Potential Application of Modified mRNA in Cardiac Regeneration.

Heart failure remains the leading cause of human death worldwide. After a heart attack, the formation of scar tissue due to the massive death of cardiomyocytes leads to heart failure and sudden death in most cases. In addition, the regenerative ability of the adult heart is limited after injury, partly due to cell-cycle arrest in cardiomyocytes. In the current post-COVID-19 era, urgently authorized modified mRNA (modRNA) vaccines have been widely used to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Therefore, modRNA-based protein replacement may act as an alternative strategy for improving heart disease. It is a safe, effective, transient, low-immunogenic, and integration-free strategy for in vivo protein expression, in addition to recombinant protein and stem-cell regenerative therapies. In this review, we provide a summary of various cardiac factors that have been utilized with the modRNA method to enhance cardiovascular regeneration, cardiomyocyte proliferation, fibrosis inhibition, and apoptosis inhibition. We further discuss other cardiac factors, modRNA delivery methods, and injection methods using the modRNA approach to explore their application potential in heart disease. Factors for promoting cardiomyocyte proliferation such as a cocktail of three genes comprising FoxM1, Id1, and Jnk3-shRNA (FIJs), gp130, and melatonin have potential to be applied in the modRNA approach. We also discuss the current challenges with respect to modRNA-based cardiac regenerative medicine that need to be overcome to apply this approach to heart disease. This review provides a short description for investigators interested in the development of alternative cardiac regenerative medicines using the modRNA platform.

Low-adhesion culture selection for human iPS cell-derived cardiomyocytes.

Despite progress in generating cardiomyocytes from pluripotent stem cells, these populations often include non-contractile cells, necessitating cardiomyocyte selection for experimental purpose. This study explores a novel cardiomyocyte enrichment mechanism: low-adhesion culture selection. The cardiac cells derived from human induced pluripotent stem cells were subjected to a coating-free low-adhesion culture using bovine serum albumin and high molecular weight dextran sulfate. This approach effectively increased the population of cardiac troponin T-positive cardiomyocytes. Similar results were obtained with commercially available low-adhesion culture dishes. Subsequently, we accessed the practicality of selection of cardiomyocytes using this phenomenon by comparing it with established methods such as glucose-free culture and selection based on puromycin resistance genes. The cardiomyocytes enriched through low-adhesion culture selection maintained autonomous pulsation and responsiveness to beta-stimuli. Moreover, no significant differences were observed in the expression of genes related to subtype commitment and maturation when compared to other selection methods. In conclusion, cardiomyocytes derived from pluripotent stem cells were more low-adhesion culture resistant than their accompanying non-contractile cells, and low-adhesion culture is an alternative method for selection of pluripotent stem cell-derived cardiomyocytes.

Head-to-head comparison of relevant cell sources of small extracellular vesicles for cardiac repair: Superiority of embryonic stem cells.

Small extracellular vesicles (sEV) derived from various cell sources have been demonstrated to enhance cardiac function in preclinical models of myocardial infarction (MI). The aim of this study was to compare different sources of sEV for cardiac repair and determine the most effective one, which nowadays remains limited. We comprehensively assessed the efficacy of sEV obtained from human primary bone marrow mesenchymal stromal cells (BM-MSC), human immortalized MSC (hTERT-MSC), human embryonic stem cells (ESC), ESC-derived cardiac progenitor cells (CPC), human ESC-derived cardiomyocytes (CM), and human primary ventricular cardiac fibroblasts (VCF), in in vitro models of cardiac repair. ESC-derived sEV (ESC-sEV) exhibited the best pro-angiogenic and anti-fibrotic effects in vitro. Then, we evaluated the functionality of the sEV with the most promising performances in vitro, in a murine model of MI-reperfusion injury (IRI) and analysed their RNA and protein compositions. In vivo, ESC-sEV provided the most favourable outcome after MI by reducing adverse cardiac remodelling through down-regulating fibrosis and increasing angiogenesis. Furthermore, transcriptomic, and proteomic characterizations of sEV derived from hTERT-MSC, ESC, and CPC revealed factors in ESC-sEV that potentially drove the observed functions. In conclusion, ESC-sEV holds great promise as a cell-free treatment for promoting cardiac repair following MI.

Differentiating the human heart: A focus on atrioventricular canal cardiomyocytes.

Bioengineering of a functional human heart continues to face many challenges, including the production of distinct cardiac cell types. Now, in Cell Stem Cell, Ye et al.1 develop AVC-like cardiomyocytes through timing- and concentration-specific activation of canonical Wnt signaling.

Focal Adhesion's Role in Cardiomyocytes Function: From Cardiomyogenesis to Mechanotransduction.

Mechanotransduction refers to the ability of cells to sense mechanical stimuli and convert them into biochemical signals. In this context, the key players are focal adhesions (FAs): multiprotein complexes that link intracellular actin bundles and the extracellular matrix (ECM). FAs are involved in cellular adhesion, growth, differentiation, gene expression, migration, communication, force transmission, and contractility. Focal adhesion signaling molecules, including Focal Adhesion Kinase (FAK), integrins, vinculin, and paxillin, also play pivotal roles in cardiomyogenesis, impacting cell proliferation and heart tube looping. In fact, cardiomyocytes sense ECM stiffness through integrins, modulating signaling pathways like PI3K/AKT and Wnt/ß-catenin. Moreover, FAK/Src complex activation mediates cardiac hypertrophic growth and survival signaling in response to mechanical loads. This review provides an overview of the molecular and mechanical mechanisms underlying the crosstalk between FAs and cardiac differentiation, as well as the role of FA-mediated mechanotransduction in guiding cardiac muscle responses to mechanical stimuli.

Cell-Cycle-Specific Autoencoding Improves Cluster Analysis of Cycling Cardiomyocytes.

BACKGROUND: Our previous analyses of cardiomyocyte single-nucleus RNA sequencing (snRNAseq) data from the hearts of fetal pigs and pigs that underwent apical resection surgery on postnatal day (P) 1 (ARP1), myocardial infarction (MI) surgery on P28 (MIP28), both ARP1 and MIP28 (ARP1MIP28), or controls (no surgical procedure or CTL) identified 10 cardiomyocyte subpopulations (clusters), one of which appeared to be primed to proliferate in response to MI. However, the clusters composed of primarily proliferating cardiomyocytes still contained noncycling cells, and we were unable to distinguish between cardiomyocytes in different phases of the cell cycle. Here, we improved the precision of our assessments by conducting similar analyses with snRNAseq data for only the 1646 genes included under the Gene Ontology term "cell cycle." METHODS: Two cardiac snRNAseq datasets, one from mice (GEO dataset number GSE130699) and one from pigs (GEO dataset number GSE185289), were evaluated via our cell-cycle-specific analytical pipeline. Cycling cells were identified via the co-expression of 5 proliferation markers (AURKB, MKI67, INCENP, CDCA8, and BIRC5). RESULTS: The cell-cycle-specific autoencoder (CSA) algorithm identified 7 cardiomyocyte clusters in mouse hearts (mCM1 and mCM3-mCM8), including one prominent cluster of cycling cardiomyocytes in animals that underwent MI or Sham surgery on P1. Five cardiomyocyte clusters (pCM1, pCM3-pCM6) were identified in pig hearts, 2 of which (pCM1 and pCM4) displayed evidence of cell cycle activity; pCM4 was found primarily in hearts from fetal pigs, while pCM1 comprised a small proportion of cardiomyocytes in both fetal hearts and hearts from ARP1MIP28 pigs during the 2 weeks after MI induction, but was nearly undetectable in all other experimental groups and at all other time points. Furthermore, pseudotime trajectory analysis of snRNAseq data from fetal pig cardiomyocytes identified a pathway that began at pCM3, passed through pCM2, and ended at pCM1, whereas pCM3 was enriched for the expression of a cell cycle activator that regulates the G1/S phase transition (cyclin D2), pCM2 was enriched for an S-phase regulator (CCNE2), and pCM1 was enriched for the expression of a gene that regulates the G2M phase transition and mitosis (cyclin B2). We also identified 4 transcription factors (E2F8, FOXM1, GLI3, and RAD51) that were more abundantly expressed in cardiomyocytes from regenerative mouse hearts than from nonregenerative mouse hearts, from the hearts of fetal pigs than from CTL pig hearts, and from ARP1MIP28 pig hearts than from MIP28 pig hearts during the 2 weeks after MI induction. CONCLUSIONS: The CSA algorithm improved the precision of our assessments of cell cycle activity in cardiomyocyte subpopulations and enabled us to identify a trajectory across 3 clusters that appeared to track the onset and progression of cell cycle activity in cardiomyocytes from fetal pigs.

Generation of 3D-Multicellular Human iPSC-Heart Organoids for the Noninvasive Assessment of Cardiac Fibrosis.

The lack of a precise noninvasive, clinical evaluation method for cardiac fibrosis hinders the development of successful treatments that can effectively work in physiological settings, where tissues and organs are interconnected and moderating drug responses. To address this challenge and advance personalized medicine, researchers have turned to human-induced pluripotent stem (iPS) cells, which can be differentiated to resemble the human heart in terms of structure, function and cellular composition. In this chapter, we present an assay protocol that uses these iPS cells to generate heart organoids for the in vitro evaluation of cardiac fibrosis. By establishing this biological platform, we pave the way for conducting phenotype evaluation and treatment screening in a multiscale approach, aiming to discover effective interventions for the treatment of cardiac fibrosis.

Culturing Cardiac Tissue Slices Under Continuous Physiological Mechanical Stretches.

Testing drugs in vivo and in vitro have been essential elements for the discovery of new therapeutics. Due to the recent advances in in vitro cell culture models, such as human-induced pluripotent stem cell-derived cardiomyocytes and 3D multicell type organoid culture methods, the detection of adverse cardiac events prior to human clinical trials has improved. However, there are still numerous therapeutics whose adverse cardiac effects are not detected until human trials due to the inability of these cell cultures to fully model the complex multicellular organization of an intact human myocardium. Cardiac tissue slices are a possible alternative solution. Myocardial slices are a 300-micron thin snapshot of the myocardium, capturing a section of the adult heart in a 1 × 1 cm section. Using a culture method that incorporates essential nutrients and electrical stimulation, tissue slices can be maintained in culture for 6 days with full viability and functionality. With the addition of mechanical stimulation and humoral cues, tissue slices can be cultured for 12 days. Here we provide detailed methods for how to culture cardiac tissue slices under continuous mechanical stimulation in the cardiac tissue culture model (CTCM) device. The CTCM incorporates four essential factors for maintaining tissue slices in culture for 12 days: mechanical stimulation, electrical stimulation, nutrients, and humoral cues. The CTCM can also be used to model disease conditions, such as overstretch-induced cardiac hypertrophy. The versatility of the CTCM illustrates its potential to be a medium-throughput screening platform for personalized drug testing.

Subtype and Lineage-Mediated Protocol for Standardizing Activin/Nodal and BMP Signaling for hiPSC-Derived Cardiomyocyte Differentiation.

The adept and systematic differentiation of embryonic stem cells (ESCs) and human-induced pluripotent stem cells (hiPSCs) to diverse lineage-prone cell types involves crucial step-by-step process that mimics the vital strategic commitment phase that is usually observed during the process of embryo development. The development of precise tissue-specific cell types from these stem cells indeed plays an important role in the advancement of imminent stem cell-based therapeutic strategies. Therefore, the usage of hiPSC-derived cell types for subsequent cardiovascular disease modeling, drug screening, and therapeutic drug development undeniably entails an in-depth understanding of each and every step to proficiently stimulate these stem cells into desired cardiomyogenic lineage. Thus, to accomplish this definitive and decisive fate, it is essential to efficiently induce the mesoderm or pre-cardiac mesoderm, succeeded by the division of cells into cardiovascular and ultimately ensuing with the cardiomyogenic lineage outcome. This usually commences from the earliest phases of pluripotent cell induction. In this chapter, we discuss our robust and reproducible step-wise protocol that will describe the subtype controlled, precise lineage targeted standardization of activin/nodal, and BMP signaling molecules/cytokines, for the efficient differentiation of ventricular cardiomyocytes from hiPSCs via the embryoid body method. In addition, we also describe techniques to dissociate hiPSCs, hiPSC-derived early cardiomyocytes for mesoderm and pre-cardiac mesoderm assessment, and hiPSC-derived cardiomyocytes for early and mature markers assessment.

Polyploidy Promotes Hypertranscription, Apoptosis Resistance, and Ciliogenesis in Cancer Cells and Mesenchymal Stem Cells of Various Origins: Comparative Transcriptome In Silico Study.

Mesenchymal stem cells (MSC) attract an increasing amount of attention due to their unique therapeutic properties. Yet, MSC can undergo undesirable genetic and epigenetic changes during their propagation in vitro. In this study, we investigated whether polyploidy can compromise MSC oncological safety and therapeutic properties. For this purpose, we compared the impact of polyploidy on the transcriptome of cancer cells and MSC of various origins (bone marrow, placenta, and heart). First, we identified genes that are consistently ploidy-induced or ploidy-repressed through all comparisons. Then, we selected the master regulators using the protein interaction enrichment analysis (PIEA). The obtained ploidy-related gene signatures were verified using the data gained from polyploid and diploid populations of early cardiomyocytes (CARD) originating from iPSC. The multistep bioinformatic analysis applied to the cancer cells, MSC, and CARD indicated that polyploidy plays a pivotal role in driving the cell into hypertranscription. It was evident from the upregulation of gene modules implicated in housekeeping functions, stemness, unicellularity, DNA repair, and chromatin opening by means of histone acetylation operating via DNA damage associated with the NUA4/TIP60 complex. These features were complemented by the activation of the pathways implicated in centrosome maintenance and ciliogenesis and by the impairment of the pathways related to apoptosis, the circadian clock, and immunity. Overall, our findings suggest that, although polyploidy does not induce oncologic transformation of MSC, it might compromise their therapeutic properties because of global epigenetic changes and alterations in fundamental biological processes. The obtained results can contribute to the development and implementation of approaches enhancing the therapeutic properties of MSC by removing polyploid cells from the cell population.

Telomerase is essential for cardiac differentiation and sustained metabolism of human cardiomyocytes.

Telomeres as the protective ends of linear chromosomes, are synthesized by the enzyme telomerase (TERT). Critically short telomeres essentially contribute to aging-related diseases and are associated with a broad spectrum of disorders known as telomeropathies. In cardiomyocytes, telomere length is strongly correlated with cardiomyopathies but it remains ambiguous whether short telomeres are the cause or the result of the disease. In this study, we employed an inducible CRISPRi human induced pluripotent stem cell (hiPSC) line to silence TERT expression enabling the generation of hiPSCs and hiPSC-derived cardiomyocytes with long and short telomeres. Reduced telomerase activity and shorter telomere lengths of hiPSCs induced global transcriptomic changes associated with cardiac developmental pathways. Consequently, the differentiation potential towards cardiomyocytes was strongly impaired and single cell RNA sequencing revealed a shift towards a more smooth muscle cell like identity in the cells with the shortest telomeres. Poor cardiomyocyte function and increased sensitivity to stress directly correlated with the extent of telomere shortening. Collectively our data demonstrates a TERT dependent cardiomyogenic differentiation defect, highlighting the CRISPRi TERT hiPSCs model as a powerful platform to study the mechanisms and consequences of short telomeres in the heart and also in the context of telomeropathies.

[Experts' consensus on mammalian cardiomyocyte regeneration].

Myocardial infarction (MI) leads to a massive loss of cardiomyocytes, resulting in pathological cardiac remodeling and heart failure. Promoting cardiomyocyte regeneration is crucial for repairing the damaged heart. It is acknowledged that regenerative cardiomyocyte derives from the existing cardiomyocytes. In recent years, advancements in this field have updated our understanding of cardiomyocyte regeneration in many aspects, including intrinsic cell source and microenvironmental characteristics, extrinsic factors, molecular biology mechanisms, and intervention strategies. Here, we report a consensus by an expert committee on the definition, characteristics, evaluation, research methods, regulatory mechanisms, and intervention measures related to mammalian cardiomyocyte regeneration. The aim is to clarify important unresolved issues in this field and to promote myocardial regeneration research and its clinical translation.

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Induced pluripotent stem cells (iPSCs) are obtained by introducing exogenous genes or adding chemicals to the culture medium to induce somatic cell differentiation. Similarly to embryonic stem cells, iPSCs have the ability to differentiate into all three embryonic cell lines. iPSCs can differentiate into cardiac muscle cells through two-dimensional differentiation methods such as monolayer cell culture and co-culture, or through embryoid body and scaffold-based three-dimensional differentiation methods. In addition, the process of iPSCs differentiation into cardiac muscle cells also requires activation or inhibition of specific signaling pathways,such as Wnt, BMP, Notch signaling pathways to mimic the development of the heart in vivo. In recent years, suspension culturing in bioreactors has been shown to produce large number of iPSCs derived cardiac muscle cells (iPSC-CMs). Before transplantation, it is necessary to purify iPSC-CMs through metabolic regulation or cell sorting to eliminate undifferentiated iPSCs, which may lead to teratoma formation. The transplantation methods for iPSC-CMs are mainly injection of cell suspension and transplantation of cell patches into the infarcted myocardium. Animal studies have shown that transplantation of iPSC-CMs into the infarcted myocardium can improve cardiac function. This article reviews the progress in preclinical studies on iPSC-CMs therapy for acute myocardial infarction and discusses the limitations and challenges of its clinical application to provide references for further clinical research and application.

Advances in 3D Bioprinted Cardiac Tissue Using Stem Cell-Derived Cardiomyocytes.

The ultimate goal of cardiac tissue engineering is to generate new muscle to repair or replace the damaged heart. This requires advances in stem cell technologies to differentiate billions of cardiomyocytes, together with advanced biofabrication approaches such as 3D bioprinting to achieve the requisite structure and contractile function. In this concise review, we cover recent progress in 3D bioprinting of cardiac tissue using pluripotent stem cell-derived cardiomyocytes, key design criteria for engineering aligned cardiac tissues, and ongoing challenges in the field that must be addressed to realize this goal.

Spatially organized cellular communities form the developing human heart.

The heart, which is the first organ to develop, is highly dependent on its form to function1,2. However, how diverse cardiac cell types spatially coordinate to create the complex morphological structures that are crucial for heart function remains unclear. Here we integrated single-cell RNA-sequencing with high-resolution multiplexed error-robust fluorescence in situ hybridization to resolve the identity of the cardiac cell types that develop the human heart. This approach also provided a spatial mapping of individual cells that enables illumination of their organization into cellular communities that form distinct cardiac structures. We discovered that many of these cardiac cell types further specified into subpopulations exclusive to specific communities, which support their specialization according to the cellular ecosystem and anatomical region. In particular, ventricular cardiomyocyte subpopulations displayed an unexpected complex laminar organization across the ventricular wall and formed, with other cell subpopulations, several cellular communities. Interrogating cell-cell interactions within these communities using in vivo conditional genetic mouse models and in vitro human pluripotent stem cell systems revealed multicellular signalling pathways that orchestrate the spatial organization of cardiac cell subpopulations during ventricular wall morphogenesis. These detailed findings into the cellular social interactions and specialization of cardiac cell types constructing and remodelling the human heart offer new insights into structural heart diseases and the engineering of complex multicellular tissues for human heart repair.

Leveraging chromatin state transitions for the identification of regulatory networks orchestrating heart regeneration.

The limited regenerative capacity of the human heart contributes to high morbidity and mortality worldwide. In contrast, zebrafish exhibit robust regenerative capacity, providing a powerful model for studying how to overcome intrinsic epigenetic barriers maintaining cardiac homeostasis and initiate regeneration. Here, we present a comprehensive analysis of the histone modifications H3K4me1, H3K4me3, H3K27me3 and H3K27ac during various stages of zebrafish heart regeneration. We found a vast gain of repressive chromatin marks one day after myocardial injury, followed by the acquisition of active chromatin characteristics on day four and a transition to a repressive state on day 14, and identified distinct transcription factor ensembles associated with these events. The rapid transcriptional response involves the engagement of super-enhancers at genes implicated in extracellular matrix reorganization and TOR signaling, while H3K4me3 breadth highly correlates with transcriptional activity and dynamic changes at genes involved in proteolysis, cell cycle activity, and cell differentiation. Using loss- and gain-of-function approaches, we identified transcription factors in cardiomyocytes and endothelial cells influencing cardiomyocyte dedifferentiation or proliferation. Finally, we detected significant evolutionary conservation between regulatory regions that drive zebrafish and neonatal mouse heart regeneration, suggesting that reactivating transcriptional and epigenetic networks converging on these regulatory elements might unlock the regenerative potential of adult human hearts.

MiR-431 promotes cardiomyocyte proliferation by targeting FBXO32 expression.

BACKGROUND: The induction of cardiomyocyte (CM) proliferation is a promising approach for cardiac regeneration following myocardial injury. MicroRNAs (miRNAs) have been reported to regulate CM proliferation. In particular, miR-431 expression decreases during cardiac development, according to Gene Expression Omnibus (GEO) microarray data. However, whether miR-431 regulates CM proliferation has not been thoroughly investigated. METHODS: We used integrated bioinformatics analysis of GEO datasets to identify the most significantly differentially expressed miRNAs. Real-time quantitative PCR and fluorescence in situ hybridization were performed to determine the miRNA expression patterns in hearts. Gain- and loss-of-function assays were conducted to detect the role of miRNA in CM proliferation. Additionally, we detected whether miR-431 affected CM proliferation in a myocardial infarction model. The TargetScan, miRDB and miRWalk online databases were used to predict the potential target genes of miRNAs. Luciferase reporter assays were used to study miRNA interactions with the targeting mRNA. RESULTS: First, we found a significant reduction in miR-431 levels during cardiac development. Then, by overexpression and inhibition of miR-431, we demonstrated that miR-431 promotes CM proliferation in vitro and in vivo, as determined by immunofluorescence assays of 5-ethynyl-2'-deoxyuridine (EdU), pH3, Aurora B and CM count, whereas miR-431 inhibition suppresses CM proliferation. Then, we found that miR-431 improved cardiac function post-myocardial infarction. In addition, we identified FBXO32 as a direct target gene of miR-431, with FBXO32 mRNA and protein expression being suppressed by miR-431. FBXO32 inhibited CM proliferation. Overexpression of FBXO32 blocks the enhanced effect of miR-431 on CM proliferation, suggesting that FBXO32 is a functional target of miR-431 during CM proliferation. CONCLUSION: In summary, miR-431 promotes CM proliferation by targeting FBXO32, providing a potential molecular target for preventing myocardial injury.

Nonsense Variant PRDM16-Q187X Causes Impaired Myocardial Development and TGF-ß Signaling Resulting in Noncompaction Cardiomyopathy in Humans and Mice.

BACKGROUND: PRDM16 plays a role in myocardial development through TGF-ß (transforming growth factor-beta) signaling. Recent evidence suggests that loss of PRDM16 expression is associated with cardiomyopathy development in mice, although its role in human cardiomyopathy development is unclear. This study aims to determine the impact of PRDM16 loss-of-function variants on cardiomyopathy in humans. METHODS: Individuals with PRDM16 variants were identified and consented. Induced pluripotent stem cell-derived cardiomyocytes were generated from a proband hosting a Q187X nonsense variant as an in vitro model and underwent proliferative and transcriptional analyses. CRISPR (clustered regularly interspaced short palindromic repeats)-mediated knock-in mouse model hosting the Prdm16Q187X allele was generated and subjected to ECG, histological, and transcriptional analysis. RESULTS: We report 2 probands with loss-of-function PRDM16 variants and pediatric left ventricular noncompaction cardiomyopathy. One proband hosts a PRDM16-Q187X variant with left ventricular noncompaction cardiomyopathy and demonstrated infant-onset heart failure, which was selected for further study. Induced pluripotent stem cell-derived cardiomyocytes prepared from the PRDM16-Q187X proband demonstrated a statistically significant impairment in myocyte proliferation and increased apoptosis associated with transcriptional dysregulation of genes implicated in cardiac maturation, including TGF-ß-associated transcripts. Homozygous Prdm16Q187X/Q187X mice demonstrated an underdeveloped compact myocardium and were embryonically lethal. Heterozygous Prdm16Q187X/WT mice demonstrated significantly smaller ventricular dimensions, heightened fibrosis, and age-dependent loss of TGF-ß expression. Mechanistic studies were undertaken in H9c2 cardiomyoblasts to show that PRDM16 binds TGFB3 promoter and represses its transcription. CONCLUSIONS: Novel loss-of-function PRDM16 variant impairs myocardial development resulting in noncompaction cardiomyopathy in humans and mice associated with altered TGF-ß signaling.

Inhibition of fatty acid oxidation enables heart regeneration in adult mice.

Postnatal maturation of cardiomyocytes is characterized by a metabolic switch from glycolysis to fatty acid oxidation, chromatin reconfiguration and exit from the cell cycle, instating a barrier for adult heart regeneration1,2. Here, to explore whether metabolic reprogramming can overcome this barrier and enable heart regeneration, we abrogate fatty acid oxidation in cardiomyocytes by inactivation of Cpt1b. We find that disablement of fatty acid oxidation in cardiomyocytes improves resistance to hypoxia and stimulates cardiomyocyte proliferation, allowing heart regeneration after ischaemia-reperfusion injury. Metabolic studies reveal profound changes in energy metabolism and accumulation of α-ketoglutarate in Cpt1b-mutant cardiomyocytes, leading to activation of the α-ketoglutarate-dependent lysine demethylase KDM5 (ref. 3). Activated KDM5 demethylates broad H3K4me3 domains in genes that drive cardiomyocyte maturation, lowering their transcription levels and shifting cardiomyocytes into a less mature state, thereby promoting proliferation. We conclude that metabolic maturation shapes the epigenetic landscape of cardiomyocytes, creating a roadblock for further cell divisions. Reversal of this process allows repair of damaged hearts.

Spatially resolved multiomics of human cardiac niches.

The function of a cell is defined by its intrinsic characteristics and its niche: the tissue microenvironment in which it dwells. Here we combine single-cell and spatial transcriptomics data to discover cellular niches within eight regions of the human heart. We map cells to microanatomical locations and integrate knowledge-based and unsupervised structural annotations. We also profile the cells of the human cardiac conduction system1. The results revealed their distinctive repertoire of ion channels, G-protein-coupled receptors (GPCRs) and regulatory networks, and implicated FOXP2 in the pacemaker phenotype. We show that the sinoatrial node is compartmentalized, with a core of pacemaker cells, fibroblasts and glial cells supporting glutamatergic signalling. Using a custom CellPhoneDB.org module, we identify trans-synaptic pacemaker cell interactions with glia. We introduce a druggable target prediction tool, drug2cell, which leverages single-cell profiles and drug-target interactions to provide mechanistic insights into the chronotropic effects of drugs, including GLP-1 analogues. In the epicardium, we show enrichment of both IgG+ and IgA+ plasma cells forming immune niches that may contribute to infection defence. Overall, we provide new clarity to cardiac electro-anatomy and immunology, and our suite of computational approaches can be applied to other tissues and organs.