Transverse Aortic Constriction Mice Research Articles

Abstract P3078: The Stimulator Of Interferon Genes (STING) Modulates Adverse Cardiac Remodeling Induced By Cardiac Pressure Overload: New Role Of Endothelial Cell Sting In Systolic Dysfunction

Introduction: Cardiac inflammation is a hallmark of heart failure (HF) defined by T cell infiltration and increased expression of pro-inflammatory cytokines. Type I Interferon (IFN-I), a cytokine induced upon activation of STING, is increased in the heart after myocardial infarction and transverse aortic constriction (TAC). We reported that absence of endothelial cell (EC) STING results in an IFN-I dependent decline in T cell extravasation to inflammatory sites. We hypothesized that STING contributes to adverse cardiac remodeling induced by TAC and EC STING regulates systolic dysfunction through modulating cardiac inflammation. Methods: Male wildtype (WT), STING -/- and inducible EC specific STING -/- mice (Cad5 ERTCre2+/- STING fl/fl treated i.p. with 85mg/kg Tamoxifen), or oil treated and Cre - littermate WT controls were subjected to TAC or Sham surgery. Systolic function by echocardiography and left ventricular (LV) hypertrophy, fibrosis, and capillary density by histology were analyzed four weeks post-surgery and T cell activation was characterized in mediastinal draining lymph nodes (MdLN) by flow cytometry. Results: In contrast to WT, STING -/- mice did not show a decline in ejection fraction (EF). LV hypertrophy was decreased in TAC STING -/- mice compared to TAC WT mice. Isolectin staining showed that capillary density was higher in STING -/- mice in response to TAC as compared to the expected capillary loss observed in WT TAC mice. The frequency of CD4+CD62 lo CD44 hi cells in the MdLN, indicative of effector T cell activation, was decreased in STING -/- mice compared to WT mice in response to TAC. Similar analyses of Cad5 ERTCre2+/- STING fl/fl mice showed that EC STING -/- mice did not show a decline in EF in response to TAC, in contrast to their littermate WT controls. However, no changes were observed in LV hypertrophy, fibrosis or capillary density between groups and activation of MdLN effector T cells was also comparable in both groups. Conclusion: STING contributes to adverse cardiac remodeling induced by TAC. While EC STING contributes to systolic dysfunction through a yet unknown mechanism, STING in other cell compartments contributes to cardiac hypertrophy, capillary density and T cell activation in response to TAC.

TFRC in cardiomyocytes promotes macrophage infiltration and activation during the process of heart failure through regulating Ccl2 expression mediated by hypoxia inducible factor-1α.

Cardiac hypertrophy is an initiating link to Heart failure (HF) which still seriously endangers human health. Transferrin receptor (TFRC), which promotes iron uptake through the transferrin cycle, is essential for cardiac function. However, whether TFRC is involved in the process of pathological cardiac hypertrophy is not clear. Transverse aortic constriction (TAC) mouse model and mice primary cardiomyocytes treated with isoproterenol (ISO) or phenylephrine (PHE) were used to mimic cardiac hypertrophy in vivo and in vitro. Single cell RNA sequence data from heart tissues of TAC-model mice was obtained from the Gene Expression Omnibus (GEO) database, and was analyzed with R package Seurat. TFRC expression and macrophage infiltration in the heart tissue were tested by immunofluorescent staining. Macrophage polarization was detected by Flow Cytometry. TFRC expressions were detected by qRT-PCR, Western blot, and ELISA. TFRC expression is increased in the pathological cardiac hypertrophy of mice model and positively associated with macrophage infiltration. Furthermore, TFRC in cardiomyocytes recruits and activates macrophages by secreting C-C motif ligand 2 (Ccl2) in the mice heart tissue with TAC surgery or in the primary cardiomyocytes stimulated with ISO or PHE to induce myocardial hypertrophy in vitro. Moreover, we find that TFRC promotes Ccl2 expression in cardiomyocytes via regulating signal transducer and activator of transcription 3 (STAT3). In addition, we find that increased TFRC expression in the HF tissue is regulated by hypoxia-inducible factor-1α (HIF-1α). This in-depth study shows that TFRC in cardiomyocytes promotes HF development through inducing macrophage infiltration and activation via the STAT3-Ccl2 signaling, and TFRC expression in cardiomyocytes is regulated by HIF-1α during HF. This study firstuncovers the role of TFRC in cardiomyocytes on macrophage infiltration and activation during HF.

T-Cell MyD88 Is a Novel Regulator of Cardiac Fibrosis Through Modulation of T-Cell Activation.

Cardiac inflammation in heart failure is characterized by the presence of damage-associated molecular patterns, myeloid cells, and T cells. Cardiac damage-associated molecular patterns provide continuous proinflammatory signals to myeloid cells through TLRs (toll-like receptors) that converge onto the adaptor protein MyD88 (myeloid differentiation response 88). These induce activation into efficient antigen-presenting cells that activate T cells through their TCR (T-cell receptor). T-cell activation results in cardiotropism, cardiac fibroblast transformation, and maladaptive cardiac remodeling. T cells rely on TCR signaling for effector function and survival, and while they express MyD88 and damage-associated molecular pattern receptors, their role in T-cell activation and cardiac inflammation is unknown. We performed transverse aortic constriction in mice lacking MyD88 in T cells and analyzed remodeling, systolic function, survival, and T-cell activation. We profiled wild type versus Myd88-/- mouse T cells at the transcript and protein level and performed several functional assays. Analysis of single-cell RNA-sequencing data sets revealed that MyD88 is expressed in mouse and human cardiac T cells. MyD88 deletion in T cells resulted in increased levels of cardiac T-cell infiltration and fibrosis in response to transverse aortic constriction. We discovered that TCR-activated Myd88-/- T cells had increased proinflammatory signaling at the transcript and protein level compared with wild type, resulting in increased T-cell effector functions such as adhesion, migration across endothelial cells, and activation of cardiac fibroblast. Mechanistically, we found that MyD88 modulates T-cell activation and survival through TCR-dependent rather than TLR-dependent signaling. Our results outline a novel intrinsic role for MyD88 in limiting T-cell activation that is central to tune down cardiac inflammation during cardiac adaptation to stress.

Identification of TGF-β-related genes in cardiac hypertrophy and heart failure based on single cell RNA sequencing.

Heart failure (HF) remains a huge medical burden worldwide. Pathological cardiac hypertrophy is one of the most significant phenotypes of HF. Several studies have reported that the TGF-β pathway plays a double-sided role in HF. Therefore, TGF-β-related genes (TRGs) may be potential therapeutic targets for cardiac hypertrophy and HF. However, the roles of TRGs in HF at the single-cell level remain unclear. In this study, to analyze the expression pattern of TRGs during the progress of cardiac hypertrophy and HF, we used three public single-cell RNA sequencing datasets for HF (GSE161470, GSE145154, and GSE161153), one HF transcriptome data (GSE57338), and one hypertrophic cardiomyopathy transcriptome data (GSE141910). Weighted gene co-expression network analysis (WGCNA), functional enrichment analysis and machine learning algorithms were used to filter hub genes. Transverse aortic constriction mice model, CCK-8, wound healing assay, quantitative real-time PCR and western blotting were used to validate bioinformatics results. We observed that cardiac fibroblasts (CFs) and endothelial cells showed high TGF-β activity during the progress of HF. Three modules (royalblue, brown4, and darkturquoize) were identified to be significantly associated with TRGs in HF. Six hub genes (TANC2, ADAMTS2, DYNLL1, MRC2, EGR1, and OTUD1) showed anomaly trend in cardiac hypertrophy. We further validated the regulation of the TGF-β-MYC-ADAMTS2 axis on CFs activation in vitro. This study identified six hub genes (TANC2, ADAMTS2, DYNLL1, MRC2, EGR1, and OTUD1) by integrating scRNA and transcriptome data. These six hub genes might be therapeutic targets for cardiac hypertrophy and HF.

Divergent effects of myostatin inhibition on cardiac and skeletal muscles in a mouse model of pressure overload.

The transforming growth factor-β (TGF-β) superfamily member, myostatin, is a negative regulator of muscle growth and may contribute to adverse cardiac remodeling. Whether suppressing myostatin could benefit pressure-overloaded heart remains unclear. We investigated the effects of pharmacological inhibition of myostatin on cardiac fibrosis and hypertrophy in a mouse model of pressure overload induced by transverse aortic constriction (TAC). Two weeks after the surgery, TAC and sham mice were randomly divided into groups receiving mRK35, a monoclonal anti-myostatin antibody, or vehicle (PBS) for 8 wk. Significant progressive cardiac hypertrophy was observed in TAC mice, as reflected by the increased wall thickness, ventricular weight, and cross-sectional area of cardiomyocytes. In the groups treated with mRK35, compared with sham mice, cardiac fibrosis was increased in TAC mice, accompanied with elevated mRNA expression of fibrotic genes. However, among the TAC mice, mRK35 did not reduce cardiac hypertrophy or fibrosis. Body weight, lean mass, and wet weights of tibialis anterior and gastrocnemius muscle bundle were increased by mRK35. When compared with the TAC-PBS group, the TAC mice treated with mRK35 demonstrated greater forelimb grip strength and a larger mean size of gastrocnemius fibers. Our data suggest that mRK35 does not attenuate cardiac hypertrophy and fibrosis in a TAC mouse model but has positive effects on muscle mass and muscle strength. Anti-myostatin treatment may have therapeutic value against muscle wasting in cardiac vascular disease.NEW & NOTEWORTHY Recent research has highlighted the importance of inhibiting TGF-β signaling in mitigating cardiac dysfunction and remodeling. As myostatin belongs to the TGF-β family, we evaluated the impact of myostatin inhibition using mRK35 in TAC-operated mice. Our data demonstrate that mRK35 significantly increased body weight, muscle mass, and muscle strength but did not attenuate cardiac hypertrophy or fibrosis. Pharmacological inhibition of myostatin may provide therapeutic benefits for the management of muscle wasting in cardiovascular diseases.

NSD2 promotes pressure overload-induced cardiac hypertrophy via activating circCmiss1/TfR1/ferroptosis signaling

Heart failure typically occurs early in the clinical course of sustained cardiac hypertrophy that is accompanied by maladaptive remodeling of the heart. It is critical to discover new mechanisms and effective therapeutic targets to prevent and cure pathological cardiac hypertrophy. The objective of the study was to evaluate the effects of circRNAs on NSD2-induced ventricular remodeling. We screened the dysregulated circRNAs in normal or NSD2−/− C57BL/6 mice with or without transverse aortic constriction (TAC), and found that circCmss1 significantly increased in normal TAC mice, but decreased in NSD2−/− TAC mice. Angiotensin II(Ang II)induced neonatal cardiomyocyte hypertrophy in vitro and the pressure overload-induced cardiac hypertrophy in vivo can be reduced by Knocking down circCmss1. We further investigated the downstream signaling of circCmss1 in the progression of NSD2-promoted ventricular remodeling and discovered that circCmss1 could interact with a transcription factor EIF4A3 and induce the expression of transferrin receptor 1 (TfR1), thus activating the ferroptosis in cardiomyocytes. This study highlights the significance of NSD2 activation of circCmss1/EIF4A3/TfR1 as therapeutic targets for treating pathological myocardial hypertrophy.

The Action and Mechanism of Trehalose on GATA4 Autophagy Degradation and Ventricular Remodeling.

To probe the effect of trehalose on myocardial hypertrophy and its specific molecular mechanism. C57BL/6J male mice were divided into four subgroups: Sham operation subgroup (Sham), negative sham subgroup (Sham+Trehalose), transverse aortic constriction (TAC), and trehalose treatment subgroup (TAC+Trehalose). Immediately after the TAC operation, trehalose at a dose of 10 mg/kg was given daily via gavage. After four weeks, changes in cardiac function were evaluated using ultrasound to measure EF (ejection fraction), FS (fractional shortening), IVRT (isovolumic relaxation time), MPI (myocardial performance index), Tau (isovolumic relaxation time constant), LVESP (left ventricular end-systolic pressure), and EDPVR (end-diastolic pressure-volume relationship). The profiles of autophagy-associated proteins (p62, LC3II/I, and Beclin-1) and GATA4 protein in mice myocardial tissues were assessed by Western blotting (WB). Myocardial cells were classified from TAC mice into five groups: Control, Trehalose, Phenylephrine (PE), PE+Trehalose, and PE+Trehalose+autophagy inhibitor chloroquine groups. In the PE group, cardiomyocytes were treated with 50 μmol/L PE. Then, the cells were treated with trehalose (100 μmol/L), trehalose (100 μmol/L)+autophagy (20 μmol/L) for 24 hours respectively. The Control group was treated with the same amount of normal saline. Flow cytometry was utilized to detect myocardial cell apoptosis in each subgroup. The alterations in apoptosis and autophagy-correlated proteins (p62, LC3II/I, and Beclin-1) were assessed by WB. Additionally, the level of GATA4 protein upstream of autophagy was estimated. Furthermore, the expression levels of pro-apoptotic proteins Bad, BAX, Cleaved-caspase-3, and anti-apoptotic protein Bcl-2 were examined by WB. The TAC operation significantly augmented myocardial hypertrophy, heart weight-to-body weight ratio, and myocardial cell apoptosis in mice (p < 0.05). Trehalose significantly improved cardiac hypertrophy, cardiomyocyte apoptosis, and cardiac function decline in mice. Additionally, it also significantly enhanced autophagy in mouse cardiac tissues (p < 0.05). At the cellular level, trehalose significantly decreased PE-elicited apoptosis and promoted the protein expressions of Beclin-1 and LC3 II/I in cardiomyocytes while significantly dampening the profiles of p62 and GATA4 in cells. The effect of trehalose and chloroquine treatment was significantly greater than that of the trehalose group. Trehalose significantly abates myocardial hypertrophy and pressure overload-induced cardiomyocyte apoptosis in mice. The cardioprotective effect of trehalose on enhanced autophagy is attributed, at least in part, to the promotion of autophagic degradation of GATA4.

Long non-coding RNA KCND1 protects hearts from hypertrophy by targeting YBX1

Cardiac hypertrophy is a common structural remodeling in many cardiovascular diseases. Recently, long non-coding RNAs (LncRNAs) were found to be involved in the physiological and pathological processes of cardiac hypertrophy. In this study, we found that LncRNA KCND1 (LncKCND1) was downregulated in both transverse aortic constriction (TAC)-induced hypertrophic mouse hearts and Angiotensin II (Ang II)-induced neonatal mouse cardiomyocytes. Further analyses showed that the knockdown of LncKCND1 impaired cardiac mitochondrial function and led to hypertrophic changes in cardiomyocytes. In contrast, overexpression of LncKCND1 inhibited Ang II-induced cardiomyocyte hypertrophic changes. Importantly, enhanced expression of LncKCND1 protected the heart from TAC-induced pathological cardiac hypertrophy and improved heart function in TAC mice. Subsequent analyses involving mass spectrometry and RNA immunoprecipitation assays showed that LncKCND1 directly binds to YBX1. Furthermore, overexpression of LncKCND1 upregulated the expression level of YBX1, while silencing LncKCND1 had the opposite effect. Furthermore, YBX1 was downregulated during cardiac hypertrophy, whereas overexpression of YBX1 inhibited Ang II-induced cardiomyocyte hypertrophy. Moreover, silencing YBX1 reversed the effect of LncKCND1 on cardiomyocyte mitochondrial function and its protective role in cardiac hypertrophy, suggesting that YBX1 is a downstream target of LncKCND1 in regulating cardiac hypertrophy. In conclusion, our study provides mechanistic insights into the functioning of LncKCND1 and supports LncKCND1 as a potential therapeutic target for pathological cardiac hypertrophy.

Bioinformatics exploration of potential common therapeutic targets for systemic and pulmonary arterial hypertension-induced myocardial hypertrophy.

Systemic and pulmonary arterial hypertension (PAH) can induce left and right ventricular hypertrophy, respectively, but common therapeutic targets for both left and right hypertrophy are limited. In this study, we attempt to explore potential common therapeutic targets and screen out potential target drugs for further study. Cardiac mRNA expression profiles in mice with transverse aortic constriction (TAC) and pulmonary arterial constriction (PAC) are obtained from online databases. After bioinformatics analyses, we generate TAC and PAC mouse models to validate the phenotypes of cardiac remodelling as well as the identified hub genes. Bioinformatics analyses show that there are 214 independent differentially expressed genes (DEGs) in GSE136308 (TAC related) and 2607 independent DEGs in GSE30922 (PAC related), while 547 shared DEGs are associated with the function of the extracellular matrix (ECM) or involved in the PI3K-Akt signaling pathway, cytokine-cytokine receptor interactions, and ECM-receptor interactions. We identifyd Fn1, Il6, Col1a1, Igf1, Col1a2, Timp1, Col3a1, Cd44, Ctgf and Postn as hub genes of the shared DEGs, and most of them are associated with myocardial fibrosis. Those hub genes and phenotypes of cardiac remodelling are validated in our TAC and PAC mouse models. Furthermore, we identify dehydroisoandrosterone (DHEA), iloprost and 4,5-dianilinophthalimide (DAPH) as potential therapeutic drugs targeting both left and right ventricular hypertrophy and validate the effect of DHEA. These findings suggest that DHEA could be an effective drug for pressure overload-induced left or right ventricular hypertrophy by regulating the shared hub differentially expressed genes associated with fibrosis.

Expression and role of SerpinE2 in cardiac fibrosis

Cardiac fibrosis is a common histological feature of a broad spectrum of cardiovascular diseases including myocardial infarction. Proteases and their inhibitors are known to be involved in the regulation of extracellular matrix deposition and remodeling. SerpinE2 is a serine protease inhibitor expressed within the myocardium, which targets thrombin and the proteases of the plasminergic system. This inhibitor has been shown to be involved in lung fibrosis and could be an actor of cardiac remodeling. To study SerpinE2 expression in human left ventricle biopsies, from organ donors (control) and recipient explanted patients. To evaluate SerpinE2 role in cardiac remodeling and fibrosis in the mouse TAC (transverse aortic constriction) model. LV biopsies from organ donors and recipient explanted patients were characterized by histology and classified. SerpinE2 mRNA and protein expression in human samples were assessed by RT-qPCR and Western-Blot after pulverization in liquid nitrogen. Immunofluorescence was used to characterize SerpinE2 expression within paraffin embedded biopsies sections. Wild-type (WT) and SerpinE2-deficient (KO) mice were subjected to 27G TAC model and cardiac function was followed weekly by echocardiography Doppler. After 4 weeks, mice were sacrificed, hearts were harvested and frozen in liquid nitrogen. Heart cryosections were then used for histological fibrosis analysis (Sirius red) or for RNA extraction and RT-qPCR. We found that SerpinE2 expression (mRNA and protein) was significantly increased (×2.5) in the infarcted zone of the biopsies from the LV of explanted patients, compared with non-infarcted area and control donors. SerpinE2 mRNA expression in these samples was also strongly correlated with the expression on type I and III collagens, and TGF-ß. Immunofluorescence assays showed that SerpinE2 protein was mainly detected within the fibrotic areas of the explanted donors. Echocardiography Doppler of 27G mice showed significant decrease in KO animals LV mass and thickness compared with WT counterparts. A significant 2.3 times decrease in type I collagen mRNA expression was observed in KO mice but this was not confirmed by Sirius red staining in heart sections. These data suggest that SerpinE2 could be an actor and/or a biomarker or cardiac fibrosis.

Catestatin Protects Against Diastolic Dysfunction by Attenuating Mitochondrial Reactive Oxygen Species Generation.

Background Catestatin has been reported as a pleiotropic cardioprotective peptide. Heart failure with preserved ejection fraction (HFpEF) was considered a heterogeneous syndrome with a complex cause. We sought to investigate the role of catestatin in HFpEF and diastolic dysfunction. METHODS AND RESULTS Administration of recombinant catestatin (1.5 mg/kg/d) improved diastolic dysfunction and left ventricular chamber stiffness in transverse aortic constriction mice with deoxycorticosterone acetate pellet implantation, as reflected by Doppler tissue imaging and pressure-volume loop catheter. Less cardiac hypertrophy and myocardial fibrosis was observed, and transcriptomic analysis revealed downregulation of mitochondrial electron transport chain components after catestatin treatment. Catestatin reversed mitochondrial structural and respiratory chain component abnormality, decreased mitochondrial proton leak, and reactive oxygen species generation in myocardium. Excessive oxidative stress induced by Ru360 abolished catestatin treatment effects on HFpEF-like cardiomyocytes in vitro, indicating the beneficial role of catestatin in HFpEF as a mitochondrial ETC modulator. The serum concentration of catestatin was tested among 81 patients with HFpEF and 76 non-heart failure controls. Compared with control subjects, serum catestatin concentration was higher in patients with HFpEF and positively correlated with E velocity to mitral annular e' velocity ratio, indicating a feedback compensation role of catestatin in HFpEF. Conclusions Catestatin protects against diastolic dysfunction in HFpEF through attenuating mitochondrial electron transport chain-derived reactive oxygen species generation. Serum catestatin concentration is elevated in patients with HFpEF, probably as a relatively insufficient but self-compensatory mechanism.

Metformin Collaborates with PINK1/Mfn2 Overexpression to Prevent Cardiac Injury by Improving Mitochondrial Function.

Both mitochondrial quality control and energy metabolism are critical in maintaining the physiological function of cardiomyocytes. When damaged mitochondria fail to be repaired, cardiomyocytes initiate a process referred to as mitophagy to clear defective mitochondria, and studies have shown that PTEN-induced putative kinase 1 (PINK1) plays an important role in this process. In addition, previous studies indicated that peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is a transcriptional coactivator that promotes mitochondrial energy metabolism, and mitofusin 2 (Mfn2) promotes mitochondrial fusion, which is beneficial for cardiomyocytes. Thus, an integration strategy involving mitochondrial biogenesis and mitophagy might contribute to improved cardiomyocyte function. We studied the function of PINK1 in mitophagy in isoproterenol (Iso)-induced cardiomyocyte injury and transverse aortic constriction (TAC)-induced myocardial hypertrophy. Adenovirus vectors were used to induce PINK1/Mfn2 protein overexpression. Cardiomyocytes treated with isoproterenol (Iso) expressed high levels of PINK1 and low levels of Mfn2, and the changes were time dependent. PINK1 overexpression promoted mitophagy, attenuated the Iso-induced reduction in MMP, and reduced ROS production and the apoptotic rate. Cardiac-specific overexpression of PINK1 improved cardiac function, attenuated pressure overload-induced cardiac hypertrophy and fibrosis, and facilitated myocardial mitophagy in TAC mice. Moreover, metformin treatment and PINK1/Mfn2 overexpression reduced mitochondrial dysfunction by inhibiting ROS generation leading to an increase in both ATP production and mitochondrial membrane potential in Iso-induced cardiomyocyte injury. Our findings indicate that a combination strategy may help ameliorate myocardial injury by improving mitochondrial quality.

Guanxinning injection ameliorates cardiac remodeling in HF mouse and 3D heart spheroid models via p38/FOS/MMP1-mediated inhibition of myocardial hypertrophy and fibrosis

BackgroundHeart failure (HF) is a cardiovascular disease with high morbidity and mortality. Guanxinning injection (GXNI) is clinically used for the treatment of coronary heart disease, but its therapeutic efficacy and potential mechanism for HF are poorly understood. This study aimed to evaluate the therapeutic potential of GXNI on HF, with a special focus on its role in myocardial remodeling. Methods3D cardiac organoids and transverse aortic constriction (TAC) mouse models were established and utilized. Heart function and pathology were evaluated by echocardiography, hemodynamic examination, tail-cuff blood pressure and histopathology. Key targets and pathways regulated by GXNI in HF mouse heart were revealed via RNA-seq and network pharmacology analysis, and were verified by RT-PCR, Western blot, immunohistochemistry and immunofluorescence. ResultsGXNI significantly inhibited cardiac hypertrophy and cells death. It protected mitochondrial function in cardiac hypertrophic organoids and markedly improved cardiac function in HF mice. Analysis of GXNI-regulated genes in HF mouse hearts revealed that IL-17A signaling in fibroblasts and the corresponding p38/c-Fos/Mmp1 pathway prominently mediated cardiac. Altered expressions of c-Fos, p38 and Mmp1 by GXNI in heart tissues and in cardiac organoids were validated by RT-PCR, WB, IHC, and IF. H&E and Masson staining confirmed that GXNI substantially ameliorated myocardial hypertrophy and fibrosis in HF mice and in 3D organoids. ConclusionGXNI inhibited cardiac fibrosis and hypertrophy mainly via down-regulating p38/c-Fos/Mmp1 pathway, thereby ameliorating cardiac remodeling in HF mice. Findings in this study provide a new strategy for the clinical application of GXNI in the treatment of heart failure.

Atrioventricular node dysfunction in pressure overload-induced heart failure-Involvement of the immune system and transcriptomic remodelling.

Heart failure is associated with atrioventricular (AV) node dysfunction, and AV node dysfunction in the setting of heart failure is associated with an increased risk of mortality and heart failure hospitalisation. This study aims to understand the causes of AV node dysfunction in heart failure by studying changes in the whole nodal transcriptome. The mouse transverse aortic constriction model of pressure overload-induced heart failure was studied; functional changes were assessed using electrocardiography and echocardiography and the transcriptome of the AV node was quantified using RNAseq. Heart failure was associated with a significant increase in the PR interval, indicating a slowing of AV node conduction and AV node dysfunction, and significant changes in 3,077 transcripts (5.6% of the transcriptome). Many systems were affected: transcripts supporting AV node conduction were downregulated and there were changes in transcripts identified by GWAS as determinants of the PR interval. In addition, there was evidence of remodelling of the sarcomere, a shift from fatty acid to glucose metabolism, remodelling of the extracellular matrix, and remodelling of the transcription and translation machinery. There was evidence of the causes of this widespread remodelling of the AV node: evidence of dysregulation of multiple intracellular signalling pathways, dysregulation of 109 protein kinases and 148 transcription factors, and an immune response with a proliferation of neutrophils, monocytes, macrophages and B lymphocytes and a dysregulation of 40 cytokines. In conclusion, inflammation and a widespread transcriptional remodelling of the AV node underlies AV node dysfunction in heart failure.

Cadherin-11-Interleukin-6 Signaling between Cardiac Fibroblast and Cardiomyocyte Promotes Ventricular Remodeling in a Mouse Pressure Overload-Induced Heart Failure Model

Heart failure is a serious and life-threatening disease worldwide. Cadherin-11 (Cad-11) is highly expressed in the heart and closely associated with inflammation. There is currently limited understanding on how Cad-11 contributes to cardiac remodeling and its underline molecular mechanism. We found an increased expression of Cad-11 in biopsy heart samples from heart failure patients, suggesting a link between Cad-11 and heart failure. To determine the role of Cad-11 in cardiac remodeling, Cad-11-deficient mice were used in a well-established mouse transverse aortic constriction (TAC) model. Loss of Cad11 greatly improved pressure overload-induced LV structural and electrical remodeling. IL (interleukin)-6 production was increased following TAC in WT mice and this increase was inhibited in cadherin-11-/- mice. We further tested the effect of IL-6 on myocyte hypertrophy and fibrosis in a primary culture system. The addition of hCad-11-Fc to cultured cardiac fibroblasts increased IL-6 production and fibroblast cell activation, whereas neutralizing IL-6 with an IL-6 antibody resulted in alleviating the fibroblast activation induced by hCad-11-Fc. On the other hand, cardiomyocytes were promoted to cardiomyocyte hypertrophy when cultured in condition media collected from cardiac fibroblasts stimulated by hCad-11-Fc.Similarly, neutralizing IL-6 prevented cardiomyocyte hypertrophy. Finally, we found that MAPKs and CaMKII-STAT3 pathways were activated in both hCad-11-Fc stimulated fibroblasts and cardiomyocytes treated with hCad-11-Fc stimulated fibroblast condition medium. IL-6 neutralization inhibited such MAPK and CaMKII-STAT3 signaling activation. These data demonstrate that Cad-11 functions in pressure overload-induced ventricular remodeling through inducing IL-6 secretion from cardiac fibroblasts to modulate the pathophysiology of neighboring cardiomyocytes.

Contribution of skeletal muscle-specific microRNA-133b to insulin resistance in heart failure.

Insulin resistance (IR) is one of the hallmarks of heart failure (HF). Abnormalities in skeletal muscle (SM) metabolism have been identified in patients with HF. However, the underlying mechanisms of IR development in SM in HF are poorly understood. Herein, we hypothesize that HF upregulates miR-133b in SM and in turn alters glucose metabolism and the propensity toward IR. Mitochondria isolated from SM of mice with HF induced by transverse aortic constriction (TAC) showed lower respiration and downregulation of muscle-specific components of the tricarboxylic acid (TCA) cycle, AMP deaminase 1 (AMPD1), and fumarate compared with those from control animals. RNA-Seq and subsequent qPCR validation confirmed upregulation of SM-specific microRNA (miRNA), miR-133b, in TAC versus sham animals. miR-133b overexpression alone resulted in significantly lower mitochondrial respiration, cellular glucose uptake, and glycolysis along with lower ATP production and cellular energy reserve compared with the scramble (Scr) in C2C12 cells. miR-133b binds to the 3'-untranslated region (UTR) of KLF15, the transcription factor for the insulin-sensitive glucose transporter, GLUT4. Overexpression of miR-133b lowers GLUT4 and lowers pAkt in presence of insulin in C2C12 cells. Finally, lowering miR-133b in primary skeletal myocytes isolated from TAC mice using antagomir-133b reversed the changes in KLF15, GLUT4, and AMPD1 compared with the scramble-transfected myocytes. Taken together, these data demonstrate a role for SM miR-133b in altered glucose metabolism in HF and suggest the therapeutic potential in HF to improve glucose uptake and glycolysis by restoring GLUT4 abundance. The data uncover a novel mechanism for IR and ultimately SM metabolic abnormalities in patients with HF.NEW & NOTEWORTHY Heart failure is associated with systemic insulin resistance and abnormalities in glucose metabolism but the underlying mechanisms are poorly understood. In the skeletal muscle, the major peripheral site of glucose utilization, we observe an increase in miR-133b in heart failure mice, which reduces the insulin-sensitive glucose transporter (GLUT4), glucose uptake, and metabolism in C2C12 and in myocytes. The antagomir for miR-133b restores GLUT4 protein and markers of metabolism in skeletal myocytes from heart failure mice demonstrating that miR-133b is an exciting target for systemic insulin resistance in heart failure and an important player in the cross talk between the heart and the periphery in the heart failure syndrome.

A time-series minimally invasive transverse aortic constriction mouse model for pressure overload-induced cardiac remodeling and heart failure.

Transverse aortic constriction (TAC) is a widely-used animal model for pressure overload-induced cardiac hypertrophy and heart failure (HF). The severity of TAC-induced adverse cardiac remodeling is correlated to the degree and duration of aorta constriction. Most studies of TAC are performed with a 27-gauge needle, which is easy to cause a tremendous left ventricular overload and leads to a rapid HF, but it is accompanied by higher mortality attributed to tighter aortic arch constriction. However, a few studies are focusing on the phenotypes of TAC applied with a 25-gauge needle, which produces a mild overload to induce cardiac remodeling and has low post-operation mortality. Furthermore, the specific timeline of HF induced by TAC applied with a 25-gauge needle in C57BL/6 J mice remains unclear. In this study, C57BL/6 J mice were randomly subjected to TAC with a 25-gauge needle or sham surgery. Echocardiography, gross morphology, and histopathology were applied to evaluate time-series phenotypes in the heart after 2, 4, 6, 8, and 12 weeks. The survival rate of mice after TAC was more than 98%. All mice subjected to TAC maintained compensated cardiac remodeling during the first two weeks and began to exhibit heart failure characteristics after 4 weeks upon TAC. At 8 weeks post-TAC, the mice showed severe cardiac dysfunction, hypertrophy, and cardiac fibrosis compared to sham mice. Moreover, the mice raised a severe dilated HF at 12 weeks. This study provides an optimized method of the mild overload TAC-induced cardiac remodeling from the compensatory period to decompensatory HF in C57BL/6 J mice.

Genetic knockout of prestin limits cardiac hypertrophy in a murine pressure overload model.

Up until recently, Prestin (Slc26a5) was considered to be exclusively expressed in the outer hair cells of the inner ear, where it serves as a voltage-to-force convertor and mediates electromotility. Our recent studies demonstrate that prestin is expressed in the heart, where it amplifies cardiac motor function. Due to prestin's ability to modulate cardiac contractility, we hypothesize that it plays a pivotal role in pathological cardiac hypertrophy. We subjected wild-type (WT) and Slc26a5-/- mice to sham operation or chronic pressure overload induced by transverse aortic constriction (TAC) surgery. Cardiac structure and function were assessed by echocardiography. We performed single cell Ca2+ transients and cell shortening recordings on isolated cardiomyocytes. Atomic force microscopy, a high-resolution technique, was also used to quantify the Young's modulus. After 8 weeks of TAC, the expression of prestin was markedly elevated in WT TAC mice relative to WT sham mice. To test whether the increased expression is maladaptive or cardioprotective, Slc26a5-/- mice underwent the same surgeries. Slc26a5-/- sham mice had depressed cardiac function relative to WT sham mice. In contrast and unexpectedly, 4 weeks after TAC, Slc26a5-/- mice exhibited blunted cardiac structural remodeling and more robust cardiac function as compared to WT TAC mice. Moreover, we found that cardiomyocytes from Slc26a5-/- mice showed more normalized Ca2+ transients and cellular shortening relative to cardiomyocytes from WT TAC mice. Our findings demonstrate that knockout of Slc26a5 is cardioprotective in a murine model of pressure overload, highlighting its crucial role in both physiological and pathological conditions. Overexpression of prestin in cardiac hypertrophy may significantly alter the mechanical properties of cardiomyocytes, leading to worsening in hypertrophy-induced cardiac dysfunction. Our study suggests that targeting prestin may represent a viable therapeutic avenue for cardiac hypertrophy.

PS-B02-1: UPREGULATION OF KEY GENES ELN AND TGFB3 WERE ASSOCIATED WITH THE SEVERITY OF CARDIAC HYPERTROPHY

Background: Hypertension induced cardiac hypertrophy is one of the most common pre-diseases of heart failure. This study aimed to identify the key pathogenic genes in the process of disease. Methods: GSE18224 was re-analyzed and obtained the differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were carried out. Networks of transcription factor (TF)-mRNA, microRNA (miRNA)-mRNA and Protein-Protein interaction (PPI) were constructed, and key module was further screened out from PPI network. GSE36074 dataset and our constructed transverse aortic constriction (TAC) mouse model were both used for validating expression of genes in the module. Finally, the correlation between the genes and biomarkers of cardiac hypertrophy were evaluated. Results: Totally, there were 348 DEGs in GSE18224, which were mainly enriched in biological process including collagen fibril organization, cellular response to transforming growth factor beta stimulus and were involved in ECM-receptor interaction and Oxytocin signaling pathway, etc. There were 387 miRNAs targeted to 257 DEGs, while 177 TFs targeted to 71 DEGs. The PPI network contains 222 nodes and 770 edges, and 18 genes were screened out in the module. After validation, eight genes, which were also significantly upregulated in GSE36074, were selected out from the 18 DEGs. Two of the eight DEGs including Eln and Tgfb3 were also significantly upregulated in our mouse model of myocardial hypertrophy. Finally, the expression of Eln and Tgfb3 were both positively correlated with level of the disease biomarkers. Conclusion: Upregulated key genes Eln and Tgfb3 were positively correlated with the severity of cardiac hypertrophy, which will provide potential therapeutic targets for the disease.

PS-B10-10: MRI CEREBROVASCULAR CHARACTERIZATION IN A MOUSE MODEL OF HYPERTENSION-INDUCED BRAIN INJURY

Hypertension has been recognized as the main modifiable risk factor for vascular cognitive impairment (VCI), dementias and neurodegenerative pathologies as Alzheimer Disease (AD). As we previously demonstrated, Transverse aortic constriction (TAC) is a relevant mouse model of cerebral injury and cognitive impairment induced by vascular factors. In this work we characterize the effect of TAC procedure on cerebral injury leveraging Magnetic Resonance Imaging (MRI), and investigate the relationship between the alterations found by MRI and histological, cognitive and cardiac parameters of injury, to establish a framework of translational imaging biomarkers to be applied in human. We subjected C57Bl/6J to TAC procedure, inducing severe cerebral hypertension. Thereafter, the brain injury was characterized by macro and microstructural MRI; cerebral blood flow (CBF) was analyzed by perfusion MRI. All neuroimaging scans were performed on a small-animal dedicated 7 Tesla MRI. The cognitive phenotype was characterized by Morris Water Maze (MWM) and Novel Object Recognition (NOR), while cardiac and carotid remodeling was assessed by ultrasound imaging. Finally, brains were analyzed for histological alterations in terms of microvascular density and integrity. Structural MRI showed white matter microstructural degradation in the left Fimbria of TAC mice, evidenced by a reduction of Fractional Anisotropy, Mean Diffusivity and Radial Diffusiity, coupled with bilateral hypothalamic enlargement (Figure A). Hemodynamic alterations resulted in reduced global, cortical and hypothalamic CBF (Figure B). The histological microvascular analysis evidenced capillary rarefaction, reduced capillary-pericyte coverage and Blood-Brain Barrier leakiness in the neocortex (Figure C). Finally, we evaluated whether cerebral MRI parameters of injury were correlated with cardiac, carotid and cognitive alterations: CBF was significantly correlated with mouse performance at Probe trial of MWM test and correlated to cardiac remodeling and diastolic dysfunction. Diastolic disfunction was correlated also to hypothalamic swelling, and cardiac remodeling was significantly correlated to microstructural alterations in the fimbria. Finally, CBF was correlated to bilateral carotid remodeling. In conclusion we have developed a mouse model of hypertension-induced brain damage that recapitulates typical traits of the human pathology and a pipeline of analysis that will ease allow to appreciate findings resulting from novel molecular targeting of the brain and translate them from bench to bedside. JOURNAL/jhype/04.03/00004872-202301001-01152/figure1/v/2023-10-24T163949Z/r/image-jpeg