Heart Cells Research Articles

Abstract P355: Multiomic Analyses Reveal Sex-specific Mechanisms Governing Cardiac Fibrosis.

Cardiac fibrosis—the deposition of excess extracellular matrix in the heart—occurs during pathological cardiac remodelling and precedes cardiac dysfunction and heart failure. Experimental and clinical data show that both cardiac fibrosis and heart failure exhibit sex-specific differences, particularly in the absence of ischemic injury. However, no study to date has systematically examined this, or the hormonal mechanisms driving these differences, using high-resolution single-cell and spatial omics. To address this gap, we applied single-cell and spatial omics to map the quality and distribution of non-ischemic cardiac fibrosis in human and mouse hearts. Further, to query the impact of sex hormones on cardiac fibrosis development, we analysed hearts of castrated (CAST) and ovariectomized (OVX) mice, which were ectopically infused with angiotensin-II (AngII). Using single cell transcriptomic data of hypertensive mouse hearts, we identified a fibroblast population driving cardiac fibrosis with a unique gene signature. We confirmed the presence of these cells in multiple mouse models and aged donor human hearts. Spatial mapping of fibrosis showed a sex-specific distribution of fibrosis, specifically in the development of perivascular fibrosis in the heart. Hypertensive male mice exhibited extensive perivascular fibrosis, and coronary artery adventitial hypertrophy, which was absent in females. However, OVX resulted in the development of perivascular fibrosis in hypertensive mice suggesting a cardioprotective role of estrogen. Confirming these observations, spatial transcriptomic analyses showed distinct fibrotic signatures driving perivascular and interstitial fibrosis, and transcriptomic differences between females and males. Our findings provide fundamental new insights towards the importance of biological sex in driving cardiac fibrosis, pointing to new mechanisms that may be manipulated to ameliorate development of heart failure.

P227 MACROPHAGE TO MYOFIBROBLAST TRANSITION IN THE DEVELOPMENT OF DIASTOLIC DYSFUNCTION

Diastolic dysfunction (DD) and heart failure (HF) ravage a significant portion of the world's population. These end-stage diseases are most often precluded by hypertension (HTN). Cardiac fibrosis is one of the major factors in the degradation of cardiac function. There are multiple cellular players involved in fibrosis—including, macrophages. Indeed, in the past decade these cells have been shown to differentiate into professional fibrotic cells, myofibroblasts, a process termed macrophage to myofibroblast transition (MMT), in models of kidney injury. Despite this, the role of MMT in fibrotic remodeling in HTN and DD remains unknown. As such, we seek to investigate this phenomenon in the HTN heart and determine the role it plays in the pathological progression of HTN to DD. We utilized cardiac ultrasound and direct left ventricular (LV) pressure catheterization to gauge diastolic function. These gold-standard methods allowed us to assess cardiac function during HTN and investigate the heart at the transition to DD. Furthermore, flow cytometry was utilized to identify MMT cells (α-SMA+, CD68+) in the heart. These methods gave insight into the role that MMT plays in the degradation of heart function during HTN that inevitably leads to DD and HF. Cardiac ultrasound revealed that E/A and deceleration time (DcT) were greatly decreased in DOCA mice 10 days after the start of HTN. Further, LV pressure catheterization revealed decreased cardiac function, as shown by decreased -dp/dt. Finally, HTN and normal hearts were harvested and leukocytes isolated. Indeed α-SMA and CD68 staining showed a significant increase in MMT cells in the HTN heart. This study has shown a rapid onset of DD inside of DOCA + salt HTN mice. In addition, there was a significant increase of MMT cells in these same hearts, elucidating a novel mechanism of cardiac fibrosis. Future studies will aim to parse out the gravity of MMT in HTN to find new pharmaceutical targets against this debilitating disease.

The Interplay between Mechanoregulation and ROS in Heart Physiology, Disease, and Regeneration.

Cardiovascular diseases are currently the most common cause of death in developed countries. Due to lifestyle and environmental factors, this problem is only expected to increase in the future. Reactive oxygen species (ROS) are a key player in the onset of cardiovascular diseases but also have important functions in healthy cardiac tissue. Here, the interplay between ROS generation and cardiac mechanical forces is shown, and the state of the art and a perspective on future directions are discussed. To this end, an overview of what is currently known regarding ROS and mechanosignaling at a subcellular level is first given. There the role of ROS in mechanosignaling as well as the interplay between both factors in specific organelles is emphasized. The consequences at a larger scale across the population of heart cells are then discussed. Subsequently, the roles of ROS in embryogenesis, pathogenesis, and aging are further discussed, exemplifying some aspects of mechanoregulation. Finally, different models that are currently in use are discussed to study the topics above.

Impacts of Plu kaow (Houttuynia cordata Thunb.) Ethanolic Extract on Diabetes and Dyslipidemia in STZ Induced Diabetic Rats: Phytochemical Profiling, Cheminformatics Analyses, and Molecular Docking Studies.

The increasing prevalence of diabetes and dyslipidemia poses significant health challenges, impacting millions of people globally and leading to high rates of illness and death. This study aimed to explore the potential antidiabetic and hypolipidemic effects of Plu kaow (Houttuynia cordata Thunb.) ethanolic extract (PK) in streptozotocin (STZ) induced diabetic rats, focusing on its molecular mechanisms. Diabetes was induced in fasting Long Evans rats using streptozotocin (65 mg/kg b. w.), with glibenclamide (5 mg/kg/day) used as the standard experimental drug. The treated groups received oral supplementation of PK (500 mg/kg/day) for 28 days. The study evaluated blood glucose levels, lipid status, body weight, liver, kidney, and heart function biomarkers, antioxidant activity, and histological examination of various organs. Additionally, untargeted metabolomics, cheminformatics, and molecular docking were employed to elucidate the probable mechanisms of action of PK. Based on metabolomic profiling data, the PK was found to contain various putative antidiabetic agents such as kaempferol 7-neohesperidoside, isochlorogenic acid C, rutin, datiscin, and diosmin and they have been proposed to significantly (p < 0.001) reduce blood glucose levels and modulated hyperlipidemia. PK also improved the tested liver, kidney, and heart function biomarkers and reversed damage to normal pancreatic, liver, kidney, and heart cells in histological analysis. In conclusion, PK shows promise as a potential treatment or management option for diabetes and hyperlipidemia, as well as their associated complications in diabetic rats.

The role of doxorubicin in the formation of cardiotoxicity is a consensus statement. Part II. Cardiotoxicity of doxorubicin unrelated to myocytes and cardioprotection strategy (review)

Introduction. The use of doxorubicin in clinical practice has shown cumulative and dose-dependent toxic effects on cardiomyocytes, leading to an increase of mortality risk among patients with cancer and as a resulting to restrictions in the indications for its use.Text. A dangerous adverse reaction of doxorubicin is cardiomyopathy, leading to congestive heart failure. Cardiotoxicity is based on at least several pathophysiological mechanisms (described in more detail in the first part of the review), leading to damage to cardiomyocytes as a result of oxidative stress with the formation of free radicals, dysfunction of mitochondria, autophagy, release of nitric oxide and inflammatory mediators, as well as changes in gene expression and proteins leading to apoptosis. The current (second) part of the review provides detailed information on the actual understanding of the pathophysiological mechanisms underlying the described cardiotoxicity, the effect of doxorubicin on other heart cells. The use of cardioprotective strategies will reduce the severity and likelihood of developing cardiotoxicity. This article describes strategies based on reducing the maximum cumulative dose, changing the speed of doxorubicin administration, using pegylated liposomal formulations and cardioprotective agents, as well as exercise.Conclusion. Despite the huge number of scientific papers devoted to various aspects of cardiotoxicity of doxorubicin, its prevention and treatment, this issue requires more careful study and development of more advanced methods of early diagnosis, prevention and more effective therapy the complication.

An investigation on the cardioprotective potential of lichen compound protocetraric acid by H2O2-induced toxicity in H9c2 rat heart cells through in vitro and in silico analysis.

Worldwide, cardiovascular diseases (CVDs) are the leading cause of death and require treatment and prevention. Lichens are symbiotic organisms that are known to produce unique secondary metabolites and have been used as folk medicines. The aim of the study is to emphasize the importance of lichens in improving heart health, with the objective of investigating protocetraric acid, a lichen metabolite, for its antioxidant and cardioprotective potential by using in vitro and in silico techniques. Protocetraric acid (PRC) was isolated, characterized, and tested for antioxidant properties using six assays. In cardiovascular investigations, hydroxymethylglutaryl-coenzymeA reductase (HMGCR), angiotensin-converting enzyme inhibitory, and fibrinolytic capacities, along with enzyme inhibitory kinetics studies, were carried out. In silico toxicology and molecular docking analysis were done to determine the binding sites on target proteins. The cytoprotective ability of PRC was evaluated by H2O2-induced toxicity in H9c2 rat heart cells. Out of six lichens, the extract of F. caperata showed comparatively stronger antioxidant activity in terms of 1,1-diphenyl-2-picryl hydrazil (DPPH), scavenging of nitric oxide (SNO), and ferric reducing potential (FRAP) equivalent values. PRC showed significant antioxidant properties, and with respect to cardiovascular studies, PRC exhibited 86% HMGCR and 82% ACE inhibition, while 57% fibrinolysis at 320µM concentration. Inhibitory kinetic tests of PRC showed competitive and uncompetitive HMGCR and ACE inhibition types respectively. PRC showed minimum binding energies of - 7.9, - 8.9, and - 9.0kcal/mol with 1HWK, 1O8A, and 4BZS. The H9c2 cell line pre-treated with PRC was found to reduce H2O2 toxicity as well as increase cell viability. Protocetraric acid is a potent compound that has been experimentally shown to have hypocholesterolemic, hypotensive, and cardioprotective properties for treating cardiovascular diseases.

CureSCi Metadata Catalog-finding and harmonizing studies for secondary analysis of hydroxyurea use for sickle cell disease.

Sickle cell disease (SCD) is a rare group of inherited red blood cell disorders that affect hemoglobin, resulting in serious multi-system complications. The limited number of patients available to participate in research studies can inhibit investigating sophisticated relationships. Secondary analysis is a research method that involves using existing data to answer new research questions. Data harmonization enables secondary analysis by combining data across studies, especially helpful for rare disease research where individual studies may be small. The National Heart, Lung, and Blood Institute Cure Sickle Cell Initiative (CureSCi) Metadata Catalog is a web-based tool to identify SCD study datasets for conducting data harmonization and secondary analysis. We present a proof-of-concept secondary analysis to explore factors associated with discontinuation of hydroxyurea, a safe and effective first line SCD therapy, to illustrate the utility of the CureSCi Metadata Catalog to expedite and enable more robust SCD research. We performed secondary analysis of SCD studies using a multi-step workflow: develop research questions, identify study datasets, identify variables of interest, harmonize variables, and establish an analysis method. A harmonized dataset consisting of eight predictor variables across five studies was created. Secondary analysis involved a generalized linear model was employed to identify factors that significantly impact hydroxyurea discontinuation. The CureSCi Metadata Catalog provided a platform to efficiently find relevant studies and design a harmonization strategy to prepare data for secondary analysis. Multivariate analysis of the harmonized identified that patients who are older, are female, had a history of blood transfusion therapy, had episodes of acute chest syndrome, and had the SC sickle cell genotype are more likely to stop hydroxyurea treatment. This secondary analysis provides a template for how the CureSCi Metadata Catalog expedites dataset discovery of sickle cell studies for identifying relationships between variables or validating existing findings.

Machine Learning in Identifying Marker Genes for Congenital Heart Diseases of Different Cardiac Cell Types.

Congenital heart disease (CHD) represents a spectrum of inborn heart defects influenced by genetic and environmental factors. This study advances the field by analyzing gene expression profiles in 21,034 cardiac fibroblasts, 73,296 cardiomyocytes, and 35,673 endothelial cells, utilizing single-cell level analysis and machine learning techniques. Six CHD conditions: dilated cardiomyopathy (DCM), donor hearts (used as healthy controls), hypertrophic cardiomyopathy (HCM), heart failure with hypoplastic left heart syndrome (HF_HLHS), Neonatal Hypoplastic Left Heart Syndrome (Neo_HLHS), and Tetralogy of Fallot (TOF), were investigated for each cardiac cell type. Each cell sample was represented by 29,266 gene features. These features were first analyzed by six feature-ranking algorithms, resulting in several feature lists. Then, these lists were fed into incremental feature selection, containing two classification algorithms, to extract essential gene features and classification rules and build efficient classifiers. The identified essential genes can be potential CHD markers in different cardiac cell types. For instance, the LASSO identified key genes specific to various heart cell types in CHD subtypes. FOXO3 was found to be up-regulated in cardiac fibroblasts for both Dilated and hypertrophic cardiomyopathy. In cardiomyocytes, distinct genes such as TMTC1, ART3, ARHGAP24, SHROOM3, and XIST were linked to dilated cardiomyopathy, Neo-Hypoplastic Left Heart Syndrome, hypertrophic cardiomyopathy, HF-Hypoplastic Left Heart Syndrome, and Tetralogy of Fallot, respectively. Endothelial cell analysis further revealed COL25A1, NFIB, and KLF7 as significant genes for dilated cardiomyopathy, hypertrophic cardiomyopathy, and Tetralogy of Fallot. LightGBM, Catboost, MCFS, RF, and XGBoost further delineated key genes for specific CHD subtypes, demonstrating the efficacy of machine learning in identifying CHD-specific genes. Additionally, this study developed quantitative rules for representing the gene expression patterns related to CHDs. This research underscores the potential of machine learning in unraveling the molecular complexities of CHD and establishes a foundation for future mechanism-based studies.

Development of a Bmi1+ Cardiac Mouse Progenitor Immortalized Model to Unravel the Relationship with Its Protective Vascular Endothelial Niche.

The adult mammalian heart has been demonstrated to be endowed with low but real turnover capacity, especially for cardiomyocytes, the key functional cell type. The source, however, of that turnover capacity remains controversial. In this regard, we have defined and characterized a resident multipotent cardiac mouse progenitor population, Bmi1+DR (for Bmi1+ Damage-Responsive cells). Bmi1+DR is one of the cell types with the lowest ROS (Reactive Oxygen Species) levels in the adult heart, being particularly characterized by their close relationship with cardiac vessels, most probably involved in the regulation of proliferation/maintenance of Bmi1+DR. This was proposed to work as their endothelial niche. Due to the scarcity of Bmi1+DR cells in the adult mouse heart, we have generated an immortalization/dis-immortalization model using Simian Vacuolating Virus 40-Large Antigen T (SV40-T) to facilitate their in vitro characterization. We have obtained a heterogeneous population of immortalized Bmi1+DR cells (Bmi1+DRIMM) that was validated attending to different criteria, also showing a comparable sensitivity to strong oxidative damage. Then, we concluded that the Bmi1-DRIMM population is an appropriate model for primary Bmi1+DR in vitro studies. The co-culture of Bmi1+DRIMM cells with endothelial cells protects them against oxidative damage, showing a moderate depletion in non-canonical autophagy and also contributing with a modest metabolic regulation.

Lineage tracing using Wnt2b-2A-CreERT2 knock-in mice reveals the contributions of Wnt2b-expressing cells to novel subpopulations of mesothelial/epicardial cell lineages during mouse development.

Mesothelial and epicardial cells give rise to various types of mesenchymal cells via epithelial (mesothelial)-to-mesenchymal transition during development. However, the genes controlling the differentiation and diversification of mesothelial/epicardial cells remain unclear. Here, we examined Wnt2b expression in the embryonic mesothelium and epicardium and performed lineage tracing of Wnt2b-expressing cells by using novel Wnt2b-2A-CreERT2 knock-in and LacZ-reporter mice. Wnt2b was expressed in mesothelial cells covering visceral organs, but the expression was restricted in their subpopulations. Wnt2b-expressing cells labeled at embryonic day (E) 10.5 were distributed to the mesothelium and mesenchyme in the lungs, abdominal wall, stomach, and spleen in Wnt2b2A-CreERT2/+;R26RLacZ/+ mice at E13.0. Wnt2b was initially expressed in the proepicardial organ (PEO) at E9.5 and then in the epicardium after E10.0. Wnt2b-expressing PEO cells labeled at E9.5 differentiated into a small fraction of cardiac fibroblasts and preferentially localized at the left side of the postnatal heart. LacZ+ epicardium-derived cells labeled at E10.5 differentiated into a small fraction of fibroblasts and smooth muscle cells in the postnatal heart. Taken together, our results reveal novel subpopulations of PEO and mesothelial/epicardial cells that are distinguishable by Wnt2b expression and elucidate the unique contribution of Wnt2b-expressing PEO and epicardial cells to the postnatal heart.

Abstract Mo042: Poison Peptides May Contribute to Dysregulation of SERCA in Heart Failure

Proteolysis increases in heart disease, resulting in an accumulation of degraded membrane protein fragments in heart cells. Previous studies have shown that diverse hydrophobic alpha helices can bind and non-specifically inhibit the cardiac calcium pump SERCA. Thus, we hypothesized that partially degraded fragments of transmembrane proteins may directly inhibit SERCA or compete with native peptide regulators of the pump, like such as phospholamban. The resulting dysregulation of SERCA may contribute to calcium mishandling in heart failure. To test these hypotheses, purified cell membranes from non-failing or dilated cardiomyopathy human hearts were subjected to SDS-PAGE and mass spectrometry to identify protein fragments smaller than 26 kD. 1800 proteins were detected, of which 92 were upregulated &gt;3 fold in the failing heart samples, consistent with increased production of small fragments from these membrane proteins. 9 of the upregulated fragments containing transmembrane segments were selected for further analysis based on sequence hydrophobicity and structural similarity to endogenous micropeptide regulators of SERCA. Of these 9 fragments, 6 showed ER localization, suggesting potential for a toxic interaction with SERCA. The relative binding of these candidates to SERCA was quantified with FRET microscopy. Fragments of three membrane proteins (SEC61B, VAMP3, VAMP8) showed an avid interaction with SERCA. To measure SERCA inhibition, we utilized a calcium activity assay was performed usingin which HEK293T cells were transfected with SERCA, ryanodine receptor, candidate peptides and rCepia as an ER calcium indicator. ER-calcium level was normalized to minimum fluorescence after caffeine addition and an ionomycin induced maximum fluorescence after addition of ionomycin and calcium and caffeine induced minimum. Unregulated Candidate poison peptides were compared to SERCA alone and SERCA-PLB phospholamban controls were also performed. . SEC61B demonstrated the a strongest inhibition, most (similar to phospholamban, ) while VAMP3 and VAMP8’s effect was more modest. This type of non-nativeThus, we propose accumulation of toxic transmembrane peptide fragments may inhibition could be contributinginhibit SERCA and contribute to the calcium mishandling seen in heart failure. Future experiments will be performed to quantify the relative abundance of the fragments compared to endogenous regulatory micropeptides.

Single-Cell RNA-Seq Analysis of Hearts in Patients with Fetal Tetralogy of Fallot.

To explore the cytological characteristics of tetralogy of Fallot (TOF), we collected samples and investigated the differences in the cytological classification between normal fetal hearts and fetal hearts with congenital defects. We then performed single-cell sequencing analysis to search for possible differential genes of disease markers. Here, the right ventricles of a heart sample with TOF and a healthy human fetal heart sample were analyzed through single-cell sequencing. Data quality control filtering, comparison, quantification, and identification of recovered cells on the raw data were performed using Cell Ranger, thereby ultimately obtaining gene expression matrices for each cell. Subsequently, Seurat was used for cell filtration, standardization, cell subgroup classification, differential expression gene analysis of each subgroup, and marker gene screening. Bioinformatic analysis identified 9,979 and 15,224 cells from the healthy and diseased samples, respectively, with an average read depth of 25,000/cell. The cardiomyocyte cell populations, derived from the abnormal samples identified through the first-level graph-based analysis, were separated into six distinct cell clusters. Our study provides some information on TOF in a fetus, which can offer a new reference for the early detection and treatment of TOF by comparing defective heart cells with normal heart cells.

Abstract We008: Hallmark Maturation of the Human Pluripotent Stem Cells-derived Cardiovascular Progenitors in Myocardial Infarcted Large Animal Model

For the goal of regenerative cardiology, cellular therapy may be used to restore injured heart muscle. The molecular mechanisms behind the engraftment and maturation of these transplanted cells, however, remain poorly understood. Thus, to obtain a comprehensive understanding of the complex transcriptional landscapes within their transplanted spatial context, we utilized spatial transcriptomics in this study. We have previously demonstrated the safety and effectiveness of transplanting our cardiovascular progenitors (CVPs) produced from laminin-221 into pig hearts that had suffered myocardial infarction (MI). Here, we undertake 10x spatial transcriptomics in chronic MI pig models at 1-, 4-, and 12 weeks post-transplantation to further our understanding of these cells in vivo. With confidence, we can distinguish between the transcripts from the pigs and the humans in the infarcted region. By using human-specific anti-Ku80 antibodies for immunohistochemical labeling of the pig tissue samples, this in silico prediction was verified. The CVPs' engraftment and longevity in the infracted region were shown by the long-term identification and staining of human cells at various time points. It is interesting to note that after 12 weeks, ribosomal and mitochondrial activity were higher than at 1 and 2 weeks. These results are consistent with the hallmarks of cardiac maturation, in which cells transit from immature fetal-like cells to mature cardiomyocytes (CMs) by switching their metabolic energy source from glucose to fatty acids. Also, by comparing the heart-specific genes, including MYH6 and MYH7, we were able to quantify the myosin isoform switches in the xenograft over time. Furthermore, to delineate the fate of the transplanted cells, we performed a ligand-receptor interaction analysis. Intriguing, we identified the midkine protein as a possible secreted human paracrine factor that enhances angiogenesis in the graft. In conclusion, we have shown the successful engraftment and maturation of human CVPs toward CMs using spatial transcriptomic technology. These data will be used to understand other aspects of regenerative cardiology such as the mechanism of VTs and in vivo cellular repair mechanisms. A shiny app atlas has been generated to share our transcriptomics data for all to explore gene expression of human transplanted cells in myocardial infarcted pig hearts.

Abstract Mo054: Long Noncoding RNA LINC00667 Downregulation Leads to SCN5A Alternative Splicing and Arrhythmogenesis

Background: The voltage-gated sodium channel SCN5A, which encodes the Nav1.5 protein, is integral to the propagation of electrical signals in cardiac tissue. SCN5A expression is diminished in cardiomyopathy, significantly increasing the susceptibility to arrhythmias. This reduction in channel activity can be attributed to decreased mRNA stability coupled with aberrant mRNA splicing. The underlying mechanisms driving the dysregulation of mRNA splicing under pathophysiological conditions remain poorly understood. Objective: This study explores the role of long noncoding RNA LINC00667 in the abnormal alternative splicing of SCN5A mRNA. Methods: LINC00667 expression was analyzed using public databases, human heart tissue, iPSC-derived cardiomyocytes (iPSCMs), and cell lines. The impact of SCN5A on action potentials and sodium currents was studied in iPSCMs. LINC00667 and SCN5A expressions were measured using qRT-PCR, while Nav1.5 protein levels were assessed through Western blot. Alternative splicing of SCN5A mRNA was examined using PCR with exon-specific primers. Gene ontology and in silico analysis identified RNA binding proteins (RBPs) potentially interacting with LINC00667. RNA immunoprecipitation (RIP) assays confirmed direct binding with key splice factors. Group differences were assessed using 2-tailed Student’s t-tests, with P &lt; 0.05 indicating significance. Results: LINC00667, found at chromosome 18p11.31, is expressed in the human heart and associated cells, including cardiomyocytes. Its levels are inversely related to arrhythmic risk, decreasing in heart failure and ischemic cardiomyopathy as well as under hypoxic conditions. Gene ontology linked RBPs to SCN5A mRNA splicing, with RBM25, RBM5, and TIA1 identified as LINC00667 interactors. LINC00667 reduction affected regions of SCN5A mRNA encompassing Exons 6, 24, and 28, which could potentially be linked to decreased function/expression or increased unfolded protein response (UPR). Abnormal RNA splicing decreased SCN5A-encoded sodium current, reduced action potential upstroke velocity, and shortened AP duration without altering resting membrane potential. Conclusion: Decreased LINC00667 releases splicing factors that result in abnormal SCN5A mRNA splicing and reduced sodium current. Since LINC00667 is reduced in cardiomyopathy, overexpression may be antiarrhythmic by preventing sodium channel downregulation.

Abstract Or111: Titin cleavage disrupts sarcomere-adhesion tensional homeostasis triggering cardiomyocyte arrhythmia in vitro

The cardiac tissue experiences several forms of mechanical stress, including contraction of cardiomyocytes and forces derived from the stiffness of extracellular matrix (ECM). Preserving homeostatic mechanical forces is crucial for a healthy heart, yet the mechanisms involved are not well-explored. Our study delves into cardiomyocyte responses following the sudden release of mechanical tension across titin, a giant sarcomere protein crucial for setting cardiomyocyte stiffness. Through the expression of virally transduced TEV protease (TEVp) in neonatal mouse cardiomyocytes (NMCMs), we induced the cleavage of mutant titin including the specific TEV protease recognition site (TEVs) within the mechanically active I-band region of the protein. Titin cleavage was validated by 1.8% polyacrylamide SDS-PAGE electrophoresis, revealing a primary band at ~2.2 MDa in TEV-positive NMCMs, corresponding to digested titin fragment A-M. This cleavage, confirmed at 2 days post-infection (dpi) with ~80% efficiency, reached 100% by 5 dpi. Consequently, the Young’s modulus of affected cardiomyocytes dropped by 50%, as determined by Atomic Force Spectroscopy. Sarcomere disarray increased over time, as evidenced by immunofluorescence, yet this effect did not induce cell death or proliferation. Cell viability was confirmed by the fact that affected cardiomyocytes are able to contract spontaneously, although titin cleavage resulted in reduced beating amplitude and induced arrhythmic behavior. Considering this arrhythmogenic situation, we tested cell-cell adhesion through dissociation assays, which revealed significantly reduced intercellular adhesion in affected cardiomyocytes. RNAseq confirmed involvement of pathways related to heart contraction, anchoring junctions, and cell junctions. Specifically, dysregulated genes associated with gap-junction and costamere complexes were observed at 5 dpi. Alterations of adhesion were further confirmed by reductions in connexin 43 and focal adhesion length in immunofluorescence experiments, which also showed mislocalization of important adhesive proteins. In conclusion, titin cleavage in neonatal cardiomyocytes leads to sarcomere disarray, perturbed cell-cell adhesion and arrhythmia. These findings highlight the importance of full mechanical integrity of the titin filament in living cardiomyocytes.

Abstract We013: Modeling Sex and Stress using Induced Pluripotent Stem Cell Derived Cardiomyocytes

Despite causing 1 in 3 deaths worldwide, cardiovascular disease and the heart have only recently been studied in women. Contrary to previous belief, the female heart is not just a smaller replica of a male heart. Almost all heart diseases show sexual differences in risk, treatment, and symptoms. Even with these differences there is a lack of female models in animal and cellular studies. Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are a powerful human model to test baseline and stressed sex differences at the cellular level. We hypothesized that using stressors that mimic disease conditions (drugs, substrate stiffness) in multiple male and female hiPSC-CMs will result in differential sex-based responses. We ran Traction Force Microscopy on 3 male (n = 424 cells, 3 batches) and 3 female (n = 318 cells, 3 batches) cell lines on 10 kPa polyacrylamide hydrogels. Male cells had 42% higher twitch force (p = 8.8E-6) with 25% higher contraction (p = 1.77E-5) and relaxation (p = 0.0006) velocities. However, female cells had a larger spread area (823 +/- 17.32 µm 2 vs. 756 +/- 21.64 µm 2 , p = 0.0018). To validate hiPSC-CMs as a model we compared preliminary RNA-seq to publicly available Heart Cell Atlas sc/snRNA-seq data sets of primary ventricular cardiomyocytes. They had similar differentially expressed genes (DEGs) as our hiPSC-CMs. Furthermore, DEGs had significantly enriched gene sets including a 25-fold enrichment in hypertrophic cardiomyopathy (FDR = 1.6E-7), 23-fold in dilated cardiomyopathy (FDR = 1.6E-7), and 16-fold in cardiac muscle contraction (FDR = 6.3E-4). We analyzed stiffness by differentiating and culturing hiPSC-CMs on 10 kPa and 100 kPa stiffness hydrogels. Our RNA-seq analysis at day 10 and 30 after differentiation found 7000 DEGs at 10 kPa while only 556 DEGs at 100 kPa. We looked hypercontractility caused stres and performed RNA-seq on day 60 wild type and hypercontractile mutant hiPSC-CMs. The wild type was dosed with positive inotrope levosimendan to induce hypercontractility while the hypercontractile mutant was dosed with negative inotrope verapamil to lower contractility. Hypercontractility had significant upregulation of the cellular response to stress including ER stress, stress-activated MAPK cascades, stress fibers, and cardiac muscle hypertrophy in response to stress. These differences can identify possible gene targets for preventing cardiovascular disease and inform personalized medicine.

Abstract Tu095: Cycloastragenol Alleviates Cardiac Fibrosis through Inhibiting Pyroptosis Activated by NLRP3 Inflammasome

Cardiac fibrosis is cardiac interstitial remodeling characterized by an enhanced extracellular matrix deposition. Acute sympathetic stress causes cardiac cells to immediately undergo pyroptosis, which induces cardiac inflammation and damage, and ultimately results in cardiac fibrosis. Our previous study has demonstrated the inhibitory effects of cycloastragenol (CAG), an active form of astragaloside IV isolated from Astragalus membranaceus, on isoproterenol (ISO)-induced cardiac fibrosis through blocking the NLRP3 signaling pathway. It is yet unknown how CAG affects pyroptosis of cardiac cells and whether its ability to prevent cardiac fibrosis coincides with this process. In this study, 5 mg/kg ISO was subcutaneously injected into wild-type (WT), NLRP3 knockout (KO), and GSDMD KO mice for 7 days to induce cardiac fibrosis. CAG (125 mg/kg/d) was administrated to the mice intragastrically at the start of the ISO injection. Primary cardiomyocytes and cardiac fibroblasts were isolated from neonatal rat hearts and treated with 10 μM ISO either with or without CAG. The results show that CAG alleviates cardiac fibrosis in WT mice, while lost this efficacy in NLRP3 KO mice. GSDMD KO mice do not develop ISO-induced cardiac fibrosis, indicating the key role of pyroptosis in cardiac fibrosis. Pyroptosis mainly occurs in mouse hearts during the initial stages of ISO induction (1 and 24 hours after ISO injection), and CAG significantly reduces pyroptosis of heart cells in WT mice, while CAG is unable to prevent pyroptosis in NLRP3 KO mouse hearts. The TGF-/Smads signaling pathway is also inhibited by CAG in WT mice but not in NLRP3 KO mice. Additionally, CAG prevents the in vitro pyroptosis of cardiomyocytes induced by ISO directly and cardiac fibroblasts caused by LPS+ATP. These results suggest that the principal mechanism of the anti-cardiac fibrosis effect of CAG mainly depends on its inhibition of pyroptosis mediated by NLRP3 inflammasome.

Altered lipid metabolism promoting cardiac fibrosis is mediated by CD34+ cell-derived FABP4+ fibroblasts

Hyperlipidemia and hypertension might play a role in cardiac fibrosis, in which a heterogeneous population of fibroblasts seems important. However, it is unknown whether CD34+ progenitor cells are involved in the pathogenesis of heart fibrosis. This study aimed to explore the mechanism of CD34+ cell differentiation in cardiac fibrosis during hyperlipidemia. Through the analysis of transcriptomes from 50,870 single cells extracted from mouse hearts and 76,851 single cells from human hearts, we have effectively demonstrated the evolving cellular landscape throughout cardiac fibrosis. Disturbances in lipid metabolism can accelerate the development of fibrosis. Through the integration of bone marrow transplantation models and lineage tracing, our study showed that hyperlipidemia can expedite the differentiation of non-bone marrow-derived CD34+ cells into fibroblasts, particularly FABP4+ fibroblasts, in response to angiotensin II. Interestingly, the partial depletion of CD34+ cells led to a notable reduction in triglycerides in the heart, mitigated fibrosis, and improved cardiac function. Furthermore, immunostaining of human heart tissue revealed colocalization of CD34+ cells and fibroblasts. Mechanistically, our investigation of single-cell RNA sequencing data through pseudotime analysis combined with in vitro cellular studies revealed the crucial role of the PPARγ/Akt/Gsk3β pathway in orchestrating the differentiation of CD34+ cells into FABP4+ fibroblasts. Through our study, we generated valuable insights into the cellular landscape of CD34+ cell-derived cells in the hypertrophic heart with hyperlipidemia, indicating that the differentiation of non-bone marrow-derived CD34+ cells into FABP4+ fibroblasts during this process accelerates lipid accumulation and promotes heart failure via the PPARγ/Akt/Gsk3β pathway.

Correlation between plasma biochemical parameters and cardio-hepatic iron deposition in thalassemia major patients

Introduction Major Thalassemia patients suffer from iron overload and organ damage, especially heart and liver damage. Early diagnosis and treatment with a chelator can reduce the complications and mortality of iron overload. Therefore, we aimed to investigate the biochemical and hematological predictors as an alternative and indirect indicator of iron deposition in heart and liver cells in comparison with the MRI T2* method as the gold standard. Material and method MRI T2* was evaluated in the heart and liver tissues of 62 major beta-thalassemia patients undergoing regular transfusion and chelator therapy. Biochemical and hematological factors were also measured, including serum ferritin, serum electrolytes, liver enzymes, hemoglobin, blood glucose, and serum magnesium. The correlation between these factors was assessed using statistical evaluations. Result Serum ferritin had a positive and significant correlation with liver siderosis based on MRI T2* (p-value = .015), and no significant association was observed with cardiac siderosis (p-value = .79). However, there was a significant positive correlation between cardiac iron deposition and fasting blood sugar level (p-value = −.049), and plasma level of liver enzymes (alanine aminotransferase (ALT) (p-value = .001), aspartate aminotransferase (AST ((p-value = .01)). Moreover, there was a significant negative correlation between cardiac iron overload and plasma magnesium level (p-value = .014). According to MRI T2*, there was no significant correlation between cardiac and hepatic iron overload (p value = .36). Conclusion An increase in blood sugar or liver enzymes and a decrease in serum magnesium was associated with an increase in cardiac iron overload based on MRI T2*. Liver iron overload based on MRI T2* had a significant correlation with serum ferritin.

Pyruvate Kinase M2: A Potential Regulator of Cardiac Injury Through Glycolytic and Non-glycolytic Pathways.

Adult animals are unable to regenerate heart cells due to postnatal cardiomyocyte cycle arrest, leading to higher mortality rates in cardiomyopathy. However, reprogramming of energy metabolism in cardiomyocytes provides a new perspective on the contribution of glycolysis to repair, regeneration, and fibrosis after cardiac injury. Pyruvate kinase (PK) is a key enzyme in the glycolysis process. This review focuses on the glycolysis function of PKM2, although PKM1 and PKM2 both play significant roles in the process after cardiac injury. PKM2 exists in both low-activity dimer and high-activity tetramer forms. PKM2 dimers promote aerobic glycolysis but have low catalytic activity, leading to the accumulation of glycolytic intermediates. These intermediates enter the pentose phosphate pathway to promote cardiomyocyte proliferation and heart regeneration. Additionally, they activate adenosine triphosphate (ATP)-sensitive K + (K ATP ) channels, protecting the heart against ischemic damage. PKM2 tetramers function similar to PKM1 in glycolysis, promoting pyruvate oxidation and subsequently ATP generation to protect the heart from ischemic damage. They also activate KDM5 through the accumulation of αKG, thereby promoting cardiomyocyte proliferation and cardiac regeneration. Apart from glycolysis, PKM2 interacts with transcription factors like Jmjd4, RAC1, β-catenin, and hypoxia-inducible factor (HIF)-1α, playing various roles in homeostasis maintenance, remodeling, survival regulation, and neovascularization promotion. However, PKM2 has also been implicated in promoting cardiac fibrosis through mechanisms like sirtuin (SIRT) 3 deletion, TG2 expression enhancement, and activation of transforming growth factor-β1 (TGF-β1)/Smad2/3 and Jak2/Stat3 signals. Overall, PKM2 shows promising potential as a therapeutic target for promoting cardiomyocyte proliferation and cardiac regeneration and addressing cardiac fibrosis after injury.