Methylcellulose improves dissociation quality of adult human primary cardiomyocytes.

Obtaining high-quality adult human primary cardiomyocytes (hPCM) have been technically challenging due to isolation-induced biochemical and mechanical stress. Building upon a previous tissue slicing-assisted digestion method, we introduced polymers into the digestion solution to reduce mechanical damage to cells. We found that low-viscosity methylcellulose (MC) significantly improved hPCM viability and yield. Mechanistically, it protected cells from membrane damage, which led to decreased apoptosis and mitochondrial reactive oxygen species production. MC also improved the electrophysiological properties of hPCMs by maintaining the density of sodium channels. The effects on cell viability and cell yield effects were not recapitulated by MC of larger viscosities, other cellulose derivatives, nor shear protectants polyethylene glycol and polyvinyl alcohol. Finally, MC also enhanced the isolation efficiency and the culture quality of hPCMs from diseased ventricular myocardium, expanding its potential applications. Our findings showed that the isolation quality of hPCMs can be further improved through the addition of a polymer, rendering hPCMs a more reliable cellular model for cardiac research.

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.

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.

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.

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.

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.

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.