Transverse Aortic Constriction Surgery Research Articles

MYSM1 Mediates Cardiac Parthanatos and Hypertrophy by Deubiquitinating PARP1.

Cardiac hypertrophy constitutes the primary pathological basis for heart failure and exerts a considerable influence on morbidity and mortality. Deubiquitinating enzymes are crucial regulators of protein degradation and play a pivotal role in cardiac pathophysiology. This study aimed to clarify the involvement of a deubiquitinating enzyme, MYSM1 (Myb-like, SWIRM, and MPN domains 1), in cardiac hypertrophy and to explore the underlying mechanism. Cardiac hypertrophy was developed by angiotensin II or transverse aortic constriction surgery. Cardiomyocyte-specific knockdown of MYSM1 was accomplished using the adeno-associated virus serotype 9-cTNT-Mysm1-shRNA. Co-immunoprecipitation combined with liquid chromatography-tandem mass spectrometry analysis was utilized to identify potential substrates of MYSM1. First, we discovered that the expression of MYSM1 increases during cardiac hypertrophy. MYSM1 knockdown mitigated cardiac dysfunction and hypertrophy after angiotensin II administration. Cardiomyocyte-specific knockdown of MYSM1 with adeno-associated virus serotype 9 alleviated myocardial dysfunction and hypertrophy caused by transverse aortic constriction surgery. Through co-immunoprecipitation and LC-MS, poly (ADP-ribose) polymerase 1 (PARP1) was identified as a potential substrate protein of MYSM1. PARP1 initiates a novel form of programmed cell death termed parthanatos, which is characterized by excessive PARylation, nuclear translocation of AIF, and depletion of nicotinamide adenine dinucleotide. MYSM1 deubiquitinates and stabilizes PARP1 in an MPN domain-dependent manner. In addition, MYSM1 mediates cardiac hypertrophy through PARP1-dependent cardiomyocyte parthanatos. This study identified the role of the MYSM1-PARP1 axis in mediating cardiac hypertrophy and suggested that MYSM1 is a promising pharmacological target for the treatment of cardiac hypertrophy.

Esaxerenone Attenuates Cardiac Hypertrophy in a Pressure Overload Model in Mice

Esaxerenone, a non-steroidal mineralocorticoid receptor (MR) blocker, exhibits high selectivity for MR. While clinically used as an anti-hypertensive drug, its impact on cardiac remodeling remains poorly understood. This study investigated the effect of esaxerenone on pressure overload-induced cardiac hypertrophy in mice.Eight-week-old C57BL/6 mice underwent either transverse aortic constriction (TAC) or sham surgery. Animals were divided into 2 groups: 0.003% (3.0 mg/kg) Esaxerenone-fed (EX) and normal-fed (CNT) groups (n = 64, Sham/CNT = 12, Sham/EX = 13, TAC/CNT = 18, TAC/EX = 21). Cardiac gene expressions were analyzed using quantitative real-time PCR.Food intake and body weight variations showed no significant differences between CNT and EX groups during the 2-week experimental period. The mortality rate from 24 hours after TAC surgery to the end of the experiment was 30.8% in the CNT group, however, all mice survived following TAC surgery in EX group. CNT group showed a remarkable increase in heart weight/tibial length ratio 2 weeks after TAC compared with the Sham group. The EX group demonstrated a significant decrease in HW/TL following TAC surgery (-23.4%, P = 0.041). Masson's trichrome staining revealed that the TAC/CNT group had a significantly higher proportion of fibrotic area than the Sham/CNT group. However, the TAC/EX group had a slightly lower proportion of fibrotic area than the TAC/CNT group. In cardiac gene expression analysis, ANP and Collagen 3a1 were upregulated in the TAC group but were significantly reduced following treatment with esaxerenone.Esaxerenone attenuates cardiac hypertrophy and hypertrophy-related gene expression, resulting in improved survival in a pressure overload model in mice.

Downregulation of HDAC2 attenuates ventricular arrhythmias in a mouse model of cardiac hypertrophy through upregulation of KCHIP2 expression.

Decrease in repolarizing K+ currents, particularly the fast component of transient outward K+ current (Ito,f), prolongs action potential duration (APD) and predisposes the heart to ventricular arrhythmia during cardiac hypertrophy. Histone deacetylases (HDACs) have been suggested to participate in the development of cardiac hypertrophy, and class I HDAC inhibition has been found to attenuate pathological remodeling. This study investigated the potential therapeutic effects of HDAC2 on ventricular arrhythmia in pressure overload-induced cardiac hypertrophy. An in vivo cardiac hypertrophic model was produced by performing transverse aortic constriction (TAC) surgery, and an in vitro cardiomyocyte hypertrophy model by stimulating neonatal rat ventricular myocytes (NRVMs) with phenylephrine (PE). HDAC2 expression was upregulated in TAC mouse hearts and in PE-stimulated cardiomyocytes. Susceptibility to ventricular arrhythmia was increased in TAC mice, while Ito,f was decreased and APD was prolonged in TAC cardiomyocytes. Heart-specific knockdown of HDAC2 (HKD) by RNA interference increased Ito,f, shortened APD and decreased susceptibility to ventricular arrhythmia. Concomitantly, HKD increased the expression of the obligatory β subunit of Ito,f, KChIP2, which is downregulated in hypertrophic hearts. The effects of HKD on KChIP2 expression, Ito,f and APD were also observed in PE-stimulated cardiomyocytes. Mechanistically, HKD increased H3K4me3 abundance and H3K4me3 enrichment at the Kcnip2 promoter in cardiomyocytes. HKD also decreased the expression of KDM5, the H3K4me3 demethylase, which resulted in H3K4me3 upregulation. While investigating the regulatory mechanisms underlying the effect of HDAC2 on KDM5 stability, we identified CNOT4 as the active KDM5 ubiquitinase in cardiomyocytes. HKD increased CNOT4 expression and CNOT4-KDM5 interactions, and thus enhanced the polyubiquitinated degradation of KDM5. HDAC2 inhibition serves as a novel therapeutic strategy for preventing cardiac hypertrophy-associated electrophysiological remodeling. Furthermore, we identified a novel signaling pathway of CNOT4-mediated KDM5 degradation contributing to the upregulation of H3K4me3-mediated KChIP2 expression in response to HDAC2 inhibition.

Adrenomedullin 2/Intermedin Exerts Cardioprotective Effects by Regulating Cardiomyocyte Mitochondrial Function.

Adrenomedullin 2 (AM2) plays critical roles in regulating blood pressure and fluid balance. However, the specific involvement of AM2 in cardiac hypertrophy has not been comprehensively elucidated, warranting further investigation into its molecular mechanisms and therapeutic implications. Cardiac hypertrophy was induced in adult mice lacking AM2 (AM2-/-) using transverse aortic constriction surgery. Comprehensive cardiac morphology, function, histology, and transcriptome/metabolome analyses were conducted. Signal transduction underlying AM2 stimulation in the cardiomyocytes was explored. The absence of endogenous AM2 led to the development of severe heart failure after transverse aortic constriction surgery, which was characterized by alterations in the mitochondrial morphology and function associated with glycolysis and the tricarboxylic acid cycle in the heart and cardiomyocytes of transverse aortic constriction-operated AM2-/- mice. AM2 stimulation was associated with the receptor-modifying factor RAMP2 (receptor activity-modifying protein 2), which primarily transduces signals through the MAPK pathway and affects the expression of genes involved in glycolysis, β-oxidation, and oxidative phosphorylation. The administration of exogenous AM2 alleviated heart failure following transverse aortic constriction. AM2 crucially regulates mitochondrial functions associated with the glycolysis and tricarboxylic acid cycles in the cardiomyocytes, thereby exerting a protective effect on the heart under pressure overload conditions.

YTHDF3-mediated FLCN/cPLA2 axis improves cardiac fibrosis via suppressing lysosomal function.

Cardiac fibrosis characterized by aberrant activation of cardiac fibroblasts impairs cardiac contractile and diastolic functions, inducing the progression of the disease towards its terminal phase, resulting in the onset of heart failure. Therefore, the inhibition of cardiac fibrosis has become a promising treatment for cardiac diseases. The ovarian follicle-stimulating hormone folliculin (FLCN) plays a significant role in various biological processes, such as lysosome function, mitochondrial synthesis, angiogenesis, ciliogenesis and autophagy. Severe heart failure was observed in FLCN knockout mice. In this study, we investigated the role of FLCN in cardiac fibrosis and its potential mechanisms. The mice were subjected to transverse aortic constriction (TAC) surgery. Myocardial fibrosis developed in the mice 8 weeks after surgery. We showed that the protein and mRNA expression levels of FLCN were significantly decreased in TAC mice. Similar results were observed in primary mouse cardiac fibroblasts treated with Ang-II, an in vitro cardiac fibrosis model, suggesting that FLCN is involved in the pathological process of cardiac fibrosis. We demonstrated that overexpression of FLCN inhibited lysosome function in cardiac fibroblasts. Furthermore, overexpression of FLCN protected the heart from TAC-induced pathological cardiac fibrosis. We revealed that FLCN bound to the cPLA2 protein, increased its activity, regulated lysosomal function, and promoted membrane permeabilisation in cardiac fibroblasts during cardiac fibrosis. Knockdown of cPLA2 blocked the antifibrotic effect of FLCN in cardiac fibrosis. In addition, we found that the reduced expression of FLCN in cardiac fibrosis resulted from the modulation of YTHDF3-regulated m6A methylation of FLCN mRNA. The overexpression of YTHDF3 alleviated the production of collagens and improved cardiac structure and function in TAC mice. YTHDF3 inhibited proliferation and differentiation and regulated lysosomal function in mouse cardiac fibroblasts, whereas these effects were abolished by FLCN knockdown. We conclude that FLCN undergoes YTHDF3-regulated m6A modification and interacts with cPLA2 to improve lysosomal function in cardiac fibroblasts, highlighting its role in myocardial fibrosis therapy. These results suggest that FLCN and YTHDF3 could serve as potential therapeutic targets for cardiac fibroblast treatment.

PTN secreted by cardiac fibroblasts promotes myocardial fibrosis and inflammation of pressure overload-induced hypertrophic cardiomyopathy through the PTN-SDC4 pathway.

Hypertrophic cardiomyopathy (HCM) is characterized by unexplained left ventricular hypertrophy (LVH) with key pathologic processes including myocardial necrosis, fibrosis, inflammation, and hypertrophy, which are involved in heart failure (HF), stroke, and even sudden death. Our aim was to explore the communication network among various cells in the heart of transverse aortic constriction (TAC) surgery induced HCM mice. Single-cell RNA-seq data of GSE137167 was downloaded from the Gene Expression Omnibus (GEO) database. Seurat was used to perform the standard workflow. CellChat was utilized to compute the cell-cell interaction network and analyze the ligand-receptor pairs. Weighted gene co-expression network analysis (WGCNA) was conducted to identify gene co-expression modules. In vitro and in vivo studies were performed to verify bioinformatic analysis findings through real-time quantitative PCR (RT-qPCR), Edu staining, transwell assay, western blot, immunofluorescence assay, CCK-8, hematoxylin and eosin (H&E) staining, and echocardiography based on TAC mouse model. Our results showed that after TAC surgery, the interaction between cardiac fibroblasts and macrophages was very common, and the increasing pleiotrophin (PTN) ligand secreted by cardiac fibroblasts could promote the self-proliferation or invasion for myocardial fibrosis as well as stimulate the inflammatory response of macrophages to contribute TAC surgery induced HCM through acting on Syndecan 4 (SDC4) receptor. Our study demonstrates that PTN derived from cardiac fibroblasts may play potential role in pressure overload-induced HCM through activating the PTN-SDC4 pathway in cardiac fibroblasts and macrophages, which may be a potential therapeutic target for pressure overload-induced HCM patients.

SerpinB1 targeting safeguards against pathological cardiac hypertrophy and remodelling by suppressing cardiomyocyte pyroptosis and inflammation initiation.

While the pivotal role of inflammation in pathological cardiac hypertrophy and remodelling is widely acknowledged, the mechanisms triggering inflammation initiation remain largely obscure. This study aims to elucidate the role and mechanism of serpin family B member 1 (SerpinB1) in pro-inflammatory cardiomyocyte pyroptosis, heart inflammation, and cardiac remodelling. C57BL/6J wild-type, inducible cardiac-specific SerpinB1 overexpression or knockout mice underwent transverse aortic constriction (TAC) surgery. Cardiac hypertrophy and remodelling were assessed through echocardiography and histology. Cardiomyocyte pyroptosis and heart inflammation were monitored. Adeno-associated virus 9 -mediated gene manipulations and molecular assays were employed to explore the mechanisms through which SerpinB1 regulates cardiomyocyte pyroptosis and heart inflammation. Finally, recombinant mouse SerpinB1 protein (rSerpinB1) was administrated both in vivo through osmotic minipump delivery and in vitro to investigate the therapeutic potential of SerpinB1 in cardiac remodelling. Myocardial SerpinB1 overexpression was up-regulated shortly upon TAC or phenylephrine challenge, with no further elevation during prolonged hypertrophic stimuli. It is important to note that cardiac-specific overexpression of SerpinB1 markedly attenuated TAC-induced cardiac remodelling, while deletion of SerpinB1 exacerbated it. At the mechanistic level, SerpinB1 gain-of-function inhibited cardiomyocyte pyroptosis and inflammation in hypertrophic hearts; the protective effect was nullified by overexpression of either cleaved N-terminal gasdermin D or cleaved caspase-1. Co-immunoprecipitation and confocal assays confirmed that SerpinB1 directly interacts with caspase-1 in cardiomyocytes. Remarkably, rSerpinB1 replicated the cardioprotective effect against cardiac hypertrophy and remodelling. SerpinB1 safeguards against pathological cardiac hypertrophy and remodelling by impeding cardiomyocyte pyroptosis to suppress inflammation initiation, achieved through interaction with caspase-1 to inhibit its activation. Targeting SerpinB1 could represent a novel therapeutic strategy for treating pathological cardiac hypertrophy and remodelling.

Fucoidan Attenuates Cardiac Remodeling by Inhibiting Galectin-3 Secretion, Fibrosis, and Inflammation in a Mouse Model of Pressure Overload.

Fucoidan, a sulfated polysaccharide derived from marine algae, is known for its antioxidant and immunomodulatory properties. Galectin-3 (Gal-3), a protein associated with cardiovascular fibrosis, has been identified as a potential therapeutic target in cardiac remodeling. This study aimed to evaluate whether fucoidan could inhibit Gal-3 activity and mitigate cardiac remodeling in a mouse model of pressure overload-induced cardiac hypertrophy. To test this hypothesis, we used transverse aortic constriction (TAC) surgery to induce pressure overload in normotensive mice, replicating the pathological features of cardiac hypertrophy. Mice were treated with fucoidan at a dose of 1.5 or 7.5 mg/kg/day. In vivo assessments of cardiac function, fibrosis, inflammation, and Gal-3 expression were performed. Pressure overload led to significant upregulation of serum Gal-3 levels, increased cardiac collagen deposition, and elevated markers of fibrosis and inflammation. In mice treated with fucoidan, these effects were significantly attenuated. Fucoidan treatment prevented the upregulation of Gal-3, reduced collagen deposition, and decreased inflammatory cell infiltration, suggesting an inhibition of both fibrosis and inflammation. Fucoidan effectively mitigated the adverse effects of pressure overload in this mouse model, including reduced Gal-3 expression, fibrosis, and inflammation. These findings suggest that fucoidan holds promise as a therapeutic agent for preventing or delaying cardiac remodeling and associated complications, such as fibrosis and inflammation, in pressure overload-induced cardiac hypertrophy. Further research is needed to explore the underlying mechanisms and clinical applicability of fucoidan in cardiac disease.

Loss of GATAD1 in cardiomyocyte does not cause cardiomyopathy in mice.

GATA zinc finger domain containing 1 (GATAD1) is an as-yet uncharacterized zinc finger domain protein, which was initially identified as a histone 3 trimethylated at lysine 4 (H3K4me3) interactor. A recessive mutation in GATAD1 is associated with adult-onset dilated cardiomyopathy and heart failure, suggesting that GATAD1 is critical for maintaining normal cardiac structure and function. However, little is known as to the specific role of GATAD1 in cardiomyocytes. A mammalian Gatad1 knockout model has yet to be generated for investigating its specific role in the heart. To address this, we generated a Gatad1 cardiomyocyte-specific knockout (cKO) mouse model. Gatad1 cKO mutants exhibited normal cardiac function during the aging process up to 18 months of age. Unlike the abnormal nuclei shape observed in patients carrying GATAD1 mutations, the nuclei shape of cardiomyocytes remained unaffected by the loss of Gatad1. Furthermore, Gatad1 cKO mice responded normally to pressure overload induced by transverse aortic constriction (TAC) surgery. Together, these observations suggest that deletion of Gatad1 in cardiomyocytes does not induce cardiomyopathy during aging or affect the response to pressure overload stress in mice.

Antrodia cinnamomea triterpenoids attenuate cardiac hypertrophy via the SNW1/RXR/ALDH2 axis.

Aldehyde dehydrogenase 2 (ALDH2), a pivotal enzyme in the metabolism of toxic aldehydes produced by oxidative stress, has been demonstrated to play a cardioprotective role in cardiovascular diseases. Antrodia cinnamomea triterpenoids (ACT) is a medicinal mushroom with anti-inflammatory and antioxidant properties, and our previous study found that ACT can exert anti-fatty liver effects by regulating ALDH2. This study aimed to elucidate the impact of ACT and its monomer on cardiac hypertrophy and investigate the relationship between its pharmacological mechanism and ALDH2. Through examining cardiac morphology and expression levels of hypertrophic biomarkers, ACT significantly reduced myocardial hypertrophy induced by angiotensin II (Ang II) and transverse aortic constriction (TAC)surgery in wild-type mice, but not in ALDH2 knockout mice. In vitro, ACT and its monomeric dehydrosulphurenic acid (DSA) inhibited the hypertrophic phenotype of Ang II-stimulated neonatal cardiac myocytes (NRCMs) in an ALDH2-dependent manner. Regarding the pharmacological mechanism, it was observed that ACT and DSA restored ALDH2 expression and activity in myocardial tissues of WT-Ang II/TAC mice and Ang II-induced NRCMs. Furthermore, it inhibited oxidative stress and improved mitochondrial quality control (MQC) homeostasis in an ALDH2-dependent manner. We screened SNW1, a transcriptional coactivator, as a DSA-binding protein by "target fishing" and cellular enthusiasm transfer assay techniques and validated that SNW1 promoted ALDH2 transcription and translation levels through synergistic interaction with the transcription factor RXR. In conclusion, the findings demonstrate that ACT/DSA upregulates ALDH2 expression via regulating SNW1/RXR, thereby inhibiting oxidative stress and maintaining MQC homeostasis, and then protects against cardiac hypertrophy.

KLF2-dependent transcriptional regulation safeguards the heart against pathological hypertrophy.

Our previous single-cell RNA sequencing study in the adult human heart revealed that cardiomyocytes from both the atrium and ventricle display high activities of Krüppel-like factor 2 (KLF2) regulons. However, the role of the transcription factor KLF2 in cardiomyocyte biology remains largely unexplored. We employed transverse aortic constriction surgery in male C57BL/6 J mice to develop an in vivo model of cardiac hypertrophy, and generated different in vitro cardiac hypertrophy models in neonatal rat ventricular myocytes and human embryonic stem cell-derived cardiomyocytes. Our results demonstrated a significant reduction in KLF2 expression during the progression of cardiac hypertrophy. In vitro, Klf2 deficiency exacerbates cardiac hypertrophy and enhances hypertrophic reprogramming, while KLF2 overexpression attenuates cardiac hypertrophy and reverses hypertrophic transcriptome reprogramming. Mechanistically, combined RNA-seq and cleavage under targets & tagmentation (CUT&Tag) analysis revealed that KLF2 exerts its protective effects by directly regulating a set of genes associated with cardiac hypertrophy. In vivo, KLF2 overexpression specifically in cardiomyocytes effectively prevents TAC-induced cardiac hypertrophy in mice. Additionally, we found that simvastatin elevates KLF2 expression in cardiomyocytes, which subsequently alleviates cardiomyocyte hypertrophy. This study provides the first evidence that transcription factor KLF2 serves as a negative regulator of cardiac hypertrophy. Our findings highlight the therapeutic potential of enhancing KLF2 expression, particularly through simvastatin administration, as a promising strategy in the treatment of cardiac hypertrophy.

Abstract 4137996: Effects of Empagliflozin on diuresis in heart failure: Results from the CINNAMON-study and in-vivo experiments

Background and Purpose: Heart failure is associated with renal dysfunction suggesting a pathophysiological link between heart and kidney. Empagliflozin, a SGLT2 inhibitor, showed beneficial effects on both cardiovascular and renal endpoints. However, mechanistically, it is unclear if empagliflozin-dependent kidney protection is mediated via inhibition of tubular SGLT2 or more indirectly via improved cardiac function. We hypothesized that Empagliflozin treatment improves left ventricular ejection fraction (LVEF) independent of diuretic effect of renal SGLT2 inhibition. Methods: We evaluated NTproBNP, hematocrit, hemoglobin and bodyweight in patients with HF with reduced (n=32) and preserved ejection fraction (n=59) after 30 and 180 days (prospective, single-arm CINNAMON-study, DRKS00031101). Furthermore, we conducted transverse aortic constriction (TAC) or sham surgery in C57BL/6J (wildtype, WT) and SGLT2 deficient mice (SGLT2 -/- ). Animals received either Empagliflozin (10 mg/kg bodyweight) or vehicle by daily oral gavage. Water intake and output was evaluated by metabolic cages at 10 weeks and cardiac function by echocardiography. Results: Empagliflozin treatment reduced NT-proBNP levels and increased hematocrit and hemoglobin levels especially in patients with reduced ejection fraction (Fig. A-C). Surprisingly, there was no change in body weight (Fig. D), which would be expected if the diuretic effects were dominant. In mice after 10 weeks, echocardiography confirmed TAC induced pressure-overload, leading to reduced LVEF. Empagliflozin attenuated changes in LVEF (Fig. G) and improved diastolic dysfunction (isovolumetric relaxation time, data not shown). To test if direct inhibition of SGLT2 in the heart and kidney is mechanistically involved, TAC surgery was repeated in SGLT2-deficient mice (SGLT2-KO). In fact, exposure to TAC resulted in comparable reduction of LVEF in SGLT2-KO and Empagliflozin prevented this deterioration similar to WT mice (Fig. G vs. I). Surprisingly, Empagliflozin did not increase drinking and urine volume neither in wildtype nor in SGLT2 deficient mice, suggesting a mechanism of cardioprotection independent of diuretic effects. Conclusion and Outlook: This is the first study investigating the role of SGLT2 in Empagliflozin treated patients and mice with heart failure. Importantly, empagliflozin treatment prevented deterioration of LVEF independent of the presence of SGLT2 and diuresis.

Abstract 4135476: The Cardiomyocyte Hypertrophy Inhibitor RFN-409, Identified by High Throughput Screening Assay, Suppresses Pressure Overload-induced Systolic Dysfunction in Mice by Suppressing p38 Activity

Purpose: When the heart is exposed to stresses such as myocardial infarction or hypertension, it undergoes compensatory hypertrophy in response. However, continuation of the stress causes this compensatory mechanism to fail, and eventually systolic dysfunction or decompensated heart failure occur. As the hypertrophy of individual cardiomyocytes has been observed in this process, controlling cardiomyocyte hypertrophy is a potential target the prevention and treatment of heart failure. In this study, we constructed a high throughput screening (HTS) assay using cardiomyocyte hypertrophy as an index parameter. Compounds that inhibit cardiomyocyte hypertrophy were selected from our low molecular compound library. Methods and Results: In the primary screening, cultured rat primary cardiomyocytes were treated with each compound at a final concentration of 1 µM and then stimulated with 30 µM phenylephrine (PE) for 48 hours. These cells were subjected to fluorescent immunostaining with α-actinin, and cardiomyocyte area was measured using an ArrayScan™ system. The hypertrophy inhibition rate (%) of each compound was calculated as [(PE(+) - compound) / (PE(+) - PE(-))] × 100. The compounds with a hypertrophy inhibition rate greater than 50% and less than 150% were selected as hit compounds. In the secondary screening, these hit compounds were evaluated based on the dose-dependency of cardiomyocyte hypertrophy inhibition and the inhibition of the mRNA levels of the cardiac hypertrophy response genes ANF and BNP using real-time PCR. From the 269 low molecular-weight compounds in the original compound library, eight were selected through the primary and secondary screenings. Among them, we focused on Reference Number 409 (RFN-409). Western blotting indicated that RFN-409 inhibited PE-induced p38 activation. Next, we investigated the effect of RFN-409 on heart failure. Eight-week-old male C57 BL/6J mice were subjected to transverse aortic constriction (TAC) surgery and then randomly assigned to intraperitoneal treatment with RFN-409 (3, 10 mg/kg) or vehicle for eight weeks. RFN-409 at 10 mg/kg significantly prevented TAC-induced increase in left ventricular posterior wall thickness and decrease in left ventricular fractional shortening. Discussion: RFN-409 suppressed TAC-induced development of heart failure, at least partially by inhibiting p38 activity. These findings suggest that RFN-409 may be an effective agent for heart failure therapy.

PINK1-mediated mitophagy attenuates pathological cardiac hypertrophy by suppressing the mtDNA release-activated cGAS-STING pathway.

Sterile inflammation is implicated in the development of heart failure (HF). Mitochondria plays important roles in triggering and maintaining inflammation. Mitophagy is important for regulation of mitochondrial quality and maintenance of cardiac function under pressure overload. The association of mitophagy with inflammation in HF is largely unclear. As PINK1 is a central mediator of mitophagy, our objective was to investigate its involvement in cardiac hypertrophy, and the effect of PINK1-mediated mitophagy on cGAS-STING activation during cardiac hypertrophy. PINK1 knockout and cardiac-specific PINK1-overexpressing transgenic mice were created and subsequently subjected to transverse aortic constriction (TAC) surgery. In order to explore whether PINK1 regulates STING-mediated inflammation during HF, PINK1/STING (stimulator of interferon genes) double-knockout mice were created. Pressure overload was induced by TAC. Our findings indicate a significantly decline in PINK1 expression in TAC-induced hypertrophy. Cardiac hypertrophic stimuli caused the release of mitochondrial DNA (mtDNA) into the cytosol, activating the cGAS-STING signaling, which in turn initiated cardiac inflammation and promoted the progression of cardiac hypertrophy. PINK1 deficiency inhibited mitophagy activity, promoted mtDNA release, and then drove the overactivation of cGAS-STING signaling, exacerbating cardiac hypertrophy. Conversely, cardiac-specific PINK1 overexpression protected against hypertrophy thorough inhibition of the cGAS-STING signaling. Double-knockout mice revealed that the effects of PINK1 on hypertrophy were dependent on STING. Our findings suggest that PINK1-mediated mitophagy plays a protective role in pressure overload-induced cardiac hypertrophy via inhibiting the mtDNA-cGAS-STING pathway.

Fibroblast-derived miR-425-5p alleviates cardiac remodelling in heart failure via inhibiting the TGF-β1/Smad signalling.

The pathological activation of cardiac fibroblasts (CFs) plays a crucial role in the development of pressure overload-induced cardiac remodelling and subsequent heart failure (HF). Growing evidence demonstrates that multiple microRNAs (miRNAs) are abnormally expressed in the pathophysiologic process of cardiovascular diseases, with miR-425 recently reported to be potentially involved in HF. In this study, we aimed to investigate the effects of fibroblast-derived miR-425-5p in pressure overload-induced HF and explore the underlying mechanisms. C57BL/6 mice were injected with a recombinant adeno-associated virus specifically designed to overexpress miR-425-5p in CFs, followed by transverse aortic constriction (TAC) surgery. Neonatal mouse CFs (NMCFs) were transfected with miR-425-5p mimics and subsequently stimulated with angiotensin II (Ang II). We found that miR-425-5p levels were significantly downregulated in HF mice and Ang II-treated NMCFs. Notably, fibroblast-specific overexpression of miR-425-5p markedly inhibited the proliferation and differentiation of CFs, thereby alleviating myocardial fibrosis, cardiac hypertrophy and systolic dysfunction. Mechanistically, the cardioprotective actions of miR-425-5p may be achieved by targeting the TGF-β1/Smad signalling. Interestingly, miR-425-5p mimics-treated CFs could also indirectly affect cardiomyocyte hypertrophy in this course. Together, our findings suggest that fibroblast-derived miR-425-5p mitigates TAC-induced HF, highlighting miR-425-5p as a potential diagnostic and therapeutic target for treating HF patients.

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.

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.

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.

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.