Cardiac Cell Populations Research Articles

Heart regeneration from the whole-organism perspective to single-cell resolution.

Cardiac regenerative potential in the animal kingdom displays striking divergence across ontogeny and phylogeny. Here we discuss several fundamental questions in heart regeneration and provide both a holistic view of heart regeneration in the organism as a whole, as well as a single-cell perspective on intercellular communication among diverse cardiac cell populations. We hope to provide valuable insights that advance our understanding of organ regeneration and future therapeutic strategies.

Rescue of mitochondrial dysfunction through alteration of extracellular matrix composition in barth syndrome cardiac fibroblasts

Fibroblast-ECM (dys)regulation is associated with a plethora of diseases. The ECM acts as a reservoir of inflammatory factors and cytokines that mediate molecular mechanisms within cardiac cell populations. The role of ECM-mitochondria crosstalk in the development and progression of cardiac disorders remains uncertain. We evaluated the influence of ECM produced by stromal cells from patients with the mitochondrial cardiomyopathy (Barth syndrome, BTHS) and unaffected healthy controls on cardiac fibroblast (CF) metabolic function. To do this, cell-derived matrices CDMs were generated from BTHS and healthy human pluripotent stem cell-derived CFs (hPSC-CF) and used as cell culture substrates. BTHS CDMs negatively impacted the mitochondrial function of healthy hPSC-CFs while healthy CDMs improved mitochondrial function in BTHS hPSC-CFs. Mass spectrometry comparisons identified 5 matrisome proteins differentially expressed in BTHS compared to healthy CDM. Our results highlight a key role for the ECM in disease through its impact on mitochondrial function.

Abstract Mo007: Molecular Basis of Sepsis-Induced Cardiomyopathy Under Anthracycline Treatment: Insights from Mouse Model

Anthracyclines constitute a fundamental component in numerous paediatric chemotherapy regimens. However, its myelosuppressive & cardiotoxic nature put patients at risk of sepsis and sepsis-induced cardiomyopathy (SIC). When anthracycline-treated patients develop SIC, successful treatment of sepsis is sufficient for the restoration of cardiac function, giving rise to the 'transient cardiac dysfunction' phenomenon. Although transient it poses compromised clinical outcomes & the mechanism driving this phenomenon remains elusive. We hypothesise that the molecular regulators of the phenomenon lie within cardiac cell populations & is controlled by transcriptional activation. Thus, we aim to model 'transient cardiac dysfunction' in mice and conduct transcriptomics on their heart tissues. ‘Transient cardiac dysfunction’ was modelled in male juvenile C57BL6 mice treated with a cumulative of 10mg/kg doxorubicin followed by sepsis induction via cecal ligation & perforation (CLP) to induce SIC. To recapitulate ‘transient cardiac dysfunction’, antibiotics were administered 20h post-CLP to treat sepsis & restore cardiac function. Controls against doxorubicin (saline treatment) was established. Echocardiography and biological sample curation were conducted at pre-SIC (before CLP), onset-SIC (10h post-CLP) & post-SIC (60h post-CLP). Sepsis was verified based on procalcitonin fold change as compared to pre-SIC (n=3-6). Procalcitonin was 15±1.6 folds higher in dox- (n=3) & 10±0.39 folds higher in saline-treated (n=3) mice at onset-SIC. The infection was more aggressive in dox-treated mice as procalcitonin remained elevated at post-SIC (17.1±2.0, n=4), whereas in saline-treated mice, procalcitonin was reduced by post-SIC (7.9±1.0, n=3). In saline-treated mice, LVEF(%) was reduced at onset-SIC (49.0%±3.0, n=7) from pre-SIC (58.1±1.1, n=7) and recovered to baseline by post-SIC (61.8±1.7, n=5). In the contrary, LVEF in dox-treated mice (n=7) was increased at onset-SIC (62.5±1.7) from pre-SIC (56.0±0.8) & sustained till post-SIC (60.1±1.0), suggesting cardioprotection. Normal distribution was verified before statistical hypothesis testing. In conclusion, ‘transient cardiac dysfunction’ was modelled in saline-treated mice, & novel cardio protective effect was observed in dox-treated mice. Moving forward, longitudinal snRNA-seq will be conducted on whole hearts to identify cardiac cell populations & genes responsible for ‘transient cardiac dysfunction’ & cardioprotection.

Abstract Mo090: Altered Cardiac Cell Populations in Hypoplastic Left Heart Syndrome

Background: Congenital heart disease (CHD) affects ~1% of infants. Hypoplastic left heart syndrome (HLHS), a severe form of CHD in which the left ventricle is underdeveloped, is associated by a 15% incidence of heart failure by 6 years of age. Only 6% of HLHS patients have a genetic cause identified on exome sequencing, limiting the ability of patients to receive a diagnosis and potentially benefit from targeted treatments. Hypothesis: Novel HLHS genes are differentially expressed in HLHS cardiac tissues, or in cells with differential abundance in HLHS hearts. Goals: To use single nucleus RNA sequencing of HLHS patient cardiac tissues to identify candidate HLHS genes. Methods: Single nucleus RNA sequencing (nucSeq) was performed on paired left and right ventricular tissues (LV, RV) from 9 participants with HLHS. Left ventricular cardiac tissues from children (4) and adults (12) without CHD were used for comparison. Filtering with CellBender and Solo were used to remove low-quality nuclei. Analysis was performed in R using the Seurat package. Results: HLHS LV tissues had a higher proportion of cardiomyocytes than pediatric or adult control tissues, (78.7% vs 64.1%, p=0.005; 78.7 % vs 47.0%, p=1.8E-08, respectively), and a lower proportion of mural cells (3.1% vs 9.3%, p=3.9E-05; 3.1 % vs 23.5%, p=7.3E-15, respectively). By contrast, there were similar proportions of endothelial cells in the HLHS, pediatric and adult tissues (7.1%, 8.8% and 7.9%, respectively; p=0.56). Within HLHS tissues, the LV had a higher proportion of cardiomyocytes than the RV (78.7% vs 51.2%, p=4.8E-04) while the proportion of endothelial cells were similar in the LV and RV (7.3% and 9.1%, respectively; p=0.33). LV tissues from HLHS participants 2-18 years of age had fewer capillary endothelial cells than pediatric controls (31.2% vs 57.8%, p=0.001), and gene markers of HLHS endothelial cells included PBX1 , which is required for Hox D-3 mediated angiogenesis, as well as ARHGAP26 and RORA, inhibitors of VEGF signaling. Conclusion: HLHS cardiac tissues have a higher proportion of cardiomyocytes in the LV despite a smaller chamber size. Differences in gene expression that accompany the differences in cell proportion could identify novel HLHS genes as well as potential therapeutic targets.

Abstract Tu102: The Role of the β IV -spectrin/STAT3 Complex in Regulating Ischemic Cardiac Remodeling

Adverse fibrotic remodeling contributes to high morbidity and mortality in patients following myocardial infarction (MI). The healing process after MI requires necrotic tissue to be cleared and replaced with fibrosis, which is driven by the coordinated effort of several distinct cardiac cell populations, including macrophages and cardiac fibroblasts (CFs). While previous studies have focused on identifying the stimuli that drives fibrotic remodeling in the infarcted myocardium, the contribution of specific cell populations to the signaling cascade and the manner of how these communication networks are tuned in response to chronic stress remains unclear. Prior work has identified a role for the cytoskeletal protein, β IV -spectrin, in coordinating the activity of the transcription factor, STAT3, with implications for CF activation and profibrotic signaling. Specifically, it was found that mice expressing truncated β IV -spectrin ( qv 3J ) are resistant to cardiac fibrosis in the setting of chronic pressure overload, with maintained β IV -spectrin expression and STAT3 localization/activity in CFs. Further, β IV -spectrin deficiency causes increased secretion of profibrotic and proinflammatory factors, including matrix metallopeptidases (MMPs). Therefore, we hypothesized that stress-induced loss of β IV -spectrin and subsequent dysregulation of STAT3 signaling in CFs proximal to the infarct promotes fibroblast activation, an important first step in repairing the infarct region. In this study, we subjected WT and qv 3J mice to permanent ligation of the left anterior descending artery and assessed cardiac function and survival through 28 days. A subset of mice were sacrificed at 7 days for histology, qPCR, and flow cytometry. Compared to WT, qv 3J mice showed a significant decrease in 28-day survival with increased mortality in the first week post-MI. Autopsy identified increased incidence of cardiac rupture in qv 3J animals. Immunohistochemistry revealed impaired scar formation at 7 days post-MI in qv 3J compared to WT. Quantitative PCR showed altered expression of matrix metallopeptidases (MMPs 3, 8, 9, and 14) in the qv 3J hearts at day 7. MMP activity was assessed in whole heart lysates using gelatin-zymography. Collagen uptake was assessed in CFs and bone marrow-derived macrophages isolated from WT and qv 3J mice. Our work identifies a novel role of the spectrin-based pathway in facilitating fibrotic remodeling in response to ischemic stress.

The effect of aerobic-resistance training on the population of cardiac multipotent cells, thickness, and diameter of the ventricular wall through the c-kit pathway in male rats during the growth stages

Background. The purpose of the present study was to investigate the effect of aerobic-resistance training on the population of cardiac multipotent cells, thickness, and diameter of the ventricular through the C-Kit pathway of male rats during the growth stages. Methods. Overall, 30 male Wistar rats in three age groups of 2 (n = 10), 8 (n = 10), and 96 (n = 10) weeks were accidentally divided into exercise and control groups. Resistance training programs (resistance ladder, 3 days a week) and aerobics (treadmill running, 3 days a week) were performed for 6 weeks. Hematoxylin-Eosin staining was done the heart tissue and histological images were prepared. Afterward, C-Kit gene expression was examined by the real-time polymerase chain reaction method. The statistical method of one-way analysis of variance (P ≥ 0.05) and Tukey’s test were used at a significant level (P ≥ 0.01). Results. The trained neonatal group had higher values in heart weight to body weight index, and the trained neonatal and trained young adult groups had higher values in left ventricular thickness (P ≥ 0.01). The decrease in left ventricular internal diameter in the trained young adult group was significant compared to the control group (P ≥ 0.01). There was a significant increase in C-Kit gene expression in trained young adult and trained old groups (P ≥ 0.01). Conclusion. Aerobic-resistance training can be an effective stimulus for increasing the number of cardiac stem cells and creating structural adaptations of the heart, including increasing thickness and ventricular diameter in neonatal and young adult groups. Practical Implications. Participation in aerobic-resistance training programs leads to the improvement of the cardiac structure and an increase in cardiac stem cells.

Left Ventricular Dilation Resulting from the Absence of Either Rgs2 or Rgs5 Is Associated with Increased Immune Cell Infiltration in the Myocardium

Sustained sympathoexcitation is a hallmark of heart failure, with equally sustained activation of downstream signaling pathways involving Gs- and Gi/o-coupled β-adrenergic receptors (βAR) stimulating cAMP production and PKA activation. A critical mechanism controlling G protein-mediated βAR signaling is the activity of regulator of G protein signaling (RGS) proteins, including members of the B/R4 subfamily, which accelerate the termination of Gq/11 and Gi/o protein signaling. Previous studies by us and others reported that moderation of Gi/o activity separately by RGS2 and RGS5 of the R4 subfamily is key to maintaining normal ventricular rhythm. The loss of one or both R4 RGS proteins also results in left ventricular (LV) dilation and reduced ejection fraction. Here, we tested the hypothesis that the LV structural remodeling is due to the disruption of the coordinated activity of RGS2 and 5 resulting in sensitization of βAR signaling. To test this hypothesis, we challenged wild type (WT), Rgs2 KO ,Rgs5 KO, and Rgs2/5 double knockout (dbKO) adult male mice with sub-chronic infusion of isoproterenol (ISO, 30 mg/kg/day, 3 days) via a peritoneal osmotic minipump implantation. Rgs2 KO and Rgs5 KO mice showed increased LV chamber size at baseline, which was unchanged by ISO administration. In saline-treated animals, the loss of Rgs2 increased Rgs5 mRNA expression, while Rgs5 absence increased Rgs4 mRNA expression. ISO infusion induced concentric cardiac hypertrophy in WT mice, as evidenced by increased heart weight/tibia length (HW/TL) ratio and cardiomyocyte cross sectional area. While ISO infusion increased interstitial fibrosis in midventricular tissue and HW/TL ratio in dbKO mice, it did not affect cardiomyocyte cross sectional area or exacerbate LV dilation, despite increased expression of the cardiac fetal gene Nppa as well as the sarcomeric protein α-actinin and Ca2+/calmodulin-dependent kinase II (CaMKII). Cardiact tissue from saline-treated Rgs2 KO and Rgs5 KO mice had markedly increased CD45+ cells vs WT, while tissue from Rgs5 KO mice showed increased CD68+ cells, as revealed by immunohistochemistry. Taken together, these results indicate that ventricular remodeling seen in Rgs2 KO, Rgs5 KO, and Rgs2/5 dbKO mice is insensitive to sustained βAR stimulation but may be due to increased cardiac resident immune cell population. This work was supported by NIH R56 DK132859-01A1 and R01 HL139754, and Craig H. Neilsen Foundation Grant 382566 to Patrick Osei-Owusu. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cardiac Progenitor Cell Metabolism.

Metabolism has emerged recently as an important determinant of stem cell function. Changes in metabolic signaling pathways precede changes in stem cell molecular and functional response. Pluripotent stem cells are highly proliferative and known to exhibit increased glycolysis. Similarly, adult stem cells reside in tissue niches in a quiescent state operating via glycolysis. Upon activation, adult stem cell metabolism transitions from glycolysis to oxidative phosphorylation which coincides with reduced proliferation and multilineage potential. In the heart, different populations of cardiac progenitor cells (CPCs) have been identified. CPCs regenerative potential is linked to changes in metabolic characteristics of cells, impacting cardiac repair following injury. Here, we discuss the methodologies for isolation and characterization of a novel cardiac progenitor cell population from the heart including measurement its metabolic features.

Urokinase-Type Plasminogen Activator Receptor Regulates Prosurvival and Angiogenic Properties of Cardiac Mesenchymal Stromal Cells.

One of the largest challenges to the implementation of cardiac cell therapy is identifying selective reparative targets to enhance stem/progenitor cell therapeutic efficacy. In this work, we hypothesized that such a target could be an urokinase-type plasminogen activator receptor (uPAR)-a glycosyl-phosphatidyl-inositol-anchored membrane protein, interacting with urokinase. uPAR is able to form complexes with various transmembrane proteins such as integrins, activating intracellular signaling pathway and thus regulating multiple cell functions. We focused on studying the CD117+ population of cardiac mesenchymal progenitor cells (MPCs), expressing uPAR on their surface. It was found that the number of CD117+ MPCs in the heart of the uPAR-/- mice is lower, as well as their ability to proliferate in vitro compared with cells from wild-type animals. Knockdown of uPAR in CD117+ MPCs of wild-type animals was accompanied by a decrease in survival rate and Akt signaling pathway activity and by an increase in the level of caspase activity in these cells. That suggests the role of uPAR in supporting cell survival. After intramyocardial transplantation of uPAR(-) MPCs, reduced cell retention and angiogenesis stimulation were observed in mice with myocardial infarction model compared to uPAR(+) cells transplantation. Taken together, the present results appear to prove a novel mechanism of uPAR action in maintaining the survival and angiogenic properties of CD117+ MPCs. These results emphasize the importance of the uPAR as a potential pharmacological target for the regulation of reparative properties of myocardial mesenchymal progenitor cells.

Cardiac cellular diversity and functionality in cardiac repair by single-cell transcriptomics.

Cardiac repair after myocardial infarction (MI) is orchestrated by multiple intrinsic mechanisms in the heart. Identifying cardiac cell heterogeneity and its effect on processes that mediate the ischemic myocardium repair may be key to developing novel therapeutics for preventing heart failure. With the rapid advancement of single-cell transcriptomics, recent studies have uncovered novel cardiac cell populations, dynamics of cell type composition, and molecular signatures of MI-associated cells at the single-cell level. In this review, we summarized the main findings during cardiac repair by applying single-cell transcriptomics, including endogenous myocardial regeneration, myocardial fibrosis, angiogenesis, and the immune microenvironment. Finally, we also discussed the integrative analysis of spatial multi-omics transcriptomics and single-cell transcriptomics. This review provided a basis for future studies to further advance the mechanism and development of therapeutic approaches for cardiac repair.

Single-nuclei multiomic analyses identify human cardiac lymphatic endothelial cells associated with coronary arteries in the epicardium

Cardiac lymphatic vessels play important roles in fluid homeostasis, inflammation, disease, and regeneration of the heart. The developing cardiac lymphatics in human fetal hearts are closely associated with coronary arteries, similar to those in zebrafish hearts. We identify a population of cardiac lymphatic endothelial cells (LECs) that reside in the epicardium. Single-nuclei multiomic analysis of the human fetal heart reveals the plasticity and heterogeneity of the cardiac endothelium. Furthermore, we find that VEGFC is highly expressed in arterial endothelial cells and epicardium-derived cells, providing a molecular basis for the arterial association of cardiac lymphatic development. Using a cell-type-specific integrative analysis, we identify a population of cardiac lymphatic endothelial cells marked by the PROX1 and the lymphangiocrine RELN and enriched in binding motifs of erythroblast transformation specific (ETS) variant (ETV) transcription factors. We report the invivo molecular characterization of human cardiac lymphatics and provide a valuable resource to understand fetal heart development.

Uncovering the Heterogeneity of Cardiac Lin-KIT+ Cells: A scRNA-seq Study on the Identification of Subpopulations.

The reparative potential of cardiac Lin-KIT+ (KIT) cells is influenced by their population, but identifying their markers is challenging due to changes in phenotype during in vitro culture. Resolving this issue requires uncovering cell heterogeneity and discovering new subpopulations. Single-cell RNA sequencing (scRNA-seq) can identify KIT cell subpopulations, their markers, and signaling pathways. We used 10× genomic scRNA-seq to analyze cardiac-derived cells from adult mice and found 3 primary KIT cell populations: KIT1, characterized by high-KIT expression (KITHI), represents a population of cardiac endothelial cells; KIT2, which has low-KIT expression (KITLO), expresses transcription factors such as KLF4, MYC, and GATA6, as well as genes involved in the regulation of angiogenic cytokines; KIT3, with moderate KIT expression (KITMOD), expresses the cardiac transcription factor MEF2C and mesenchymal cell markers such as ENG. Cell-cell communication network analysis predicted the presence of the 3 KIT clusters as signal senders and receivers, including VEGF, CXCL, and BMP signaling. Metabolic analysis showed that KIT1 has the low activity of glycolysis and oxidative phosphorylation (OXPHOS), KIT2 has high glycolytic activity, and KIT3 has high OXPHOS and fatty acid degradation activity, indicating distinct metabolic adaptations of the 3 KIT populations. Through the systemic infusion of KIT1 cells in a mouse model of myocardial infarction, we observed their involvement in promoting the formation of new micro-vessels. In addition, in vitro spheroid culture experiments demonstrated the cardiac differentiation capacity of KIT2 cells.

Abstract P2102: The Basic Helix-loop-helix Transcription Factor Tcf21 Regulates Fibroblast Cell States In The Adult Murine Heart

Objective: Transcription factor 21 (Tcf21) is a basic helix-loop-helix protein that is essential for normal mesodermal organogenesis and its loss is incompatible with life. In the developing heart, epicardial progenitor cells differentiate to either vascular smooth muscle cells or fibroblasts, the latter of which is dependent on Tcf21 expression. In the adult heart, Tcf21 is expressed by the majority of fibroblasts but is downregulated in response to fibrotic stimuli as the fibroblast differentiates to a myofibroblast. In atherosclerosis, smooth muscle cells that undergo phenotypic modulation towards a fibroblast-like cell acquire expression of Tcf21. These findings led to the hypothesis that Tcf21 could be a regulator of fibroblast cell-fate in the adult mammalian heart and contribute to cardiac fibrosis. Methods and Results: To test this hypothesis, we generated tamoxifen-inducible genetic mouse models to study cardiac fibroblast Tcf21 deletion and overexpression in vivo . Acute and long-term Tcf21 ablation in the adult heart did not promote differentiation to myofibroblasts. After long-term ablation, no differences were detected on cardiac interstitial cell populations, cardiac structure or function and extracellular matrix deposition. In contrast, in response to fibrotic stimuli, overexpression of Tcf21 suppressed differentiation to myofibroblasts with reduced expression of contractile proteins. This was accompanied by reduced cardiac fibrosis. Conclusions: The expression of Tcf21 in the healthy fibroblast is not required to maintain a ‘resting’ fibroblast cell-state. In disease, Tcf21 functions as a suppressor of fibroblast differentiation to the contractile myofibroblast and its overexpression reduces the fibrotic burden on the heart.

Return of the Tbx5; lineage-tracing reveals ventricular cardiomyocyte-like precursors in the injured adult mammalian heart

The single curative measure for heart failure patients is a heart transplantation, which is limited due to a shortage of donors, the need for immunosuppression and economic costs. Therefore, there is an urgent unmet need for identifying cell populations capable of cardiac regeneration that we will be able to trace and monitor. Injury to the adult mammalian cardiac muscle, often leads to a heart attack through the irreversible loss of a large number of cardiomyocytes, due to an idle regenerative capability. Recent reports in zebrafish indicate that Tbx5a is a vital transcription factor for cardiomyocyte regeneration. Preclinical data underscore the cardioprotective role of Tbx5 upon heart failure. Data from our earlier murine developmental studies have identified a prominent unipotent Tbx5-expressing embryonic cardiac precursor cell population able to form cardiomyocytes, in vivo, in vitro and ex vivo. Using a developmental approach to an adult heart injury model and by employing a lineage-tracing mouse model as well as the use of single-cell RNA-seq technology, we identify a Tbx5-expressing ventricular cardiomyocyte-like precursor population, in the injured adult mammalian heart. The transcriptional profile of that precursor cell population is closer to that of neonatal than embryonic cardiomyocyte precursors. Tbx5, a cardinal cardiac development transcription factor, lies in the center of a ventricular adult precursor cell population, which seems to be affected by neurohormonal spatiotemporal cues. The identification of a Tbx5-specific cardiomyocyte precursor-like cell population, which is capable of dedifferentiating and potentially deploying a cardiomyocyte regenerative program, provides a clear target cell population for translationally-relevant heart interventional studies.

3D bioprinted alginate-gelatin hydrogel patches containing cardiac spheroids recover heart function in a mouse model of myocardial infarction

Epicardial transplantation of 3D bioprinted patches represents a promising protective strategy against infarction-induced myocardial damage. We previously showed that 3D bioprinted tissues containing cardiac spheroids [in alginate/gelatin (AlgGel) hydrogels] promoted cell viability/function and endothelial cell tubular self-assembly. Here, we hypothesise that bioprinted cardiac spheroid patches improve cardiac function after myocardial infarction (MI). To determine treatment effects of hydrogel alone or with cells, MI mice were transplanted with: (i) AlgGel acellular patches, (ii) AlgGel with freely suspended cardiac cells, (iii) AlgGel with cardiac spheroids. We included control MI mice (no treatment) and mice undergoing sham surgery. We performed measurements to 28 days including echocardiography, flow cytometry and transcriptomic analyses. Our results measured median baseline (pre-surgery) left ventricular ejection fraction (LVEF%) for all mice at 66%. Post-surgery, LVEF% was 58% for Sham (non-infarcted) and 41% for MI (no treatment) mice. Patch transplantation increased LVEF%: 55% (acellular; p = 0.012), 59% (cells; p = 0.106), 64% (spheroids; p = 0.010). Flow cytometry demonstrated host cardiac tissue immune cell population changes with treatments. RNAseq transcriptomes demonstrated similar gene expression profiles for Sham and mice treated with cardiac spheroid patches. Extrusion 3D bioprinting permits hydrogel patch generation even preserving microtissue cardiac spheroids directly suspended in the bioink. Inflammatory and genetic mechanisms may play important roles in regulating host responses after patch transplantation in infarcted hearts. Future studies are needed to elucidate the possible immune cell and gene expression-related molecular mechanisms underlying these initial findings.

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart.

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more than half of the total cardiac cell population, and they contribute to adaptive compensations upon myocardial injury, including inflammatory responses, tissue repair, and scar formation. To study the post-MI cardiac microenvironment, single-cell RNA sequencing (scRNA-seq) is widely used to identify different cardiac cell types and intercellular communications. Among the procedures of scRNA-seq sample preparation, preparing the cell suspension is one of the most critical steps, because the cell viability can affect the quality of the scRNA-seq results. Therefore, we designed an experimental protocol for preparing a non-cardiomyocyte cell suspension from post-MI mouse hearts with an extra focus on improving the cell viability by choosing mild digestive enzymes, controlling the digestion time, and applying fluorescence-activated cell sorting (FACS). Finally, we isolated CD45+ cells from the non-cardiomyocyte cell suspension obtained through this protocol, and then we performed scRNA-seq.

Defining cardiac cell populations and relative cellular composition of the early fetal human heart.

While the adult human heart is primarily composed of cardiomyocytes, fibroblasts, endothelial and smooth muscle cells, the cellular composition during early development remains largely unknown. Reliable identification of fetal cardiac cell types using protein markers is critical to understand cardiac development and delineate the cellular composition of the developing human heart. This is the first study to use immunohistochemistry (IHC), flow cytometry and RT-PCR analyses to investigate the expression and specificity of commonly used cardiac cell markers in the early human fetal heart (8-12 post-conception weeks). The expression of previously reported protein markers for the detection of cardiomyocytes (Myosin Heavy Chain (MHC) and cardiac troponin I (cTnI), fibroblasts (DDR2, THY1, Vimentin), endothelial cells (CD31) and smooth muscle cells (α-SMA) were assessed. Two distinct populations of cTnI positive cells were identified through flow cytometry, with MHC positive cardiomyocytes showing high cTnI expression (cTnIHigh) while MHC negative non-myocytes showed lower cTnI expression (cTnILow). cTnI expression in non-myocytes was further confirmed by IHC and RT-PCR analyses, suggesting troponins are not cardiomyocyte-specific and may play distinct roles in non-muscle cells during early development. Vimentin (VIM) was expressed in cultured ventricular fibroblast populations and flow cytometry revealed VIMHigh and VIMLow cell populations in the fetal heart. MHC positive cardiomyocytes were VIMLow whilst CD31 positive endothelial cells were VIMHigh. Using markers investigated within this study, we characterised fetal human cardiac populations and estimate that 75-80% of fetal cardiac cells are cardiomyocytes and are MHC+/cTnIHigh/VIMLow, whilst non-myocytes comprise 20-25% of total cells and are MHC-/cTnILow/VIMHigh, with CD31+ endothelial cells comprising ~9% of this population. These findings show distinct differences from those reported for adult heart.

Abstract 11965: Cellular Senescence Produces Diastolic Dysfunction and Cardiac Hypertrophy With Aging in Mice

Background: Age is a major risk factor for heart failure with preserved ejection fraction (HFpEF); however, the underlying mechanisms are poorly understood. Cellular senescence is an aging and stress related response; senescent cells (SCs) secrete bioactive substances that produce a variety of pathological changes. The role of senescence in HFpEF is unknown. Objectives: To study the effect of targeted deletion of SCs on cardiac function, structure and left ventricular (LV) pressure in an aging mouse model of HFpEF. Methods: We studied INK ATTAC mice, in which SCs undergo targeted clearance following treatment with a dimerizing agent, AP20187 (AP). Mice were treated with blinded AP or vehicle from 12 months to 18 months of age. Cardiac function and structure were studied with echocardiography. Contractility parameters were measured with a Millar catheter. The percentage of SCs in different cardiac-cell populations was analyzed with the use of Fluorescence-Activated Cell Sorting (FACS). Results: From 12 to 18 months, LV mass/LV diameter at end diastole (LVDd) and the LV anterior wall thickness at end diastole (LVAWd) increased in the vehicle group while they decreased in the AP group (LV mass/LVDd: %Δvehicle=+7.9± 3%, %ΔAP=-2±1.2%, P=0.005; LVAWd: %Δvehicle=+5.3±2.5%, %ΔAP=-3.5±1.6%, P=0.007) (Fig1A, B). E/e’ (echo) and isovolumic relaxation time indicated diastolic dysfunction in vehicle mice, and improved with AP (Fig1C, D). GFP bicistronic with p16 indicated SCs; FACS-purified fibroblasts (FBs) showed reduced GFP fluorescence (P=0.056) in the AP group, whereas immune and endothelial cells were not affected, suggesting that the anti-HFpEF effects of SC-clearance might be mediated through the clearance of senescent FBs (Fig1E). Conclusions: Our study points to a significant role of SCs, likely senescent FBs, in the pathophysiology of HFpEF. Modulating senescence might provide the basis for novel therapeutic approaches to HFpEF prevention and/or therapy.

Single-Cell Transcriptional Profiling Reveals Low-Level Tragus Stimulation Improves Sepsis-Induced Myocardial Dysfunction by Promoting M2 Macrophage Polarization.

Background Sepsis can lead to multiple organ damage, of which the heart is one of the most vulnerable organs. Vagal nerve stimulation can reduce myocardial injury in sepsis and improve survival rates. However, the potential impact of low-level tragus stimulation and disparate cell populations on sepsis-induced myocardial dysfunction remains undetermined. Methods A cardiac single-cell transcriptomic approach was used for characterizing cardiac cell populations that form the heart. Single-cell mRNA sequencing data were used for selecting all cardiac macrophages from CD45+ cells. Then, echocardiography, western blot, flow cytometry, immunofluorescence, and immunohistochemistry were performed to verify the single-cell mRNA sequencing results. Results Using single-cell mRNA sequencing data, we uncovered the multiple cell populations contributing to myocardial injury in sepsis under low-level tragus stimulation, thereby illustrating a comprehensive map of the cardiac cellular landscape. Pseudotiming analysis in single-cell sequencing showed that low-level vagal nerve stimulation played an anti-inflammatory role by promoting cardiac monocytes into M2 macrophages, which significantly increased α7nAChR expression in heart tissues. Echocardiography assessment indicated that low-level vagal nerve stimulation could also improve cardiac functions in mice with sepsis-induced myocardial dysfunction. In addition, the heart tissues of mice from the sepsis group with low-level tragus stimulation had significantly lower interleukin-1β expression levels than those from the sepsis group. Flow cytometry analysis showed that different acetylcholine concentrations promoted cardiac monocytes into M2 macrophages in in vitro experiments. Conclusion Low-level tragus stimulation could improve sepsis-induced myocardial dysfunction by promoting cardiac monocytes to M2 macrophages.

Dissecting the cardiac cell landscape in myocarditis

Abstract Introduction Myocarditis is the prototypical cardiac inflammatory disease, with an incidence of 20–30 per 100000 individuals. Acute myocarditis potentially develops into lethal inflammatory cardiomyopathy in a substantial fraction of patients, rendering myocarditis and its sequelae the underlying reason for many heart transplants and 10–20% of cardiovascular sudden deaths in young adults. Different pathological routes had been described to lead to myocardial inflammation (eg. viral, bacterial or parasitic infection, autoimmune processes, toxins). Moreover, genetic predisposition and environmental factors such as the microbiome influence the progression of the disease. Therefore, it is plausible that the inherent complexity of the disease hampers the elucidation of effective therapeutic interventions because the molecular mechanisms underlying this severe heart condition are – to a large extent – incompletely understood. Therefore, it is important to elucidate the molecular pathways governing the interplay between immune cells and stromal cells in the cardiac microenvironment in myocarditis. Methods To dissect the processes involved in myocarditis progression, we took advantage of the T cell receptor transgenic mouse model of spontaneous autoimmune myocarditis (TCRM) were all mice develop acute myocarditis around 4 weeks of age that progresses to lethal dilated cardiomyopathy in 50% of the mice. To analyse the mechanisms involved in cardiac inflammation in mouse and human patients, we used single cell and single nucleus transcriptomic approaches in combination with high dimensional flow cytometry, confocal microscopy and echocardiographic analysis of the cardiac function. To examine the cardiac landscape in humans we applied the same analysis methodologies to right ventricle endomyocardial biopsies from myocarditis or heart transplantation patients (control). Results Our analysis shows that dynamic changes occur in the cardiac microenvironment during the course of myocarditis; these changes involve not only the recruitment of immune cells expressing pro-inflammatory signatures to the myocardium (T cells, inflammatory monocytes, neutrophils) but also the alteration of transcriptional programs in non-immune cells including fibroblast, endothelial cells and cardiomyocytes. The analysis of endomyocardial biopsies from myocarditis patients revealed similar changes in the cardiac cell population's transcriptome. Conclusion Our study reveal that the transcriptional landscape of cardiac cells is strongly influenced by the inflammatory microenvironment. Notably, during the acute phase of myocarditis pathways supporting inflammation and fibrosis overrule circuits involved in cardiac homeostasis. Moreover, we show that the analysis of endomyocardial biopsies by single-nucleus RNAseq represents a powerful tool to unveil the processes involved in cardiac diseases. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ERC personal grant to Burkhard Ludewig