Modeling drug-induced mitochondrial toxicity with human primary cardiomyocytes.

Mitochondrial toxicity induced by therapeutic drugs is a major contributor for cardiotoxicity, posing a serious threat to pharmaceutical industries and patients' lives. However, mitochondrial toxicity testing is not incorporated into routine cardiac safety screening procedures. To accurately model native human cardiomyocytes, we comprehensively evaluated mitochondrial responses of adult human primary cardiomyocytes (hPCMs) to a nucleoside analog, remdesivir (RDV). Comparison of their response to human pluripotent stem cell-derived cardiomyocytes revealed that the latter utilized a mitophagy-based mitochondrial recovery response that was absent in hPCMs. Accordingly, action potential duration was elongated in hPCMs, reflecting clinical incidences of RDV-induced QT prolongation. In a screen for mitochondrial protectants, we identified mitochondrial ROS as a primary mediator of RDV-induced cardiotoxicity. Our study demonstrates the utility of hPCMs in the detection of clinically relevant cardiac toxicities, and offers a framework for hPCM-based high-throughput screening of cardioprotective agents.

INO80-Dependent Remodeling of Transcriptional Regulatory Network Underlies the Progression of Heart Failure.

BACKGROUND: Progressive remodeling of cardiac gene expression underlies decline in cardiac function, eventually leading to heart failure. However, the major determinants of transcriptional network switching from normal to failed hearts remain to be determined. METHODS: In this study, we integrated human samples, genetic mouse models, and genomic approaches, including bulk RNA sequencing, single-cell RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, to identify the role of chromatin remodeling complex INO80 in heart homeostasis and dysfunction. RESULTS: The INO80 chromatin remodeling complex was abundantly expressed in mature cardiomyocytes, and its expression further increased in mouse and human heart failure. Cardiomyocyte-specific overexpression of Ino80, its core catalytic subunit, induced heart failure within 4 days. Combining RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, we revealed INO80 overexpression-dependent reshaping of the nucleosomal landscape that remodeled a core set of transcription factors, most notably the MEF2 (Myocyte Enhancer Factor 2) family, whose target genes were closely associated with cardiac function. Conditional cardiomyocyte-specific deletion of Ino80 in an established mouse model of heart failure demonstrated remarkable preservation of cardiac function. CONCLUSIONS: In summary, our findings shed light on the INO80-dependent remodeling of the chromatin landscape and transcriptional networks as a major mechanism underlying cardiac dysfunction in heart failure, and suggest INO80 as a potential preventative or interventional target.

Coordinated regulation of vegetative phase change by brassinosteroids and the age pathway in Arabidopsis.

Vegetative phase change in plants is regulated by a gradual decline in the level of miR156 and a corresponding increase in the expression of its targets, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. Gibberellin (GA), jasmonic acid (JA), and cytokinin (CK) regulate vegetative phase change by affecting genes in the miR156-SPL pathway. However, whether other phytohormones play a role in vegetative phase change remains unknown. Here, we show that a loss-of-function mutation in the brassinosteroid (BR) biosynthetic gene, DWARF5 (DWF5), delays vegetative phase change, and the defective phenotype is primarily attributable to reduced levels of SPL9 and miR172, and a corresponding increase in TARGET OF EAT1 (TOE1). We further show that GLYCOGEN SYNTHASE KINASE3 (GSK3)-like kinase BRASSINOSTEROID INSENSITIVE2 (BIN2) directly interacts with and phosphorylates SPL9 and TOE1 to cause subsequent proteolytic degradation. Therefore, BRs function to stabilize SPL9 and TOE1 simultaneously to regulate vegetative phase change in plants.

Isolation of porcine adult cardiomyocytes: Comparison between Langendorff perfusion and tissue slicing-assisted enzyme digestion.

Tissue slicing-assisted digestion (TSAD) of adult cardiomyocytes has shown significant improvements over conventional chunk methods. However, it remains unclear how this method compares to Langendorff perfusion, the current standard of adult cardiomyocyte isolation. Using adult Bama minipigs, we performed cardiomyocyte isolation via these two distinct methods, and compared the resulting cellular quality, including viability, cellular structure, gene expression, and electrophysiological properties, of cardiomyocytes from 3 distinct anatomical regions, namely the left ventricle, right ventricle, and left atrial appendage. Our results revealed largely indistinguishable cell quality in all of the measured parameters. These findings suggest that that TSAD can be reliably used to isolate adult mammalian cardiomyocytes as a reliable alternative to perfusion in cardiomyocyte isolation from larger mammals, particularly when Langendorff perfusion is not feasible.

Spatial Multiplexed Protein Profiling of Cardiac Ischemia-Reperfusion Injury.

BACKGROUND: Reperfusion therapy is critical to myocardial salvage in the event of a myocardial infarction but is complicated by ischemia-reperfusion injury (IRI). Limited understanding of the spatial organization of cardiac cells, which governs cellular interaction and function, has hindered the search for targeted interventions minimizing the deleterious effects of IRI. METHODS: We used imaging mass cytometry to characterize the spatial distribution and dynamics of cell phenotypes and communities in the mouse left ventricle following IRI. Heart sections were collected from 12 cardiac segments (basal, mid-cavity, apical, and apex of the anterior, lateral, and inferior wall) and 8 time points (before ischemia [I-0H], and postreperfusion [R-0H, R-2H, R-6H, R-12H, R-1D, R-3D, R-7D]), and stained with 29 metal-isotope-tagged antibodies. Cell community analysis was performed on reconstructed images, and the most disease-relevant cell type and target protein were selected for intervention of IRI. RESULTS: We obtained a total of 251 multiplexed images, and identified 197 063 single cells, which were grouped into 23 distinct cell communities based on the structure of cellular neighborhoods. The cellular architecture was heterogeneous throughout the ventricular wall and exhibited swift changes following IRI. Analysis of proteins with posttranslational modifications in single cells unveiled 13 posttranslational modification intensity clusters and highlighted increased H3K9me3 (tri-methylated lysine 9 of histone H3) as a key regulatory response in endothelial cells during the middle stage of IRI. Erasing H3K9 methylation, by silencing its methyltransferase Suv39h1 or overexpressing its demethylase Kdm4d in isolated endothelial cells, attenuated cardiac dysfunction and pathological remodeling following IRI. in vitro, H3K9me3 binding significantly increased at endothelial cell function-related genes upon hypoxia, suppressing tube formation, which was rescued by inhibiting H3K9me3. CONCLUSIONS: We mapped the spatiotemporal heterogeneity of cellular phenotypes in the adult heart upon IRI, and uncovered H3K9me3 in endothelial cells as a potential therapeutic target for alleviating pathological remodeling of the heart following myocardial IRI.

Mitochondrial Dysfunction in Cardiac Diseases and Therapeutic Strategies.

Mitochondria are the main site of intracellular synthesis of ATP, which provides energy for various physiological activities of the cell. Cardiomyocytes have a high density of mitochondria and mitochondrial damage is present in a variety of cardiovascular diseases. In this paper, we describe mitochondrial damage in mitochondrial cardiomyopathy, congenital heart disease, coronary heart disease, myocardial ischemia-reperfusion injury, heart failure, and drug-induced cardiotoxicity, in the context of the key roles of mitochondria in cardiac development and homeostasis. Finally, we discuss the main current therapeutic strategies aimed at alleviating mitochondrial impairment-related cardiac dysfunction, including pharmacological strategies, gene therapy, mitochondrial replacement therapy, and mitochondrial transplantation. It is hoped that this will provide new ideas for the treatment of cardiovascular diseases.

Functional isolation, culture and cryopreservation of adult human primary cardiomyocytes.

Cardiovascular diseases are the most common cause of death globally. Accurately modeling cardiac homeostasis, dysfunction, and drug response lies at the heart of cardiac research. Adult human primary cardiomyocytes (hPCMs) are a promising cellular model, but unstable isolation efficiency and quality, rapid cell death in culture, and unknown response to cryopreservation prevent them from becoming a reliable and flexible in vitro cardiac model. Combing the use of a reversible inhibitor of myosin II ATPase, (-)-blebbistatin (Bleb), and multiple optimization steps of the isolation procedure, we achieved a 2.74-fold increase in cell viability over traditional methods, accompanied by better cellular morphology, minimally perturbed gene expression, intact electrophysiology, and normal neurohormonal signaling. Further optimization of culture conditions established a method that was capable of maintaining optimal cell viability, morphology, and mitochondrial respiration for at least 7 days. Most importantly, we successfully cryopreserved hPCMs, which were structurally, molecularly, and functionally intact after undergoing the freeze-thaw cycle. hPCMs demonstrated greater sensitivity towards a set of cardiotoxic drugs, compared to human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Further dissection of cardiomyocyte drug response at both the population and single-cell transcriptomic level revealed that hPCM responses were more pronouncedly enriched in cardiac function, whereas hiPSC-CMs responses reflected cardiac development. Together, we established a full set of methodologies for the efficient isolation and prolonged maintenance of functional primary adult human cardiomyocytes in vitro, unlocking their potential as a cellular model for cardiovascular research, drug discovery, and safety pharmacology.

Assessing Drug-Induced Mitochondrial Toxicity in Cardiomyocytes: Implications for Preclinical Cardiac Safety Evaluation.

Drug-induced cardiotoxicity not only leads to the attrition of drugs during development, but also contributes to the high morbidity and mortality rates of cardiovascular diseases. Comprehensive testing for proarrhythmic risks of drugs has been applied in preclinical cardiac safety assessment for over 15 years. However, other mechanisms of cardiac toxicity have not received such attention. Of them, mitochondrial impairment is a common form of cardiotoxicity and is known to account for over half of cardiovascular adverse-event-related black box warnings imposed by the U.S. Food and Drug Administration. Although it has been studied in great depth, mitochondrial toxicity assessment has not yet been incorporated into routine safety tests for cardiotoxicity at the preclinical stage. This review discusses the main characteristics of mitochondria in cardiomyocytes, drug-induced mitochondrial toxicities, and high-throughput screening strategies for cardiomyocytes, as well as their proposed integration into preclinical safety pharmacology. We emphasize the advantages of using adult human primary cardiomyocytes for the evaluation of mitochondrial morphology and function, and the need for a novel cardiac safety testing platform integrating mitochondrial toxicity and proarrhythmic risk assessments in cardiac safety evaluation.

Postnatal state transition of cardiomyocyte as a primary step in heart maturation.

Postnatal heart maturation is the basis of normal cardiac function and provides critical insights into heart repair and regenerative medicine. While static snapshots of the maturing heart have provided much insight into its molecular signatures, few key events during postnatal cardiomyocyte maturation have been uncovered. Here, we report that cardiomyocytes (CMs) experience epigenetic and transcriptional decline of cardiac gene expression immediately after birth, leading to a transition state of CMs at postnatal day 7 (P7) that was essential for CM subtype specification during heart maturation. Large-scale single-cell analysis and genetic lineage tracing confirm the presence of transition state CMs at P7 bridging immature state and mature states. Silencing of key transcription factor JUN in P1-hearts significantly repressed CM transition, resulting in perturbed CM subtype proportions and reduced cardiac function in mature hearts. In addition, transplantation of P7-CMs into infarcted hearts exhibited cardiac repair potential superior to P1-CMs. Collectively, our data uncover CM state transition as a key event in postnatal heart maturation, which not only provides insights into molecular foundations of heart maturation, but also opens an avenue for manipulation of cardiomyocyte fate in disease and regenerative medicine.

Deciphering Cardiac Biology and Disease by Single-Cell Transcriptomic Profiling.

By detecting minute molecular changes in hundreds to millions of single cells, single-cell RNA sequencing allows for the comprehensive characterization of the diversity and dynamics of cells in the heart. Our understanding of the heart has been transformed through the recognition of cellular heterogeneity, the construction of regulatory networks, the building of lineage trajectories, and the mapping of intercellular crosstalk. In this review, we introduce cardiac progenitors and their transcriptional regulation during embryonic development, highlight cellular heterogeneity and cell subtype functions in cardiac health and disease, and discuss insights gained from the study of pluripotent stem-cell-derived cardiomyocytes.

Dysregulation of interaction between LOXhigh fibroblast and smooth muscle cells contributes to the pathogenesis of aortic dissection.

Rationale: While cell-cell interaction plays a critical role in physiology and disease, a comprehensive understanding of its dynamics in vascular homeostasis and diseases is yet absent. Methods: Here, by use of single-cell RNA-sequencing and multi-color staining, we delineate the cellular composition and spatial characterization of human aorta with or without aortic dissection (AD). Results: Scrutinization of cell subtype alterations revealed significantly changed fibroblast (FB)-smooth muscle cell (SMC) interactions in AD. Of these cellular interactions, LOXhigh fibroblast (fibroblast subtype 2, FB2) in diseased state exerted the most pronounced effects on pathological deterioration of SMCs in AD. In addition, pharmacologically targeting the BMP (bone morphogenetic protein) signaling pathway effectively suppressed FB2 state transition and reduced AD incidence in mice. Finally, COL5A1 (collagen type V alpha 1 chain), one of the secreted proteins released from FB2, was significantly higher in the plasma of AD patients than in control patients, suggesting its potential use as a biomarker for AD diagnosis. Conclusions: Our work not only identified a pivotal role of a specific FB subtype in AD progression, but also shed light on cell interaction dynamics in vascular diseases.

Profiling of immune features to predict immunotherapy efficacy.

Immune checkpoint blockade (ICB) therapies exhibit substantial clinical benefit in different cancers, but relatively low response rates in the majority of patients highlight the need to understand mutual relationships among immune features. Here, we reveal overall positive correlations among immune checkpoints and immune cell populations. Clinically, patients benefiting from ICB exhibited increases for both immune stimulatory and inhibitory features after initiation of therapy, suggesting that the activation of the immune microenvironment might serve as the biomarker to predict immune response. As proof-of-concept, we demonstrated that the immune activation score (IS Δ) based on dynamic alteration of interleukins in patient plasma as early as two cycles (4-6 weeks) after starting immunotherapy can accurately predict immunotherapy efficacy. Our results reveal a systematic landscape of associations among immune features and provide a noninvasive, cost-effective, and time-efficient approach based on dynamic profiling of pre- and on-treatment plasma to predict immunotherapy efficacy.

Human Pluripotent Stem Cell-Derived Cardiac Cells: Application in Disease Modeling, Cell Therapy, and Drug Discovery.

Cardiac diseases are the leading cause of deaths worldwide; however, to date, there has been limited progress in the development of therapeutic options for these conditions. Animal models have been the most extensively studied methods to recapitulate a wide variety of cardiac diseases, but these models exhibit species-specific differences in physiology, metabolism and genetics, which lead to inaccurate and unpredictable drug safety and efficacy results, resulting in drug attrition. The development of human pluripotent stem cell (hPSC) technology in theory guarantees an unlimited source of human cardiac cells. These hPSC-derived cells are not only well suited for traditional two-dimensional (2-D) monoculture, but also applicable to more complex systems, such as three-dimensional (3-D) organoids, tissue engineering and heart on-a-chip. In this review, we discuss the application of hPSCs in heart disease modeling, cell therapy, and next-generation drug discovery. While the hPSC-related technologies still require optimization, their advances hold promise for revolutionizing cell-based therapies and drug discovery.

Reading the heart at single-cell resolution.

The burgeoning field of single-cell transcriptomics augments our ability to scrutinize organ systems at unprecedented resolutions. Single-cell RNA sequencing (scRNA-seq) and analytical techniques have shed light on the cellular heterogeneity, developmental trajectories, intercellular communications of the cardiac system, and thus contributed much to the understanding of cardiac development, homeostasis and disorders. Although generalized protocols are well established for scRNA-seq pipelines, customized sample preparation, quality control, and data interpretation are still needed in cardiac research. In this article, we highlight major steps that impact data quality in scRNA-seq experiments, with particular focus on sample and data processing of cardiomyocytes. We also summarize popular applications of scRNA-seq, outlining general tools, caveats and examples in cardiac research.

Transcriptional Profiling of Single Cardiomyocytes in Health and Disease.

PURPOSE OF REVIEW: Cardiomyocytes are the chief cell type in the heart, and are central to the pathogenesis of many cardiac diseases. Increasing recognition of its cellular, molecular, and functional heterogeneity prompted us to review the latest advancements in cardiac health and disease at single-cell resolution. RECENT FINDINGS: Single-cell RNA profiling of cardiac lineage commitment events uncovered immense heterogeneity amongst ostensibly homogeneous cell populations. Classic cardiac transcription factors and new regulatory genes exhibit cell subtype-specific and temporally controlled expression patterns that serve the phenotypic changes in development, disease progression, and regeneration. Dissection of dynamically changing cell-cell communications and cardiac cell plasticity offers new opportunities in disease intervention and cardiac repair. Finally, updates in research models, platforms, and pipelines are continuously increasing the efficiency and reliability in data generation and interpretation. Transcriptional profiling of cardiac lineage cells, especially cardiomyocytes, has tremendously enriched our knowledge of the cellular milieu and the transcriptional network in the heart. Implementing technical standards and interrogating underexplored research areas will further our understanding of this organ and increase the likelihood of finding tractable therapeutic targets.

Single-cell analysis of murine fibroblasts identifies neonatal to adult switching that regulates cardiomyocyte maturation.

Cardiac maturation lays the foundation for postnatal heart development and disease, yet little is known about the contributions of the microenvironment to cardiomyocyte maturation. By integrating single-cell RNA-sequencing data of mouse hearts at multiple postnatal stages, we construct cellular interactomes and regulatory signaling networks. Here we report switching of fibroblast subtypes from a neonatal to adult state and this drives cardiomyocyte maturation. Molecular and functional maturation of neonatal mouse cardiomyocytes and human embryonic stem cell-derived cardiomyocytes are considerably enhanced upon co-culture with corresponding adult cardiac fibroblasts. Further, single-cell analysis of in vivo and in vitro cardiomyocyte maturation trajectories identify highly conserved signaling pathways, pharmacological targeting of which substantially delays cardiomyocyte maturation in postnatal hearts, and markedly enhances cardiomyocyte proliferation and improves cardiac function in infarcted hearts. Together, we identify cardiac fibroblasts as a key constituent in the microenvironment promoting cardiomyocyte maturation, providing insights into how the manipulation of cardiomyocyte maturity may impact on disease development and regeneration.

Single-Cell Reconstruction of Progression Trajectory Reveals Intervention Principles in Pathological Cardiac Hypertrophy.

BACKGROUND: Pressure overload-induced pathological cardiac hypertrophy is a common predecessor of heart failure, the latter of which remains a major cardiovascular disease with increasing incidence and mortality worldwide. Current therapeutics typically involve partially relieving the heart's workload after the onset of heart failure. Thus, more pathogenesis-, stage-, and cell type-specific treatment strategies require refined dissection of the entire progression at the cellular and molecular levels. METHODS: By analyzing the transcriptomes of 11,492 single cells and identifying major cell types, including both cardiomyocytes and noncardiomyocytes, on the basis of their molecular signatures, at different stages during the progression of pressure overload-induced cardiac hypertrophy in a mouse model, we characterized the spatiotemporal interplay among cell types, and tested potential pharmacological treatment strategies to retard its progression in vivo. RESULTS: We illustrated the dynamics of all major cardiac cell types, including cardiomyocytes, endothelial cells, fibroblasts, and macrophages, as well as those of their respective subtypes, during the progression of disease. Cellular crosstalk analysis revealed stagewise utilization of specific noncardiomyocytes during the deterioration of heart function. Specifically, macrophage activation and subtype switching, a key event at middle-stage of cardiac hypertrophy, was successfully targeted by Dapagliflozin, a sodium glucose cotransporter 2 inhibitor, in clinical trials for patients with heart failure, as well as TD139 and Arglabin, two anti-inflammatory agents new to cardiac diseases, to preserve cardiac function and attenuate fibrosis. Similar molecular patterns of hypertrophy were also observed in human patient samples of hypertrophic cardiomyopathy and heart failure. CONCLUSIONS: Together, our study not only illustrated dynamically changing cell type crosstalk during pathological cardiac hypertrophy but also shed light on strategies for cell type- and stage-specific intervention in cardiac diseases.

Single-cell reconstruction of the adult human heart during heart failure and recovery reveals the cellular landscape underlying cardiac function.

Owing to the prevalence and high mortality rates of cardiac diseases, a more detailed characterization of the human heart is necessary; however, this has been largely impeded by the cellular diversity of cardiac tissue and limited access to samples. Here, we show transcriptome profiling of 21,422 single cells-including cardiomyocytes (CMs) and non-CMs (NCMs)-from normal, failed and partially recovered (left ventricular assist device treatment) adult human hearts. Comparative analysis of atrial and ventricular cells revealed pronounced inter- and intracompartmental CM heterogeneity as well as compartment-specific utilization of NCM cell types as major cell-communication hubs. Systematic analysis of cellular compositions and cell-cell interaction networks showed that CM contractility and metabolism are the most prominent aspects that are correlated with changes in heart function. We also uncovered active engagement of NCMs in regulating the behaviour of CMs, exemplified by ACKR1+-endothelial cells, injection of which preserved cardiac function after injury. Beyond serving as a rich resource, our study provides insights into cell-type-targeted intervention of heart diseases.

Single-cell reconstruction of differentiation trajectory reveals a critical role of ETS1 in human cardiac lineage commitment.

BACKGROUND: Cardiac differentiation from human pluripotent stem cells provides a unique opportunity to study human heart development in vitro and offers a potential cell source for cardiac regeneration. Compared to the large body of studies investigating cardiac maturation and cardiomyocyte subtype-specific induction, molecular events underlying cardiac lineage commitment from pluripotent stem cells at early stage remain poorly characterized. RESULTS: In order to uncover key molecular events and regulators controlling cardiac lineage commitment from a pluripotent state during differentiation, we performed single-cell RNA-Seq sequencing and obtained high-quality data for 6879 cells collected from 6 stages during cardiac differentiation from human embryonic stem cells and identified multiple cell subpopulations with distinct molecular features. Through constructing developmental trajectory of cardiac differentiation and putative ligand-receptor interactions, we revealed crosstalk between cardiac progenitor cells and endoderm cells, which could potentially provide a cellular microenvironment supporting cardiac lineage commitment at day 5. In addition, computational analyses of single-cell RNA-Seq data unveiled ETS1 (ETS Proto-Oncogene 1) activation as an important downstream event induced by crosstalk between cardiac progenitor cells and endoderm cells. Consistent with the findings from single-cell analysis, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) against ETS1 revealed genomic occupancy of ETS1 at cardiac structural genes at day 9 and day 14, whereas ETS1 depletion dramatically compromised cardiac differentiation. CONCLUSION: Together, our study not only characterized the molecular features of different cell types and identified ETS1 as a crucial factor induced by cell-cell crosstalk contributing to cardiac lineage commitment from a pluripotent state, but may also have important implications for understanding human heart development at early embryonic stage, as well as directed manipulation of cardiac differentiation in regenerative medicine.