Background:Differential growth between the left (LV) and right ventricles (RV) is a cornerstone of normal heart morphogenesis after birth, leading to the relatively larger and dominant LV over RV in the adult heart regarding size and function. Yet, little is known about the factors that regulate this chamber-specific growth. Methods:We used both loss-and gain-of-function mouse models, achieved through genetic or pharmacological manipulation of IRE1α or Xbp1 in cardiomyocytes. We also used primary cultured neonatal cardiomyocytes to explore the roles of IRE1α, spliced Xbp1 (sXbp1: activated form), and newly identified sXbp1 downstream targets. In addition, we generated heart-specific mosaic mutant mouse models using CRISPR/Cas9/AAV9-based somatic mutagenesis to elucidate the roles of sXbp1 downstream targets in cardiomyocytes. Results:Pharmacological inactivation of IRE1α and genetic depletion of Xbp1 resulted in a smaller LV size, due to decreased cardiomyocyte proliferation and hypertrophic growth, as well as increased cardiomyocyte death. These effects were not observed in the RV. Cardiomyocyte-specific induction of IRE1α or sXbp1 led to increased ventricular size in both ventricles, through enhanced cardiomyocyte proliferation and hypertrophic growth in both LV and RV, and reduced apoptosis in the RV. We identified two ER resident transmembrane proteins, Vimp and Rpn2, as direct binding partners of sXbp1 in targeted gene regulation at the chromatin level. CRISPR/Cas9/AAV9-based somatic mutagenesis mouse models for Vimp and Rpn2 revealed that both genes regulate cardiomyocyte proliferation, hypertrophic growth, and death. We also observed accumulated misfolded proteins in these two mutant hearts. Conclusions We demonstrate that the IRE1α-Xbp1-Vimp/Rpn2 axis regulates differential ventricular size between LV and RV during postnatal development by orchestrating cardiomyocyte proliferation, hypertrophic growth, and death through regulating protein homeostasis. Clinical Perspective:What Is New: IRE1α-Xbp1 axis is dominantly activated in the LV cardiomyocyte during the postnatal period in mouse heart.IRE1α-Xbp1 mediated ER stress signaling increases cardiomyocyte proliferation and hypertrophic growth and decreases apoptosis in the postnatal period.Activated Xbp1 directly regulates LV-specific cardiomyocyte protein homeostasis via interaction with ER membrane targeted Vimp and Rpn2.What Are the Clinical Implications?: Differential heart growth patterns between the LV and RV are critical for normal morphogenesis and function of each ventricle.Control of protein homeostasis by modulating ER stress signaling could be a potential therapeutic approach for single-chamber heart diseases.

Heart failure with preserved ejection fraction (HFpEF) and metabolic dysfunction-associated steatotic liver disease (MASLD) are increasingly prevalent, interrelated conditions driven by the global rise in obesity and metabolic syndrome. Once viewed in isolation, HFpEF and MASLD are now recognized as organ-specific manifestations of shared systemic metabolic dysfunction. Evidence from the past decade highlights not only overlapping risk factors but also a dynamic, bidirectional inter-organ crosstalk between the liver and the heart that shapes their natural history. In this Review, we explore the epidemiological and mechanistic basis of the MASLD-HFpEF connection, focusing on shared metabolic drivers such as lipotoxicity, meta-inflammation and oxidative stress. We also discuss emerging liver-derived mediators, including hepatokines, metabolites and extracellular vesicles, that influence cardiac structure and function. Finally, we highlight diagnostic and therapeutic strategies relevant to both conditions and propose a multiorgan framework to improve their clinical recognition and management. Understanding the liver-heart axis is key to rethinking cardiometabolic disease beyond organ silos and towards more integrated, mechanism-based approaches.

BACKGROUND:Metabolic dysfunction-associated steatohepatitis (MASH) has become a major global health burden yet effective pharmacological treatments remain scarce. Lipotoxicity is one of the central drivers of MASH, but the molecular regulators that control lipid metabolism homeostasis in MASH remain incompletely defined. OBJECTIVE:To discover and characterise key regulators of hepatic lipotoxicity that drive MASH progression and to delineate their mechanistic and therapeutic relevance. DESIGN:We integrated multiple RNA-seq datasets from murine and human MASH to identify candidate genes linked to disease-associated metabolic signatures. Functional and mechanistic studies were performed using primary hepatocytes, hepatocyte-specific transgenic and knockout mice and an AAV8-based post-onset knockdown model. Protein interactions and subcellular dynamics were evaluated using co-immunoprecipitation-mass spectrometry, structural modelling and colocalisation analysis. The small molecule of monooxygenase DBH like 1 (MOXD1) inhibitor candidates was screened by the artificial intelligence (AI) model, and their anti-MASH capacity was evaluated in vitro and in vivo. RESULTS:We identified MOXD1 as a previously unrecognised gene tightly associated with MASH transcriptional programmes. Hepatocyte MOXD1 significantly exacerbated MASH phenotypes. Mechanistically, MOXD1 directly interacted with the ACOX1-PEX5 translocation complex, promoting ACOX1 trafficking to peroxisomes to block lipolysis, lipophagy. We further identified four key MOXD1 residues required for ACOX1 binding and resultant pro-MASH capacity. Importantly, based on the AI model and interacting details of MOXD1-ACOX1, we identified a small molecule rM15 that directly binds to MOXD1 and blocks its interaction with ACOX1. Notably, rM15 robustly protected against hepatocyte lipid accumulation and suppressed diet-induced MASH progression in vivo. CONCLUSION:This study identifies MOXD1 as a previously unrecognised regulator of hepatic fatty-acid homeostasis and a key driver of MASH pathogenesis. Targeting the MOXD1-ACOX1 axis offers a promising therapeutic strategy for MASH.

Stem-cell-based cardiac repair holds promise for the infarcted myocardium, yet the in vivo molecular behavior of transplanted cells is poorly understood. Using time series spatial transcriptomics, we profiled human pluripotent stem-cell-derived cardiovascular progenitors engrafted into a pig model. We show that the engrafted cardiovascular progenitors progressively upregulated genes associated with cardiac maturation, oxidative metabolism, calcium handling and fibrosis resolution. Cell-cell communication analysis identified Midkine (MDK), secreted by the human xenograft, as a key regulator of host neovascularization. We validated this using immunohistochemistry, lentiviral MDK overexpression and functional assays that demonstrated enhanced endothelial cell migration and increased CD31+ vascular density in vivo. A publicly available interactive Shiny atlas of spatial and temporal transcriptomic data from myocardial infarction pig hearts with human xenografts is provided. These findings advance mechanistic understanding of stem-cell-mediated cardiac repair and identify MDK as a tractable target for therapeutic angiogenesis in ischemic heart disease.

Cardiac hypertrophy is associated with an increased risk of mortality, largely due to growth of myocytes in response to pathological stimulation. Post-transcriptional regulation plays an important role in maintaining cell homeostasis during pathological remodeling, such as modification, alternative splicing, and degradation. While most of current literature explores the role of transcriptional regulation during cardiac hypertrophic response, the role of targeted mRNA degradation remains unknown. Using a BRIC-Seq (5’bromo-uridine immunoprecipitation-chase deep-sequencing) in normal and hypertrophic cardiomyocytes we found a global shift in RNA stability. In addition, GO analysis have identified that the mRNA with altered half-life is involved in cardiac hypertrophy, inflammation and metabolic processes without any impact on classic nonsense mediated decay (NMD) targets, suggesting a previously uncharacterized cardiac hypertrophic stress induced transcriptome remodeling at the level of mRNA degradation. Among the known factors involved in mRNA degradation, we found only Upf1 (Up-frameshift protein 1), but not other Upf family members, is significantly induced in hypertrophic cardiomyocytes and failing mouse hearts, suggesting the changes in RNA stability during hypertrophy may be an Upf1-dependent but NMD-independent mechanism. Using cultured cardiomyocytes, we have identified that loss of Upf1 expression led to cardiac hypertrophy while restoring expression of Upf1 protected phenylephrine induced cardiac hypertrophy based on marker gene expression. In vivo, we further demonstrated loss of Upf1 exacerbates myocardial infarction induced cardiac pathogenesis using AAV9 mediated Upf1 inactivation, suggesting that Upf1 mediated mRNA stability regulation plays an important role in cardiac pathological remodeling. Mechanistically, we have validated that Upf1 interacts directly with RBFox1, a cardiac enriched RNA binding protein, using targeted co-IP analysis and proximity ligation assay, and this interaction is disrupted upon hypertrophy stimulation. Lastly, we found that RBFox1 could provide target specificity in this novel Upf1 dependent, yet NMD independent RNA decay during cardiac hypertrophy. In summary, we have identified a global shift of mRNA stability in stressed myocytes regulated by Upf1-RBFox1 complex, targeted manipulation of the stress regulated mRNA stability could potentially provide new therapeutic targets for cardiac disease.

Introduction: Cardiomyocyte maturation is a postnatal heart development that requires precise molecular, metabolic, structural, and electrophysiological changes to sustain the increased workload of the adult heart. Previous studies demonstrate that alternative RNA splicing is a key regulatory mechanism during this process. However, the full landscape of isoform switch and its role in functional specialization in adult cardiomyocyte remain largely unexplored. This study aims to provide a comprehensive landscape of isoform utilization during heart maturation. Methods: Using PacBio Iso-Seq, we profiled full-length transcriptomic changes in mouse left ventricles across neonatal to postnatal developmental stages. Isoform expression and usage ratios were analyzed to identify the genes with significant isoform switch. Functional validation of selected isoforms was performed in hESC-CMs. Results: While the total number of genes expressed remains relatively stable, the total number of full-length transcripts decreased from E18 to P49, indicating a progressive reduction in transcriptomic complexity at the isoform level. PCA analysis of isoform-specific expression and ratios shows stage-dependent clustering, highlighting that cardiac isoform dynamics is a defining feature for postnatal heart maturation. We identified at least 50 genes showing significant isoform switches across the maturation stages. In addition, an isoform of PDLIM5 is found to have exon 5B inclusion (PDLIM5-Ex5B) and its expression is increased in adult mouse heart but reversed to neonatal level in the mouse failing heart (n=3, p<0.05 compared to P49 and Sham control). hESC-CMs expressing PDLIM5-Ex5B improved sarcomere organization, enhanced binucleation, and increased expression of maturation markers. In contrast, isoforms lacking exon 5B were less effective in promoting cardiomyocyte maturation features. These findings further confirm that alternative splicing-mediated isoform switch is critical for cardiomyocyte structural and functional maturation. Conclusions: This study provides a comprehensive landscape of RNA splicing and isoform switching events during heart maturation, highlighting their importance in defining postnatal heart maturation stages and achieving functional specialization. Our results serve as a valuable resource for exploring maturation-related RNA splicing regulators and may represent a potential splicing-targeted therapy for heart diseases.

AIMS:Alternative mRNA splicing is a significant part of transcriptome reprogramming during the pathological manifestation of heart diseases. Earlier studies have identified a muscle-specific isoform of RBFox1 (RNA binding fox-1 homolog 1) to be a key RNA splicing regulator in pressure overload induced heart failure. However, the physiological impact of RBFox1 in myocardial infarction (MI), and the downstream mRNA alternative splicing events during MI induced cardiac remodelling remains unknown. METHODS AND RESULTS:Here we found RBFox1 expression was significantly decreased in Sprague-Dawley rat hearts post MI. Restoring the expression of RBFox1 prevented cardiac remodelling and dysfunction post MI characterized by improved cardiac function, reduced hypertrophy and fibrosis, associated with attenuated induction of cardiac stress marker genes. In cultured cardiomyocytes, expression of RBFox1 was sufficient to prevent hypoxia induced cell death measured by TUNEL staining and cleaved caspase 3, while inactivation of RBFox1 aggravated cardiac cell death. Mechanistically, we identified RBFox1 expression affected a broad spectrum of gene expression in post-MI hearts. In addition, a hypoxia-sensitive alternative splicing variant of Mbnl1 (Muscleblind-like 1) mRNA was identified to be regulated by RBFox1, resulting in the expression of a cell death related Mbnl1 isoform with 12 amino-acid deletion at the C-terminus (Mbnl1-ΔExon7). Strikingly, the selective inhibition of Mbnl1 Exon7 inclusion using anti-sense oligo protected the heart from myocardial infarction induced injury in vivo. CONCLUSION:In summary, we have established a cardio-protective role of RBFox1 in myocardial infarction induced cardiac remodelling and dysfunction. Restoration of RBFox1 expression, and targeted modulation of its downstream alternative splicing target Mbnl1, is a potential therapeutic approach for cardiac dysfunction and remodelling in MI injured heart.

Cardiomyocyte postnatal maturation is a critical step of the mammalian heart development continuum, involving a myriad of phenotypic changes at morphological, molecular, and functional levels. While the phenotypic hallmarks of cardiac maturation are well characterized, the molecular mechanisms that govern this maturation process are still poorly defined. This review aims to explore the recent findings on how post-transcriptional regulations orchestrate the fetal-to-adult cardiomyocyte transition and to highlight their clinical implications for cardiac diseases and regeneration medicine. The molecular regulations of cardiac maturation are distinct from the gene regulatory network implicated in embryonic stages of cardiac development. RNA alternative splicing and the resulting isoform switching events are significant part of the post-transcriptional reprogramming during the transitional stage of maturation, driving functional refinement through a network of RNA-binding proteins. Cardiomyocytes undergo significant changes in structure, physiology, metabolic activity, and proliferative capacities during fetal to adult maturation. Recent findings highlight the importance of post-transcriptional regulation in this process, in particular RNA alternative splicing and isoform switch. Understanding the post-transcriptional regulatory mechanisms, including key molecular players that contribute to the fetal-to-adult transition, can provide a new conceptual framework for cardiac development, diseases, and regenerative medicine.

Metabolic dysfunction-associated steatohepatitis (MASH) is a globally prevalent but intractable disease lacking effective pharmacotherapies. Here, we performed an integrated multilayered screening for pathogenic genes and druggable targets for MASH. We identified the subclass of metabolite-sensing G protein-coupled receptors, specifically GPR31, a critical contributor to MASH occurrence, which, to our knowledge, was previously uncharacterized. Mechanistically, Gαi3 is the essential downstream effector for the pro-MASH efficiency of GPR31 via glycosylation-dependent interaction with GPR31 and extra activation of PKCδ-MAPK signaling. Hepatocyte-specific GPR31 deficiency robustly blocked hepatic lipotoxicity and fibrosis in a mouse model of diet-induced MASH, whereas expression of the GPR31 transgene aggravated MASH development. Of translational importance, we developed a small-molecule inhibitor, named G4451, that specifically inhibits the GPR31-Gαi3 interaction by targeting the GPR31 conformational transition. Encouragingly, oral administration of G4451 effectively blocked MASH progression in preclinical models in both rodents and nonhuman primates. Collectively, the present study provides proof of concept that interference with GPR31 constitutes an attractive therapeutic strategy for MASH.

Previous single-cell and single-nucleus heart atlases, often limited by small sample sizes, lack the statistical power needed for phenotype association analysis, particularly for cardiovascular diseases and cardiac aging. To address this, we integrated data from 436 samples across 12 single-cell studies, harmonized the corresponding sample metadata, and constructed a comprehensive heart atlas comprising 355,762 cells and 1,436,719 nuclei. Consensus annotation identified 10 broad cell types and 54 fine-grained subsets. Associating gene expression patterns and cell type proportions with phenotypic data, we identified NRG1 -expressing endocardial cells linked to multiple cardiac diseases and found that interferon (IFN) response signatures mark aging in multiple heart cell types. Importantly, we also developed PopComm, a novel computational method for inferring ligand–receptor (LR) interactions from population-scale single-cell data and quantifying interaction strength for individual samples. Using PopComm, we revealed a close association between the IFN response state and altered cell–cell communication during cardiac aging.

Post-injury remodeling is a complex process involving temporal specific cellular interactions in the injured tissue where the resident fibroblasts play multiple roles. Here, we performed single-cell and spatial transcriptome analysis in human and mouse infarcted hearts to dissect the molecular basis of these interactions. We identified a unique fibroblast subset with high CD248 expression, strongly associated with extracellular matrix remodeling. Genetic Cd248 deletion in fibroblasts mitigated cardiac fibrosis and dysfunction following ischemia/reperfusion. Mechanistically, CD248 stabilizes type I transforming growth factor beta receptor and thus upregulates fibroblast ACKR3 expression, leading to enhanced T cell retention. This CD248-mediated fibroblast–T cell interaction is required to sustain fibroblast activation and scar expansion. Disrupting this interaction using monoclonal antibody or chimeric antigen receptor T cell reduces T cell infiltration and consequently ameliorates cardiac fibrosis and dysfunction. Our findings reveal a CD248+ fibroblast subpopulation as a key regulator of immune–fibroblast cross–talk and a potential therapy to treat tissue fibrosis. Using single-cell and spatial molecular profiling of infarcted mouse and human hearts, Li, Ni, Wang and colleagues identify a subset of cardiac fibroblasts expressing CD248 that plays a critical role in fibroblast–T cell interaction, and show that disrupting this interaction with monoclonal antibodies or anti-CD248 chimeric antigen receptor T cells results in reduced cardiac fibrosis and improved function.

Introduction: Heart Failure with Preserved Ejection Fraction (HFpEF) is a rising unmet medical need with limited effective treatment. Chronically elevated catecholamine level is a hallmark of metabolic disorders and heart failure. However, the role of catecholamine in the pathogenesis of HFpEF remains unknown. We have previously identified Glutamyl-prolyl-tRNA Synthetase 1(EPRS) as a gene significantly involved in adrenal gland growth post chronic isoproterenol treatment and a novel regulator for catecholamine synthesis through regulating key enzymes expression at post-transcriptional level through its noncanonical function. Methods: High fat diet (HFD) and N [W] -nitro-1-arginine methyl ester (L-NAME) were utilized to induce HFpEF in C57B/L mice and the catecholamine synthesizing protein expression changes in adrenal gland were measured. An adrenal gland specific EPRS knockout mouse model was established through crossing EPRSfl/fl with PNMT-Cre (EPRS-aKO) and subjected with L-NAME/HFD induced HFpEF. Cardiac contractile and diastolic functions were determined. Metabolic analysis including glucose tolerance and insulin tolerance tests were exploited to examine the metabolic impact of ERPS inactivation in adrenal gland. Masson Trichrome staining was performed to determine cardiac fibrosis. Exercise endurance test and protein nitrosative stress levels were tested. Results: After 14 weeks of L-NAME/HFD challenge, we observed a significant induction of EPRS as well as catecholamine synthesizing enzymes protein expression in adrenal gland. EPRS-aKO mice were protected from obesity and diabetes compared to their control littermates based on both glucose tolerance and insulin tolerance tests. Further, EPRS-aKO mice were protected from L-NAME/HFD induced cardiac dysfunction based on improved left ventricular diastolic function and decreased expression of pathological markers as well as improved exercise endurance level. EPRS inhibition decreased cardiac hypertrophic response based on decreased left ventricle weight/tibia length ratio. Inactivation of EPRS in adrenal gland protected HFpEF associated protein nitrosative stress based on protein S-nitrosylation analysis. Conclusion: Our results demonstrate that EPRS plays an important role in HFpEF pathogenesis through regulation of adrenal-heart crosstalk. Targeting of the newly identified EPRS- adrenal- heart crosstalk may serve as a basis for novel therapies for HFpEF.

Rationale: Heart failure with preserved ejection fraction (HFpEF) is a major unmet medical need with limited effective treatments. A significant contributing factor to HFpEF, a multifactorial disease, is underlying metabolic dysfunction. While much of the prior research has been on glucose and fatty acid metabolic defects in the pathogenesis of HFpEF, other metabolic activities remain under investigated. Methods: System-based metabolomics and targeted mass spectrometry were employed to analyze serum and tissue samples from a deep-phenotyped human HFpEF cohort. A preclinical mouse model of HFpEF was developed by combined administration of a high-fat diet (HFD) and the nitric oxide (NO) synthase inhibitor N[w]-nitro-l-arginine methyl ester (L-NAME). The branched-chain amino acid (BCAA) catabolic activities were enhanced by genetic inactivation of branched-chain ketoacid-dehydrogenase kinase (BCKDK) or treatment with BT2 (3,6-dichlorobenzo[b]thiophene-2-carboxylic acid), a highly selective inhibitor of BCKDK. Cardiac function, myocardial remodeling and insulin signaling in the left ventricle were assessed across all experimental cohorts. Results: The systems-based metabolomics analysis of the deep-phenotyped HFpEF and non-HFpEF patients revealed that abnormal circulating BCAA levels were significantly associated with adverse outcomes. In the rodent model of HFpEF, significant impairment of BCAA catabolic activities in the heart and abnormal circulating BCAA levels were also observed. In adult mice, inducible knockout of BCKDK, the rate-limiting negative regulator of BCAA catabolic flux, markedly augmented BCAA catabolic activities. Compared with the controls, BCKDK inactivation blunted diastolic dysfunction, cardiac hypertrophy and myocardial remodeling in response to chronic treatment with HFD/L-NAME. This functional amelioration was associated with improved insulin signaling in the myocardium and reduced S-nitrosylation of cardiac proteins, without any impact on systemic blood pressure. Finally, pharmacological inhibition of BCKDK in HFpEF mice significantly reversed the diastolic dysfunction and cardiac hypertrophy associated with HFpEF. Conclusions: Our study provides the first proof-of-concept evidence that global catabolic impairment of BCAAs is an important pathogenic contributor and metabolic signature of HFpEF and restoring BCAA catabolic flux could be an efficacious therapeutic strategy for HFpEF.