Cardiomyocyte Clusters Research Articles

Single cell multi-omics analysis unveils a distinct mechanism of exercise-mediated cardioprotection in ischemic cardiomyopathy

Abstract Background Exercise is pivotal in heart disease prevention, with proven cardioprotective effects in clinical trials. However, its precise molecular mechanisms remain elusive. Recent advancements in single-cell omics have revealed cellular heterogeneity within heart tissues, offering a deeper understanding of the pathophysiology of cardiac diseases. Purpose To investigate the exercise-induced physiological changes in heart tissue at the single-cell level and elucidate the molecular mechanisms underlying its cardioprotective effect on ischemic cardiomyopathy. Methods Murine myocardial infarction (MI) model was established by ligating the left ascending coronary artery, and a six-week voluntary wheel running exercise regimen was implemented to investigate the effect of aerobic exercise on ischemic cardiomyopathy. Integrated multi-omics analyses of single-cell DNA methylation profiling, single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq) and single-nucleus RNA sequencing (snRNA-seq) were conducted to clarify the molecular mechanism of exercise-mediated cardioprotection at single-cell resolution. Overexpression and short hairpin RNA-mediated knockdown of gene X were conducted for murine MI model using cardiomyocyte-specific Myo-AAV2a vector. snRNA-seq of heart tissues from heart failure patients was performed to validate the clinical significance of the expression of gene X in cardiomyocytes. Results Exercise attenuates pathological cardiac remodeling, inducing physiological hypertrophy in non-infarcted areas while suppressing pathological remodeling of cardiomyocytes in infarct border zones. Single-cell epigenetic analysis unveiled intricate molecular mechanisms underlying exercise-induced transcriptome changes, providing insights into the gene regulatory networks modulated by exercise in the heart. In addition, integrated with snRNA-seq, we identified exercise-specific cardiomyocyte clusters, highlighting the substantial role of gene X in the molecular mechanism of exercise. Functional studies confirmed the essential role of gene X in exercise-induced cardioprotection, as its overexpression suppressed the adverse remodeling after MI and knockdown abolished the beneficial effects of exercise. Furthermore, snRNA-seq of heart failure patients revealed that higher expression of gene X was associated with ventricular reverse remodeling and favorable clinical outcomes, underscoring its significance in the pathophysiology of patients with heart failure. Conclusion Our study elucidates exercise-specific gene expression regulation networks in ischemic cardiomyopathy by unraveling the cellular heterogeneity and molecular changes associated with exercise. We identified gene X as a central player in exercise-mediated cardioprotection and further confirmed its significance in clinical settings, providing a potential therapeutic target for heart failure.

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

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." 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). 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. 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.

An optimized protocol for routine development of cell culture from adult oyster, Crassostrea madrasensis.

Marine molluscan cell lines, required for virus screening and cultivation, form essential tools for developing health management strategies for these animals in the blue economy. Moreover, they are also crucial to develop cultivated seafood. As there is no valid marine molluscan cell line, primary cell cultures are relied upon for all investigations. A sound protocol for generating primary cell cultures from molluscs is entailed, but existing protocols often involve heavy antibiotic usage and depuration that invariably affect gene expression and cell health. This work presents an easy-to-adopt, time-saving protocol using non-depurated mollusc Crassostrea madrasensis, which requires only initial antibiotic treatment and minimal exposure or no use of antibiotics in the cell culture medium. The important experimental considerations for arriving at this protocol have been elucidated. Accordingly, sodium hypochlorite and neomycin sulfate were chosen for disinfecting tissues. The study is the first to use shrimp cell culture medium (SCCM) as a cell culture medium for molluscan cell culture. Despite being osmoconformers, the oysters exhibited stable intracellular osmotic conditions and pH, which, when provided in vitro, promoted effective cardiomyocyte formation. The cell viability could be enhanced using 10% fetal bovine serum (FBS), but healthy cell culture could also be obtained using SCCM without FBS. The optimized culture conditions allowed for regular beating cardiomyocyte clusters that could be retained for a month. Limited cell proliferation, as shown by the BrdU assay, demands further interventions, such as possibly producing induced pluripotent stem cells. The optimized protocol and culture conditions also align with some requirements for producing cultivated meat from marine molluscs.

Abstract 15500: Identifying Cardiomyocytes at Specific Cell-Cycle Stages by a New Cell-Cycle-Specific snRNAseq Analytic Method

Background: Deciphering the cardiomyocyte cell cycle is a critical question in studying heart development and regeneration. We already collected the single-nuclei RNA sequencing (snRNAseq) data from fetal pig hearts and from pigs that underwent apical resection on postnatal day (P) 1 (AR P1 ) followed by myocardial infarction (MI) on P28 (MI P28 ); in these hearts, robust cardiomyocyte proliferation was confirmed. Research questions: Although our previous snRNAseq analyses found cardiomyocyte clusters that primed to proliferate, each of these clusters only appeared at one specific time point; furthermore, these clusters still contain a significant amount of non-cycling cell; and thus, we were unable to distinguish between cardiomyocytes in different phases of the cell cycle. Approach: In this report, we designed a new Autoencoder that only utilized 1646 cell-cycle-specific genes to cluster cardiomyocytes and applied the Autoencoder on pig heart GEO datasets number GSE185289. Results: Five cardiomyocyte clusters (pCM1+2, pCM3-pCM6) were identified. Five proliferation markers (AURKB, MKI67, INCENP, CDCA8, and BRCA5) highly co-expressed in pCM1+2; also, the top enriched gene ontologies of pCM1+2 were all related to cell-cycle. PCM1+2 percentages were ~6% in fetal hearts and ~2-3% in hearts from ARP1MIP28 pigs during the two weeks after MI induction but were nearly undetectable in all other experimental groups and at all other time points. We also identified four transcription factors (E2F8, FOXM1, GLI3, and RAD51) that explicitly coexpressed within pCM1. Also, a pseudo-time trajectory was constructed in Fetal cardiomyocyte, which began at pCM3 (G1/S phase, CCND2 localization), passed through pCM2 (S phase, CCNE2 localization), and ended at pCM1 (M phase, CCNB2 localization). Conclusions: Applying the more precise cell-cycle specific Autoencoder, we found three clusters that likely represented cardiomyocytes at three different cell-cycle stages.

Analysis of cardiac single-cell RNA-sequencing data can be improved by the use of artificial-intelligence-based tools

Single-cell RNA sequencing (scRNAseq) enables researchers to identify and characterize populations and subpopulations of different cell types in hearts recovering from myocardial infarction (MI) by characterizing the transcriptomes in thousands of individual cells. However, the effectiveness of the currently available tools for processing and interpreting these immense datasets is limited. We incorporated three Artificial Intelligence (AI) techniques into a toolkit for evaluating scRNAseq data: AI Autoencoding separates data from different cell types and subpopulations of cell types (cluster analysis); AI Sparse Modeling identifies genes and signaling mechanisms that are differentially activated between subpopulations (pathway/gene set enrichment analysis), and AI Semisupervised Learning tracks the transformation of cells from one subpopulation into another (trajectory analysis). Autoencoding was often used in data denoising; yet, in our pipeline, Autoencoding was exclusively used for cell embedding and clustering. The performance of our AI scRNAseq toolkit and other highly cited non-AI tools was evaluated with three scRNAseq datasets obtained from the Gene Expression Omnibus database. Autoencoder was the only tool to identify differences between the cardiomyocyte subpopulations found in mice that underwent MI or sham-MI surgery on postnatal day (P) 1. Statistically significant differences between cardiomyocytes from P1-MI mice and mice that underwent MI on P8 were identified for six cell-cycle phases and five signaling pathways when the data were analyzed via Sparse Modeling, compared to just one cell-cycle phase and one pathway when the data were analyzed with non-AI techniques. Only Semisupervised Learning detected trajectories between the predominant cardiomyocyte clusters in hearts collected on P28 from pigs that underwent apical resection (AR) on P1, and on P30 from pigs that underwent AR on P1 and MI on P28. In another dataset, the pig scRNAseq data were collected after the injection of CCND2-overexpression Human-induced Pluripotent Stem Cell-derived cardiomyocytes (CCND2hiPSC) into injured P28 pig heart; only the AI-based technique could demonstrate that the host cardiomyocytes increase proliferating by through the HIPPO/YAP and MAPK signaling pathways. For the cluster, pathway/gene set enrichment, and trajectory analysis of scRNAseq datasets generated from studies of myocardial regeneration in mice and pigs, our AI-based toolkit identified results that non-AI techniques did not discover. These different results were validated and were important in explaining myocardial regeneration.

Cardiomyocyte Cell-Cycle Regulation in Neonatal Large Mammals: Single Nucleus RNA-Sequencing Data Analysis via an Artificial-Intelligence-Based Pipeline.

Adult mammalian cardiomyocytes have very limited capacity to proliferate and repair the myocardial infarction. However, when apical resection (AR) was performed in pig hearts on postnatal day (P) 1 (ARP1) and acute myocardial infarction (MI) was induced on P28 (MIP28), the animals recovered with no evidence of myocardial scarring or decline in contractile performance. Furthermore, the repair process appeared to be driven by cardiomyocyte proliferation, but the regulatory molecules that govern the ARP1-induced enhancement of myocardial recovery remain unclear. Single-nucleus RNA sequencing (snRNA-seq) data collected from fetal pig hearts and the hearts of pigs that underwent ARP1, MIP28, both ARP1 and MI, or neither myocardial injury were evaluated via autoencoder, cluster analysis, sparse learning, and semisupervised learning. Ten clusters of cardiomyocytes (CM1–CM10) were identified across all experimental groups and time points. CM1 was only observed in ARP1 hearts on P28 and was enriched for the expression of T-box transcription factors 5 and 20 (TBX5 and TBX20, respectively), Erb-B2 receptor tyrosine kinase 4 (ERBB4), and G Protein-Coupled Receptor Kinase 5 (GRK5), as well as genes associated with the proliferation and growth of cardiac muscle. CM1 cardiomyocytes also highly expressed genes for glycolysis while lowly expressed genes for adrenergic signaling, which suggested that CM1 were immature cardiomyocytes. Thus, we have identified a cluster of cardiomyocytes, CM1, in neonatal pig hearts that appeared to be generated in response to AR injury on P1 and may have been primed for activation of CM cell-cycle activation and proliferation by the upregulation of TBX5, TBX20, ERBB4, and GRK5.

HESC derived cardiomyocyte biosensor to detect the different types of arrhythmogenic properties of drugs

In the present work, we introduce a new cell-based biosensor for detecting arrhythmias based on a novel utilization of the combination of the Atomic Force Microscope (AFM) lateral force measurement as a nanosensor with a dual 3D cardiomyocyte syncytium. Two spontaneously coupled clusters of cardiomyocytes form this.The syncytium's functional contraction behavior was assessed using video sequences analyzed with Musclemotion ImageJ/Fiji software, and immunocytochemistry evaluated phenotype composition.The application of caffeine solution induced arrhythmia as a model drug, and its spontaneous resolution was monitored by AFM lateral force recording and interpretation and calcium fluorescence imaging as a reference method describing non-synchronized contractions of cardiomyocytes.The phenotypic analysis revealed the syncytium as a functional contractile and conduction cardiac behavior model. Calcium fluorescence imaging was used to validate that AFM fully enabled to discriminate cardiac arrhythmias in this in vitro cellular model. The described novel 3D hESCs-based cellular biosensor is suitable to detect arrhythmic events on the level of cardiac contractile and conduction tissue cellular model. The resulting biosensor allows for screening of arrhythmogenic properties of tailored drugs enabling its use in precision medicine.

Abstract 9528: Apical Resection in Newborn Pig Hearts Extends the Regenerative Window of Cardiomyocyte

Background: The neonatal porcine heart possesses proliferative cardiomyocyte capacity to recover heart function and structure after an acute myocardial injury during two days of birth. In this study, we have extended the myocardial regenerative window to postnatal day 28 (P28) by a double injury pig model (AR P1 MI P28 ), which performed apical resection at P1 (AR P1 ) followed by LAD ligation at P28 (MI P28 ). However, the molecular mechanism of regenerative window extension is unknown. Methods: The AR P1 MI P28 hearts showed no visible sign of myocardial damage or malfunction 4 weeks after LAD ligation. The nuclear RNA contents were isolated from hearts that received none, singe, and double injury hearts. They performed single nuclei RNA-seq to characterize the gene-expression profile of the AR P1 MI P28 in cardiomyocytes. Results: Gene-expression profiles revealed that regenerating AR P1 MI P28 hearts contained different subpopulations of cardiomyocytes early after 2 nd injury. Louvain clustering identified the six cardiomyocyte clusters (CM1-CM6). The CM6, CM3, and CM2 clusters have mainly comprised fetal (92.4%), CTL-P1 (95.7%), and CTL-P56 (96.2%) cardiomyocytes. In contrast, the CM1 cluster has comprised heterogeneous cardiomyocyte groups from all other animal groups, including less than 1% of fetal and CTL-P1 cardiomyocytes. The CM5 and CM4 clusters have mainly comprised AR P1 MI P28 -P35 (double injury hearts harvested at P35) cardiomyocytes, 97.8% and 68.6%, respectively. Sparse modeling analysis revealed that AR and MI injury, both alone and in combination, appeared to extend the regenerative capacity of cardiomyocytes and perturb cardiomyocyte maturation. Conclusion: We have demonstrated that the unique CM5 cluster appeared only after the 2 nd injury of AR P1 MI P28 -P35 hearts. This cell cluster was characterized by a highly expressed isoenzyme of the glycolytic enzyme pyruvate kinase M2 (Pkm2) and skeletal muscle filamentous gene Nebulin. Transient expression of the CM5 cluster induces by the AR P1 might contribute to the sustained remuscularization and myocardial function recovery following an acute MI in large mammals.

Abstract MP227: Single Cell RNA Sequencing Analysis Reveals Insulin Signaling Defects In Cardiomyocytes Derived From A Novel Mouse Model Of Heart Failure With Preserved Ejection Fraction

Heart failure with preserved ejection fraction (HFpEF) is an emerging form of heart failure worldwide with no effective therapies in contrast with heart failure with reserved ejection fraction (HFrEF). To simulate multiple risk-factors associated with HFpEF in clinic, we developed a HFpEF mouse model by introducing cardiac hypertrophy with transverse aortic constriction (TAC) in ObOb ( Lep ob/ob ) mice, which has intrinsic systemic metabolic dysfunctions including obesity and insulin resistance. We first validated pathological changes in diastolic but not systolic parameters in the Ob-TAC vs. Ob-sham mice up to 10 weeks post-TAC by echocardiography. To evaluate the global transcriptome change in difference cell types, we conducted single nuclei RNA sequencing (snRNA-seq) from whole hearts of lean mice (c57), ObOb, and Ob-TAC mice (male only). 10x genomic 3’ GEM kit was used to generate the cDNA library and sequencing was done by Novaseq SP platform. A total of 13k nuclei were recovered from QC, nFeature RNA (&lt 2500) and mitochondrial gene (&lt 5%) filtering. By UMAP dimension reduction analysis, we annotated major cardiac cell types in the integrated snRNA-seq dataset, including 3 clusters of Cardiomyocytes (CMs). By pathway analysis of the differentially expressed genes in each CM clusters, we found that insulin resistance and glucagon pathway were enriched among the up regulated genes in CMs in HFpEF vs. lean control, while cell migration, signal transduction including insulin substrates were down regulated. Thus, we hypothesized that the altered crosstalk between glucagon and insulin signaling might contribute to the development of HFpEF in this mouse modal. This hypothesis was validated in a proof-of-concept study showing significant improvement of HFpEF features by inhibiting the glucagon receptors post-TAC with injection of a glucagon receptor antagonist.

Emergent synchronous beating behavior in spontaneous beating cardiomyocyte clusters

We investigated the dominant rule determining synchronization of beating intervals of cardiomyocytes after the clustering of mouse primary and human embryonic-stem-cell (hES)-derived cardiomyocytes. Cardiomyocyte clusters were formed in concave agarose cultivation chambers and their beating intervals were compared with those of dispersed isolated single cells. Distribution analysis revealed that the clusters’ synchronized interbeat intervals (IBIs) were longer than the majority of those of isolated single cells, which is against the conventional faster firing regulation or “overdrive suppression.” IBI distribution of the isolated individual cardiomyocytes acquired from the beating clusters also confirmed that the clusters’ IBI was longer than those of the majority of constituent cardiomyocytes. In the complementary experiment in which cell clusters were connected together and then separated again, two cardiomyocyte clusters having different IBIs were attached and synchronized to the longer IBIs than those of the two clusters’ original IBIs, and recovered to shorter IBIs after their separation. This is not only against overdrive suppression but also mathematical synchronization models, such as the Kuramoto model, in which synchronized beating becomes intermediate between the two clusters’ IBIs. These results suggest that emergent slower synchronous beating occurred in homogeneous cardiomyocyte clusters as a community effect of spontaneously beating cells.

Effects of docosahexaenoic acid or arachidonic acid supplementation on gene expression and contractile force of rat cardiomyocytes in primary culture

While fatty acids play essential roles in the physiology of the myocardium, conventional culture media contain little lipid. We previously revealed that rat neonatal myocardium mainly contains docosahexaenoic (DHA), linoleic (LA), and arachidonic (AA) acids as polyunsaturated fatty acids (PUFAs), and these contents in cultured cardiomyocytes derived from fetal rats were markedly lower than those in the neonatal myocardium. In this study, we first assessed the effects of supplementation of DHA, LA, or AA on the fatty acid contents and the percentage change of contractile area in primarily cultured rat cardiomyocytes. Based on this assessment, we then evaluated the effects of DHA or AA supplementation on mRNA expression and further directly measured the contractile force of cardiomyocytes with the supplementations. This study revealed that percentage change of contractile area was maximized under 20 μM DHA or 50 μM AA supplementation while LA supplementation did not affect this contraction index, and that a widespread upregulation tendency of the mRNA expression related to differentiation, maturity, fatty acid metabolism, and cell adhesion was seen in the cultured cardiomyocytes with supplementation of DHA or AA. In particular, upregulation of the gene expression of cellular adhesion molecules connexin43 and N-cadherin were remarkable, whereas the effects on differentiation and maturation were less pronounced. Correspondingly, the increase of the percentage change of the contractile area of cardiomyocyte clusters in culture dishes with the supplementations was significant, whereas the enhancement of the contractile force was modest. These results suggest that supplementation of DHA or AA to the fetal cardiomyocyte culture may play effective roles in preventing the de-differentiation of the cardiomyocytes in culture and that the enhancement of the contractile performance may be mainly attributed to the improvement of intercellular connection.

Abstract 17402: Single Nuclei RNA-sequencing of Human Hypertrophic Cardiomyopathy Myectomy Samples Reveals Common Novel Mechanisms of Pathogenesis and Potential Therapeutic Targets Regardless of Genotype

Introduction: Hypertrophic cardiomyopathy (HCM) is a common inherited cardiovascular disease, often resulting in left ventricular outflow tract obstruction, relieved by surgical myectomy. Current treatments are largely palliative and do not target the root causes. Understanding the molecular drivers of the disease could lead to alternative treatment strategies through identification of novel therapeutic targets. Methods: We performed single nuclei RNA-sequencing (snRNA-seq) on thousands of nuclei from 9 patient myectomy samples and septal tissue from 4 unused donor hearts selected randomly without regard to genotype to identify the cell populations and determine the gene expression patterns in individual cells. Each sample was processed individually using Seurat v3 for quality control and normalization. Next, all 13 samples were integrated into a combined dataset for clustering and differential gene expression analysis to identify markers specific to each cluster and to assign cell identities. Results: Our results revealed several clusters of cardiomyocytes with differences in sarcomeric and metabolic gene expression. Several fibroblast populations were also observed. Numerous genes were differentially expressed between the HCM and normal samples. For example, RARRES1 expression was observed in many of the fibroblast populations in the normal samples, but was absent in the HCM samples. RARRES1 is involved in regulating fatty acid metabolism and autophagy, both of which are altered in HCM. Additionally, expression of PLA2G2A was absent in the HCM samples but was present in almost every cell type in the normal controls. PLA2G2A is involved in suppression of RTK mediated hypertrophic signaling, impacts lipid signaling, and has tumor suppressor properties. Thus, both RARRES1 and PLA2G2A may represent novel targets in HCM. Conclusions: This approach reveals novel potential therapeutic targets within common final HCM pathological pathways independent of genotype that have the potential to guide development of alternative treatment strategies. Further analysis of larger datasets using this approach will likely identify even more common pathway targets and identify additional common mechanisms in the pathogenesis of obstructive HCM.

RNA‐Binding Protein QKI is a critical pre‐RNA splicing regulator for cardiac development and function

RNA‐binding protein QKI (also known as Quaking) belongs to hnRNP K‐homology (KH)‐domain protein family and is implicated a novel regulator in pre‐RNA splicing. As previously shown, the QKI homolog how potentially regulates heart lumen formation and function in fruit fly. However, QKI’s function in mammalian heart development is largely unknown. We found that Qki specifically expressed in mouse developing hearts as early as E7.0 and this high‐level expression was maintained in the hearts to adulthood. Consistent with this finding, by using human embryonic stem cells (hESCs)‐cardiomyocyte differentiation system, QKI expression was found up‐regulated at Day 6, a critical time frame that cardiogenic progenitor transition into early cardiomyocytes. To determine the biological function of QKI in cardiogenesis, we generated hESCsQKIdef by using CRISPR/Cas9 gene editing technology and analyzed the physiological impact of QKI‐deficiency to cardiomyocyte differentiation and maturation. As expected, we did not find defect in pluripotency in hESCsQKIdef, but the contractile function was dramatically reduced in hESCsQKIdef‐derived cardiomyocytes. Based on scRNA‐seq and clustering analyses, there was no significant difference in the distribution of the clusters when compared hESCsQKIdef‐cardiomyocyte group to normal control group, suggesting that the cardiomyocyte differentiation was largely unaffected in hESCsQKIdef. However, scRNA‐seq demonstrated a handful of genes encoding sarcomere proteins were significantly altered in cardiomyocyte clusters, which included ACTN2, ACTA1, MYH7, and MYL2, etc., indicating a significant defect in cardiomyocyte maturation. Bulk RNA‐seq data not only further confirmed this notion, but also provided additional depth for detailed analysis on the QKI‐mediated pre‐RNA splicing events. In conjunction with QKI‐binding motif search and RNA immunoprecipitation (RIP) analysis, we were able to demonstrate that QKI was a key pre‐RNA splicing regulator for sets of genes involved in cardiac myofibrillogenesis and cardiac excitation‐contraction (E‐C) coupling, which ultimately impacted on cardiomyocyte maturation and function. Additional analysis of cardiac developmental defects in Qkibgeo mutant mice further validated that QKI was an indispensable splicing regulator in cardiovascular development. Our results not only revealed a novel mechanism by which QKI regulates pre‐mRNA splicing in cardiogenesis, but also implied that QKI was likely involved in the pathogenesis of certain forms of congenital heart defects and cardiomyopathies.Support or Funding InformationThis work was supported in part by National Institute of Health P01HL134599

Design and characterization of an electroconductive scaffold for cardiomyocytes based biomedical assays.

Cardiovascular diseases (CVD) are a major cause of mortality worldwide. Accessibility to heart tissue is limited due to sampling issues and lack of appropriate culture conditions. In addition, animal models are not an ideal choice for physiological, pharmacological, and fundamental evaluations in the cardiovascular field due to interspecies differences. Hence, there is an inevitable need for functional in vitro cardiac models. In this study, we have synthesized a novel electroconductive scaffold comprised of cardiac extracellular matrix (ECM) derived pre-cardiogel (pCG) blended with polypyrrole (Ppy). Our data revealed that 2.5% (w/v) pyrrole (Py) had the highest possible Py ratio that provided pCG-Ppy gel formation. The prepared mixture was fabricated into a scaffold by using the freeze-dried method. The scaffolds had open interconnected pores that ranged from 55±24μm for the cardiogel (CG)-Ppy to 74±26μm for the CG scaffolds, with no alterations in vital ECM components of collagen, polysaccharides, and glycosaminoglycans (GAGs). Incorporation of Ppy increased the CG stiffness with a final complex modulus from 80 pa to 140 pa. The CG-Ppy group had significantly greater electrical conductivity than the CG group. Scaffolds supported neonatal mouse cardiomyocyte (NMCM) adhesion, viability, cardiac-specific gene expression, and spontaneous beating up to 14days after seeding. Among the fabricated hydrogels, the CG-Ppy group resulted in the synchronous beating of cardiomyocyte clusters and upregulation of cardiac genes involved in cardiac muscle contraction (cardiac troponin T [cTNT]) and cardiomyocyte electrical coupling (connexin 43 [Cx43]). Thus, this ECM-based electro-conductive scaffold might provide a promising substrate for constructing in vitro cardiac models for drug testing, disease modeling, developmental studies, and cardiac regenerative approaches.

Enterovirus Persistence in Cardiac Cells of Patients With Idiopathic Dilated Cardiomyopathy Is Linked to 5’ Terminal Genomic RNA-Deleted Viral Populations With Viral-Encoded Proteinase Activities

Group B enteroviruses are common causes of acute myocarditis, which can be a precursor of chronic myocarditis and dilated cardiomyopathy, leading causes of heart transplantation. To date, the specific viral functions involved in the development of dilated cardiomyopathy remain unclear. Total RNA from cardiac tissue of patients with dilated cardiomyopathy was extracted, and sequences corresponding to the 5' termini of enterovirus RNAs were identified. After next-generation RNA sequencing, viral cDNA clones mimicking the enterovirus RNA sequences found in patient tissues were generated in vitro, and their replication and impact on host cell functions were assessed on primary human cardiac cells in culture. Major enterovirus B populations characterized by 5' terminal genomic RNA deletions ranging from 17 to 50 nucleotides were identified either alone or associated with low proportions of intact 5' genomic termini. In situ hybridization and immunohistological assays detected these persistent genomes in clusters of cardiomyocytes. Transfection of viral RNA into primary human cardiomyocytes demonstrated that deleted forms of genomic RNAs displayed early replication activities in the absence of detectable viral plaque formation, whereas mixed deleted and complete forms generated particles capable of inducing cytopathic effects at levels distinct from those observed with full-length forms alone. Moreover, deleted or full-length and mixed forms of viral RNA were capable of directing translation and production of proteolytically active viral proteinase 2A in human cardiomyocytes. We demonstrate that persistent viral forms are composed of B-type enteroviruses harboring a 5' terminal deletion in their genomic RNAs and that these viruses alone or associated with full-length populations of helper RNAs could impair cardiomyocyte functions by the proteolytic activity of viral proteinase 2A in cases of unexplained dilated cardiomyopathy. These results provide a better understanding of the molecular mechanisms that underlie the persistence of EV forms in human cardiac tissues and should stimulate the development of new therapeutic strategies based on specific inhibitors of the coxsackievirus B proteinase 2A activity for acute and chronic cardiac infections.

Biodegradable Self-Powered Electronics

Electronics used for transient implantable medical devices have some specific requirements that are more restrictive than for other applications, such as high reliability, controllable lifetime, biocompatibility or biodegradability [1]. However, the internal heat, capacity loss, and battery failure are the common problems of traditional power sources for the electronics. Meanwhile, the it requires second surgery to remove the devices after the accomplishment of their work cycles. In this context, electronic systems entirely built with biodegradable materials and self-powered sources are of growing interest for their potential applications in systems that can be integrated with living tissue and used for diagnostic and/or therapeutic purposes during certain physiological processes. TENGs and PENGs have superior performance in converting biomechanical energy and could convert electrical energy in cardiac pacemaker, health monitoring, cell or tissue engineering. Therefore, several works have been done studying degradable self-powered electronics for biomedical engineering in our group [2-4]. In 2016, Zheng et al. reported the first biodegradable triboelectric nanogenerator (BD-TENG) for short-term in vivo biomechanical energy conversion. Enabled by the design of a multilayer structure that is composed of biodegradable polymers (BDPs) and resorbable metals, the BD-TENG can be degraded and resorbed in an animal body after completing its work cycle without any adverse long-term effects. It is demonstrated the potential of BD-TENG as a power source for transient implantable medical devices. When applying BD-TENG to power two complementary micrograting electrodes, a DC-pulsed electric field (EF) was generated (1 Hz, 10 V/mm), and the nerve cell growth was successfully orientated, which was crucial for neural repair. Soon afterwards, Jiang et al. developed a fully bioabsorbable BN-TENGs in vivo using natural materials. It provided a new design idea for TENG and other energy harvesters using natural materials. The operation time of BN-TENG in vivo and in vitro could be modulated from days to weeks by modification of silk fiber encapsulation film. Using the proposed BN-TENG as a voltage source, the beating rates of dysfunctional cardiomyocyte clusters are accelerated, and the consistency of cell contraction is improved. This provides a new and valid solution to treat some heart diseases such as bradycardia and arrhythmia. Immediately after that, we fabricated a serial of biodegradable (BD) iTENGs and effectively tuned their degradation process in vivo by employing Au nanorods (AuNRs), which responded to the near-infrared (NIR) light sensitively. The degradation of our BD-iTENG could be triggered and come into effect very quickly with rational manipulation. Moreover, the output voltage could be applied on fibroblast cells and significantly accelerate cell migration across the scratch, which was very beneficial to wound healing process. In summary, we have fabricated biodegradable self-powered electronics which could harvest biomechanical energy and convert electrical energy into biomedical engineering, such as cardiac pacemaker, health monitoring and cell or tissue engineering.

Biodegradable Self-Powered Electronics and Application in Biomedical Engineering

Electronics used for transient implantable medical devices have some specific requirements that are more restrictive than for other applications, such as high reliability, controllable lifetime, biocompatibility or biodegradability [1]. However, the internal heat, capacity loss, and battery failure are the common problems of traditional power sources for the electronics. Meanwhile, the it requires second surgery to remove the devices after the accomplishment of their work cycles. In this context, electronic systems entirely built with biodegradable materials and self-powered sources are of growing interest for their potential applications in systems that can be integrated with living tissue and used for diagnostic and/or therapeutic purposes during certain physiological processes. TENGs and PENGs have superior performance in converting biomechanical energy and could convert electrical energy in cardiac pacemaker, health monitoring, cell or tissue engineering. Therefore, several works have been done studying degradable self-powered electronics for biomedical engineering in our group [2-4]. In 2016, Zheng et al. reported the first biodegradable triboelectric nanogenerator (BD-TENG) for short-term in vivo biomechanical energy conversion. Enabled by the design of a multilayer structure that is composed of biodegradable polymers (BDPs) and resorbable metals, the BD-TENG can be degraded and resorbed in an animal body after completing its work cycle without any adverse long-term effects. It is demonstrated the potential of BD-TENG as a power source for transient implantable medical devices. When applying BD-TENG to power two complementary micrograting electrodes, a DC-pulsed electric field (EF) was generated (1 Hz, 10 V/mm), and the nerve cell growth was successfully orientated, which was crucial for neural repair. Soon afterwards, Jiang et al. developed a fully bioabsorbable BN-TENGs in vivo using natural materials. It provided a new design idea for TENG and other energy harvesters using natural materials. The operation time of BN-TENG in vivo and in vitro could be modulated from days to weeks by modification of silk fiber encapsulation film. Using the proposed BN-TENG as a voltage source, the beating rates of dysfunctional cardiomyocyte clusters are accelerated, and the consistency of cell contraction is improved. This provides a new and valid solution to treat some heart diseases such as bradycardia and arrhythmia. Immediately after that, we fabricated a serial of biodegradable (BD) iTENGs and effectively tuned their degradation process in vivo by employing Au nanorods (AuNRs), which responded to the near-infrared (NIR) light sensitively. The degradation of our BD-iTENG could be triggered and come into effect very quickly with rational manipulation. Moreover, the output voltage could be applied on fibroblast cells and significantly accelerate cell migration across the scratch, which was very beneficial to wound healing process. In summary, we have fabricated biodegradable self-powered electronics which could harvest biomechanical energy and convert electrical energy into biomedical engineering, such as cardiac pacemaker, health monitoring and cell or tissue engineering.

Evidence for the impact of BAG3 on electrophysiological activity of primary culture of neonatal cardiomyocytes.

Homeostasis of proteins involved in contractility of individual cardiomyocytes and those coupling adjacent cells is of critical importance as any abnormalities in cardiac electrical conduction may result in cardiac irregular activity and heart failure. Bcl2-associated athanogene 3 (BAG3) is a stress-induced protein whose role in stabilizing myofibril proteins as well as protein quality control pathways, especially in the cardiac tissue, has captured much attention. Mutations of BAG3 have been implicated in the pathogenesis of cardiac complications such as dilated cardiomyopathy. In this study, we have used an in vitro model of neonatal rat ventricular cardiomyocytes to investigate potential impacts of BAG3 on electrophysiological activity by employing the microelectrode array (MEA) technology. Our MEA data showed that BAG3 plays an important role in the cardiac signal generation as reduced levels of BAG3 led to lower signal frequency and amplitude. Our analysis also revealed that BAG3 is essential to the signal propagation throughout the myocardium, as the MEA data-based conduction velocity, connectivity degree, activation time, and synchrony were adversely affected by BAG3 knockdown. Moreover, BAG3 deficiency was demonstrated to be connected with the emergence of independently beating clusters of cardiomyocytes. On the other hand, BAG3 overexpression improved the activity of cardiomyocytes in terms of electrical signal amplitude and connectivity degree. Overall, by providing more in-depth analyses and characterization of electrophysiological parameters, this study reveals that BAG3 is of critical importance for electrical activity of neonatal cardiomyocytes.

A microfluidic device for noninvasive cell electrical stimulation and extracellular field potential analysis.

We developed a device that can quickly apply versatile electrical stimulation (ES) signals to cells suspended in microfluidic channels and measure extracellular field potential simultaneously. The device could trap cells onto the surface of measurement electrodes for ES and push them to the downstream channel after ES by increasing pressure for continuous measurement. Cardiomyocytes, major functional cells in heart, together with human fibroblast cells and human umbilical vein endothelial cells, were tested with the device. Extracellular field potential signals generated from the cells were recorded. We found that under electrical stimulation, cardiomyocytes were triggered to alter their field potential, while non-excitable cells were not triggered. Hence this device can noninvasively distinguish electrically excitable cells from electrically non-excitable cells. Results have also shown that increased cardiomyocyte cell number led to increased magnitude and occurrence of the cell responses. This relationship could be used to detect the viable cells in a cardiac tissue. Application of variable ES signals on different cardiomyocyte clusters has shown that the application of ES clearly boosted cardiomyocytes electrical activities according to the stimulation frequency. In addition, we confirmed that the device can apply ES onto and detect the electrical responses from a mixed cell cluster; the responses from the mixed cluster is dependent on the ratio of cardiomyocytes. These results demonstrated that our device could be used as a tool to optimize ES conditions to facilitate the functional engineered cardiac tissue development.

Probing Collective Mechanoadaptation in Cardiomyocyte Development by Plasma Lithography Patterned Elastomeric Substrates.

Understanding how the mechanical microenvironment affects cardiomyocyte development is crucial to the creation of in vitro models for studying heart physiology and pathophysiology. This knowledge will also facilitate the design of biomaterials and tissue scaffolds utilized in the generation of functional tissue constructs for regenerative medicine and drug screening applications. Here, plasma lithography patterning of elastomeric substrates is exploited for creating microtissues composed of neonatal cardiomyocytes and investigating their attributes in different mechanical microenvironments. Restriction of the cellular outgrowth in line patterns results in cardiomyocytes developing into multicellular clusters and collectively adapting to geometric confinement and substrate stiffness. Immunofluorescence microscopy, video microscopy, and force spectroscopy show that the size and shape of the cardiomyocyte clusters, as well as sarcomere length, fiber alignment, beating amplitude, and beating frequency of the cardiomyocytes, are regulated by the microenvironmental cues. Computational analysis reveals that the mechanical stress at the cluster-substrate interface strongly correlates with the characteristics of the cardiomyocytes. Taken together, our results underscore a collective mechanoadaptation scheme in cardiac development.