Transverse Aortic Constriction Research Articles

Temporal dynamics of cardiac remodelling in pressure overload: the key role of visceral adipose tissue

Abstract Background Myocardial fibrosis is a prominent pathological feature of cardiac remodelling in pressure overload. The underlying mechanisms of cardiac remodelling are complex including mechanical, inflammatory and neurohormonal factors. We have recently uncovered a pathological crosstalk between visceral adipose tissue (VAT) and heart in biological aging characterized by profibrotic proteins in both the VAT secretome and the plasma contributing to myocardial interstitial fibrosis. We hypothesize that a model of pressure overload induced by transverse aortic constriction (TAC) is responsible for VAT remodelling and a VAT profibrotic secretome, which in turn worsens cardiac fibrosis and alters cardiac function. Purpose To explore the effect of pressure overload on VAT and identify a crosstalk between heart and VAT. Methods We divided wild-type male mice (5-month-old, n=10/group) into two groups: a visceral Lipectomy group and a Sham group two weeks before a moderate TAC (26G; i.e. gradient between left ventricle and aorta of 30mmHg) or a sham surgery. We sacrificed the mice at 1- and 8-week after TAC. We performed in vivo metabolic tests, echocardiography, and invasive hemodynamics, followed by ex vivo tissue analysis. We then analysed the time-course of the cardiac transcriptomic signature in TAC vs control and the impact of visceral lipectomy in both groups. Results Our results show that TAC induced not only cardiac remodelling affecting the structure and function of the heart, but also VAT fibrosis and inflammation together with activation of adrenergic receptors (Adrb3). VAT became a major source of profibrotic (TGFb and osteopontin) and proinflammatory (TNFa, IL6 and PAI1) factors as early as 1 week after TAC. Interestingly, lipectomy prevented cardiac remodelling and dysfunction. After 1 week of TAC, cardiac transcriptomic profile showed a similar upregulation of profibrotic and prohypertrophic genes in TAC groups in presence or absence of lipectomy. After 8 weeks of TAC, lipectomy prevented the upregulation of those genes and transcriptomic profile of TAC+lipectomy was similar to sham group (Figure). PCR analysis confirmed the lower expression of profibrotic (TGFb, Osteopontin), prohypertrophic (Nppa) and contractility genes (Adr1b) in TAC+Lipectomy vs TAC group after 8 weeks of TAC. Conclusion Our results suggest that VAT is detrimental to cardiac structure and function by releasing a profibrotic and a proinflammatory secretome that aggravates the cardiac remodelling induced by TAC. Conversely, lipectomy prevents the early development of TAC-induced cardiac fibrosis.

Effects of empagliflozin on cardio-renal interaction in heart failure with reduced ejection fraction: results from in-vivo experiments and the CINNAMON-study

Abstract Background Heart failure is often accompanied by renal dysfunction, which indicates a pathophysiological connection between the heart and kidneys. Empagliflozin, a SGLT2 inhibitor, showed beneficial effects on both cardiovascular and renal endpoints. Mechanistically, however, it is unclear whether the empagliflozin-dependent renal protection is mediated via the inhibition of renal SGLT2 or rather indirectly via improved cardiac function. Interestingly, in the EMPEROR trials, there was a significant interaction of empagliflozin-dependent kidney protection with systolic contractile dysfunction. We hypothesized that Empagliflozin treatment ameliorates heart failure in response to chronic pressure overload in mice leading to improved renal function. We correlated these findings with data from our prospective, single-armed trial. Methods Transverse aortic constriction (TAC) or sham surgery was performed in C57BL/6J (wildtype, WT) and SGLT2 deficient mice (SGLT2-/-). Animals received either Empagliflozin (10 mg/kg bodyweight) or vehicle by daily oral gavage. Cardiac function was evaluated by echocardiography and kidney function by transdermal FITC-Sinistrin measurement (GFR), urinary albumin/creatinine ratio (UACR) and Siriusred fibrosis staining. Results After 10 weeks, echocardiography confirmed TAC induced pressure-overload, leading to reduced left ventricular ejection fraction (LVEF) and diastolic dysfunction. Empagliflozin attenuated changes in LVEF (Fig. A) and improved diastolic dysfunction (data not shown). Interestingly, at 10 weeks, TAC reduced GFR, increased UACR and tended to increase renal fibrosis, which was attenuated by empagliflozin (Fig. B, C and D). To test if direct inhibition of SGLT2 in the heart and kidney is mechanistically involved, TAC surgery was repeated in SGLT2-deficient mice (SGLT2-/-). In fact, exposure to TAC resulted in comparable reduction of LVEF in SGLT2-/- mice and Empagliflozin prevented this deterioration similar to WT mice (Fig. E vs. A). Surprisingly, Empagliflozin also prevented GFR deterioration 10 weeks after TAC in mice lacking SGLT2 (SGLT2-/-) with comparable magnitude as in WT mice (Fig. F), suggesting that the reno-protective effect of Empagliflozin was independent from renal SGLT2 inhibition. The effect of empagliflozin on the heart and kidney was also investigated in patients with HF (prospective, single-arm CINNAMON-study, DRKS00031101). After 180 days of Empagliflozin treatment (10 mg), there was a significant improvement in LVEF, NT-proBNP levels and KCCQ-12 Scores (Fig. G-I) and the effects on UACR and GFR are subject of investigation. Conclusion This is the first study investigating the role of SGLT2 in Empagliflozin-dependent kidney protection of mice that develop heart failure after TAC. Importantly, empagliflozin treatment prevented deterioration of LVEF and GFR independent of the presence of SGLT2 in mice and improved cardiac markers and quality of life in patients.

The effect of afterload on right ventricular pacing-induced left ventricular dysfunction: a translational study

Abstract Introduction Right ventricular (RV) pacing (RVP) impairs left ventricular (LV) function. Nevertheless, the interaction between arterial afterload and RVP-associated LV dysfunction has not been investigated yet. Purpose We sought to examine the effect of afterload on RVP-evoked LV hemodynamic changes in patients with aortic stenosis (AS) undergoing transcatheter aortic valve implantation (TAVI) as well as in a small animal model of sustained LV pressure overload (PO). Methods Thirty-five patients with AS and a previously implanted pacemaker underwent detailed echocardiographic examination immediately before TAVI and also on the second postoperative day. The ultrasound measurements were carried out during intrinsic, narrow QRS rhythm and during frequency-matched RVP as well. Global myocardial work index (GMWI) was calculated to assess LV contractility. In parallel, LV pressure-volume analysis was performed in Wistar rats with transverse aortic constriction (TAC) or sham operation (Sham) during intrinsic narrow QRS rhythm (termed as TAC-Vsense and Sham-Vsense) as well as during RVP (termed as TAC-Vpace and Sham-Vpace). RVP was achieved in rats by an octopolar electrophysiology catheter placed in the RV. Results RVP decreased LV contractility in AS patients before and after TAVI as well. Nevertheless, the extent of RVP-evoked LV dysfunction was alleviated following pressure unloading (ΔGMWI: -37±7 vs. -25±8%, before TAVI vs. after TAVI, P<0.05). Similarly, although RVP was associated with reduced LV contractility in both the TAC (the slope of the maximum rate of LV pressure rise-end-diastolic volume relation [dPdtmax-EDV]: 92±8 vs. 53±6mmHg/s/µl, TAC-Vsense vs. TAC-Vpace; P<0.05) and Sham groups (dPdtmax-EDV: 74±5 vs. 60±9 mmHg/s/µl, Sham-Vsense vs. Sham-Vpace; P<0.05), the negative inotropic effect of RVP was found to be stronger in the TAC compared to the Sham group (ΔdPdtmax-EDV: -42±4 vs. -19±6%; TAC vs. Sham, P<0.01). Furthermore, reduction of the maximal rate of LV pressure decrease (ΔdPdtmin: -20±3 vs. -5±5%, TAC vs. Sham, P<0.01) and prolongation of the active relaxation time constant (Δτ: 22±3 vs. 6±3%, TAC vs. Sham, P<0.01) also occurred to a greater extent in case of sustained LV PO, indicating augmented diastolic dysfunction. Conclusion Our translational results indicate that RVP-induced LV dysfunction might be afterload dependent.

Intact DNA repair in differentiated cardiomyocytes is essential for maintaining cardiac function in response to pressure overload in mice

Abstract Introduction DNA in every cell is continuously damaged and DNA repair mechanisms are essential for protection against DNA damage-induced aging-related diseases. For example, deficient repair of endogenously generated DNA damage in mice with cardiomyocyte-restricted inactivation of Xpg, is associated with progressive heart failure. Purpose Here we tested the hypothesis that unrepaired DNA damage in differentiated cardiomyocytes increases cardiac vulnerability in response to hemodynamic overload. Methods To increase the burden of spontaneous DNA damage, we generated mice with cardiomyocyte-restricted inactivation of DNA repair endonuclease XPG. At 8 weeks of age, αMHC-Xpgc/- and control (Ctrl) mice were subjected to pressure overload by transverse aortic constriction (TAC). Eight weeks after TAC, left ventricular (LV) function was assessed using echocardiography and hemodynamic measurements, followed by molecular and histological analyses. Results Cardiomyocyte-restricted inactivation of Xpg resulted in systolic as well as diastolic LV dysfunction (Table). TAC-induced LV hypertrophy is similar in both groups (Ctrl 38%; αMHC-Xpgc/- 34%). In Ctrl mice, LV hypertrophy was accompanied by minimal LV dilation and modest changes in systolic and diastolic LV function. Conversely, TAC in αMHC-Xpgc/- produced severe LV dysfunction and resulted in overt congestive heart failure, demonstrated by aggravation of LV dilation, and marked increases in LV end-diastolic pressure, left atrial weight and lung fluid weight, which was accompanied by further increases in the expression levels of the hypertrophic marker genes atrial natriuretic peptide and beta-myosin heavy chain (Table). Moreover, lectin staining revealed a decrease in capillary density and TUNEL staining revealed further elevated levels of myocardial apoptosis. In addition, a significant increase of myocardial collagen content was observed. Conclusion Cardiomyocyte-restricted loss of DNA repair protein XPG increases cardiac vulnerability to develop heart failure in response to pressure overload. These findings underscore the importance of genomic stability for maintenance of cardiac function, not only during basal conditions, but also in response to a pathological stimulus.

ATM: a protein involved in glucose and lipid metabolism in the heart

Abstract Backround Post-mitotic cells, such as neurons and cardiomyocytes, cannot repair DNA lesions with DNA replication and rely for their survival on efficient sensors and effectors that orchestrate the DNA damage response (DDR). The Ataxia Telangiectasia Mutated (ATM) protein kinase is the most important sensor of oxidative stress and the DNA damage response (DDR) and is implicated in cellular metabolism. It is believed that while transient DNA damage and DDR can temporarily improve cardiovascular function, persistent activation of DDR (and ATM) might promote the onset and development of heart failure (HF). Purpose We hypothesised that ATM might control and regulate energy metabolism in the heart under stress to promote DNA repair. Methods We analyzed the effects of ATM inactivation on cardiomyocyte hypertrophy, cardiac function, DDR and metabolism in hearts from wild-type (Atm+/+) or Atm-mutated (Atm-/-) mice under sham conditions or after pressure overload by transverse aortic constriction (TAC). Results ATM inactivation in Atm-/- mice induced cardiomyocyte hypertrophy, fetal gene expression re-activation and a specific metabolomic signature in the heart, characterized by significant accumulation of pyruvate, branched chain amino-acids, short-medium acyl-carnitines and metabolites of tricarboxylic acid cycle. The levels of the glycolytic enzymes, hexokinase-2 (HK2) and phosphofructokinase (PFK), were elevated. pyruvate was trapped in the cytosol because mitochondrial carriers were suppressed and the enzymes that process pyruvate were dysregulated. Because of pyruvate metabolic block, fatty acids oxidation was inefficient and resulted in the accumulation of acyl-carnitines and insulin resistance. These metabolic changes were amplified by TAC, which rapidly induced heart failure in Atm-/- mice. ATM inactivation also increased basal and TAC-induced genomic stress in cardiomyocytes, as shown by the levels of p-γ-H2AX, 8-oxodG glycosylase (OGG1/2) and apurinic site nuclease (APE1). These results prove that ATM rewires the metabolism of cardiac cells by inducing glycolysis and fatty acids oxidation. ATM stimulates glycolysis to repair DNA lesions and protect the heart against stress-induced dysfunction. The metabolic block due to ATM inactivation accelerates heart failure. Conclusions Our data suggest that ATM favors the metabolic flexibility of the heart by stimulating glucose entry and balancing glucose catabolism with fatty acid oxidation, thus preventing mechanical stress-induced cardiac systolic dysfunction.

Integrating single-cell sequencing and genetic lineage tracing reveals endothelial plasticity during atrial remodeling

Abstract Background Atrial fibrillation (AF), as the most burdensome cardiac arrhythmia, could lead to the significant mortality and morbidity. Atrial fibrosis is a well-recognized pathophysiological factor of the development and progression of AF, which also hampers its treatment. However, the underlying mechanisms of this intricate process remain largely unknown. Purpose This study sought to reveal the cellular composition of AF substrate and investigate the vital contributors to atrial structural remodeling. Methods Left atrial appendages from three patients with AF undergoing surgical ablation and three individuals with sinus rhythm undergoing heart transplantation were subjected to 10X single-cell RNA sequencing (scRNA-seq). Another five public scRNA-seq or snRNA-seq datasets of potentially remodeled right or left appendages were retrieved across three publications. The vital contributors of atrial structural remodeling and their characteristics were identified by intercellular communication analysis and trajectory inference. Transverse aortic constriction (TAC) was used to induce atrial remodeling in Cdh5-CreERT2;RFP mice, followed by scRNA-seq of endothelial cells (ECs). Human primary atrial ECs and fibroblasts were isolated for cell-cell interaction experiments. ECs-specific mouse models of Transforming growth factor beta 1 (TGFB1) (knockout and overexpression) were generated. Electrophysiological and histology analyses were performed to determine the consequences of ECs-derived TGFB1 gain and loss of function in the atrium. Results The comprehensive analysis of different scRNA-seq datasets revealed that ECs played an important regulatory role in the homeostasis of atrial substrate and displayed remarkable mesenchymal activation during atrial remodeling, which was further confirmed by TAC mice. Immunofluorescence staining and high-content screening suggested that the complete transition from ECs to mesenchymal cells slightly contributed to atrial remodeling. However, TGFB1 secreted from mesenchymal activated ECs significantly promoted activation, proliferation, and collagen secretion of fibroblasts. We discovered that overexpression of TGFB1 in ECs of mice significantly increased atrial fibrosis, and thus prolonging AF duration. Besides, knockout of TGFB1 in ECs of mice underwent TAC significantly attenuated the atrial fibrosis and decreased the rate of AF. Conclusion Our work demonstrates that mesenchymal ECs-derived Tgfb1 plays an important role in atrial remodeling by regulating the fibroblast function. EC-specific interventions were suggested to facilitate the development of novel strategies for mitigating AF substrate remodeling.

Cardiac secreted-HSP90alpha in cardiomyocyte hypertrophy by N-Cadherin mediated signaling under pressure overload

Abstract Background Left ventricular hypertrophy (LVH) in response to pressure overload is strongly associated with adverse cardiovascular outcomes. Heat shock protein 90 (HSP90) has been reported to be involved in cardiac remodeling under stress. Aims Here, we evaluate whether serum HSP90α (an isoform of HSP90) increases and associates with cardiac hypertrophy in patients with hypertension or severe aortic stenosis (AS), and explore the potential mechanisms in experimental pressure overload mouse model. Methods Serum HSP90α levels in patients with hypertension or aortic stenosis (AS) were determined by ELISA. Pressure overload mouse model was built by a transverse aortic constriction (TAC). Cardiac function was assessed by echocardiography. Cardiac hypertrophy was examined by histology, western blot and RNA analysis. Results Serum levels of HSP90α were higher in patients with hypertension or AS than in the control group, respectively, and the levels positively correlated with LVH. HSP90α levels also increased in heart tissues of patients with obstructive hypertrophic cardiomyopathy (HCM), and mice after TAC, in parallel with the hypertrophic markers. TAC induced the enhanced expression and secretion of HSP90α from cardiomyocytes and cardiac fibroblasts. Knockdown of HSP90α or blockade of extracellular HSP90α (eHSP90α) prevent the development of LVH under pressure overload in vivo and in vitro. By Nano-HPLC-MS/MS analysis, we identified eHSP90α interacted with N-cadherin in cardiomyocyte surface, which induced the activation of β-catenin and promoted β-catenin to bind TCF7 (a member of TCF/Lef family) in nucleus, enhanced the transcription of hypertrophic genes, contributing to cardiac hypertrophy and dysfunction under pressure overload. Conclusions Our data demonstrated that cardiac secreted-HSP90α promoted myocardial hypertrophy by N-Cadherin mediated β-catenin/TCF7 signaling under pressure overload. It indicates the therapeutic potential of targeting HSP90α-initiated signaling against cardiac hypertrophy and dysfunction under pressure overload.

Vaccination targeting senescence-associated transmembrane protein alleviated cardiovascular pathology in the mice

Abstract Introduction Accumulation of senescent cells is observed in various organs including vasculature, heart and white adipose tissue with age, and known to become a substrate of pathophysiology of various cardiovascular-metabolic diseases. We have previously developed a vaccine to eliminate senescent cells, so-called "senolytic vaccine" by targeting Glycoprotein Nonmetastatic Melanoma Protein B (GPNMB) as a cellular-senescence-associated cell surface protein. We have demonstrated that GPNMB senolytic vaccine could remove senescent cells and alleviate the pathology cardiovascular-metabolic diseases. Senescence-associated adhesion molecule 1 (SAAM-1) is also known as a cell-surface membrane to increase its expression during cellular senescence in endothelial cells as well as the development of cardiovascular diseases. Here, we identified SAAM-1 as another senescence antigen and created a vaccine to eliminate senescent cells by targeting SAAM-1. Purpose This study was purposed to evaluate the efficacy of SAAM-1 vaccine for the treatment of cardiovascular diseases in mice models. Methods As a pressure overload-induced heart failure (HF) model, C57BL/6N wild type (WT) mice were underwent transverse aortic constriction (TAC) or sham opretaion at 13 weeks of age. SAAM-1 or control vaccination were given at 8 and 10 weeks of age before operation. Echocardiography examination was conducted two weeks after TAC operation, followed by tissue sampling 3 weeks after the examination. As an atherosclerosis model, ApoE KO mice were subjected to a high-fat diet (HFD) starting at 4 weeks of age. Vaccination of ApoE KO mice was administered at 8 weeks of age with SAAM-1-vaccine or control-vaccine, followed by sacrifice 4 weeks later for further analysis. ApoE-KO mice were also crossed with p16-mediated Luciferase expressing mice (p16-Luc mice) and subjected to generation of atherosclerosis model. Results SAAM-1 expression was significantly increased in left ventricle (LV) in TAC mice but its increase was suppressed by SAAM-1 vaccination. Interestingly, both worsening systolic function and dilatation of LV by pressure overload were significantly alleviated by SAAM-1 vaccination. Furthermore, expression level of senescent markers including p16 and p21, as well as several inflammation markers were increased in failing heart along with development of cardiac fibrosis, but SAAM-1 vaccine could significantly attenuate this pathological change. In ApoE KO mice, SAAM-1 vaccine decreased expression level of several inflammation markers in the mice, along with a decreasing p16 signal in the aorta. Conclusions Targeting SAAM-1 for vaccination would be a novel and promising approach to alleviating age-related cardiovascular disease.

SNORD3A: a new possible circulating biomarker of human heart failure

Abstract Background Heart failure (HF) is a complex clinical syndrome and the leading cause of mortality, morbidity and hospitalization in industrialized countries. Human cardiac biopsies are invaluable resources for identifying cardiac signaling pathways, however blood fractions and peripheral blood mononuclear cells (PBMCs) could be used as a source of surrogate biomarkers of signaling pathway activation in human heart failure. Purpose In the present study we aimed to identify novel circulating biomarkers mirroring human myocardial signaling in HF and to study the role played by SNORD3A in cardiomyocyte survival and death. Methods Cardiac left ventricle samples and PBLs from 8 HF patients undergoing heart transplantation and 2 control patients (CTRL) were analyzed by RNA sequencing (RNASeq) to identify transcripts that were regulated in both hearts and PBLs. Five identified transcripts, KCNQOT1, MIAT, SCRN1, MALAT1 and SNORD3A, were then validated by quantitative real-time PCR in PBMCs and in LVs. Next, we analyzed the expression of the Small Nucleolar RNA (snoRNA) SNORD3A in LVs and PBMCs from 8-week-old wild type C57BL/6 mice with pressure overload-induced HF by transverse aortic constriction (TAC). Sham-operated mice (sham) were used as controls. To further investigate the role of SNORD3A in cardiomyocyte function and survival, SNORD3A antisense oligonucleotides (SNOaso) and scramble control sequences (GFPaso) were synthesized and tested in cardiomyoblasts H9C2 cells under normoxic or hypoxic conditions to evaluate SNORD3A transcription levels, cell death and protein synthesis. Results SNORD3A expression was significantly increased in both LVs and PBMCs from HF patients compared to control subjects. Consistent with these results, SNORD3A levels were increased both in PBMCs and LVs from TAC mice compared to sham. In vitro hypoxia significantly increased SNORD3A expression levels in H9C2 cells, compared to normoxia. After SNOaso transfection, SNORD3A transcripts were downregulated, and they were further reduced following 3-hour-hypoxia. Depletion of SNORD3A levels increased cardiomyocyte death and reduced protein synthesis in H9C2 cells. Conclusions Our data suggest that SNORD3A might be involved in the regulation of cardiomyocyte survival in HF and could be useful for diagnostic and prognostic applications in HF.

Stress memory of heart failure in hematopoietic niche promotes recurrence via adipocytic skewing of mesenchymal stromal cells

Abstract Background Heart failure (HF) is marked by recurrent acute decompensations, which poses significant treatment challenges in advanced stages. Our preceding work demonstrated that hematopoietic stem cells (HSCs) from HF-experienced mice induce cardiac fibrosis and dysfunction when transplanted, as a result of epigenetic alterations favoring myeloid hematopoiesis and impeding the differentiation of monocytes into cardiac mature macrophages. These findings hint at epigenetic modifications in HSCs as potential drivers of HF recurrence, suggesting their manipulation as a novel therapeutic strategy. However, the exact mechanisms by which HF-associated stress leads to these epigenetic changes remain unclear. Given the crucial role of bone marrow mesenchymal stromal cells (MSCs) in maintaining HSC stemness, this study explores the impact of cardiac stress on MSC function and its subsequent effect on HSCs. Purpose This study aims to elucidate the mechanisms behind the phenotypic changes in HSCs and to identify novel therapeutic targets to mitigate HF recurrence by investigating the role of MSCs in the hematopoietic niche under cardiac stress. Methods & Results Initially, we examined MSC phenotypic changes during HF using bulk and single-cell RNA sequencing. Results from transverse aortic constriction (TAC) model mice revealed a shift towards adipocyte differentiation under cardiac stress. Histological analyses confirmed an increase in adipocytes in TAC mice compared to controls, suggesting a stress-induced predisposition of MSCs towards adipocytic lineage commitment. This trend was further supported by ex vivo models using MSCs isolated from TAC mice. Notably, the proportion of adipocyte lineage-specific MSCs correlated with cardiac dysfunction severity. Subsequently, we assessed the impact of HF-conditioned MSCs on HF development by transplanting healthy HSCs with MSCs from control or TAC mice. Mice receiving HF-conditioned MSCs developed HF, evidenced by reduced ejection fraction and cardiac fibrosis. In these mice, a notable increase in HSC proliferation and a relative increase of myeloid cells within the peripheral blood were observed, along with an increase in proinflammatory cardiac macrophages. These findings suggest HF induces a "stress memory" in bone marrow MSCs. To test the potential of inhibiting adipocyte differentiation of MSCs in erasing this stress memory, we administered MSC phenotype-modulating agents to TAC mice, resulting in improved cardiac function and the disappearance of adipocytes from the bone marrow. Conclusions This study sheds light on how cardiac stress affects the bone marrow niche, leading to adipocytic skewing of MSCs and subsequent alterations in HSCs, thereby exacerbating cardiac dysfunction. The potential of targeting bone marrow niche components in HF treatment is highlighted.

Deletion of mitochondrial DNA methyltransferase 1 contributes the development of murine heart failure under pressure overloaded

Abstract Background Mitochondrial DNA (mitDNA) has similarities to bacterial DNA, which contains inflammatogenic unmethylated CpG motifs. We found that mitDNA that escapes from autophagy causes inflammatory response and heart failure under pressure overload. DNA methyltransferase 1 (DNMT1) maintains methylation patterns in somatic cells. Deletion of DNMT1 results in embryonic lethality in mice and mitotic catastrophe in cultured cells. Additional possible translational start sites (ATG1, ATG2) are located upstream of the commonly accepted nuclear DNMT1 translational start site (ATG3) and there is a conserved mitochondrial translocation signal sequence between ATG1 and ATG3. The role of mitochondrial DNMT1 in the development of heart failure remains to be elucidated. Purpose The purpose of this study is to examine the in vivo role of mitochondrial DNMT1 in the heart under pressure overload. Methods We generated HA-tagged DNMT1 constructs, 1) starting from ATG1 (termed as whole) 2) from ATG3 (nuclear) and 3) from ATG3 and mutated in the nuclear translocation sequence (mitochondrial) and transfected HEK293 cells with the constructs. Localization of HA-tagged proteins was assessed by immunocytochemistry. We also generated mitochondrial DNMT1-deficient mice by inserting mutations into the ATG1 and ATG2. The mice were subjected to pressure overload by means of transverse aortic constriction (TAC) to induce heart failure. Cardiac remodelling was assessed by echocardiography as well as histological analyses 4 weeks after operation. Results Whole, nuclear and mitochondrial DMNT1 translocated to both mitochondria and nuclei, only nucleus and only mitochondria, respectively. Mitochondrial DNMT1-deficient mice were born at Mendelian frequency and grew to adulthood. Those showed no cardiac phenotypes under baseline conditions. However, the mice exhibited severe systolic dysfunction, indicated by left ventricular fractional shortening, 2 weeks after surgery compared with control littermates (control littermates 47.9% versus Mitochondrial DNMT1-deficient mice 32.3%, p<0.05). Intermuscular cell infiltration was observed in TAC-operated mitochondrial DNMT1-deficient hearts. Conclusions Mitochondrial DNA methyltransferase 1 plays a protective role in failing hearts and mitDNA methylation might be a therapeutic target to treat patients with heart failure.

HECT domain containing-3 (HECTD3) E3 ubiquitin ligase attenuates cardiac ischemia and hypertrophy in mice

Abstract HECT domain containing 3 (HECTD3) is one of the E3 ubiquitin ligases, the cardiac function of which is not completely understood yet. We aimed here to understand the role of HECTD3 in cardiomyocytes using loss- and gain-of-function approaches in in vitro and in vivo models. Interestingly, we found that HECTD3 was significantly downregulated in cardiac tissue obtained from human patients suffering from cardiac hypertrophy and ischemia. We thus employed loss- and gain-of-function approaches in neonatal rat cardiomyocytes (NRVCMs) to get further insights into potential cardiac function of HECTD3. Overexpression of HECTD3 in NRVCMs attenuated cardiomyocyte hypertrophy and lipopolysaccharide (LPS) or interferon-γ (IFN- γ) induced cellular inflammation; its knockdown on the other hand aggravated these effects. Mechanistically, using Yeast two-hybrid screen, mass-spectrometry, and transcriptomics analyses, we identified Small Ubiquitin like Modifier 2 (SUMO2) Signal Transducer and Activator of Transcription-1 (STAT1) as novel substrates for HECTD3, regulation of which resulted into attenuation of hypertrophy and inflammation, respectively. To translate these in vitro results in vivo, we employed Adeno-associated virus-9 (AAV-9) mediated gene therapy approach to overexpress HECTD3 specifically in the heart of mouse models of cardiac ischemia due to permanent ligation of the left anterior descending artery (LAD), and cardiac hypertrophy due to transverse aortic constriction (TAC). Overexpression of HECTD3 in mice, though moderate but significantly improved cardiac function upon ischemic or hypertrophic stress compared to respective control groups. Importantly, in vitro findings were consistent in vivo, where AAV-9 mediated overexpression of HECTD3 substantially reduced cardiac hypertrophy and fibrosis, improved cardiac function as assessed by echocardiography, inhibited activation of STAT1-mediated downstream signaling, and attenuated infiltration of inflammatory cells to the heart after TAC or LAD ligation. This data indicates that HECTD3 is a novel modulator of cardiac ischemia, hypertrophy, and inflammation via regulation of SUMO2 and STAT1-signaling. In conclusion, we report here a novel cardioprotective mechanism involving the E3 ubiquitin ligase HECTD3, which exerts anti-hypertrophic and anti-inflammatory effects via dual regulation of SUMO2 and its SUMOylating target STAT1. We believe that this "dual pathway" inhibition exhibits translational importance and could further be exploited for the prevention and/or therapy of heart failure due to cardiac ischemia or hypertrophy.

Deletion of nephrocystin 4 exacerbates cardiac function and survival rate in a mouse model of cardio-renal syndrome

Abstract Background Cardiorenal syndrome (CRS), characterized by cardiac and renal dysfunction, is an increasing health problem associated with high mortality rate. Nephrocystin 4 (NPHP4) gene is the major cause of end-stage renal disease in children. We have reported NPHP4 single nucleotide polymorphism is the genetic risk for CRS and subsequent cardiovascular events in general population. However, the functional role of Nphp4 in the development of CRS has never been examined yet. Methods and Results We generated systemic Nphp4 knockout (KO) mice deleting exon 3 to 8 using CRISPR-Cas9 system. RNA sequence analysis of heart and kidney tissues demonstrated that gene-sets related to mitochondrial function were significantly downregulated in Nphp4 KO mice compared to their wild-type (WT) littermates. Nphp4 KO mice showed less cardiac hypertrophy, however, exacerbated cardiac function and survival compared to WT mice in a CRS model induced by thoracic transverse aortic constriction. Nphp4 located in endosome and cell membrane in H9C2 cardiomyocytes. Immunoprecipitation confirmed protein interaction between Nphp4 and HECT-type E3 ligase Itch. Furthermore, we found that Itch targeted Lats2 and insulin-like growth factor-1 receptor in cardiomyocytes. Knockdown of Nphp4 activated Hippo signaling and worsened mitochondrial function in H9C2 cardiomyocytes through the inhibition of Itch-dependent Lats2 degradation. Knockdown of Nphp4 inhibited insulin-like growth factor-1 signaling through the disturbance of Itch-mediated endosome function. Conclusions Deletion of Nphp4 impaired compensatory cardiac hypertrophy and exacerbated cardiac function and survival in a mouse CRS model through mitochondrial dysfunction. Nphp4 mediated Hippo signaling and insulin-like growth factor-1 signaling through the interaction with Itch. NPHP4 could be the novel therapeutic target in CRS.figure1figure2

Inhibiting chondroitin sulfate synthase 1 ameliorates hyperplastic arterial remodelling

Abstract Introduction Hyperplastic arterial remodelling is characterized by intima-media thickening, lumen narrowing, inflammation and increased extracellular matrix (ECM) deposition. Chondroitin sulfate (CS) glycosaminoglycans, the unbranched polysaccharide chains of proteoglycans, are important components of ECM. We previously found CS accumulated in adversely remodelled heart and artery, aggravating cardiac deterioration. Objective We aim to explore a therapeutic strategy to interfere with CS synthesis and investigate its potential in ameliorating hyperplastic arterial remodelling. Methods Through single-cell RNA sequencing of non-cardiomyocytes cardiac cells from mouse myocardial infarction model, chondroitin sulfate synthase 1 (CHSY1) was identified as the primary CS synthase and was increased during disease progression. A Chsy1 knockout (KO) mouse strain was generated. Fibrosis and arterial remodelling were induced using: (i) 4 weeks of angiotensin II/phenylephrine (AngII/PE) infusion through subcutaneous osmotic minipumps; and (ii) transverse aortic constriction surgery. To assess the therapeutic potential of CHSY1 inhibition in ameliorating hyperplastic arterial remodelling, Chsy1 was knockdown by dsiRNA post disease onset. Mechanism of action was investigated using primary vascular smooth muscle cells (VSMCs). Results Chsy1 KO reduced CS and its stereoisomer dermatan sulfate in the heart to 42 ± 22% and 36 ± 7% respectively, whereas other types of glycosaminoglycans remain intact. KO mice were protected against phenotypes of hyperplastic arterial remodelling induced by the two disease models in both the coronary and carotid arteries. For example, 90.1% of coronary arterial fibrosis and 79.7% of macrophage infiltration induced by AngII/PE was declined in KO mice compared to WT mice, while cardiac function as stroke volume was augmented by 41.3%. Concomitantly, carotid arterial wall thickening was lower by 36.5% and distensibility was higher by 36.7%, validating Chsy1 as a potent target. As a therapeutic approach, weekly intravenous injection of dsiRNA achieved Chsy1 reduced to 30.2 ± 5.5% in the carotid artery and 72.5 ± 4.7% in the heart. Post disease onset, Chsy1 KD therapy reduced fibrosis by 59.9% in the coronary artery (P = 0.006) and improved stroke volume by 25.6% (P = 0.036). The structural integrity and function of carotid arteries were well-reserved, manifesting as improved distensibility (29%, P = 0.016), increased elastin/perimeter ratio (15%, P = 0.002), and reduced macrophage infiltration (38%, P = 0.029). In vitro, CHSY1 inhibition suppressed migration and cytokine production of macrophages, fibroblast-to-myofibroblast transition, and VSMC hyperproliferation. NOTCH3 signalling was found to be positively correlated with CHSY1 modulation. Conclusions We demonstrated that inhibiting CHSY1 is necessary and sufficient to block CS synthesis. Chsy1 KD by dsiRNA is an effective therapy to ameliorate hyperplastic arterial remodelling.

ALDH2 mediates the effects of sodium-glucose cotransporter 2 inhibitors (SGLT2i) on improving cardiac remodeling

BackgroundSodium-glucose cotransporter-2 inhibitors (SGLT2i) are now recommended for patients with heart failure, but the mechanisms that underlie the protective role of SGLT2i in cardiac remodeling remain unclear. Aldehyde dehydrogenase 2 (ALDH2) effectively prevents cardiac remodeling. Here, the key role of ALDH2 in the efficacy of SGLT2i on cardiac remodeling was studied.MethodsAnalysis of multiple transcriptomic datasets and two-sample Mendelian randomization were performed to find out the differentially expressed genes between pathological cardiac hypertrophy models (patients) and controls. A pathological cardiac hypertrophy mouse model was established via transverse aortic constriction (TAC) or isoproterenol (ISO). Cardiomyocyte-specific ALDH2 knockout mice (ALDH2CMKO) and littermate control mice (ALDH2flox/flox) were generated to determine the critical role of ALDH2 in the preventive effects of dapagliflozin (DAPA) on cardiac remodeling. RNA sequencing, gene knockdown or overexpression, bisulfite sequencing PCR, and luciferase reporter assays were performed to explore the underlying molecular mechanisms involved.ResultsOnly ALDH2 was differentially expressed when the differentially expressed genes obtained via Mendelian analysis and the differentially expressed genes obtained from the multiple transcriptome datasets were combined. Mendelian analysis revealed that ALDH2 was negatively related to the severity of myocardial hypertrophy in patients. DAPA alleviated cardiac remodeling in mouse hearts subjected to TAC or ISO. ALDH2 expression was reduced, whereas ALDH2 expression was restored by DAPA in hypertrophic hearts. Cardiomyocyte specific ALDH2 knockout abolished the protective role of DAPA in preventing cardiac remodeling. ALDH2 expression and activity were increased in DAPA-treated neonatal rat primary cardiomyocytes (NRCMs), H9C2 cells and AC16 cells. Moreover, DAPA upregulated ALDH2 in peripheral blood mononuclear cells (PBMCs) from patients with type 2 diabetes. Sodium/proton exchanger 1 (NHE1) inhibition contributed to the regulation of ALDH2 by DAPA. DAPA suppressed the production of reactive oxygen species (ROS), downregulated DNA methyltransferase 1 (DNMT1) and subsequently reduced the ALDH2 promoter methylation level. Further studies revealed that DAPA enhanced the binding of nuclear transcription factor Y, subunit A (NFYA) to the promoter region of ALDH2, which was due to the decreased promoter methylation level of ALDH2.ConclusionsThe upregulation of ALDH2 plays a critical role in the protection of DAPA against cardiac remodeling. DAPA enhances the binding of NFYA to the ALDH2 promoter by reducing the ALDH2 promoter methylation level through NHE1/ROS/DNMT1 pathway.Graphical abstract

Metabolic pathways for removing reactive aldehydes are diminished in the skeletal muscle during heart failure.

Muscle wasting is a serious complication in heart failure patients. Oxidative stress and inflammation are implicated in the pathogenesis of muscle wasting. Oxidative stress leads to the formation of toxic lipid peroxidation products, such as 4-hydroxy-2-nonenal (HNE), which covalently bind with proteins and DNA and activate atrophic pathways. Whether the formation of lipid peroxidation products and metabolic pathways that remove these toxic products are affected during heart failure-associated skeletal muscle wasting has never been studied. Male C57BL/6J mice were subjected to sham and transverse aortic constriction (TAC) surgeries for 4, 8 or 14weeks. Different skeletal muscle beds were weighed, and the total cross-sectional area of the gastrocnemius muscle was measured via immunohistochemistry. Muscle function and muscle stiffness were measured by a grip strength meter and atomic force microscope, respectively. Atrophic and inflammatory marker levels were measured via qRT‒PCR. The levels of acrolein and HNE-protein adducts, aldehyde-removing enzymes, the histidyl dipeptide-synthesizing enzyme carnosine synthase (CARNS), and amino acid transporters in the gastrocnemius muscle were measured via Western blotting and qRT‒PCR. Histidyl dipeptides and histidyl dipeptide aldehyde conjugates in theGastrocnemius and soleus muscles were analyzed by LC/MS-MS. Body weight, gastrocnemius muscle and soleus muscle weights and the total cross-sectional area of the gastrocnemius musclewere decreased after 14weeks of TAC. Heart weight, cardiac function, grip strength and muscle stiffness were decreased in the TAC-operated mice. Expression of the atrophic and inflammatory markers Atrogin1 and TNF-α, respectively, was increased ~ 1.5-2fold in the gastrocnemius muscle after 14weeks of TAC (p < 0.05 and p = 0.004 vs sham). The formation of HNE and acrolein protein adducts was increased, and the expression of the aldehyde-removing enzyme aldehyde dehydrogenase (ALDH2) was decreased in the gastrocnemius muscle of TAC mice. Carnosine (sham: 5.76 ± 1.3 vs TAC: 4.72 ± 0.7nmol/mg tissue, p = 0.04) and total histidyl dipeptide levels (carnosine and anserine; sham: 11.97 ± 1.5 vs TAC: 10.13 ± 1.4nmol/mg tissue, p < 0.05) were decreased in the gastrocnemius muscle of TAC mice. Depletion of histidyl dipeptides diminished the aldehyde removal capacity of the atrophic gastrocnemius muscle. Furthermore, CARNS and TAUT protein expression were decreased in the atrophic gastrocnemius muscle. Our data reveals that reduced expression of ALDH2 and depletion of histidyl dipeptides in the gastrocnemius muscle during heart failure leads to the accumulation of toxic aldehydes and might contribute to muscle wasting.

PFKP inhibition protects against pathological cardiac hypertrophy by regulating protein synthesis

Metabolic reprogramming precedes most alterations during pathological cardiac hypertrophy and heart failure (HF). Recent studies have revealed that Phosphofructokinase, platelet (PFKP) has a wealth of metabolic and non-metabolic functions. In this study, we explored the role of PFKP in cardiac hypertrophic growth and HF. The expression level of PFKP was elevated both in pathological cardiac remodeling mouse model challenged by transverse aortic constriction (TAC) surgery and in the neonatal rat cardiomyocytes (NRCMs) stimulated by phenylephrine (PE). In global PFKP knockout (PFKP-KO) mice, cardiac hypertrophy was ameliorated under TAC surgery, while overexpression of PFKP by intravenous injection of adeno-associated virus 9 (AAV9) under the cardiac troponin T (cTnT) promoter worsened myocardial hypertrophy and fibrosis. In NRCMs, small interfering RNA (SiRNA) knockdown or adenovirus (Adv) overexpression of PFKP was employed and the intervention of PFKP showed a similar phenotype. Mechanistically, immunoprecipitation combined with liquid chromatography-tandem mass spectrometry (IP-MS/MS) analysis was used to identify the interacting proteins of PFKP. Eukaryotic translation initiation factor 2 subunit beta (EIF2S2) was identified as the downstream target of PFKP. In the PE-stimulated NRCM hypertrophy model and mouse TAC model, knocking down EIF2S2 after PFKP overexpression reduced the synthesis of new proteins and alleviated the hypertrophy phenotype. Our findings illuminate that PFKP participates in pathological cardiac hypertrophy partly by regulating protein synthesis through EIF2S2, which provides a new clue for the involvement of metabolic intermediates in signal transduction.

Long noncoding RNA AK144717 exacerbates pathological cardiac hypertrophy through modulating the cellular distribution of HMGB1 and subsequent DNA damage response

DNA damage induced by oxidative stress during cardiac hypertrophy activates the ataxia telangiectasia mutated (ATM)-mediated DNA damage response (DDR) signaling, in turn aggravating the pathological cardiomyocyte growth. This study aims to identify the functional associations of long noncoding RNA (lncRNAs) with cardiac hypertrophy and DDR. The altered ventricular lncRNAs in the mice between sham and transverse aortic constriction (TAC) group were identified by microarray analysis, and a novel lncRNA AK144717 was found to gradually upregulate during the development of pathological cardiac hypertrophy induced by TAC surgery or angiotensin II (Ang II) stimulation. Silencing AK144717 had a similar anti-hypertrophic effect to that of ATM inhibitor KU55933 and also suppressed the activated ATM-DDR signaling induced by hypertrophic stimuli. The involvement of AK144717 in DDR and cardiac hypertrophy was closely related to its interaction with HMGB1, as silencing HMGB1 abolished the effects of AK144717 knockdown. The binding of AK144717 to HMGB1 prevented the interaction between HMGB1 and SIRT1, contributing to the increased acetylation and then cytosolic translocation of HMGB1. Overall, our study highlights the role of AK144717 in the hypertrophic response by interacting with HMGB1 and regulating DDR, hinting that AK144717 is a promising therapeutic target for pathological cardiac growth.

Cardiac tumour necrosis factor receptor-associated factor 7 mediates the ubiquitination of apoptosis signal-regulating kinase 1 and aggravates cardiac hypertrophy.

Cardiac remodelling is a common pathophysiological process in the development of various cardiovascular diseases, but there is still a lack of effective interventions. Tumour necrosis receptor-associated factor 7 (TRAF7) belongs to the tumour necrosis factor receptor-associated factor family and plays an important role in biological processes. Previous studies have shown that TRAF7 mutations lead to congenital defects and malformations of the heart. However, the molecular mechanisms of TRAF7 in the underlying pathogenesis of pathological cardiac hypertrophy remain unknown. We aim to study the molecular mechanisms and effects of TRAF7 in cardiac remodelling and whether it has the potential to become a therapeutic target for cardiac remodelling. The pressure overload-induced cardiac hypertrophy model in mice was established via transverse aortic constriction (TAC) surgery, and cardiomyocytes were treated with phenylephrine (PE) to induce hypertrophic phenotype. Levels of cardiac dysfunction and remodelling were measured with echocardiography and tissue or cell staining. RNA sequencing, western blot, qRT-PCR, co-immunoprecipitation, and in vivo ubiquitination assays were used to explore the molecular mechanisms. The results showed that the expression of TRAF7 increased gradually during the development of hypertrophy. Accordingly, TRAF7 significantly exacerbated the PE-induced enlargement of primary neonatal Sprague-Dawley rat cardiomyocytes, whereas TRAF7 knockdown alleviated the hypertrophic phenotype in primary cardiomyocytes. Cardiac-specific overexpression of TRAF7 accelerated hypertrophic phenotype in mice and cardiac-specific Traf7 conditional knockout mice improved hypertrophic phenotype induced by TAC. Mechanistically, TRAF7 directly interacted with apoptosis signal-regulating kinase-1 (ASK1) and promoted ASK1 phosphorylation by mediating the K63-linked ubiquitination of ASK1 in response to PE stimulation, which then promoted ASK1 activation and downstream signalling during cardiac hypertrophy. Notably, the pro-hypertrophic effect of TRAF7 was largely blocked by GS4997 in vitro and cardiac-specific Ask1 conditional knockout in vivo. In summary, we identified TRAF7 as an essential regulator during cardiac hypertrophy, and modulation of the regulatory axis between TRAF7 and ASK1 could be a novel therapeutic strategy to prevent this pathological process.

Von Willebrand factor exacerbates heart failure through formation of neutrophil extracellular traps.

Heart failure (HF) is a leading cause of mortality worldwide and characterized by significant co-morbidities and dismal prognosis. Neutrophil extracellular traps (NETs) aggravate inflammation in various cardiovascular diseases; however, their function and mechanism of action in HF pathogenesis remain underexplored. This study aimed to investigate the involvement of a novel VWF-SLC44A2-NET axis in HF progression. NET levels were examined in patients with HF and mouse models of transverse aortic constriction (TAC) HF. PAD4 knockout mice and NET inhibitors (GSK-484, DNase I, NEi) were used to evaluate the role of NETs in HF. RNA sequencing was used to investigate the downstream mechanisms. Recombinant human ADAMTS13 (rhADAMTS13), ADAMTS13, and SLC44A2 knockouts were used to identify novel upstream factors of NETs. Elevated NET levels were observed in patients with HF and TAC mouse models of HF. PAD4 knockout and NET inhibitors improved the cardiac function. Mechanistically, NETs induced mitochondrial dysfunction in cardiomyocytes, inhibiting mitochondrial biogenesis via the NE-TLR4-mediated suppression of PGC-1α. Furthermore, VWF/ADAMTS13 regulated NET formation via SLC44A2. Additionally, sacubitril/valsartan amplifies the cardioprotective effects of the VWF-SLC44A2-NET axis blockade. This study established the role of a novel VWF-SLC44A2-NET axis in regulating mitochondrial homeostasis and function, leading to cardiac apoptosis and contributing to HF pathogenesis. Targeting this axis may offer a potential therapeutic approach for HF treatment.