day) or vehicle for 10 weeks. Endpoints were echocardiography, functional assessment, gravimetric studies, tail-cuff pressure, trichrome fibrosis, and flow cytometry of heart and spleen. Results: All nine analytes, including the autotaxin protein ENPP2, were higher in HFpEF than comparators. HFpEF plasma showed higher LPE O16:1, LPE O18:2, PS 38:4 and PC 36:4;O, and lower SM 39:2; O3 and PS 36:0. LPA 20:0 was 3.5-fold higher in both sexes, whereas LPA 18:2 was lower in women. Diet plus LNAME raised blood pressure, LV mass, and isovolumic relaxation time with preserved ejection fraction. PF-8380 reduced echocardiographic indices of diastolic dysfunction, fibrosis area, cardiomyocyte area, and cardiac CD11b+, CD64+, CD86+, and Ly6G+ frequencies, without altering fat or lean mass. Conclusion: In male mice with established two-hit HFpEF, autotaxin inhibition improved diastolic indices and reduced fibrosis, hypertrophy, and cardiac myeloid accumulation. Human data show altered lysophospholipid composition. Collectively, these findings nominate the autotaxin/LPA axis as a tractable therapeutic target and support further evaluation of autotaxin inhibition as a candidate disease-modifying strategy for HFpEF management.

Abstract Background: Most animal models of HCM are mouse-based, but the thin interventricular septum in mice makes it difficult to clearly distinguish pathological hypertrophy, which introduces substantial errors and constrains basic HCM research. Cats develop HCM spontaneously, and the common MYBPC3-A31P variant in cats is homologous to human mutations in both genetics and pathology, with a larger body size that makes them suitable as large-animal models. This study examines how heterozygosity or homozygosity for the p.A31P mutation (c.91G>C) in the MYBPC3 gene affects the phenotype and severity of HCM in affected cats, with the aim of establishing an ideal large-animal model for clinical risk stratification and precision diagnosis and treatment of human HCM. Methods: Forty-nine Maine Coon cats were enrolled and stratified into homozygous mutant (HOM, n=8), heterozygous mutant (HET, n=26), and wild-type (WT, n=15) groups. All cats underwent echocardiography, blood pressure measurement, physiological assessment, hematological and biochemical analyses, and cross-species sequence conservation analysis. Results: No significant differences in baseline characteristics including age and body weight were observed among groups (P>0.05). HOM cats exhibited significantly higher left ventricular outflow tract pressure gradients and greater basal septal thickness compared to WT cats (P<0.05), with HET cats showing intermediate values. Analysis of hematological and serum biochemical parameters revealed no evidence of systemic inflammation or hepatic injury. Sequence conservation analysis confirmed that the A31 residue is highly conserved across mammalian species. Conclusions: This study provides a phenotypic characterization of Maine Coon cats carrying the MYBPC3-A31P mutation, revealing marked gene-dose effects on cardiac structure and function, with homozygous individuals exhibiting more severe phenotypic features. This model serves as a large-animal translational platform that not only clarifies genotype-phenotype correlations but also supports risk stratification and precision therapeutic strategies in human HCM. Its spontaneous nature and genetic homology to human disease make it particularly valuable for bridging preclinical findings to clinical application.

Background: Atrial fibrillation (AF) burden is increasingly recognized as a determinant of clinical risk. Currently, AF burden can only be estimated using long-term rhythm monitoring. Bone morphogenetic protein 10 (BMP10) is a protein secreted from cardiac atria associated with AF and AF-related complications. This study evaluated whether BMP10 concentrations are associated with AF burden in a human atrial model: atrial engineered heart tissue (aEHT). Methods: Human induced pluripotent stem cell-derived atrial cardiomyocytes were cast into atrial engineered heart tissues (aEHTs). To mimic AF burden, mature aEHTs were optogenetically-paced at a high rate of 4 Hz, either intermittently for 4 hours every 2 days (~10% burden) or continuously for 24 hours per day (100% burden). After 18 days of high-rate pacing intervention, 7 days of recovery without pacing followed. BMP10 release was quantified by ELISA and contractile function was assessed by video analysis. EHT transcriptional remodeling in response to mimicked AF burden was assessed by RNA sequencing and the effect of recovery was analyzed by qPCR. Results: High-rate optogenetic pacing mimicking AF lead to a dynamic, burden-dependent BMP10 release: BMP10 concentrations in the medium were increased by intermittent optogenetic pacing (~10% burden) and highest under continuous optogenetic pacing (100% burden). BMP10 release declined toward control levels during recovery. Contractile dysfunction was most impaired after continuous pacing and showed only partial recovery within 7 days after pacing cessation. RNA sequencing revealed distinct burden-dependent transcriptional states. Pacing-regulated transcripts were related to BMP/TGF{beta} signaling, atrial identity, calcium handling, contractile phenotype, and electrophysiological remodeling. After recovery, BMP10 mRNA expression remained elevated despite normalization of BMP10 protein release. Conclusions: AF burden dynamically regulates BMP10 release and functional and molecular remodeling in human aEHTs. BMP10 release depicts a secreted protein-based readout of current or recent atrial high-rate stress, whereas persistent transcriptional changes indicate molecular memory of prior AF burden. These findings support BMP10 release as a burden-sensitive AF biomarker

Background: Cardiac fibrosis is a hallmark of many cardiovascular diseases, driven by sustained fibroblast activation and excessive extracellular matrix deposition, leading to myocardial stiffening and impaired contractility. Current therapies inadequately address this process. This study evaluated the antifibrotic potential of lanifibranor, a balanced pan-peroxisome proliferator-activated receptors (PPARs) agonist, in TGF-beta1-induced cardiac fibrosis. Methods: Human cardiac microtissues, along with 2D and 3D cardiac fibroblast and cardiomyocyte cultures, were used to assess cell viability, structure, metabolism, contractility, and gene expression. Results: Lanifibranor reduced TGF-beta1-induced fibrosis by limiting fibroblast activation and matrix deposition without affecting viability. In fibroblasts, these effects were associated with partial restoration of mitochondrial respiration and reduced focal adhesion maturation. In cardiac microtissues, lanifibranor improved contraction kinetics, decreased profibrotic transcriptional activity, and preserved bioenergetic homeostasis despite altered nucleotide balance. In cardiomyocytes, treatment normalized contractility and calcium handling while maintaining metabolic stability. Conclusions: Lanifibranor attenuates TGF-beta1-driven cardiac fibrosis by combining antifibrotic effects with metabolic and functional improvements in human models.

BackgroundHeart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome associated with metabolic stress, hypertension, systemic inflammation, and microvascular dysfunction. Early cell-type-specific events and intercellular communication programs that accompany disease onset and progression remain poorly defined. MethodsWe performed a longitudinal study of HFpEF progression in high-fat diet (HFD)+L-NAME mice at control/baseline (0 weeks, 0w/Ctrl), early (1w), intermediate (4w), and established (8w) stages. Metabolic, hemodynamic, exercise, echocardiographic, and single-cardiomyocyte function were assessed. Cardiac non-cardiomyocytes (non-CMs) were profiled by single-cell RNA sequencing (scRNA-seq), with bulk RNA-seq for tissue-level comparison. Endothelial remodeling was assessed in an L-NAME-independent HFD plus mild transverse aortic constriction model (HFD+mTAC) and a published human HFpEF single-nucleus RNA-seq cohort. An endothelial-macrophage adhesion assay tested whether HFpEF-mimic stress promotes endothelial activation and macrophage adhesion. ResultsIn the HFD+L-NAME model, metabolic dysfunction, hypertension, reduced exercise tolerance, abnormal diastolic filling with preserved ejection fraction, and altered cardiomyocyte calcium handling were detected by 1w and persisted through 8w. Bulk RNA-seq showed progressive remodeling, with limited change between 8w and 12w, guiding scRNA-seq timepoint selection. scRNA-seq of 94,848 cardiac non-CMs identified nine major populations with stage-dependent remodeling. Endothelial cells (ECs) were recovered in high proportion and showed an early, pronounced transcriptional response, with inflammatory, adhesion, interferon-response, migratory, and vascular-remodeling programs emerging by 1w. Related EC activation signatures were observed in HFD+mTAC and human HFpEF data. Functionally, HFpEF-mimic stress increased adhesion and chemokine expression in human ECs and enhanced macrophage adhesion. Fibroblast matrix remodeling occurred at later stages, while macrophages progressively shifted toward inflammatory states. CellChat suggested stage-dependent communication remodeling from early endothelial-immune interactions toward later macrophage-fibroblast crosstalk. ConclusionTime-resolved scRNA-seq reveals coordinated, stage-dependent remodeling of the cardiac microvascular and interstitial microenvironment during HFpEF progression. Early endothelial activation emerges before later fibroblast matrix remodeling and inflammatory macrophage remodeling, identifying candidate cell states and signaling pathways for future mechanistic investigation. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIThis study provides a time-resolved single-cell atlas of the cardiac non-cardiomyocyte compartment across baseline, early, intermediate, and established stages of HFpEF progression, rather than a single late-stage snapshot. C_LIO_LIEndothelial cells exhibit early inflammatory, adhesion, interferon-response, and vascular-remodeling programs within the first week of disease, preceding the later predominance of fibroblast matrix remodeling and inflammatory macrophage remodeling. C_LIO_LIThis endothelial activation signature is supported across two mechanistically distinct HFpEF mouse models and aligns with endothelial inflammatory and vascular-remodeling programs in human HFpEF myocardium, supporting its translational relevance. C_LI What Are the Clinical Implications?O_LIEarly endothelial activation may represent a targetable stage of HFpEF pathogenesis that arises before more established structural and fibrotic remodeling. C_LIO_LITherapeutic strategies aimed at limiting endothelial inflammatory activation or endothelial-immune interactions may help attenuate downstream vascular, immune, and stromal remodeling in HFpEF. C_LIO_LIThese findings provide a preclinical foundation for future longitudinal human studies testing whether early endothelial activation can serve as a biomarker, therapeutic target, or disease-staging feature in HFpEF. C_LI

Inositol-requiring enzyme 1a (IRE1a) is a canonical signaling factor in the unfolded protein response (UPR). In addition to this essential role (which prevents the accumulation of misfolded proteins in the endoplasmic reticulum), the endoribonuclease activity of IRE1a targets multiple mRNAs for degradation through a process called Regulated IRE1a-Dependent Decay (RIDD). The products of over 50 genes have been identified as RIDD targets; however, the biological significance of this process remains underexplored. Using publicly available datasets, we examined the fate of 27 well-characterized RIDD targets in the septal wall of heart failure patients, and in mice subject to pressure overload-induced heart failure. We show that decreased mRNA abundance from these RIDD substrate genes - an outcome consistent with RIDD induction - is commonly observed in heart failure.

Heart Failure with Preserved Ejection Fraction (HFpEF) is more prevalent in females and is associated with altered cardiac glucose metabolism. However, whether these metabolic alterations are conserved across sexes and between humans and widely used cardiometabolic mouse model of HFpEF remains unclear. We investigated species-, sex-, and ventricle-specific conserved and divergent features of HFpEF. Cardiometabolic HFpEF was induced in mice using the 'two-hit' model (high-fat diet + L-NAME), followed by assessment of cardiac function, RNA sequencing, and protein expression in the right (RV) and left (LV) ventricles. Published human HFpEF RV and LV RNA-seq datasets were reanalyzed and compared with our mouse data. Only male HFpEF mice recapitulated human phenotype of increased RV GLUT1 protein. In contrast, mouse GLUT1 was downregulated in RV of females and in the LV of both sexes, whereas GLUT4 protein remained unchanged. Cardiac PDK4 transcript and protein levels increased in the RV and LV of mice. Conversely, human PDK4 mRNA levels were reduced in the RV with HFpEF and unchanged in LV. Cardiac transcriptome analysis in mice revealed extensive alterations in LV, particularly in females, with enrichment of inflammatory pathways. Cross-species analysis demonstrated greater conservation of HFpEF-associated signatures in the RV than the LV. Furthermore, number of differentially expressed transcripts in human LV increased substantially after excluding patients with atrial fibrillation or diabetes. Overall, the RV of the 'two-hit' model more closely resembles human HFpEF. The cardiac transcriptome reflects sexual dimorphism, and conserved signatures are primarily associated with metabolic alteration, mitochondrial dysfunction, and cellular stress.

Living myocardial slices (LMS) are a highly relevant ex vivo model for investigating cardiac physiology and disease, as they preserve the native three-dimensional architecture, cellular diversity, and extracellular matrix of the heart. In addition, LMS enable longitudinal functional and molecular analyses. In this study, we established and compared two LMS culture approaches: an air-liquid interface system and a biomimetic culture system. We further examined how different slicing techniques affect tissue quality and longevity within the biomimetic setup. To develop a fibrosis model, LMS were stimulated with transforming growth factor-beta1 (TGF-beta1) and/or exposed to increased mechanical load. Tissue viability was assessed using LIVE/DEAD staining and the MTT assay, while cytotoxicity was evaluated with the LDH-Glo-TM Cytotoxicity assay. Contractile function was measured, and fibrotic remodelling was analysed using RT-qPCR, ELISA, and immunohistochemistry. Our results demonstrate that LMS cultured in the biomimetic system exhibit superior viability, structural integrity, and functional performance compared with those maintained at the air-liquid interface. Mouse LMS could be stably cultured for up to one week in the biomimetic system. Importantly, sample preparation, particularly the slicing method, had a significant impact on tissue quality and culture duration. While TGF-beta1 stimulation alone did not consistently induce fibrosis, combining TGF-beta1 treatment with increased mechanical load led to more pronounced fibrotic remodelling in LMS. These findings highlight the importance of biomechanical cues in modelling cardiac fibrosis ex vivo and support the biomimetic system as a robust platform for functional and disease-relevant studies.

Fatty acid oxidation (FAO) is a major energy source in the adult heart, and disruption of cardiac metabolism is closely associated with heart failure. Here, we investigated the effects of cardiac-specific overexpression of the H19X-encoded miR-424(322)/-503 cluster using an inducible transgenic mouse model. Sustained miR-424(322)/-503 overexpression caused rapid metabolic and functional deterioration, with early impairment of fatty acid oxidation. Short-term induction reduced FAO activity and downregulated genes involved in lipid metabolism, while glycolytic enzyme activity remained largely unchanged. Continued miR-424(322)/-503 expression subsequently led to severe dilated cardiomyopathy characterized by ventricular dilation, wall thinning, fibrosis, reduced contractility, and high mortality. Importantly, disease progression was dependent on the level and duration of miR-424(322)/-503 expression, as intermittent or lower-dose induction delayed cardiac dysfunction and prolonged survival. Withdrawal of miR-424(322)/-503 expression after the onset of dysfunction promoted substantial functional recovery. Together, these findings identify miR-424(322)/-503 as a potent regulator of cardiac metabolic reprogramming that disrupts fatty acid metabolism and drives progressive heart failure.

Transverse (t)-tubules enable rapid, synchronous Ca release required for efficient cardiac contraction by bringing L-type Ca channels into close apposition with ryanodine receptors. In heart failure with reduced ejection fraction (HFrEF), t-tubule disorganisation and loss occur alongside cardiac microtubule remodelling, contributing to impaired Ca handling and contractile dysfunction. Despite their canonical function in contraction, how t-tubules develop is unknown. Microtubules support delivery of L-type Ca channels to t-tubules via Amphiphysin-II/BIN1, yet whether microtubules directly regulate t-tubule formation and maintenance is unclear. Here, we investigated a role for microtubules in t-tubule development and homeostasis. Neonatal rat ventricular myocytes (NRVMs), which lack endogenous t-tubules, were used as a reductionist model in which BIN1 overexpression induces nascent membrane tubules. Microtubule depolymerisation with nocodazole before BIN1 overexpression impaired BIN1-driven tubule formation, reducing tubule density and length. Dynein inhibition with EHNA produced similar effects, indicating a requirement for microtubule-based motor activity during tubule elongation. Knockdown of the microtubule +TIP tracking protein CLIP-170 also reduced BIN1-driven tubule density, implicating BIN1-CLIP-170-dependent microtubule capture in tubule initiation. Microtubules were also required to maintain existing tubules. In NRVMs with established BIN1-driven tubules, microtubule depolymerisation, microtubule stabilisation or dynein inhibition each reduced tubule density and length. Consistent with this, acute microtubule depolymerisation or stabilisation disrupted native t-tubule networks in isolated adult sheep left atrial myocytes. Together, these findings identify cardiac microtubules as active regulators of t-tubule architecture. We propose that BIN1-dependent tubule formation requires CLIP-170-mediated microtubule plus-end capture and dynein-dependent elongation, while ongoing microtubule dynamics are necessary to preserve mature t-tubule structure.

Mitochondrial metabolism undergoes dramatic reprogramming during postnatal cardiac maturation, yet the enzymatic regulators that ensure continuity of TCA cycle flux in this period remain incompletely defined. FAHD1 is a mitochondrial oxaloacetate decarboxylase (ODx) with proposed roles in modulating the activity of Complex II of the electron transport chain (ETC), but its physiological relevance in vivo has remained unclear. Here, we identify FAHD1 as a critical regulator of mitochondrial function with strong impact on cardiomyocyte (CM) maturation. Using a germline Fahd1-knockout (KO) mouse model, we show that Fahd1 deficiency impairs Complex II respiration, reduces pyruvate levels, and induces a compensatory metabolic shift toward glycolysis and anabolic biosynthesis. Loss of FAHD1 disrupts sarcomere organization, delays the fetal-to-adult myosin isoform switch, and leads to left ventricle systolic dysfunction and cardiomyocyte hypertrophy. These findings highlight FAHD1 as a mitochondrial gatekeeper and potential target for modulating cardiac development and disease.

BACKGROUND: Increased left ventricular mass (LVM) at a single time point after heart transplantation (HT) predicts future adverse outcomes. However, dynamic changes in LVM could have better biological relevance and reflect adverse graft remodeling (AGR). The prognostic significance of such serial changes has not been studied. METHODS: Using an automated, electronic health record-based institutional data infrastructure, we studied 439 HT recipients with 5,563 LVM measurements. Separate Bayesian joint models estimated the simultaneous associations of current LVM and its instantaneous rate of change with graft dysfunction (GD) and mortality. A joint-model-derived remodeling score combining patient-specific deviations in LVM and slope was dichotomized to define AGR and non-AGR groups. A mixed-effects analysis of all clinical variables was performed to assess associations with LVM both between and within patients. An independent cohort of 35 patients with 79 surveillance-biopsy RNA-sequencing samples was used to examine early stress-responsive pathways associated with the remodeling score. RESULTS: LVM declined by approximately 7 g/year after transplantation, with regression attenuating over time. Sixty patients (13.7%) had GD, and 75 (17.1%) died. Higher LVM was associated with subsequent GD (hazard ratio [HR] per 10 g, 1.14; 95% credible interval [CrI], 1.02-1.28) and mortality (HR, 1.10; 95% CrI, 1.02-1.19). A more positive LVM slope was associated with GD (HR per 1 g/year, 1.21; 95% CrI, 1.06-1.42) and with cardiac allograft vasculopathy (CAV) grade 2 or 3 (HR, 1.39; 95% Crl, 1.02-1.96). LVM regressed more slowly in the AGR group (-5.8 vs -8.4 g/year), with higher GD (21.0% vs 6.4%) and mortality (24.2% vs 10.0%). Time-updated GD was associated with subsequent death (HR, 8.12; 95% Confidence Interval [CI], 4.67-14.14). Transcriptomic analysis showed enrichment of interferon-mediated signaling and vascular endothelial activation with higher remodeling scores, whereas lower scores were associated with mitochondrial and metabolic processes, ribosome biogenesis, and pathways related to tissue repair and stress responses. CONCLUSIONS: AGR is an easily accessible imaging biomarker that reflects the changes in the allograft in response to various stressors and predicts future adverse outcomes. Discovery of molecular mechanisms of AGR could lead to novel therapies to protect the allograft from chronic rejection.

Background: The His-Purkinje network drives rapid ventricular activation and is a major substrate for ventricular arrhythmias, yet it is among the least molecularly characterized cardiac compartments. Markers validated in rodents transfer poorly across species, few are confirmed at the protein level in large mammals or humans, and most lack the stability and surface accessibility that demanding applications require. Methods: We combined histology-guided laser-capture microdissection with low-input, cell-type-resolved RNA-sequencing to profile Purkinje fibers, left-ventricular cardiomyocytes and peri-Purkinje stroma from adult sheep. Differentially expressed genes were ranked by a transparent composite framework weighting expression specificity, cross-individual stability and predicted subcellular accessibility; leading candidates were validated by RT-qPCR and immunolabelling in sheep and by RT-qPCR in human myocardium. Results: RNA-sequencing resolved a Purkinje transcriptome distinct from cardiomyocytes and stroma and defined 331 concordantly enriched genes, which the composite framework ranked into stable, specific candidates spanning intracellular and cell-surface compartments. By RT-qPCR, the canonical conduction markers connexin-40/GJA5, HCN4, NEFM and MYL4 were strongly enriched in Purkinje fibers, whereas the rodent gold-standard contactin-2 was not, underscoring species divergence. Thirteen of sixteen prioritized candidates were confirmed by RT-qPCR, and immunolabelling localized MYL4, CNN1, TAGLN and DKK3 to Purkinje fibers; contactin-5 emerged as a novel transcript- and protein-validated Purkinje marker. In human myocardium, a defined subset - MYL4, connexin-40/GJA5, contactin-5 and TAGLN - was conserved, while several markers proved species-restricted. Conclusions: We provide the first genome-wide, cell-type-resolved molecular portrait of the Purkinje fiber in a large-animal model and a generalizable strategy that selects markers for specificity, stability and accessibility. The resulting resource - including the cross-species marker contactin-5 and compartment-matched candidates - supplies validated tools to identify, isolate and target Purkinje cells and demonstrates the necessity of cross-species validation.

RationaleHypertrophic cardiomyopathy (HCM) is a common inherited cardiac disorder characterized by cardiac hypertrophy, fibrosis, arrhythmias, and sudden cardiac death (SCD). Although current therapies primarily target sarcomere dysfunction, the contribution of epigenetic dysregulation to HCM pathogenesis and its therapeutic potential remain poorly understood. ObjectiveTo determine whether pharmacological inhibition of histone lysine demethylases (KDMs) with JIB-04 can prevent or reverse HCM progression and to identify the underlying epigenetic mechanisms. Methods and ResultsWe evaluated the pan-KDM inhibitor JIB-04 in Myh6R403Q/+ mice carrying the murine equivalent of the pathogenic human MYH7 R403Q mutation. JIB-04 prevented disease progression, reduced cardiac hypertrophy and fibrosis, preserved cardiac function, and completely prevented SCD in cyclosporin A- accelerated HCM. JIB-04 also reversed established disease, produced sustained therapeutic benefits after drug withdrawal, and improved cardiac function in aged mice with spontaneous HCM. Bulk RNA sequencing and ATAC-seq demonstrated partial restoration of disease-associated transcriptional programs and chromatin accessibility. Proteomic analyses identified PHF2 (KDM7C) as a candidate target of JIB-04 in both mouse and human HCM hearts. PHF2 knockdown suppressed hypertrophic, inflammatory, and fibrotic gene expression in cardiomyocytes, macrophages, and fibroblasts, respectively. Human HCM hearts exhibited increased expression of multiple JIB-04-sensitive KDMs, including PHF2. In MYH7 R403Q induced pluripotent stem cell- derived cardiomyocytes, JIB-04 normalized disease-associated gene expression, restored connexin-43 membrane localization, and improved mitochondrial respiration. Although prolonged treatment induced reversible hepatomegaly with hepatic lipid accumulation, co-administration of the antioxidant N-acetylcysteine mitigated liver toxicity while preserving the therapeutic efficacy of JIB-04. ConclusionsPharmacological KDM inhibition prevents and reverses HCM through epigenetic remodeling of disease-associated transcriptional and chromatin programs. These findings identify KDM inhibition as a promising therapeutic strategy for HCM, establish PHF2 as a candidate mediator of disease pathogenesis, and support further development of KDM-targeted therapies.

Background and AimsMutations in the desmin (DES) gene cause a variety of cardiomyopathies associated with arrhythmias, yet the electrophysiological consequences of these variants remain largely uncharacterized. The aim of this study was to investigate the pathogenic mechanisms of the de novo DES p.R406W variant, which was identified in a 9-year-old patient who suffered from severe ventricular arrhythmias and sudden cardiac death without overt structural heart disease. MethodsHuman induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the DES p.R406W variant (including the patients line) were compared to isogenic controls. Action potentials (AP) of hiPSC-CMs were recorded using patch-clamp. Furthermore, 3D engineered heart tissues (EHTs) were generated from hiPSC-CMs and their APs were recorded with sharp microelectrodes. Analytical techniques also included transmission electron microscopy (TEM) and integrated transcriptomic and proteomic profiling. Finally, a heterozygous knock-in (KI) mouse model carrying the Des p.R405W ortholog was evaluated through surface ECG, echocardiography and ex vivo cardiac optical mapping. ResultsThe DES p.R406W mutation prolonged AP duration in IM-R406W hiPSC-CMs and EHTs vs Control ones. Multi-omics analysis of EHTs revealed a dysregulation of genes and proteins involved in contractile function, cell adhesion, and electrical activity. TEM imaging revealed changes in Z-disc architecture in mutant tissues. Twenty-week-old Des p.R405W KI mice exhibited ventricular conduction slowing (prolonged QRS) and a high susceptibility to ventricular tachyarrhythmias, likely due to reentrant mechanisms. Mild hypertrophy was also observed, but only in females. ConclusionThe DES p.R406W variant is highly pathogenic, causing electrical and structural remodeling of the myocardium. This study highlights the effectiveness of hiPSC-CMs and EHTs in recapitulating the clinical phenotype of desminopathy, providing a platform for investigating the mechanisms of early-onset cardiac arrhythmias and SCD.

A pair of paralogs, Chchd3 and Chchd6, two components of mitochondrial contact site and cristae organizing system (MICOS), have been identified to be candidate pathogenetic genes in congenital heart disease (CHD). Previous research found that knockdown (KD) of the single Chchd3/6 (Chchd3) gene and other MICOS components in Drosophila impaired heart function, likely due to a deficit in mitochondrial organization, ATP production, actomyosin levels, and thus severely diminished contractility. However, the underlying mechanisms of how MICOS deficiency leads to these defects are not clear. Here, we performed genetic manipulations in the Drosophila heart to probe for possible interactions between MICOS-compromised mitochondria and other organelles and processes. We found that moderate reduction in Pink1/parkin-mediated mitophagy synergistically aggravated cardiac Chchd3 KD phenotypes, indicating a major interaction. Further, Chchd3 KD increased the level of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. Interestingly, KD of catalase (CAT) also elevated cardiac ROS levels, but surprisingly did not compromise contractility either by itself or in combination with Chchd3 KD to aggravate the cardiac phenotype. However, CAT overexpression (OE) in Chchd3 KD hearts restored contractility, but only partially, even though elevated ROS due to Chchd3 KD was fully normalized. Similarly, counteracting ER stress by overexpressing Xbp1 (or spliced mouse Xbp1) also partially rescued the heart function defects induced by Chchd3 KD. Overall, these data indicate a critical role of mitophagy and ER/oxidative stress in cardiac homeostasis involving Chchd3, which suggests that deficiency of MICOS function contributes to heart dysfunction via multiple stress responsive pathways.