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.

TBX3 transfection and nodal signal pathway inhibition promote differentiation of adipose mesenchymal stem cell to cardiac pacemaker-like cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are known as one of the best candidate cells to produce cardiac pacemaker-like cells (CPLCs). Upregulation of TBX3 transcription factor and inhibition of the nodal signal pathway have a significant role in the formation of cardiac pacemaker cells such as sinoatrial and atrioventricular nodes, which initiate the heartbeat and control the rhythm of heart contractions. This study aimed to confirm the effects of transfection of TBX3 transcription factor and inhibition of the nodal signal pathway on differentiating adipose-derived MSCs (AD-MSCs) to CPLCs. AD-MSCs were characterized using flow cytometry and three-lineage differentiation staining. METHODS: The transfection of TBX3 plasmid was carried out using lipofectamine, and inhibition of the nodal signal pathway was done using the small-molecule SB431542. The morphology of the cells was observed using a light microscope. Pacemaker-specific markers, including TBX3, Cx30, HCN4, HCN1, HCN3, and KCNN4, were evaluated using the qRT-PCR method. For protein level, TBX3 and Cx30 were evaluated using ELISA and immunofluorescence staining. The electrophysiology of cells was evaluated using a patch clamp. RESULTS: The TBX3 expression in the TBX3, SM, and TBX + SM groups significantly higher (p < 0.05) compared to the control group and cardiomyocytes. The expression of Cx40 and Cx43 genes were lower in TBX3, SM, TBX + SM groups. In contrast, Cx30 gene showed higher expression in TBX3 group. The expression HCN1, HCN3, and HCN4 genes are higher in TBX3 group. CONCLUSION: The transfection of TBX3 and inhibition of the nodal signal pathway by small-molecule SB431542 enhanced differentiation of AD-MSCs to CPLCs.

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.

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.

Advanced cell and gene therapies in cardiology.

We review the evidence for the presence of stem/progenitor cells in the heart and the preclinical and clinical data using diverse cell types for the therapy of cardiac diseases. We highlight the failure of adult stem/progenitor cells to ameliorate heart function in most cardiac diseases, with the possible exception of refractory angina. The use of pluripotent stem cell-derived cardiomyocytes is analysed as a viable alternative therapeutic option but still needs further research at preclinical and clinical stages. We also discuss the use of direct reprogramming of cardiac fibroblasts into cardiomyocytes and the use of extracellular vesicles as therapeutic agents in ischemic and non-ischemic cardiac diseases. Finally, gene therapies and genome editing for the treatment of hereditary cardiac diseases, ablation of genes responsible for atherosclerotic disease, or modulation of gene expression in the heart are discussed.

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.

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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.

Selective Induction of Molecular Assembly to Tissue-Level Anisotropy on Peptide-Based Optoelectronic Cardiac Biointerfaces.

The conduction efficiency of ions in excitable tissues and of charged species in organic conjugated materials both benefit from having ordered domains and anisotropic pathways. In this study, a photocurrent-generating cardiac biointerface is presented, particularly for investigating the sensitivity of cardiomyocytes to geometrically comply to biomacromolecular cues differentially assembled on a conductive nanogrooved substrate. Through a polymeric surface-templated approach, photoconductive substrates with symmetric peptide-quaterthiophene (4T)-peptide units assembled as 1D nanostructures on nanoimprinted polyalkylthiophene (P3HT) surface are developed. The 4T-based peptides studied here can form 1D nanostructures on prepatterned polyalkylthiophene substrates, as directed by hydrogen bonding, aromatic interactions between 4T and P3HT, and physical confinement on the nanogrooves. It is observed that smaller 4T-peptide units that can achieve a higher degree of assembly order within the polymeric templates serve as a more efficient driver of cardiac cytoskeletal anisotropy than merely presenting aligned -RGD bioadhesive epitopes on a nanotopographic surface. These results unravel some insights on how cardiomyocytes perceive submicrometer dimensionality, local molecular order, and characteristics of surface cues in their immediate environment. Overall, the work offers a cardiac patterning platform that presents the possibility of a gene modification-free cardiac photostimulation approach while controlling the conduction directionality of the biotic and abiotic components.

The effect of MiR-497 on the expression of target genes BCL-2 and LC3B on cardiomyocytes injured by hypoxia/reoxygenation.

Myocardial ischemia is easy to cause hypoxia or necrosis of myocardial cells. At present, the performance of various patients is different. Basically, it is mainly caused by chest pain or chest discomfort. Severe patients may die suddenly. Therefore, looking for effective drugs or methods to prevent and treat Cardiomyocyte injury is of great significance for clinical practice, in which the expression of regulatory gene BCL-2 and microtubule-associated protein light chain 3B (LC3B) has a certain effect on hypoxia/reoxygenation injured cardiomyocytes. To this end, the team designed a study on the effect of miR-497 on the expression of target genes BCL-2 and LC3B on cardiomyocytes injured by hypoxia/reoxygenation. In this study, a control group experiment was set up for the study. During the experiment, the cells were treated with hypoxia-reoxygenation and transfected with the corresponding miR-497 treated cells. By detecting apoptosis, the kit was used to detect cell activity and RT-PCR detection. Gene expression levels and other methods are comparatively judged. The results of this study showed that compared with the normal group 14.50±0.78, the viability of cardiomyocytes in the model group was significantly reduced (P<0.01), the amount of NO released by cardiomyocytes was reduced (P<0.01), and the protein expression in cardiomyocytes was significantly reduced (P<0.01). The experimental results of this study prove that miR-497 can alleviate the damage caused by hypoxia-reoxygenation of cardiomyocytes by regulating target genes BCL-2 and LC3B.

WDR62 variants contribute to congenital heart disease by inhibiting cardiomyocyte proliferation.

BACKGROUND: Congenital heart disease (CHD) is the most common birth defect and has high heritability. Although some susceptibility genes have been identified, the genetic basis underlying the majority of CHD cases is still undefined. METHODS: A total of 1320 unrelated CHD patients were enrolled in our study. Exome-wide association analysis between 37 tetralogy of Fallot (TOF) patients and 208 Han Chinese controls from the 1000 Genomes Project was performed to identify the novel candidate gene WD repeat-containing protein 62 (WDR62). WDR62 variants were searched in another expanded set of 200 TOF patients by Sanger sequencing. Rescue experiments in zebrafish were conducted to observe the effects of WDR62 variants. The roles of WDR62 in heart development were examined in mouse models with Wdr62 deficiency. WDR62 variants were investigated in an additional 1083 CHD patients with similar heart phenotypes to knockout mice by multiplex PCR-targeting sequencing. The cellular phenotypes of WDR62 deficiency and variants were tested in cardiomyocytes, and the molecular mechanisms were preliminarily explored by RNA-seq and co-immunoprecipitation. RESULTS: Seven WDR62 coding variants were identified in the 237 TOF patients and all were indicated to be loss of function variants. A total of 25 coding and 22 non-coding WDR62 variants were identified in 80 (6%) of the 1320 CHD cases sequenced, with a higher proportion of WDR62 variation (8%) found in the ventricular septal defect (VSD) cohort. WDR62 deficiency resulted in a series of heart defects affecting the outflow tract and right ventricle in mouse models, including VSD as the major abnormality. Cell cycle arrest and an increased number of cells with multipolar spindles that inhibited proliferation were observed in cardiomyocytes with variants or knockdown of WDR62. WDR62 deficiency weakened the association between WDR62 and the cell cycle-regulated kinase AURKA on spindle poles, reduced the phosphorylation of AURKA, and decreased expression of target genes related to cell cycle and spindle assembly shared by WDR62 and AURKA. CONCLUSIONS: WDR62 was identified as a novel susceptibility gene for CHD with high variant frequency. WDR62 was shown to participate in the cardiac development by affecting spindle assembly and cell cycle pathway in cardiomyocytes.

Heart regeneration: 20 years of progress and renewed optimism.

Cardiovascular disease is a leading cause of death worldwide, and thus there remains great interest in regenerative approaches to treat heart failure. In the past 20 years, the field of heart regeneration has entered a renaissance period with remarkable progress in the understanding of endogenous heart regeneration, stem cell differentiation for exogenous cell therapy, and cell-delivery methods. In this review, we highlight how this new understanding can lead to viable strategies for human therapy. For the near term, drugs, electrical and mechanical devices, and heart transplantation will remain mainstays of cardiac therapies, but eventually regenerative therapies based on fundamental regenerative biology may offer more permanent solutions for patients with heart failure.

Lymphangiogenesis contributes to exercise-induced physiological cardiac growth.

BACKGROUND: Promoting cardiac lymphangiogenesis exerts beneficial effects for the heart. Exercise can induce physiological cardiac growth with cardiomyocyte hypertrophy and increased proliferation markers in cardiomyocytes. However, it remains unclear whether and how lymphangiogenesis contributes to exercise-induced physiological cardiac growth. We aimed to investigate the role and mechanism of lymphangiogenesis in exercise-induced physiological cardiac growth. METHODS: Adult C57BL6/J mice were subjected to 3 weeks of swimming exercise to induce physiological cardiac growth. Oral treatment with vascular endothelial growth factor receptor 3 (VEGFR3) inhibitor SAR131675 was used to investigate whether cardiac lymphangiogenesis was required for exercise-induced physiological cardiac growth by VEGFR3 activation. Furthermore, human dermal lymphatic endothelial cell (LEC)-conditioned medium was collected to culture isolated neonatal rat cardiomyocytes to determine whether and how LECs could influence cardiomyocyte proliferation and hypertrophy. RESULTS: Swimming exercise induced physiological cardiac growth accompanied by a remarkable increase of cardiac lymphangiogenesis as evidenced by increased density of lymphatic vessel endothelial hyaluronic acid receptor 1-positive lymphatic vessels in the heart and upregulated LYVE-1 and Podoplanin expressions levels. VEGFR3 was upregulated in the exercised heart, while VEGFR3 inhibitor SAR131675 attenuated exercise-induced physiological cardiac growth as evidenced by blunted myocardial hypertrophy and reduced proliferation marker Ki67 in cardiomyocytes, which was correlated with reduced lymphatic vessel density and downregulated LYVE-1 and Podoplanin in the heart upon exercise. Furthermore, LEC-conditioned medium promoted both hypertrophy and proliferation of cardiomyocytes and contained higher levels of insulin-like growth factor-1 and the extracellular protein Reelin, while LEC-conditioned medium from LECs treated with SAR131675 blocked these effects. Functional rescue assays further demonstrated that protein kinase B (AKT) activation, as well as reduced CCAAT enhancer-binding protein beta (C/EBPß) and increased CBP/p300-interacting transactivators with E (glutamic acid)/D (aspartic acid)-rich-carboxylterminal domain 4 (CITED4), contributed to the promotive effect of LEC-conditioned medium on cardiomyocyte hypertrophy and proliferation. CONCLUSION: Our findings reveal that cardiac lymphangiogenesis is required for exercise-induced physiological cardiac growth by VEGFR3 activation, and they indicate that LEC-conditioned medium promotes both physiological hypertrophy and proliferation of cardiomyocytes through AKT activation and the C/EBPß-CITED4 axis. These results highlight the essential roles of cardiac lymphangiogenesis in exercise-induced physiological cardiac growth.

miR-340-5p Mediates Cardiomyocyte Oxidative Stress in Diabetes-Induced Cardiac Dysfunction by Targeting Mcl-1.

Diabetic cardiomyopathy (DCM) is initially characterized by early diastolic dysfunction, left ventricular remodeling, hypertrophy, and myocardial fibrosis, and it is eventually characterized by clinical heart failure. MicroRNAs (miRNAs), endogenous small noncoding RNAs, play significant roles in diabetes mellitus (DM). However, it is still largely unknown about the mechanism that links miRNAs and the development of DCM. Here, we aimed to elucidate the mechanism underlying the potential role of microRNA-340-5p in DCM in db/db mouse, which is a commonly used model of type 2 DM and diabetic complications that lead to heart failure. We first demonstrated that miR-340-5p expression was dramatically increased in heart tissues of mice and cardiomyocytes under diabetic conditions. Overexpression of miR-340-5p exacerbated DCM, which was reflected by extensive myocardial fibrosis and more serious dysfunction in db/db mice as represented by increased apoptotic cardiomyocytes, elevated ROS production, and impaired mitochondrial function. Inhibition of miR-340-5p by a tough decoy (TUD) vector was beneficial for preventing ROS production and apoptosis, thus rescuing diabetic cardiomyopathy. We identified myeloid cell leukemia 1 (Mcl-1) as a major target gene for miR-340-5p and showed that the inhibition of Mcl-1 was responsible for increased functional loss of mitochondria, oxidative stress, and cardiomyocyte apoptosis, thereby caused cardiac dysfunction in diabetic mice. In conclusion, our results showed that miR-340-5p plays a crucial role in the development of DCM and can be targeted for therapeutic intervention.

Genome-wide analyses reveal the detrimental impacts of SARS-CoV-2 viral gene Orf9c on human pluripotent stem cell-derived cardiomyocytes.

Patients with coronavirus disease 2019 (COVID-19) commonly have manifestations of heart disease. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome encodes 27 proteins. Currently, SARS-CoV-2 gene-induced abnormalities of human heart muscle cells remain elusive. Here, we comprehensively characterized the detrimental effects of a SARS-CoV-2 gene, Orf9c, on human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) by preforming multi-omic analyses. Transcriptomic analyses of hPSC-CMs infected by SARS-CoV-2 with Orf9c overexpression (Orf9cOE) identified concordantly up-regulated genes enriched into stress-related apoptosis and inflammation signaling pathways, and down-regulated CM functional genes. Proteomic analysis revealed enhanced expressions of apoptotic factors, whereas reduced protein factors for ATP synthesis by Orf9cOE. Orf9cOE significantly reduced cellular ATP level, induced apoptosis, and caused electrical dysfunctions of hPSC-CMs. Finally, drugs approved by the U.S. Food and Drug Administration, namely, ivermectin and meclizine, restored ATP levels and ameliorated CM death and functional abnormalities of Orf9cOE hPSC-CMs. Overall, we defined the molecular mechanisms underlying the detrimental impacts of Orf9c on hPSC-CMs and explored potentially therapeutic approaches to ameliorate Orf9c-induced cardiac injury and abnormalities.

Targeting ACSL1 promotes cardiomyocyte proliferation and cardiac regeneration.

BACKGROUND: Neonatal hearts have considerable regenerative potential within 7 days post birth (P7), but the rate of regeneration is extremely low after P7. Interestingly, lipid metabolism increases dramatically after P7. The similarities in these age profiles suggests a possible link between cardiac regeneration and lipid metabolism. Acyl CoA synthase long chain family member 1 (ACSL1) is the key enzyme that regulates lipid metabolism. The aim of this study was to identify the role of ACSL1 in the regeneration of cardiomyocytes. METHODS AND RESULTS: The uptake of fatty acids in hearts increased after P7; however, myocardial regeneration was decreased. We profiled an RNA-sequence array of hearts from mice of different ages, including E10.5 (embryonic stage)-, 3-, 7-, 21-, 30-, and 60-day-old mice, and found that the expression of ACSL1 was significantly increased after P7. By establishing ACSL1 knockdown mice with adeno-associated virus (AAV9). Then, we verified that knockdown of ACSL1 enhanced the capacity for myocardial regeneration both in mice and in primary cardiomyocytes. Indeed, ACSL1 knockdown in primary cardiomyocytes promoted the cell cycle progression from G0 to G2 phase by regulating specific factors, which may correlate with the activation of AKT by ACSL1 and withdrawal of FOXO1 from the nucleus. In vivo, knockdown of ACSL1 effectively restored cardiac function and myocardial regeneration in adult mice with myocardial infarction (MI). CONCLUSIONS: ACSL1 possibly induces the loss of the myocardial regenerative potential beginning at P7 in mice, and inhibition of ACSL1 effectively promoted myocardial repair after MI in mice.

Exosomal miR-17-3p Alleviates Programmed Necrosis in Cardiac Ischemia/Reperfusion Injury by Regulating TIMP3 Expression.

OBJECTIVE: Myocardial ischemia/reperfusion (I/R) injury can aggravate myocardial injury. Programmed necrosis plays a crucial role in this injury. However, the role of exosomal miRNAs in myocardial I/R injury remains unclear. Therefore, this study is aimed at exploring the function and mechanism of exosomal miR-17-3p in myocardial I/R injury. METHODS: The myocardial I/R injury animal model was established in C57BL/6 mice. Exosomes were identified using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting. Programmed necrosis was detected by PI staining. Heart function and myocardial infarct size were evaluated using echocardiography and triphenyl tetrazolium chloride (TTC) staining, respectively. Histopathological changes were visualized by hematoxylin and eosin (H&E) and Masson staining. The regulation of TIMP3 expression by miR-17-3p was verified using a dual-luciferase reporter assay. Lactate dehydrogenase (LDH) and tumor necrosis factor-α (TNF-α) levels were measured by enzyme-linked immunosorbent assays (ELISA). TIMP3 expression was measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting. RESULTS: We demonstrated that miR-17-3p was significantly downregulated in peripheral blood exosomes after cardiac I/R injury. Further analysis indicated that exosomal miR-17-3p attenuated H2O2-induced programmed necrosis in cardiomyocytes in vitro. Moreover, TIMP3 was a target for miR-17-3p. TIMP3 affected H2O2-induced programmed necrosis in cardiomyocytes. This effect was modulated by miR-17-3p in vitro. Furthermore, exosomal miR-17-3p greatly alleviated cardiac I/R injury in vivo. CONCLUSIONS: The present study demonstrated that exosomal miR-17-3p alleviated the programmed necrosis associated with cardiac I/R injury by regulating TIMP3 expression. These findings could represent a potential treatment for I/R injury.

Human iPSC-Cardiomyocytes as an Experimental Model to Study Epigenetic Modifiers of Electrophysiology.

The epigenetic landscape and the responses to pharmacological epigenetic regulators in each human are unique. Classes of epigenetic writers and erasers, such as histone acetyltransferases, HATs, and histone deacetylases, HDACs, control DNA acetylation/deacetylation and chromatin accessibility, thus exerting transcriptional control in a tissue- and person-specific manner. Rapid development of novel pharmacological agents in clinical testing-HDAC inhibitors (HDACi)-targets these master regulators as common means of therapeutic intervention in cancer and immune diseases. The action of these epigenetic modulators is much less explored for cardiac tissue, yet all new drugs need to be tested for cardiotoxicity. To advance our understanding of chromatin regulation in the heart, and specifically how modulation of DNA acetylation state may affect functional electrophysiological responses, human-induced pluripotent stem-cell-derived cardiomyocyte (hiPSC-CM) technology can be leveraged as a scalable, high-throughput platform with ability to provide patient-specific insights. This review covers relevant background on the known roles of HATs and HDACs in the heart, the current state of HDACi development, applications, and any adverse cardiac events; it also summarizes relevant differential gene expression data for the adult human heart vs. hiPSC-CMs along with initial transcriptional and functional results from using this new experimental platform to yield insights on epigenetic control of the heart. We focus on the multitude of methodologies and workflows needed to quantify responses to HDACis in hiPSC-CMs. This overview can help highlight the power and the limitations of hiPSC-CMs as a scalable experimental model in capturing epigenetic responses relevant to the human heart.

Modeling reduced contractility and impaired desmosome assembly due to plakophilin-2 deficiency using isogenic iPS cell-derived cardiomyocytes.

Loss-of-function mutations in PKP2, which encodes plakophilin-2, cause arrhythmogenic cardiomyopathy (AC). Restoration of deficient molecules can serve as upstream therapy, thereby requiring a human model that recapitulates disease pathology and provides distinct readouts in phenotypic analysis for proof of concept for gene replacement therapy. Here, we generated isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with precisely adjusted expression of plakophilin-2 from a patient with AC carrying a heterozygous frameshift PKP2 mutation. After monolayer differentiation, plakophilin-2 deficiency led to reduced contractility, disrupted intercalated disc structures, and impaired desmosome assembly in iPSC-CMs. Allele-specific fluorescent labeling of endogenous DSG2 encoding desmoglein-2 in the generated isogenic lines enabled real-time desmosome-imaging under an adjusted dose of plakophilin-2. Adeno-associated virus-mediated gene replacement of PKP2 recovered contractility and restored desmosome assembly, which was sequentially captured by desmosome-imaging in plakophilin-2-deficient iPSC-CMs. Our isogenic set of iPSC-CMs recapitulates AC pathology and provides a rapid and convenient cellular platform for therapeutic development.

Knockdown of forkhead box protein P1 alleviates hypoxia reoxygenation injury in H9c2 cells through regulating Pik3ip1/Akt/eNOS and ROS/mPTP pathway.

Forkhead box protein P1 (Foxp1) exerts an extensive array of physiological and pathophysiological impacts on the cardiovascular system. However, the exact function of myocardial Foxp1 in myocardial ischemic reperfusion injury (MIRI) stays largely vague. The hypoxia reoxygenation model of H9c2 cells (the rat ventricular myoblasts) closely mimics myocardial ischemia-reperfusion injury. This report intends to research the effects and mechanisms underlying Foxp1 on H9c2 cells in response to hypoxia (12 h)/reoxygenation (4 h) (HR) stimulation. Expressions of Foxp1 and Phosphatidylinositol 3-kinase interacting protein 1 (Pik3ip1) were both upregulated in ischemia/reperfusion (IR)/HR-induced injury. Stimulation through HR led to marked increases in cellular apoptosis, mitochondrial dysfunction, and superoxide generation in H9c2 cells, which were rescued with knockdown of Foxp1 by siRNA. Silence of Foxp1 depressed expression of Pik3ip1 directly activated the PI3K/Akt/eNOS pathway and promoted nitric oxide (NO) release. Moreover, the knockdown of Foxp1 blunted HR-induced enhancement of reactive oxygen species (ROS) generation, thus alleviating excessive persistence of mitochondrial permeability transition pore (mPTP) opening and decreased mitochondrial apoptosis-associated protein expressions in H9c2 cells. Meanwhile, these cardioprotective effects can be abolished by LY294002, NG-nitro-L-arginine methyl ester (L-NAME), and Atractyloside (ATR), respectively. In summary, our findings indicated that knockdown of Foxp1 prevented HR-induced encouragement of apoptosis and oxidative stress via PI3K/Akt/eNOS signaling activation by targeting Pik3ip1 and improved mitochondrial function by inhibiting ROS-mediated mPTP opening. Inhibition of Foxp1 may be a promising therapeutic avenue for MIRI.

Dexmedetomidine attenuates ischemia and reperfusion-induced cardiomyocyte injury through p53 and forkhead box O3a (FOXO3a)/p53-upregulated modulator of apoptosis (PUMA) signaling signaling.

Dexmedetomidine (DEX) has been reported to attenuate the ischemia and reperfusion (I/R) induced cardiomyocyte apoptosis. However, mechanisms underlying these protective effect remain to be fully elucidated. Cardiomyocyte apoptosis is associated with ischemic heart disease. Here we investigated the role of DEX in I/R -induced cardiomyocyte apoptosis. Mice and H9c2 cardiomyocyte cells were subjected to cardiomyocyte I/R injury and hypoxia/reoxygenation (H/R) injury, respectively. The roles and mechanisms of DEX on H9c2 cardiomyocyte cells and mice cardiomyocyte cells exposured to H/R or I/R injury were explored. The results showed that DEX attenuates H/R injury-induced H9c2 cell apoptosis and alleviated mitochondrial oxidative stress; it also reduced myocardial infarct size and protected the cardiac function following cardiomyocyte I/R injury. In addition, H/R and I/R injury increased p53 expression and forkhead box O3a (FOXO3a)/p53-upregulated modulator of apoptosis (PUMA) signaling in H9c2 cardiomyocyte cells and cardiomyocytes. Targeting p53 expression or FOXO3a/PUMA signaling inhibited cell apoptosis and protected against H/R injury in H9c2 cardiomyocyte cells and cardiomyocytes. Pretreatment with DEX reduced the H/R or I/R injury-induced activation of p53 expression and FOXO3a/PUMA signaling, and alleviated H/R or I/R injury-induced apoptosis and mitochondrial oxidative stress. Therefore, DEX could alleviate H/R- or I/R-induced cardiomyocytes injury by reducing cell apoptosis and blocking p53 expression and FOXO3a/PUMA signaling. Targeting p53 or/and FOXO3a/PUMA signaling could alleviate cardiomyocyte I/R injury.