Resident Cardiac Cells Research Articles

Clec4e deletion in cardiac resident cells leads to smaller infarct size and better cardiac remodeling following myocardial ischemia-reperfusion injury

Abstract Introduction Clec4e-signalling in cardiac resident cells and circulating immune cells stimulates recruitment of proinflammatory leukocytes to infarcted myocardium following ischemia reperfusion injury (I/R) and contributes to ischemic myocardial damage. Clec4e deletion is associated with reduced cardiac injury and better left ventricular structural and functional remodeling. The mechanisms of this cardioprotective effect remain unknown. Research question: We investigated whether Clec4e gene function in cardiac resident cells vs infiltrating immune cells mediates cardioprotection during myocardial ischemia-reperfusion injury. Methods We generated chimeric mice by performing bone marrow transfer in wild type (WT) C57Bl6/J and C57Bl6/J Clec4e-/- mice. Following total body irradiation using 9.5 Gy in 8w-old WT and Clec4e-/- receptor mice, 5 x 10^6 bone marrow cells originating from Clec4e-/- and WT donor mice, respectively, were injected IV. Four weeks later, mice were anesthetized and subjected to ischemia reperfusion injury (I/R) by transient occlusion of the LAD for 60 min. Troponin levels were measured at 90 min post I/R and 7T-magnetic resonance imaging with late gadolinium enhancement (LGE) for infarct size evaluation was performed at 24 hours and 28 days post I/R without LGE. Mice were euthanized at 28 days post I/R and the heart and long shaft bones were processed for postmortem immunohistochemistry and analysis of BM chimerism. Results Immunohistochemical analysis of CLEC4E expression on a bone marrow smear 8 w post bone marrow transfer confirmed successful BM chimerism (7% vs 99%, p<0.01). TnI levels at 90 minutes and LGE assessed infarct size on MRI at 24 hours were not significantly different between groups (n=17 in both groups) (42.1 vs 48.4 ng/ml, and 25.2 vs 31.5% of LVmass in Clec4e-/- mice with WT bone marrow versus in WT mice with Clec4e-/- BM, respectively) suggesting similar myocardial damage . After 28 days, mean LV end diastolic volumes (43µL vs 55µL), and mean end systolic volumes (13µL vs 25µL) were significantly (p<0,05) smaller in Clec4e-/- mice with WT bone marrow resulting in better preserved global LV function (LVEF 62% vs 47%, p<0,05, fig A). Infarct size, semiquantitatively assessed using planimetry on Sirius red stains showed a smaller infarct size in the Clec4e-/- mice with WT bone marrow (6.9% vs 19.8%, p<0.01). Figure B shows a representative example of LGE infarct size to scar tissue on MRI and Sirius red staining. Conclusion Clec4e gene deletion in cardiac resident cells, but not in infiltrating hematopoietic immune cells is associated with better functional and structural remodeling after myocardial I/R. Inhibition of Clec4e signaling via blocking antibodies or small molecule inhibitors in cardiac myocytes might be a promising cardioprotective strategy.

Abstract We073: CLEC4E Signaling in Myocardial Ischemia-Reperfusion Injury

Background: The innate immune receptor CLEC4E (C-type lectin domain family 4 member E) in cardiac resident cells and circulating leukocytes contributes to ischemic myocardial damage and adverse LV remodeling following ischemia reperfusion injury (I/R). Mice with loss of CLEC4E gene function show reduced cardiac injury and better LV remodeling but the mechanisms of this cardioprotection remain unknown. Aims: We investigated whether Clec4e gene function in cardiac resident cells vs infiltrating immune cells mediates cardioprotection during myocardial ischemia-reperfusion injury. Methods: We generated chimeric mice by IV infusion of 5 x 10 6 bone marrow cells originating from C57Bl6/J CLEC4E -/- and C57Bl6/J wild-type (WT) donor mice in 8w-old lethally irradiated (9.5 Gy) WT (n=36) and CLEC4E -/- (n=50) receptor mice, respectively. After 4 w, mice were subjected to ischemia reperfusion injury (I/R) by 60 min LAD occlusion. Troponin levels (TnI) were measured at 90 min post I/R and 7T-magnetic resonance imaging with late gadolinium enhancement (LGE) for infarct size evaluation was performed at 24 h and 28 d post I/R. Mice were euthanized at 28 days for immunohistochemistry and analysis of BM chimerism. Results: Immunohistochemistry for CLEC4E expression on bone marrow smears confirmed successful BM chimerism. At 24h post I/R, TnI levels and LGE-assessed infarct size on MRI were not significantly different between groups (42.1 vs 48.4 ng/ml, and 25.2% vs 31.5% of LVmass in CLEC4E -/- & WT BM versus in WT & CLEC4E -/- BM, respectively) suggesting similar myocardial damage . After 28 d, LV end-diastolic volumes (43µL vs 55µL), and end-systolic volumes (13µL vs 25µL) were significantly smaller in CLEC4E -/- & WT BM than in WT & CLEC4E -/- BM mice (P<0.05 for both), resulting in better preserved global LV function (LVEF 62% vs 47%, P<0.05) . Infarct size, semiquantitatively assessed using planimetry on Sirius red stains, was smaller in CLEC4E -/- & WT BM than in WT & CLEC4E -/- BM (6.9% vs 19.8%, P<0.01). Conclusion: CLEC4E gene deletion in cardiac resident cells, but not in infiltrating hematopoietic immune cells is associated with better functional and structural remodelling after myocardial I/R. Inhibition of CLEC4E signaling via blocking antibodies or small molecule inhibitors in cardiac myocytes might be a promising cardioprotective strategy.

Role of the Anaphylatoxin Receptor C5aR2 in Angiotensin II-Induced Hypertension and Hypertensive End-Organ Damage.

Complement activation may facilitate hypertension through its effects on immune responses. The anaphylatoxin C5a, a major inflammatory effector, binds to the C5a receptors 1 and 2 (C5aR1, C5aR2). We have recently shown that C5aR1-/- mice have reduced hypertensive renal injury. The role of C5aR2 in hypertension is unknown. For examination of C5aR2 expression on infiltrating and resident renal cells a tandem dye Tomato-C5aR2 knock-in reporter mouse was used. Human C5aR2 expression was analyzed in a single-cell RNAseq data set from the kidneys of hypertensive patients. Finally, we examined the effect of angiotensin II-induced hypertension in C5aR2-deficient mice. Flow cytometric analysis of leukocytes isolated from kidneys of the reporter mice showed that dendritic cells are the major C5aR2-expressing population (34%) followed by monocyte/macrophages (30%) and neutrophils (14%). Using confocal microscopy C5aR2 was not detected in resident renal or cardiac cells. In the human kidney, C5aR2 was also mainly found in monocytes, macrophages, and dendritic cells with a significantly higher expression in hypertension (P < 0.05). Unilateral nephrectomy was performed followed by infusion of Ang II (0.75 ng/g/min) and a high salt diet in wildtype (n = 18) and C5aR2-deficient mice (n = 14). Blood pressure, renal injury (albuminuria, glomerular filtration rate, glomerular and tubulointerstitial injury, inflammation), and cardiac injury (cardiac fibrosis, heart weight, gene expression) did not differ between hypertensive wildtype and C5aR2-/- mice. In summary, C5aR2 is mainly expressed in myeloid cells in the kidney in mice and humans but its deficiency has no effect on Ang II-induced hypertensive injury.

Asprosin as a potential new paracrine regulator in the heart

Abstract Funding Acknowledgements None. Background Asprosin was recently described as a novel hormone that is released by white adipose tissue upon fasting. However, a functional role of asprosin in human cardiovascular tissues has not been yet investigated at the tissue level. Asprosin is the C-terminal propeptide of the extracellular matrix (ECM) glycoprotein fibrillin-1 and is cleaved off from the pro-fibrillin-1 precursor by the endopeptidase furin. Fibrillin-1 is expressed in the heart and aorta, and FBN1 mutations are causative for aortic aneurysm formation and cardiomyopathy. Whether asprosin is also present in cardiac tissues and utilized by cardiac resident cells remains unknown. Purpose In the present study, we investigated the localization and distribution of asprosin protein in human atrial and ventricular heart tissue. Methods Human heart tissue was obtained from patients undergoing aortic valve replacement. In addition, we used ventricular tissue from mice. Cryosections from human (n=8) and mouse (n=8) heart samples were analysed by immunohistochemistry. The anti-asprosin antibodies were raised against human or mouse asprosin respectively and applied in combination with anti-CD31/PECAM, an endothelial cell marker, or anti-sarcomeric alpha-actinin, a Z-line marker. The immunofluorescence of incubated specimens was examined by confocal microscopy. Results Our data suggest that asprosin is localized in myocardium in different cell types: cardiomyocytes (Figure 1 a-d), arterial smooth muscle cells (Figure 1 a), micro- and vascular endothelial cells (Figure 1 d), and with the lowest intensity, in periarterial located epicardial adipocytes (Figure 1 e). In cardiomyocytes, incubation with anti-asprosin resulted in a high signal intensity. Asprosin immunoreactivity was observed: in intercalary discs (Figure 1 b-d), as striated pattern in the sarcoplasm, and as weak signal in the perinuclear area (Figure 1 b, c). In human tissue, the anti-asprosin signal was about four times higher in intercalated discs than in the sarcoplasm. The distribution pattern in ventricular cardiomyocytes of mice were similar. Conclusion Our results show that in human myocardium, asprosin localises mainly intracellular. Asprosin accumulations were found in intercalary discs. In short, asprosin was found at locations that are known to be crucial for regulation of cardiac function. Intracellular presence of asprosin may indicate cellular utilization by uptake from extracellular asprosin pools. Whether these pools are replenished exclusively by resident cells or by other tissues such as WAT is currently unknown. However, further research is needed to prove this hypothesis. Figure. 1. Confocal microscopy of asprosin localization in human myocardium. Cryosections incubated with anti-asprosin (red) or combined with anti-alpha actinin (sarcomeric) or anti-CD31/PECAM (green) antibodies and DAPI (blue, nuclei). SM: vascular smooth muscle cells, BV: blood vessels, Arrowheads: Intercalated disks.

Platelets Induce Cell Apoptosis of Cardiac Cells via FasL after Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is one of the leading causes of death worldwide. Cell apoptosis in the myocardium plays an important role in ischemia and reperfusion (I/R) injury, leading to cardiac damage and dysfunction. Platelets are major players in hemostasis and play a crucial role in vessel occlusion, inflammation, and cardiac remodeling after I/R. Here, we studied the impact of platelets on cell apoptosis in the myocardium using a close-chest mouse model of AMI. We found caspase-3-positive resident cardiac cells, while leukocytes were negative for caspase-3. Using two different mouse models of thrombocytopenia, we detected a significant reduction in caspase-3 positive cells in the infarct border zone after I/R injury. Further, we identified platelet FasL to induce cell apoptosis via the extrinsic pathway of Fas receptor activation of target cells. Mechanistically, hypoxia triggers platelet adhesion to FasR, suggesting that platelet-induced apoptosis is elevated after I/R. Platelet-specific FasL knock-out mice showed reduced Bax and Bcl2 expression, suggesting that platelets modulate the intrinsic and extrinsic pathways of apoptosis, leading to reduced infarct size after myocardial I/R injury. Thus, a new mechanism for how platelets contribute to tissue homeostasis after AMI was identified that should be validated in patients soon.

Immune Cells in Cardiac Injury Repair and Remodeling.

Immune cells are emerging as central cellular components of the heart which communicate with cardiac resident cells during homeostasis, cardiac injury, and remodeling. These findings are contributing to the development and continuous expansion of the new field of cardio-immunology. We review the most recent literature on this topic and discuss ongoing and future efforts to advance this field forward. Cell-fate mapping, strategy depleting, and reconstituting immune cells in pre-clinical models of cardiac disease, combined with the investigation of the human heart at the single cell level, are contributing immensely to our understanding of the complex intercellular communication between immune and non-immune cells in the heart. While the acute immune response is necessary to initiate inflammation and tissue repair post injury, it becomes detrimental when sustained over time and contributes to adverse cardiac remodeling and pathology. Understanding the specific functions of immune cells in the context of the cardiac environment will provide new opportunities for immunomodulation to induce or tune down inflammation as needed in heart disease.

Dasatinib targets c-Src kinase in cardiotoxicity.

Dasatinib is a multitargeted kinase inhibitor used for treatment of chronic myeloid leukemia and acute lymphoblastic leukemia. Unfortunately, treatment of cancer patients with some kinase inhibitors has been associated with cardiotoxicity. Cancer treatment with dasatinib has been reported to be associated with cardiotoxic side effects such as left ventricular dysfunction, heart failure, pericardial effusion and pulmonary hypertension. Here we aimed to investigate the molecular mechanisms underlying the cardiotoxicity of dasatinib. We found that among the resident cardiac cell types, cardiomyocytes were most sensitive to dasatinib-induced cell death. Exposure of cardiomyocytes to dasatinib attenuated the activity of extracellular signal-regulated kinase (ERK), which is a downstream target of dasatinib target kinase c-Src. Similar to dasatinib, c-Src depletion in cardiomyocytes compromised cardiomyocyte viability. Overexpression of dasatinib-resistant mutant of c-Src rescued the toxicity of dasatinib on cardiomyocytes, whereas forced expression of wild type c-Src did not have protective effect. Collectively, our results show that c-Src is a key target of dasatinib mediating the toxicity of dasatinib to cardiomyocytes. These findings may influence future drug design and suggest closer monitoring of patients treated with agents targeting c-Src for possible adverse cardiac effects.

Activation of cardiac macrophages, endothelial cells and fibroblasts in experimental autoimmune myocarditis

Abstract Background/Introduction Inflammation of cardiac tissue, termed myocarditis, is a common cause of dilated cardiomyopathy associated with abnormal tissue remodeling, stiffening of ventricles and heart failure. Experimental autoimmune myocarditis (EAM) represents CD4+ T cell-dependent animal model of acute myocarditis followed by development of cardiomyopathy, cardiac fibrosis and systolic dysfunction. Purpose The aim of the study was to investigate the role of cardiac macrophages, endothelial cells and fibroblasts in myocarditis and post-inflammatory cardiomyopathy in mouse model of EAM. Methods EAM was induced in BALB/c mice by immunization with alpha myosin heavy chain and complete Freund's adjuvant. Reporter mice expressing EGFP under collagen type I promoter (Coll-EGFP), transgenic αSMA-TK mice with ganciclovir-inducible ablation of proliferating myofibroblasts and Rosa26-YFP/LysM-Cre and Rosa26-YFP/LysM-Cre/Tgfbr2 fl/fl with YFP expression and Tgfbr2 deletion in myeloid cell population were used in this study. Cardiac fibroblasts and macrophages were sorted using BD FACSAria™ II Cell Sorter and analyzed for the genome transcriptomics by RNA sequencing. Echocardiography was performed on Vevo 2100 Imaging System. Cardiac fibrosis was measured as percentage of fibrotic area using Trichrome Massons's staining and by hydroxyproline assay. Cardiac hypertrophy was analyzed as means of cross-sectional cardiomyocyte area. Monocytes and endobcells were analyzed using BD FACSCanto™ II flow cytometry. Results Cardiac fibroblasts in response to acute myocarditis (d21 of EAM) showed activation of immune processes (mainly chemokine production such as Ccl6, Ccl9, Cxcl2, Cxcl3, Cxcl5, Cxcl9, Cxcl13), cytoskeletal re-organization (Cxadr, F11r, Gdpd2, Krt8, Krt19, Ptk2b, Rac2, Rhov, Rnd1, S100a9, Spire2, Was) and upregulation of genes involved in extracellular matrix turnover (Bmp7, Kng2, Lgals3, Cthrc1, Cela1, Spn) including collagens. Ablation of myofibroblasts (between d21–40 of EAM) resulted in markedly reduced heart weight and cardiomyocyte hypertrophy, attenuated expression of genes related to hypertrophy (Acta1, Actc1, Bnp, Cfl2, Pdlim5), improved stroke volume, ejection fraction and cardiac output but did not prevent development of post-inflammatory cardiac fibrosis measured at d40 of EAM. Analysis of monocytes and endothelial cells indicated excessive production of type I collagen by these cells at d21. Analysis of cardiac macrophages pointed out TGF-β-dependent expression of cytokines (Ifn, Il23a, Il10, Il12b, Cxcl1, Tnf) and theirs receptors (Cxcr1, Ccr4) at d21 of EAM. Conclusions Acute myocarditis activates proinflammatory and profibrotic responses in cardiac resident cells. Our data suggest that cardiac myofibroblasts play a particularly important role in development of post-inflammatory cardiomyopathy and heart failure. Targeting cardiac myofibroblasts might therefore represent a novel therapeutic strategy in inflammatory heart disease. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Science Centre (Poland)

Exosomes and Exosomal Cargos: A Promising World for Ventricular Remodeling Following Myocardial Infarction.

Exosomes are a pluripotent group of extracellular nanovesicles secreted by all cells that mediate intercellular communications. The effective information within exosomes is primarily reflected in exosomal cargos, including proteins, lipids, DNAs, and non-coding RNAs (ncRNAs), the most intensively studied molecules. Cardiac resident cells (cardiomyocytes, fibroblasts, and endothelial cells) and foreign cells (infiltrated immune cells, cardiac progenitor cells, cardiosphere-derived cells, and mesenchymal stem cells) are involved in the progress of ventricular remodeling (VR) following myocardial infarction (MI) via transferring exosomes into target cells. Here, we summarize the pathological mechanisms of VR following MI, including cardiac myocyte hypertrophy, cardiac fibrosis, inflammation, pyroptosis, apoptosis, autophagy, angiogenesis, and metabolic disorders, and the roles of exosomal cargos in these processes, with a focus on proteins and ncRNAs. Continued research in this field reveals a novel diagnostic and therapeutic strategy for VR.

Soluble Isoform of ST2 is a Cardiac Inducer of Collagen Deposition in an Ex Vivo Model of Obesity

IntroductionOne of the main cardiac mechanosensor is the IL‐33/ST2L complex composed by the allarmin IL‐33 released into extracellular space by damaged cells during cardiac overload and its receptor, ST2L, which their binding promotes cell survival and anti‐fibrotic signaling. After damage, to stop IL‐33/ST2L signal, resident cardiac cells release sST2, the soluble isoform of transmembrane receptor of ST2, which sequesters IL‐33, functioning as a decoy receptor and blocking its signal. In obesity, the sST2 become over express especially by hypertrophic fat cells. The aim of our study is to evaluate in an ex vivo model of obesity the role of sST2 in the promotion of collagen production by cardiac cells.MethodsTen obese nondiabetic male Zucker rats (OB) (fa/fa‐, 10 weeks of age) and 10 lean littermates (L) (Fa/+) at the age of 25 weeks were weighted and scarified according to Italian Ministry of Health Authorization (N°325/2015PR of 2015/04/05) and their heart and visceral adipose tissue (VAT) were collected for the analysis. VAT biopsies were cut in 30mg pieces and added into a co‐culture system with H9C2 for 6‐24 and 48 hours. At each time points, cells and culture medium were collected. In all samples were evaluated ST2 by ELISA and collagen were quantified by quantitative Sircol assay for measurement of both acid‐soluble and pepsin‐soluble collagens.ResultsOB cardiac biopsies showed higher protein level of both ST2 and collagen than L rats (p&lt;0.02 and p&lt;0.002 respectively) and the rat weight positively correlates with ST2 cardiac protein level (p&lt;0.05). Co‐culture data demonstrated that OB VAT promotes ST2 releases in both cardiac and fat cells, resulting an incremental increase of sST2 release in cell culture medium than L VAT in time. sST2 quantification in correspondent H9C2 co‐cultured lysate is more incremented in OB than L H9C2 after 48h.total collagen quantification in co‐culture medium resulted higher in OB than L (p&lt;0.05).ConclusionOur results demonstrated in a co‐culture system that obesity promotes sST2 increase in the heart and this promotes collagen releases by cardiac cells, suggesting sST2 role in activation of pro‐fibrotic signaling and detrimental heart remodeling.

Designer Functional Nanomedicine for Myocardial Repair by Regulating the Inflammatory Microenvironment

Acute myocardial infarction is a major global health problem, and the repair of damaged myocardium is still a major challenge. Myocardial injury triggers an inflammatory response: immune cells infiltrate into the myocardium while activating myofibroblasts and vascular endothelial cells, promoting tissue repair and scar formation. Fragments released by cardiomyocytes become endogenous “danger signals”, which are recognized by cardiac pattern recognition receptors, activate resident cardiac immune cells, release thrombin factors and inflammatory mediators, and trigger severe inflammatory responses. Inflammatory signaling plays an important role in the dilation and fibrosis remodeling of the infarcted heart, and is a key event driving the pathogenesis of post-infarct heart failure. At present, there is no effective way to reverse the inflammatory microenvironment in injured myocardium, so it is urgent to find new therapeutic and diagnostic strategies. Nanomedicine, the application of nanoparticles for the prevention, treatment, and imaging of disease, has produced a number of promising applications. This review discusses the treatment and challenges of myocardial injury and describes the advantages of functional nanoparticles in regulating the myocardial inflammatory microenvironment and overcoming side effects. In addition, the role of inflammatory signals in regulating the repair and remodeling of infarcted hearts is discussed, and specific therapeutic targets are identified to provide new therapeutic ideas for the treatment of myocardial injury.

Inflammatory degranulation of the cardiac resident mast cells suppresses the pacemaking and affects activation pattern in the sinoatrial node

BackgroundMany bacterial and viral infections may cause sepsis or/and systemic or local proinflammatory responses that lead to life-threatening cardiovascular system failure. Severe bradycardia and/or bradyarrhythmias frequently accompany systemic inflammation and contribute to hemodynamic insufficiency. Markers for immunocompetent cells have been shown to be present in the tissue of the primary heart pacemaker, the sinoatrial node (SAN). However, the role of the resident cardiac immune cells in inflammation-induced bradycardia and SAN automaticity have still not been elucidated. Materials and methodsConfocal and bright field microscopy were used to identify toluidine blue-positive cells (TPC) in the SAN. Standard microelectrode technique and optical mapping were used to record action potentials (AP) and analyze activation pattern in the SAN tissue preparation from rats in presence of secretagogue C48/80. ResultsNumerous TPC, which exhibit toluidine-accumulating granules, typical for mast cells, are revealed in the SAN region of the right atrium. The activation and degranulation of the resident mast cells by C48/80 leads to biphasic changes of the pacemaker AP rate in the SAN – an initial acceleration is followed by rate reduction. The activation of the resident mast cells causes an increase of the primary activation area in the SAN that probably underlies the AP rate increase in response to C48/80. In addition, the application of C48/80 causes the primary/leading activation point migration in the SAN and AP rate decrease is probably mediated by the shift of the primary pacemaker site to the region surrounding the coronary sinus. Moreover, inexcitable zones and conduction blocks induced in the central SAN myocardium by C48/80 underlie the suppression of automaticity. ConclusionThe degranulation of resident mast cells affects the pacemaker cardiomyocytes’ ability to generate spontaneous AP in the SAN tissue and causes shift of the dominant pacemaker site. Therefore, the activation of the resident mast cells in the SAN can be considered as a new mechanism of inflammatory induced bradyarrhythmias.

MiR-185-5p regulates the development of myocardial fibrosis

BackgroundCardiac fibrosis stiffens the ventricular wall, predisposes to cardiac arrhythmias and contributes to the development of heart failure. In the present study, our aim was to identify novel miRNAs that regulate the development of cardiac fibrosis and could serve as potential therapeutic targets for myocardial fibrosis. Methods and resultsAnalysis for cardiac samples from sudden cardiac death victims with extensive myocardial fibrosis as the primary cause of death identified dysregulation of miR-185-5p. Analysis of resident cardiac cells from mice subjected to experimental cardiac fibrosis model showed induction of miR-185-5p expression specifically in cardiac fibroblasts. In vitro, augmenting miR-185-5p induced collagen production and profibrotic activation in cardiac fibroblasts, whereas inhibition of miR-185-5p attenuated collagen production. In vivo, targeting miR-185-5p in mice abolished pressure overload induced cardiac interstitial fibrosis. Mechanistically, miR-185-5p targets apelin receptor and inhibits the anti-fibrotic effects of apelin. Finally, analysis of left ventricular tissue from patients with severe cardiomyopathy showed an increase in miR-185-5p expression together with pro-fibrotic TGF-β1 and collagen I. ConclusionsOur data show that miR-185-5p targets apelin receptor and promotes myocardial fibrosis.

Cardiomyocyte-GSK-3β deficiency induces cardiac progenitor cell proliferation in the ischemic heart through paracrine mechanisms.

Cardiomyopathy is an irreparable loss and novel strategies are needed to induce resident cardiac progenitor cell (CPC) proliferation in situ to enhance the possibility of cardiac regeneration. Here, we sought to identify the potential roles of glycogen synthase kinase-3β (GSK-3β), a critical regulator of cell proliferation and differentiation, in CPC proliferation post-myocardial infarction (MI). Cardiomyocyte-specific conditional GSK-3β knockout (cKO) and littermate control mice were employed and challenged with MI. Though cardiac left ventricular chamber dimension and contractile functions were comparable at 2 weeks post-MI, cKO mice displayed significantly preserved LV chamber and contractile function versus control mice at 4 weeks post-MI. Consistent with protective phenotypes, an increased percentage of c-kit-positive cells (KPCs) were observed in the cKO hearts at 4 and 6 weeks post-MI which was accompanied by increased levels of cardiomyocyte proliferation. Further analysis revealed that the observed increased number of KPCs in the ischemic cKO hearts was mainly from a cardiac lineage, as the majority of identified KPCs were negative for the hematopoietic lineage marker, CD45. Mechanistically, cardiomyocyte-GSK-3β profoundly suppresses the expression and secretion of growth factors, including basic-fibroblast growth factor, angiopoietin-2, erythropoietin, stem cell factor, platelet-derived growth factor-BB, granulocyte colony-stimulating factor, and vascular endothelial growth factor, post-hypoxia. In conclusion, our findings strongly suggest that loss of cardiomyocyte-GSK-3β promotes cardiomyocyte and resident CPC proliferation post-MI. The induction of cardiomyocyte and CPC proliferation in the ischemic cKO hearts is potentially regulated by autocrine and paracrine signaling governed by dysregulated growth factors post-MI. A strategy to inhibit cardiomyocyte-GSK-3β could be helpful for the promotion of in situ cardiac regeneration post-ischemic injury.

Tissue factor cytoplasmic domain exacerbates post-infarct left ventricular remodeling via orchestrating cardiac inflammation and angiogenesis

Abstract Introduction The coagulation protein tissue factor (TF) regulates inflammation and angiogenesis via its cytoplasmic domain in infection, cancer and diabetes. While TF is highly abundant in the heart and implicated in cardiac injuries and dysfunction, the contribution of its cytoplasmic domain in cardiac pathology remains unclear. Purpose We aimed to investigate the contribution of the cytoplasmic domain of TF to post-infarct myocardial injury and adverse left ventricular (LV) remodeling. Methods and results Myocardial infarction was induced by permanent occlusion of the left anterior descending coronary artery. Male mice with C57BL/Jax background were used for the study. Compared with wild-type mice, mice lacking the TF cytoplasmic domain (TFΔCT) had a higher survival rate (90.5% versus 70%, p=0.0298) during a 28-day follow-up after myocardial infarction. Among surviving mice, TFΔCT mice had better cardiac function and less LV remodeling (ESV: 114.5±13.1mL for WT, 67.06±10.8mL for TFΔCT, p&amp;lt;0.001; EDV: 146.6±12.4mL for WT, 99.97±11.71mL for TFΔCT, p&amp;lt;0.001) than wild-type mice. Bone marrow chimerism indicated that deletion of the TF cytoplasmic domain in either bone marrow-derived cells or cardiac resident cells could alleviate post-infarct cardiac dysfunction. Speckle-tracking strain analysis revealed that the overall improvement of post-infarct cardiac performance in TFΔCT mice was attributed to reduced myocardial deformation in the peri-infarct region (strain-%: 11.14±0.97 for WT, 15.34±1.10 for TFΔCT, p=0.007; strain rate-/s: 3.89±0.26 for WT, 5.18±0.21 for TFΔCT, p=0.0005). Histological analysis demonstrated that TFΔCT hearts had in the infarct area greater proliferation of endothelial cells and myofibroblasts accompanied with better scar formation. Compared with wild-type hearts, infarcted TFΔCT hearts showed less infiltration of proinflammatory cells with concomitant lower expression of protease-activated receptor-1 (PAR1)-Rac1 axis. Furthermore, infarcted TFΔCT hearts presented markedly higher peri-infarct vessel density associated with enhanced endothelial cell proliferation and higher expression of PAR2 and PAR2-associated pro-angiogenic pathway factors. Conclusions Our findings demonstrate that the TF cytoplasmic domain exacerbates post-infarct cardiac injury and adverse LV remodeling via differential regulation of inflammation and angiogenesis. Targeted inhibition of the TF cytoplasmic domain-mediated intracellular signaling may ameliorate post-infarct LV remodeling without perturbing coagulation. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): National University Health System of Singapore

Genetic deletion of aryl hydrocarbon receptor activates cardiac NLRP3-inflammasome and suppresses apoptosis in mice with acute myocardial infarction

Abstract Background and purpose There is great interest in understanding the role of immune cells in the inflammatory mechanisms associated with acute myocardial infarction (AMI). Resident cardiac immune cells are activated by damage-associated molecular patterns interacting with specific pattern recognition receptors (PRRs), triggering inflammatory responses. Activated immune cells and fibroblasts then promote prohypertrophic and profibrotic signalling, which induces cardiac hypertrophy, fibrosis and remodelling after AMI. Nucleotide-binding domain leucine-rich repeat-containing proteins (NLRs) are PRRs that have emerged as key therapeutic targets in cardiovascular disease. NLRP3 assembles the inflammasome, which serves as molecular signalling platform to activate caspase-1 and regulate maturation of the potent proinflammatory cytokine IL-1β. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor of the basic Helix-Loop-Helix-PER-ARNT-SIM superfamily that mediates responses against environmental pollutants. Beyond its role in metabolising exogenous toxins as part of an adaptive chemical response, there is growing evidence that AhR is activated by endogenous ligands under physiological conditions. AhR has been proposed as a negative regulator of NLRP3-inflammasome activation. Here we studied the effects of defective AhR signalling on apoptosis, NLRP3-inflammasome activation and survival in a mouse model of AMI. Methods and results AMI was induced in 8–10-weeks-old AhR+/+ and AhR−/− mice by ligation of the left anterior descending coronary artery. Immunofluorescence analysis revealed that the number of TUNEL-positive nuclei and the expression of the apoptotic effector cleaved caspase-3 was higher in hearts from infarcted AhR+/+ mice than from sham-operated AhR+/+ mice 24 h after surgery. A similar analysis in AhR−/− mice revealed the absence of apoptotic markers after AMI. Gene analysis showed that cardiac levels of Nlrp3, apoptosis-associated speck-like protein (Asc) and Il1b were all significantly higher in AhR−/− mice than in AhR+/+ mice 24 h after AMI. Finally, survival analysis showed that 25% of AhR+/+ mice died 72 h after AMI surgery whereas the mortality in AhR−/− mice was 100%. Conclusion Our results show that loss of AhR inhibits cardiac apoptosis and increases NLRP3-mediated inflammation 24 h after AMI, highlighting a possible role for this nuclear receptor in cardiac remodelling post-AMI. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund

Age-related defects in autophagy alter the secretion of paracrine factors from bone marrow mononuclear cells.

Bone marrow mononuclear cell therapy improves cardiac repair after myocardial infarction (MI), in-part through signaling to resident cardiac cells, such as fibroblasts, which regulate scar formation. The efficacy of cell therapy declines with age, as aging of both donor and recipient cells decreases repair responses. Autophagy regulates the microenvironment by both extracellular vesicle (EV)-dependent and independent secretion pathways. We hypothesized that age-related autophagy changes in bone marrow cells (BMCs) alter paracrine signaling, contributing to lower cell therapy efficacy. Here, we demonstrate that young Sca-1+ BMCs exhibited a higher LC3II/LC3I ratio compared to old Sca-1+ BMCs, which was accentuated when BMCs were cultured under hypoxia. To examine the effect on paracrine signaling, old cardiac fibroblasts were cultured with conditioned medium (CM) from young and old Sca-1+ BMCs. Young, but not old CM, enhanced fibroblast proliferation, migration, and differentiation, plus reduced senescence. These beneficial effects were lost when autophagy or EV secretion in BMCs was blocked pharmacologically, or by siRNA knockdown of Atg7. Therefore, both EV-dependent and -independent paracrine signaling from young BMCs is responsible for paracrine stimulation of old cardiac fibroblasts. In vivo, bone marrow chimerism of old mice with young BMCs increased the number of LC3b+ cells in the heart compared to old mice reconstituted with old BMCs. These data suggest that the deterioration of autophagy with aging negatively impacts the paracrine effects of BMCs, and provide mechanistic insight into the age-related decline in cell therapy efficacy that could be targeted to improve the function of old donor cells.

Anti-integrin \u03b1v therapy improves cardiac fibrosis after myocardial infarction by blunting cardiac PW1+ stromal cells

There is currently no therapy to limit the development of cardiac fibrosis and consequent heart failure. We have recently shown that cardiac fibrosis post-myocardial infarction (MI) can be regulated by resident cardiac cells with a fibrogenic signature and identified by the expression of PW1 (Peg3). Here we identify αV-integrin (CD51) as an essential regulator of cardiac PW1+ cells fibrogenic behavior. We used transcriptomic and proteomic approaches to identify specific cell-surface markers for cardiac PW1+ cells and found that αV-integrin (CD51) was expressed in almost all cardiac PW1+ cells (93% ± 1%), predominantly as the αVβ1 complex. αV-integrin is a subunit member of the integrin family of cell adhesion receptors and was found to activate complex of latent transforming growth factor beta (TGFβ at the surface of cardiac PW1+ cells. Pharmacological inhibition of αV-integrin reduced the profibrotic action of cardiac PW1+CD51+ cells and was associated with improved cardiac function and animal survival following MI coupled with a reduced infarct size and fibrotic lesion. These data identify a targetable pathway that regulates cardiac fibrosis in response to an ischemic injury and demonstrate that pharmacological inhibition of αV-integrin could reduce pathological outcomes following cardiac ischemia.

Crucial role of NLRP3 inflammasome in a murine model of Kawasaki disease

Kawasaki disease (KD) is a systemic febrile syndrome during childhood that is characterized by coronary arteritis. The etiopathogenesis of KD remains to be elucidated. NLRP3 inflammasome is a large multiprotein complex that plays a key role in IL-1β-driven sterile inflammatory diseases. In the present study, we investigated the role of NLRP3 inflammasome in a murine model of KD induced by Candida albicans water-soluble fraction (CAWS) and found that NLRP3 inflammasome is required for the development of CAWS-induced vasculitis. CAWS administration induced IL-1β production, caspase-1 activation, leukocyte infiltration, and fibrotic changes in the aortic root and coronary arteries, which were significantly inhibited by a deficiency of IL-1β, NLRP3, and ASC. In vitro experiments showed that among cardiac resident cells, macrophages, but not endothelial cells or fibroblasts, expressed Dectin-2, but did not produce IL-1β in response to CAWS. In contrast, CAWS induced caspase-1 activation and IL-1β production in bone marrow-derived dendritic cells (BMDCs), which were inhibited by a specific caspase-1 inhibitor and a deficiency of NLRP3, ASC, and caspase-1. CAWS induced NLRP3 and pro-IL-1β expression through a Dectin-2/Syk/JNK/NF-κB pathway, and caspase-1 activation and cleavage of pro-IL-1β through Dectin-2/Syk/JNK-mediated mitochondrial ROS generation, indicating that CAWS induces the priming and activation of NLRP3 inflammasome in BMDCs. These findings provide new insights into the pathogenesis of KD vasculitis, and suggest that NLRP3 inflammasome may be a potential therapeutic target for KD.

P6296The role of extracellular matrix protein 1 (ECM1) - a novel link between inflammation and cardiac fibrosis

Abstract Introduction Cardiac fibrosis is a severe consequence of cardiovascular disease and aging, in which we currently have no effective treatments. The mechanisms underpinning the development of cardiac fibrosis remains poorly understood. Our preliminary data suggested extracellular matrix protein 1 (ECM1) is involved in cardiac fibrosis. We therefore aimed to investigate the role of ECM1 in several fibrotic cardiac diseases. Methods Young and ageing (3m/18m) male C57BL/6 mice, and primary mouse cardiac fibroblast (cFB) cultures, commercial human cardiac fibroblasts (Hu-cFB), human coronary artery endothelial cell (HCAEC)/smooth muscle cell (HCASMC), and human cardiac myocyte (HCM) cell lines were used. Young mice were subject to myocardial infarction (MI, 3-day/28-day, n=6/6), or pressure overload (TAC, 3-day/13-week, n=4/4). Left ventricle (LV) was collected at all time-points, and at 18m (ageing; n=3). Spleen and bone marrow was extracted from young control mice. Hu-cFB cells were treated with recombinant ECM1 (20ng/ml) for either 10, 30 or 50 min, or 48h. Immunoblotting was conducted on all samples, qPCR on LV tissue only, density gradient centrifugation and multicolour flow cytometry coupled with fluorescent ECM1 mRNA in-situ hybridisation (FISH-Flow) on bone marrow cells. Results ECM1 expression was upregulated in ageing LV (mRNA 2.2±0.1-fold, p=0.0002; protein 2.0-fold, p=0.0006), day-3 post-MI (mRNA, 4.9±2.0-fold, p=0.004; protein, 3.0-fold, p=0.004), a trend of ECM1 upregulation was observed at day-28 post-MI (mRNA, 13.2±12.0-fold, p=0.003; protein, 1.8-fold, p=0.2), but no change post-TAC. Both ERK1/2 and AKT phosphorylation was upregulated 10 min post-ECM1 treatment of Hu-cFBs (ERK1/2, 2.0-fold, p&lt;0.0001; AKT, 1.9-fold, p&lt;0.0001), and Collagen-I protein expression was upregulated 48h post-ECM1 treatment (1.9-fold, p=0.004). ECM1 protein was not expressed in cFB, Hu-cFB, HCAEC, HCASMC or HCM, however ECM1 protein was highly expressed in spleen and bone marrow; to a greater extent in granulocytes compared to monocytes (p=0.004). tSNE analysis of ECM1 mRNA FISH-Flow revealed ECM1+ are highly granular, moderate to large in size, and express (to varying levels) CD45, CD11b, CD11c, F4/80, Ly6-C, Ly-6G, and FcεrI-α. However ECM1+ cells did not express markers indicative of smaller cells (CD3 or MHC II). Conclusions These data demonstrate that ECM1 plays a role in ageing and post-MI fibrosis. Although ECM1 was not produced by resident cardiac cells, it was highly expressed in spleen and bone marrow; specifically, large, granular bone marrow cell sub-types such as granulocytes and/or macrophages. Our data suggest ECM1 is expressed by cardiac infiltrating leukocytes to provoke fibroblast collagen expression in a disease specific manner; potentially via the ERK1/2 and/or AKT pathway activation. Therefore, ECM1 warrants further investigation, and may be a promising target for the treatment of fibrotic cardiac diseases. Acknowledgement/Funding John hunter hospital charitable trust, Hunter medical research institute (HMRI) grants