Induced Pluripotent Stem Cell-derived Cardiomyocytes Research Articles

SARS-CoV-2 spike receptor-binding domain is internalized and promotes protein ISGylation in human induced pluripotent stem cell-derived cardiomyocytes

Although an increased risk of myocarditis has been observed after vaccination with mRNA encoding severe acute respiratory syndrome coronavirus 2 spike protein, its underlying mechanism has not been elucidated. This study investigated the direct effects of spike receptor-binding domain (S-RBD) on human cardiomyocytes differentiated from induced pluripotent stem cells (iPSC-CMs). Immunostaining experiments using ACE2 wild-type (WT) and knockout (KO) iPSC-CMs treated with purified S-RBD demonstrated that S-RBD was bound to ACE2 and internalized into the subcellular space in the iPSC-CMs, depending on ACE2. Immunostaining combined with live cell imaging using a recombinant S-RBD fused to the superfolder GFP (S-RBD-sfGFP) demonstrated that S-RBD was bound to the cell membrane, co-localized with RAB5A, and then delivered from the endosomes to the lysosomes in iPSC-CMs. Quantitative PCR array analysis followed by single cell RNA sequence analysis clarified that S-RBD-sfGFP treatment significantly upregulated the NF-kβ pathway-related gene (CXCL1) in the differentiated non-cardiomyocytes, while upregulated interferon (IFN)-responsive genes (IFI6, ISG15, and IFITM3) in the matured cardiomyocytes. S-RBD-sfGFP treatment promoted protein ISGylation, an ISG15-mediated post-translational modification in ACE2-WT-iPSC-CMs, which was suppressed in ACE2-KO-iPSC-CMs. Our experimental study demonstrates that S-RBD is internalized through the endolysosomal pathway, which upregulates IFN-responsive genes and promotes ISGylation in the iPSC-CMs.

Loss of sodium current caused by a Brugada syndrome–associated variant is determined by patient-specific genetic background

BackgroundBrugada syndrome (BrS) is an inherited cardiac arrhythmogenic disease that predisposes patients to sudden cardiac death. It is associated with mutations in SCN5A, which encodes the cardiac sodium channel alpha subunit (NaV1.5). BrS-related mutations have incomplete penetrance and variable expressivity within families. ObjectiveThe purpose of this study was to determine the role of patient-specific genetic background on the cellular and clinical phenotype among carriers of NaV1.5_p.V1525M. MethodsWe studied sodium currents from patient-specific human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) and heterologously transfected human embryonic kidney (HEK) tsA201 cells using the whole-cell patch-clamp technique. We determined gene and protein expression by quantitative polymerase chain reaction, RNA sequencing, and western blot and performed a genetic panel for arrhythmogenic diseases. ResultsOur results showed a large reduction in INa density in hiPSC-CM derived from 2 V1525M single nucleotide variant (SNV) carriers compared with hiPSC-CM derived from a noncarrier, suggesting a dominant-negative effect of the NaV1.5_p.V1525M channel. INa was not affected in hiPSC-CMs derived from a V1525M SNV carrier who also carries the NaV1.5_p.H558R polymorphism. Heterozygous expression of V1525M in HEK-293T cells produced a loss of INa function, not observed when this variant was expressed together with H558R. In addition, the antiarrhythmic drug mexiletine rescued INa function in hiPSC-CM. SCN5A expression was increased in the V1525M carrier who also expresses NaV1.5_p.H558R. ConclusionOur results in patient-specific hiPSC-CM point to a dominant-negative effect of NaV1.5_p.V1525M, which can be reverted by the presence of NaV1.5_p.H558R. Overall, our data points to a role of patient-specific genetic background as a determinant for incomplete penetrance in BrS.

Characterisation of hypertrophic response and the effect of a GATA4-targeting compound on the phenotype of hypertrophic cardiomyopathy patient-derived hiPSC-cardiomyocytes

Abstract Introduction Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. Patient-derived human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an informative platform to study disease pathomechanisms. Transcription factor GATA4 serves a significant role in cardiomyocyte hypertrophy, and GATA4-targeted compounds inhibit hypertrophy [1]. Purpose This study aims at identifying phenotypic differences between patient-derived hiPSC-CMs and control hiPSC-CMs and at investigating the effect of a novel GATA4-targeting compound C-2021 on the patient-derived hiPSC-CMs phenotype. Methods hiPSC-CMs were differentiated from a healthy, control line (iPS(IMR90)-4; here, control hiPSC-CMs) and from HCM patient-derived hiPSCs (HCM hiPSC-CMs) that carry a mutation in the MYBPC3 gene. Two types of hypertrophic stimuli were applied to hiPSC-CMs: mechanical stretch and endothelin-1 (ET-1) treatment. For pharmacological assays, compound C-2021 (at 30 µM) or vehicle (0.1 % DMSO) were introduced to hiPSC-CMs one hour prior to the stimuli. hiPSC-CMs were exposed to cyclic equibiaxial mechanical stretching for 24 h or 72 h using Flexcell FX-5000 Tension System (Flexcell International Corporation) [2]. The expression of hypertrophic markers (such as NPPB, NPPA) and structural proteins (including α-myosin and β-myosin heavy chains; MYH6 and MYH7, respectively) was investigated with qPCR. The relative gene expression was analysed using the 2-ΔΔCt method using 18S and ACTB as reference genes. Furthermore, hiPSC-CMs were subjected to ET-1 at 100 nM for 24 h and proBNP expression was quantified using high-content analysis (HCA). Results HCM hiPSC-CMs displayed increased basal expression of NPPB (Fig. 1A-B), lower expression of MYH6 (Fig. 1C) and higher expression of MYH7 (Fig. 1D) compared to control hiPSC-CMs, which is in line with the hypertrophic disease phenotype. Control and HCM hiPSC-CMs responded to 24 h and 72 h mechanical stretching by increased NPPB expression (Fig. 1A-B). At 72 h, the increase in NPPB expression was more pronounced in the HCM hiPSC-CMs (Fig. 1B). The compound C-2021 inhibited stretch-induced NPPB expression after 72 h stretching. HCA results indicate that ET-1 induced a 17-fold increase in proBNP-positive control hiPSC-CMs, and this effect was attenuated by C-2021 treatment. Interestingly, ET-1 did not enhance proBNP expression in HCM hiPSC-CMs (Fig. 2). Conclusions Our results show that the HCM hiPSC-CMs differ from the control hiPSC-CMs in response to hypertrophic stimuli and in myosin heavy chain isoform expression. Additionally, C-2021 may have anti-hypertrophic effects in both in control and HCM hiPSC-CMs based on the downregulation of NPPB expression, and the decrease in proBNP-positive hiPSC-CMs. Further studies are needed to elucidate the exact mechanisms underlying HCM and the action of C-2021 on the HCM hiPSC-CM phenotype.

Massive expansion of functional human iPSC-derived cardiomyocytes by concomitant glycogen synthase kinase-3 beta inhibition and removal of cell-cell contact

Abstract Background Adult mammalian heart has limited capacity for regeneration. Cardiac injuries such as myocardial infarction lead to significant cardiomyocyte (CM) loss and subsequent heart failure with significant morbidity and mortality. Cell-based therapeutic approaches such as injection of stem cell-derived cells or transplantation of engineered cardiac tissue patches have shown promise to re-muscularize the damaged myocardium and enhance cardiac functions. Rationale: Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro can expand CMs modestly (< 5-fold). Results Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e. 100 to 250-fold) by glycogen synthase kinase-3β (GSK-3β) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show GSK-3β inhibition suppresses CM maturation while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10- to 25-times greater expansion beyond GSK-3β inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from Yes associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. Conclusion In summary, we uncovered a molecular interplay that enables massive hiPSC-CMs expansion for large-scale drug screening and tissue engineering applications.

Abstract 16714: An Injectable Thermo-Responsive Hydrogel as a Tool for Cardiac Tissue Engineering Efforts: In vitro and in vivo Biocompatibility Assessment

Background: Heart failure (HF) is a major contributor to the global burden of cardiovascular disease. Current treatments for HF do not regenerate muscle nor function and so cardiac transplantation remains the only definitive treatment for HF. Thus, novel therapies for HF are still needed. To overcome this limitation, we engineered an injectable thermo-responsive hydrogel that transitions from a liquid-solution to a gel-based matrix upon reaching body temperature allowing for a liquid injection rapidly followed by gel localization. Previously, we demonstrated the potential of our hydrogel for cardiac tissue applications using primary cardiomyocytes. Goal: The goal of this project is to assess the biocompatibility of the thermo-responsive hydrogel with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) and its effect on mice heart tissue. Methods: 3D cell culture: 9x104 hiPSC-CM were encapsulated for 21 days using 150μl of hydrogel solution (1.5% w/w) and cell function was assessed afterwards. Mouse intracardial injection: a left thoracotomy was performed via the fourth intercostal space in an adult C57BL/6 mouse model. Then, three injections of 10 μl of either (i) lipofectamine, (ii) hydrogel (1% w/w) or (ii) saline were injected into the left ventricle anterior wall. After injections, the thoracotomy site was closed, and the animals recovered. Four weeks post inject the tissues were harvested and prepared for studies. Results: We found that our hydrogel promotes hiPSC-CMs alignment, elongation with increased Cx43 localization and improved contraction function when compared with traditional 2D fibronectin controls. Furthermore, the in-vivo biocompatibility in mice (intracardial injection) proved that our hydrogel is well tolerated by cardiac tissue. Conclusion: Our results indicated that the injectable thermos-responsive hydrogel is a biocompatible material with potential to be used in cardiac tissue engineering efforts.

Abstract 12784: Ischemic Cardiac Autophagy/Mitophagy is Regulated by TRPML1 and Mitochondrial Reactive Oxygen Species

Introduction: In myocardial infarction (MI), activated autophagy/mitophagy helps degrade cytoplasmic macromolecules/proteins/ organelles (especially damaged mitochondria), recycle breakdown products, and prevent oxidative damage and cell death. It had reported that reactive oxygen species (ROS) activates the transcription factor EB (TFEB) via a lysosomal transient receptor potential mucolipin 1 (TRPML1) Ca 2+ efflux. TFEB is thought to be the master regulator of lysosome biogenesis. Hypothesis: We investigated whether lysosomal TRPML1 was activated by ROS and contributed to ischemic cardiac autophagy/mitophagy. Methods: MI was induced in wild type C57BL/6J mice by ligated left anterior descending coronary artery. Mice were studied at three weeks after MI surgery. Results: The lysosomal-restricted TRPML1 Ca 2+ release channel was significantly upregulated by 84.8% in cardiac border zone (BZ) and 55.4% in a remote zone after MI. As expected, dephosphorylated TFEB (i.e., active TFEB), mitophagy marker Parkin, autophagy marker LC3II, and lysosome marker LAMP1 increased substantially after MI. Autophagy degradation was upregulated as measured by SQSTM1/p62. TRPML1 protein expression was reduced in a dose-dependent manner by TRPML1 shRNA AAV9 (AAV9 was injected 1 week after MI for two weeks). The protein levels of LC3II and LAMP1 significantly decreased in MI BZ after AAV9 transfection. Mitochondrial ROS scavenger (mitoTEMPO, 1 mg/kg/day, i.p.) treatment for two weeks (starting at 1 week after MI) significantly inhibited TRPML1, LAMP1, LC3II and Parkin expression in the MI BZ. Conversely, when TRPML1 was overexpressed in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), the protein levels of LC3II/Parkin were increased. Speaking to human relevance, TRPML1 was significantly increased in human patients with ischemic cardiomyopathy. Conclusion: Autophagy and mitophagy after MI are regulated by TRPML1 and mitochondrial ROS.

Abstract 13336: Myocardial Protection of Chitosan Cardiac Muscle Patch in Myocardial Infarcted Mice

Background: Poor grafted rate of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) via direct injection into the myocardium limits the efficacy of cardiac repair after myocardial infarction (MI). Chitosan-based scaffolds has regarded as functional biomaterial for designing delivery systems in cardiac therapies. This study aimed to enhancing the potency of cardiac repair with the hiPSC-CMs loaded chitosan cardiac patch (hCCMP). Methods: The patches were fabricated with chitosan. Characterization of the chitosan patches was evaluated through the detection of swelling rate and porosity. Biocompatibility of the hiPSC-CMs loaded patches was analysed by scanning electron microscope (SEM) and CCK8 assay. The mice were randomly divided into 4 groups (n=10 for each). Animals underwent MI-inductive surgery followed by epicardium implantation of hCCMP or Empty-patch (without hiPSC-CMs); Sham and MI mice without further intervention were set as control. 4 weeks after MI, heart function was measured via echocardiography. Engraftment was evaluated with human cardiac troponin T staining. Infarction size, left ventricular anterior wall thickness, cardiac remodeling, angiogenesis, arterialization and apoptosis was detected via immunofluorescence in infarcted boder area. Results: SEM showed that the chitosan patch had regular pore size and good 3D structure; porosity was about (96.7±0.37)%. On day3, the patch swelling rate was about (715±64.55)%. SEM, CCK8 assay showed a normal cells growth in the chitosan patch. The hiPSC-CMs grafted well with chitosan patch delivery systems and was associated with significantly improvement in LV heart function, infarction size, LV anterior wall thickness, cardiac remodeling, angiogenesis and cardiomyocyte apoptosis 4 weeks after MI. Conclusions: The 3D chitosan cardiac patch with hiPSC-CMs loaded can enhance the potency of cardiac repair after MI and present a novel and promising therapic strategy.

Abstract 15451: Regulations of Nrf2 by Nf-κB in Ischemic Heart Failure

Introduction: Oxidative stress is an important pathophysiological factor in the development and progression of ischemic heart failure (IHF). Nuclear factor E2-related factor 2 (Nrf2) is a major regulator of the protective antioxidant response and has been found to decrease by 50% in IHF. However, the mechanism of Nrf2 downregulation remains incompletely understood. NF-kB is a critical transcription factor that influences many cellular processes and is activated in IHF with increased reactive oxygen species (ROS). We hypothesize that activation of NF-kB may underlie the decreased Nrf2 activity in IHF. Methods and Results: To induce IHF, we performed left anterior descending coronary artery (LAD) ligation surgery in Sprague Dawley rats and FVB/NJ mice. We found a significant reduction in cardiac function after LAD surgery in ischemia/reperfusion (I/R) mice relative to sham-operated mice using echocardiography. Tissue sample analyses reveal a crucial role of NF-kB activation in the regulation of Nrf2 in IHF. Additionally, after 2 weeks of I/R, we found that rats treated with Nrf2 overexpressed lentivirus demonstrated greater pressure development than rats with vehicle treatment. Using rat cardiac myoblast cells (H9C2) and western blot analysis, we tested the expression of cytosolic and nuclear Nrf2 and p65 (a component of NF-kB) following time- and dose-dependent treatments of H2O2. We found that p65 activation inhibited Nrf2 activity, while inhibitions of p65 nuclear translocation prevented the decrease in Nrf2 nuclear activities. We then generated a knockout of p65 in H9C2 cells and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using CRISPR-Cas9 technology to test NF-κB regulation on the Nrf2 activities, which further strengthened our previous findings. Conclusions: Taken together, our data support the critical role of NF-kB activation in the regulation of Nrf2 in IHF, providing new insights into possible molecular targets for the treatment of IHF through the regulation of the protective antioxidant response.

Abstract 15334: Laminin Alpha 2 Increases the Protective Effect of Exosomes in Human IPSC-Derived Cardiomyocytes During Ischemia-Reperfusion

Acute myocardial infarction (AMI) is a leading cause of death and disability worldwide. With early treatment, restoration of blood flow to ischemic myocardium can be achieved and therefore reduce mortality risk. Evidence suggests that mesenchymal stromal cell (MSC)-derived exosomes (EXO) promote myocardial repair after AMI due to paracrine activity. Laminin alpha 2 (LAMA2) is a major cardiac extracellular matrix (ECM) component and can be used to produce cardiac-specific EXO. Here we coated MSC-derived EXOs in LAMA2 to enhance their recognition by human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We hypothesized that LAMA2-coated MSC-EXO would confer protective effects on CMs during ischemia-reperfusion. The LAMA2-EXO had a similar yield of particles, with a size range of 130nm as the control (gelatin-EXO). No cytotoxic effect was observed in CMs after 3-day exposure to LAMA2-EXO. CMs were then treated with LAMA2-EXO two hours prior to 4h ischemia (1% O 2 , 5% CO 2 , and glucose-free media). LAMA2-EXO had a 2-fold protective effect compared to non-treatment, and after 1.5h of reperfusion (5% CO 2 , CM-enriched media) CMs treated with LAMA2-EXO recovered faster than the control group. Conversely, due to the laminin 511/411 ECM component of endothelial cells (ECs), we did not see a significant protective effect nor a difference between LAMA2-EXO vs. the control in the EC ischemia-reperfusion group. This is the first report of suggesting the specificity of LAMA2-EXO to treat CM post-exposure to ischemia-reperfusion injury. Overall, these results indicate that LAMA2-EXO can be a promising treatment to target CM survival in ischemic cardiovascular disease.

Prioritizing cardiovascular disease-associated variants altering NKX2-5 and TBX5 binding through an integrative computational approach

Cardiovascular diseases (CVDs) are the leading cause of death worldwide and are heavily influenced by genetic factors. Genome-wide association studies (GWAS) have mapped > 90% of CVD-associated variants within the non-coding genome, which can alter the function of regulatory proteins, like transcription factors (TFs). However, due to the overwhelming number of GWAS single nucleotide polymorphisms (SNPs) (>500,000), prioritizing variants for in vitro analysis remains challenging. In this work, we implemented a computational approach that considers support vector machine (SVM)-based TF binding site classification and cardiac expression quantitative trait loci (eQTL) analysis to identify and prioritize potential CVD-causing SNPs. We identified 1,535 CVD-associated SNPs that occur within TF footprints and putative enhancers in the human heart and 14,218 variants in linkage disequilibrium (LD) with genotype-dependent gene expression in cardiac tissue. Using ChIP-seq data from two essential cardiac TFs (NKX2-5 and TBX5) in human induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CM), we trained a large-scale gapped k-mer SVM (LS-GKM-SVM) model to identify CVD-associated SNPs that altered NKX2-5 and TBX5 binding sites. The model was tested by scoring human heart TF genomic footprints within putative enhancers and measuring in vitro binding through electrophoretic mobility shift assay (EMSA). Five variants predicted to alter NKX2-5 (rs59310144, rs6715570, and rs61872084) and TBX5 (rs7612445 and rs7790964) binding were prioritized for in vitro validation based on the magnitude of the predicted change in binding and because they are eQTLs in cardiac tissue. All five variants altered NKX2-5 and TBX5 DNA binding. In summary, we present a bioinformatic approach that considers tissue-specific eQTL analysis and SVM-based TF binding site classification to prioritize CVD-associated variants for in vitro experimental analysis.

Current status and emerging trends of cardiac metabolism from the past 20 years: A bibliometric study

BackgroundAbnormal cardiac metabolism is a key factor in the development of cardiovascular diseases. Consequently, there has been considerable emphasis on researching and developing drugs that regulate metabolism. This study employed bibliometric methods to comprehensively and objectively analyze the relevant literature, offering insights into the knowledge dynamics in this field. MethodsThe data source for this study was the Web of Science Core Collection (WoSCC), from which the collected data were imported into bibliometric software for analysis. ResultsThe United States was the leading contributor, accounting for 38.33 % of publications. The University of Washington and Damian J. Tyler were the most active institution and author, respectively. The American Journal of Physiology-Heart and Circulatory Physiology, Journal of Molecular and Cellular Cardiology, Cardiovascular Research, Circulation Research, and American Journal of Physiology-Endocrinology and Metabolism were highly influential journals that published numerous high-quality articles on cardiac metabolism. Common keywords in this research area included heart failure, insulin resistance, skeletal muscle, mitochondria, as well as topic words such as cardiac metabolism, fatty acid oxidation, glucose metabolism, and myocardial metabolism. Co-citation analysis has shown that research on heart failure and in vitro modeling of cardiovascular disease has gained prominence in recent years and making it a research hotspot. ConclusionResearch on cardiac metabolism is steadily growing, with a specific focus on heart failure and the interplay between mitochondrial dysfunction, insulin resistance, and cardiac metabolism. An emerging trend in this field involves the enhancement of maturation in human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) through the manipulation of cardiac metabolism.

Metoprolol disrupts inflammatory response of human cardiomyocytes via β-arrestin2 biased agonism and NF-κB signaling modulation

AimsRecent evidence supports non-class cardioprotective effects of metoprolol against neutrophil-mediated ischemia-reperfusion injury during exacerbated inflammation. Whether metoprolol exerts direct anti-inflammatory effect on cardiomyocytes is unknown. Accordingly, we aimed to investigate the direct anti-inflammatory effects of metoprolol in a cellular model of human induced pluripotent stem cell-derived cardiomyocytes (hiCMs) and to explore the role of β-arrestin2 (β-ARR2) biased agonism signaling pathway. Methods and resultshiCMs were treated with TNF-α for 24 h, followed by 4-hour treatment with metoprolol or esmolol. Electrical response of hiCMs to β1-selective blockade was assessed by microelectrode arrays technology. The effect on inflammatory and adhesion molecule expression was evaluated in wild-type and β-ARR2 silenced hiCMs. To silence β-ARR2 expression, hiCMs were transfected with a specific small interfering RNA targeting β-ARR2 mRNA and preventing its translation.TNF-α stimulation boosted the expression of IκB, NF-κB, IL1β, IL6, and VCAM1 in hiCMs. TNF-α-treated hiCMs showed similar physiological responses to metoprolol and esmolol, with no difference in field potential duration and beat period recorded. Adding metoprolol significantly decreased inflammatory response patterns in wild-type hiCMs by dampening TNF-α induced expression of NF-κB, IL1β, and IL6, but not in β-ARR2-knockout hiCMs. A similar response was not observed in presence of β1-selective blockade with esmolol. ConclusionsMetoprolol exerts a non-class direct anti-inflammatory effect on hi-CMs. β1-selective blockade with metoprolol disrupts inflammatory responses induced by TNF-α and induces significant inhibition of NF-κB signaling cascade via β-ARR2 biased agonism. If confirmed at clinical level, metoprolol could be tested and repurposed to treat cardiac inflammatory disorders.

Reduced Mitochondrial Protein Translation Promotes Cardiomyocyte Proliferation and Heart Regeneration.

The importance of mitochondria in normal heart function are well recognized and recent studies have implicated changes in mitochondrial metabolism with some forms of heart disease. Previous studies demonstrated that knockdown of the mitochondrial ribosomal protein S5 (MRPS5) by small interfering RNA (siRNA) inhibits mitochondrial translation and thereby causes a mitonuclear protein imbalance. Therefore, we decided to examine the effects of MRPS5 loss and the role of these processes on cardiomyocyte proliferation. We deleted a single allele of MRPS5 in mice and used left anterior descending coronary artery ligation surgery to induce myocardial damage in these animals. We examined cardiomyocyte proliferation and cardiac regeneration both in vivo and in vitro. Doxycycline treatment was used to inhibit protein translation. Heart function in mice was assessed by echocardiography. Quantitative real-time polymerase chain reaction and RNA sequencing were used to assess changes in transcription and chromatin immunoprecipitation (ChIP) and BioChIP were used to assess chromatin effects. Protein levels were assessed by Western blotting and cell proliferation or death by histology and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. Adeno-associated virus was used to overexpress genes. The luciferase reporter assay was used to assess promoter activity. Mitochondrial oxygen consumption rate, ATP levels, and reactive oxygen species were also analyzed. We determined that deletion of a single allele of MRPS5 in mice results in elevated cardiomyocyte proliferation and cardiac regeneration; this observation correlates with improved cardiac function after induction of myocardial infarction. We identified ATF4 (activating transcription factor 4) as a key regulator of the mitochondrial stress response in cardiomyocytes from Mrps5+/- mice; furthermore, ATF4 regulates Knl1 (kinetochore scaffold 1) leading to an increase in cytokinesis during cardiomyocyte proliferation. The increased cardiomyocyte proliferation observed in Mrps5+/- mice was attenuated when one allele of Atf4 was deleted genetically (Mrps5+/-/Atf4+/-), resulting in the loss in the capacity for cardiac regeneration. Either MRPS5 inhibition (or as we also demonstrate, doxycycline treatment) activate a conserved regulatory mechanism that increases the proliferation of human induced pluripotent stem cell-derived cardiomyocytes. These data highlight a critical role for MRPS5/ATF4 in cardiomyocytes and an exciting new avenue of study for therapies to treat myocardial injury.

Versican Promotes Cardiomyocyte Proliferation and Cardiac Repair.

The adult mammalian heart is incapable of regeneration, whereas a transient regenerative capacity is maintained in the neonatal heart, primarily through the proliferation of preexisting cardiomyocytes. Neonatal heart regeneration after myocardial injury is accompanied by an expansion of cardiac fibroblasts and compositional changes in the extracellular matrix. Whether and how these changes influence cardiomyocyte proliferation and heart regeneration remains to be investigated. We used apical resection and myocardial infarction surgical models in neonatal and adult mice to investigate extracellular matrix components involved in heart regeneration after injury. Single-cell RNA sequencing and liquid chromatography-mass spectrometry analyses were used for versican identification. Cardiac fibroblast-specific Vcan deletion was achieved using the mouse strains Col1a2-2A-CreER and Vcanfl/fl. Molecular signaling pathways related to the effects of versican were assessed through Western blot, immunostaining, and quantitative reverse transcription polymerase chain reaction. Cardiac fibrosis and heart function were evaluated by Masson trichrome staining and echocardiography, respectively. Versican, a cardiac fibroblast-derived extracellular matrix component, was upregulated after neonatal myocardial injury and promoted cardiomyocyte proliferation. Conditional knockout of Vcan in cardiac fibroblasts decreased cardiomyocyte proliferation and impaired neonatal heart regeneration. In adult mice, intramyocardial injection of versican after myocardial infarction enhanced cardiomyocyte proliferation, reduced fibrosis, and improved cardiac function. Furthermore, versican augmented the proliferation of human induced pluripotent stem cell-derived cardiomyocytes. Mechanistically, versican activated integrin β1 and downstream signaling molecules, including ERK1/2 and Akt, thereby promoting cardiomyocyte proliferation and cardiac repair. Our study identifies versican as a cardiac fibroblast-derived pro-proliferative proteoglycan and clarifies the role of versican in promoting adult cardiac repair. These findings highlight its potential as a therapeutic factor for ischemic heart diseases.

RBPMS regulates cardiomyocyte contraction and cardiac function through RNA alternative splicing.

RNA binding proteins play essential roles in mediating RNA splicing and are key post-transcriptional regulators in the heart. Our recent study demonstrated that RBPMS (RNA binding protein with multiple splicing) is crucial for cardiac development through modulating mRNA splicing, but little is known about its functions in the adult heart. In this study, we aim to characterize the post-natal cardiac function of Rbpms and its mechanism of action. We generated a cardiac-specific knockout mouse line and found that cardiac-specific loss of Rbpms caused severe cardiomyocyte contractile defects, leading to dilated cardiomyopathy and early lethality in adult mice. We showed by proximity-dependent biotin identification assay and mass spectrometry that RBPMS associates with spliceosome factors and other RNA binding proteins, such as RBM20, that are important in cardiac function. We performed paired-end RNA sequencing and RT-PCR and found that RBPMS regulates mRNA alternative splicing of genes associated with sarcomere structure and function, such as Ttn, Pdlim5, and Nexn, generating new protein isoforms. Using a minigene splicing reporter assay, we determined that RBPMS regulates target gene splicing through recognizing tandem intronic CAC motifs. We also showed that RBPMS knockdown in human induced pluripotent stem cell-derived cardiomyocytes impaired cardiomyocyte contraction. This study identifies RBPMS as an important regulator of cardiomyocyte contraction and cardiac function by modulating sarcomeric gene alternative splicing.

Point mutations in RyR2 Ca2+-binding residues of human cardiomyocytes cause cellular remodelling of cardiac excitation contraction-coupling.

CRISPR/Cas9 gene edits of cardiac ryanodine receptor (RyR2) in human-induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) provide a novel platform for introducing mutations in RyR2 Ca2+-binding residues and examining the resulting excitation contraction (EC)-coupling remodelling consequences. Ca2+-signalling phenotypes of mutations in RyR2 Ca2+-binding site residues associated with cardiac arrhythmia (RyR2-Q3925E) or not proven to cause cardiac pathology (RyR2-E3848A) were determined using ICa- and caffeine-triggered Ca2+ releases in voltage-clamped and total internal reflection fluorescence-imaged wild type and mutant cardiomyocytes infected with sarcoplasmic reticulum (SR)-targeted ER-GCaMP6 probe. (i) ICa- and caffeine-triggered Fura-2 or ER-GCaMP6 signals were suppressed, even when ICa was significantly enhanced in Q3925E and E3848A mutant cardiomyocytes; (ii) spontaneous beating (Fura-2 Ca2+ transients) persisted in mutant cells without the SR-release signals; (iii) while 5-20 mM caffeine failed to trigger Ca2+-release in voltage-clamped mutant cells, only ∼20% to ∼70% of intact myocytes responded respectively to caffeine; (iv) and 20 mM caffeine transients, however, activated slowly, were delayed, and variably suppressed by 2-APB, FCCP, or ruthenium red. Mutating RyR2 Ca2+-binding residues, irrespective of their reported pathogenesis, suppressed both ICa- and caffeine-triggered Ca2+ releases, suggesting interaction between Ca2+- and caffeine-binding sites. Enhanced transmembrane calcium influx and remodelling of EC-coupling pathways may underlie the persistence of spontaneous beating in Ca2+-induced Ca2+ release-suppressed mutant myocytes.

Efficacy of exon-skipping therapy for DMD cardiomyopathy with mutations in actin binding domain 1

Exon-skipping therapy is a promising treatment strategy for Duchenne muscular dystrophy (DMD), which is caused by loss-of-function mutations in the DMD gene encoding dystrophin, leading to progressive cardiomyopathy. In-frame deletion of exons 3-9 (Δ3-9), manifesting a very mild clinical phenotype, is a potential targeted reading frame for exon-skipping by targeting actin-binding domain 1 (ABD1); however, the efficacy of this approach for DMD cardiomyopathy remains uncertain. In this study, we compared three isogenic human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing Δ3-9, frameshifting Δ3-7, or intact DMD. RNA sequencing revealed a resemblance in the expression patterns of mechano-transduction-related genes between Δ3-9 and wild-type samples. Furthermore, we observed similar electrophysiological properties between Δ3-9 and wild-type hiPSC-CMs; Δ3-7 hiPSC-CMs showed electrophysiological alterations with accelerated CaMKII activation. Consistently, Δ3-9 hiPSC-CMs expressed substantial internally truncated dystrophin protein, resulting in maintaining F-actin binding and desmin retention. Antisense oligonucleotides targeting exon 8 efficiently induced skipping exons 8-9 to restore functional dystrophin and electrophysiological parameters in Δ3-7 hiPSC-CMs, bringing the cell characteristics closer to those of Δ3-9 hiPSC-CMs. Collectively, exon-skipping targeting ABD1 to convert the reading frame to Δ3-9 may become a promising therapy for DMD cardiomyopathy.

In need of age-appropriate cardiac models: Impact of cell age on extracellular matrix therapy outcomes.

Aging is the main risk factor for cardiovascular disease (CVD). As the world's population ages rapidly and CVD rates rise, there is a growing need for physiologically relevant models of aging hearts to better understand cardiac aging. Translational research relies heavily on young animal models; however, these models correspond to early ages in human life, therefore cannot fully capture the pathophysiology of age-related CVD. Here, we first investigated the transcriptomic and proteomic changes that occur with human cardiac aging. We then chronologically aged human induced pluripotent stem cell-derived cardiomyocytes (iCMs) and showed that 14-month-old iCMs exhibited a similar aging profile to the human CMs and recapitulated age-related disease hallmarks. Using aged iCMs, we studied the effect of cell age on the young extracellular matrix (ECM) therapy, an emerging approach for myocardial infarction (MI) treatment and prevention. Young ECM decreased oxidative stress, improved survival, and post-MI beating in aged iCMs. In the absence of stress, young ECM improved beating and reversed aging-associated expressions in 3-month-old iCMs while causing the opposite effect on 14-month-old iCMs. The same young ECM treatment surprisingly increased SASP and impaired beating in advanced aged iCMs. Overall, we showed that young ECM therapy had a positive effect on post-MI recovery; however, cell age was determinant in the treatment outcomes without any stress conditions. Therefore, "one-size-fits-all" approaches to ECM treatments fail, and cardiac tissue engineered models with age-matched human iCMs are valuable in translational basic research for determining the appropriate treatment, particularly for the elderly.

Anthracycline Toxicity: Light at the End of the Tunnel?

Anthracycline-induced cardiotoxicity (AIC) is a serious and common side effect of anthracycline therapy. Identification of genes and genetic variants associated with AIC risk has clinical potential as a cardiotoxicity predictive tool and to allow the development of personalized therapies. In this review, we provide an overview of the function of known AIC genes identified by association studies and categorize them based on their mechanistic implication in AIC. We also discuss the importance of functional validation of AIC-associated variants in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to advance the implementation of genetic predictive biomarkers. Finally, we review how patient-specific hiPSC-CMs can be used to identify novel patient-relevant functional targets and for the discovery of cardioprotectant drugs to prevent AIC. Implementation of functional validation and use of hiPSC-CMs for drug discovery will identify the next generation of highly effective and personalized cardioprotectants and accelerate the inclusion of approved AIC biomarkers into clinical practice.

An Anterior Second Heart Field Enhancer Regulates the Gene Regulatory Network of the Cardiac Outflow Tract.

Conotruncal defects due to developmental abnormalities of the outflow tract (OFT) are an important cause of cyanotic congenital heart disease. Dysregulation of transcriptional programs tuned by NKX2-5 (NK2 homeobox 5), GATA6 (GATA binding protein 6), and TBX1 (T-box transcription factor 1) have been implicated in abnormal OFT morphogenesis. However, there remains no consensus on how these transcriptional programs function in a unified gene regulatory network within the OFT. We generated mice harboring a 226-nucleotide deletion of a highly conserved cardiac enhancer containing 2 GATA-binding sites located ≈9.4 kb upstream of the transcription start site of Nkx2-5 (Nkx2-5∆enh) using CRISPR-Cas9 gene editing and assessed phenotypes. Cardiac defects in Nkx2-5∆enh/∆enh mice were structurally characterized using histology and scanning electron microscopy, and physiologically assessed using electrocardiography, echocardiography, and optical mapping. Transcriptome analyses were performed using RNA sequencing and single-cell RNA sequencing data sets. Endogenous GATA6 interaction with and activity on the NKX2-5 enhancer was studied using chromatin immunoprecipitation sequencing and transposase-accessible chromatin sequencing in human induced pluripotent stem cell-derived cardiomyocytes. Nkx2-5∆enh/∆enh mice recapitulated cyanotic conotruncal defects seen in patients with NKX2-5, GATA6, and TBX1 mutations. Nkx2-5∆enh/∆enh mice also exhibited defects in right Purkinje fiber network formation, resulting in right bundle-branch block. Enhancer deletion reduced embryonic Nkx2-5 expression selectively in the right ventricle and OFT of mutant hearts, indicating that enhancer activity is localized to the anterior second heart field. Transcriptional profiling of the mutant OFT revealed downregulation of important genes involved in OFT rotation and septation, such as Tbx1, Pitx2, and Sema3c. Endogenous GATA6 interacted with the highly conserved enhancer in human induced pluripotent stem cell-derived cardiomyocytes and in wild-type mouse hearts. We found critical dose dependency of cardiac enhancer accessibility on GATA6 gene dosage in human induced pluripotent stem cell-derived cardiomyocytes. Our results using human and mouse models reveal an essential gene regulatory network of the OFT that requires an anterior second heart field enhancer to link GATA6 with NKX2-5-dependent rotation and septation gene programs.