To Transverse Aortic Constriction Research Articles

Matairesinol Blunts Adverse Cardiac Remodeling and Heart Failure Induced by Pressure Overload by Regulating Prdx1 and PI3K/AKT/FOXO1 Signaling

BackgroundPathological cardiac remodeling is a critical process leading to heart failure, characterized primarily by inflammation and apoptosis. Matairesinol (Mat), a key chemical component of Podocarpus macrophyllus resin, exhibits a wide range of pharmacological activities, including anti-hydatid, antioxidant, antitumor, and anti-inflammatory effects. PurposeThis study aims to investigate whether Matairesinol alleviate cardiac hypertrophy and remodeling caused by pressure overload and to elucidate its mechanism of action. MethodsAn in vitro pressure loading model was established using neonatal rat cardiomyocytes treated with angiotensin Ⅱ, while an in vivo model was created using C57 mice subjected to transverse aortic constriction (TAC). To activate the PI3K/Akt/FoxO1 pathway, Ys-49 was employed. Moreover, small interfering RNA (siRNA) and short hairpin RNA (shRNA) were utilized to silence Prdx1 expression both in vitro and in vivo. Various techniques, including echocardiography, wheat germ agglutinin (WGA) staining, HE staining, PSR staining, and Masson trichrome staining, were used to assess cardiac function, cardiomyocyte cross-sectional area, and fibrosis levels in rats. Apoptosis in myocardial tissue and in vitro was detected by TUNEL assay, while reactive oxygen species (ROS) content in tissues and cells was measured using DHE staining. Furthermore, the affinity of Prdx1 with Mat and PI3K was analyzed using computer-simulated molecular docking. Western blotting and RT-PCR were utilized to evaluate Prdx1 levels and proteins related to apoptosis and oxidative stress, as well as the mRNA levels of cardiac hypertrophy and fibrosis-related indicators. ResultsMat significantly alleviated cardiac hypertrophy and fibrosis induced by TAC, preserved cardiac function, and markedly reduced cardiomyocyte apoptosis and oxidative damage. In vitro, mat attenuated ang Ⅱ - induced hypertrophy of nrvms and activation of neonatal rat fibroblasts. Notably, activation of the PI3K/Akt/FoxO1 pathway and downregulation of Prdx1 expression were observed in TAC mice; however, these effects were reversed by Mat treatment. Furthermore, Prdx1 knockdown activated the PI3K/Akt/FoxO1 pathway, leading to exacerbation of the disease. Molecular docking indicated that Molecular docking indicated that Mat upregulated Prdx1 expression by binding to it, thereby inhibiting the PI3K/Akt/FoxO1 pathway and protecting the heart by restoring Prdx1 expression levels. ConclusionMatairesinol alleviates pressure overload-induced cardiac remodeling both in vivo and in vitro by upregulating Prdx1 expression and inhibiting the PI3K/Akt/FoxO1 pathway. This study highlights the therapeutic potential of Matairesinol in the treatment of cardiac hypertrophy and remodeling, providing a promising avenue for future research and clinical application.

TNIP3 protects against pathological cardiac hypertrophy by stabilizing STAT1

Pathological cardiac hypertrophy is one of the major risk factors of heart failure and other cardiovascular diseases. However, the mechanisms underlying pathological cardiac hypertrophy remain largely unknown. Here, we identified the first evidence that TNFAIP3 interacting protein 3 (TNIP3) was a negative regulator of pathological cardiac hypertrophy. We observed a significant upregulation of TNIP3 in mouse hearts subjected to transverse aortic constriction (TAC) surgery and in primary neonatal rat cardiomyocytes stimulated by phenylephrine (PE). In Tnip3-deficient mice, cardiac hypertrophy was aggravated after TAC surgery. Conversely, cardiac-specific Tnip3 transgenic (TG) mice showed a notable reversal of the same phenotype. Accordingly, TNIP3 alleviated PE-induced cardiomyocyte enlargement in vitro. Mechanistically, RNA-sequencing and interactome analysis were combined to identify the signal transducer and activator of transcription 1 (STAT1) as a potential target to clarify the molecular mechanism of TNIP3 in pathological cardiac hypertrophy. Via immunoprecipitation and Glutathione S-transferase assay, we found that TNIP3 could interact with STAT1 directly and suppress its degradation by suppressing K48-type ubiquitination in response to hypertrophic stimulation. Remarkably, preservation effect of TNIP3 on cardiac hypertrophy was blocked by STAT1 inhibitor Fludaradbine or STAT1 knockdown. Our study found that TNIP3 serves as a novel suppressor of pathological cardiac hypertrophy by promoting STAT1 stability, which suggests that TNIP3 could be a promising therapeutic target of pathological cardiac hypertrophy and heart failure.

Abstract 1038: Map Kinase Phosphatase-5 Deficiency Impairs Macrophage Efferocytosis And Exacerbates Late-stage Atherosclerosis

Objective: Atherosclerosis is a vascular inflammatory disease that is a major cause of death in Westernized society. Late-stage atherosclerosis is characterized by a vulnerable plaque, which consists of a thin fibrous cap and a large necrotic core that is prone to rupture. Efferocytosis is a repair mechanism that clears apoptotic cells within the plaque, contributing to plaque stability. In late-stage atherosclerotic conditions efferocytosis is impaired and promotes rupture. MAPK phosphatase-5 (MKP5) regulates various processes such as immune diseases and cardiac fibrosis. The goal of this project is to identify the role of MKP5 in late-stage atherosclerotic plaque stability. Approach/Results: To induce late-stage atherosclerosis, male and female LDLR -/- and LDLR -/- MKP5 -/- mice were subjected to transverse aortic constriction (TAC) surgery and fed on a highfat diet (western diet with 1.25% cholesterol) for 12 weeks. Our data show that MKP5 deficiency exacerbates plaque instability, through decreased cap thickness, decreased collagen content, increased lesion size and necrosis in the plaque, and severe coronary lesions. Furthermore, there are higher instances of intraplaque hemorrhage in the right carotids of the LDLR -/- MKP5 -/- mice. Mechanistically, TUNEL staining of the aortic roots was performed to determine efferocytotic capabilities in vivo. Results showed fewer macrophage-associated TUNEL-positive cells in LDLR -/- MKP5 -/- aortic roots. To further investigate this mechanism, bone-marrow-derived macrophages (BMDMs) isolated from WT and MKP5 -/- mice were incubated with apoptotic Jurkat cells and then analyzed to determine the engulfment of apoptotic cells by the macrophages via confocal microscopy and flow cytometry. Results showed that efferocytosis was significantly inhibited in MKP5 -/- BMDMs. Bulk-RNA sequencing analysis revealed that the expression of efferocytotic genes in MKP5 -/- BMDMs was decreased at basal and oxLDL stimulated conditions. Conclusion: Results suggest that MKP5 deficiency impairs efferocytosis, exacerbates atherosclerosis, and causes late-stage plaque to move toward unstable phenotypes.

IL-12p40 deletion reduces M1 macrophage polarization and alleviates cardiac remodeling via regulating Th17 cells differentiation, but not γδT 17 cells, in TAC mice

BackgroundThe interleukin (IL) −12 p40 subunit is the common subunit of IL-12 and IL-23. It affects the immune inflammatory response, which may be closely related to cardiac remodeling. In this study, the regulatory effect of IL-12p40 knockout (KO) on cardiac remodeling was investigated, and the underlying mechanism was explored. Methods and resultsMice were subjected to transverse aortic constriction (TAC) to establish a model of cardiac remodeling. First, IL-12p40 was deleted to observe its effects on cardiac remodeling and cardiac inflammation, and the results showed that IL-12p40 deletion reduced both T helper 17 (Th17) and γδT17 cell differentiation, decreased proinflammatory macrophage differentiation, alleviated cardiac remodeling, and relieved cardiac dysfunction in TAC mice. Next, we explored whether IL-17 regulated TAC-induced cardiac remodeling, and the results showed that IL-17 neutralization alleviated proinflammatory macrophage differentiation and cardiac remodeling in IL-12p40 knockout mice and WT mice. Neutralization with cluster of differentiation 4 receptor (CD4) and γδ T-cell receptor (γδTCR) antibodies inhibited pro-inflammatory macrophage polarization and improved cardiac remodeling, and CD4 neutralizing antibody (NAb) had more significant effects. Finally, adoptive transfer of Th17 cells aggravated proinflammatory macrophage differentiation and cardiac remodeling in TAC-treated CD4 KO mice, while neutralization with the IL-12p40 antibody alleviated these pathological changes. ConclusionMainly Th17 cells but not γδT17 cells secrete IL-17, which mediates IL-12p40, promotes the polarization of proinflammatory macrophages, and exacerbates cardiac remodeling in TAC mice. IL-12p40 may be a potential target for the prevention and treatment of cardiac remodeling.

Anserine, a Histidine-Containing Dipeptide, Suppresses Pressure Overload-Induced Systolic Dysfunction by Inhibiting Histone Acetyltransferase Activity of p300 in Mice.

Anserine, an imidazole dipeptide, is present in the muscles of birds and fish and has various bioactivities, such as anti-inflammatory and anti-fatigue effects. However, the effect of anserine on the development of heart failure remains unknown. We cultured primary cardiomyocytes with 0.03 mM to 10 mM anserine and stimulated them with phenylephrine for 48 h. Anserine significantly suppressed the phenylephrine-induced increases in cardiomyocyte hypertrophy, ANF and BNP mRNA levels, and histone H3K9 acetylation. An in vitro histone acetyltransferase (HAT) assay showed that anserine directly suppressed p300-HAT activity with an IC50 of 1.87 mM. Subsequently, 8-week-old male C57BL/6J mice were subjected to transverse aortic constriction (TAC) and were randomly assigned to receive daily oral treatment with anserine-containing material, Marine Active® (60 or 200 mg/kg anserine) or vehicle for 8 weeks. Echocardiography revealed that anserine 200 mg/kg significantly prevented the TAC-induced increase in left ventricular posterior wall thickness and the decrease in left ventricular fractional shortening. Moreover, anserine significantly suppressed the TAC-induced acetylation of histone H3K9. These results indicate that anserine suppresses TAC-induced systolic dysfunction, at least in part, by inhibiting p300-HAT activity. Anserine may be used as a pharmacological agent for human heart failure therapy.

Mac-1 deficiency ameliorates pressure overloaded heart failure through inhibiting macrophage polarization and activation

Persistent pressure overload commonly leads to pathological cardiac hypertrophy and remodeling, ultimately leading to heart failure (HF). Cardiac remodeling is associated with the involvement of immune cells and the inflammatory response in pathogenesis. The macrophage-1 antigen (Mac-1) is specifically expressed on leukocytes and regulates their migration and polarization. Nonetheless, the involvement of Mac-1 in cardiac remodeling and HF caused by pressure overload has not been determined. The Mac-1-knockout (KO) and wild-type (WT) mice were subjected to transverse aortic constriction (TAC) for 6 weeks. Echocardiography and pressure–volume loop assessments were used to evaluate cardiac function, and cardiac remodeling and macrophage infiltration and polarization were estimated by histopathology and molecular techniques. The findings of our study demonstrated that Mac-1 expression was markedly increased in hearts subjected to TAC treatment. Moreover, compared with WT mice, Mac-1-KO mice exhibited dramatically ameliorated TAC-induced cardiac dysfunction, hypertrophy, fibrosis, oxidative stress and apoptosis. The potential positive impacts may be linked to the inhibition of macrophage infiltration and M1 polarization via reductions in NF-kB and STAT1 expression and upregulation of STAT6. In conclusion, this research reveals a new function of Mac-1 deficiency in reducing pathological cardiac remodeling and HF caused by pressure overload. Additionally, inhibiting Mac-1 could be a potential treatment option for patients with HF in a clinical setting.

Cardioprotection by the adiponectin receptor agonist ALY688 in a preclinical mouse model of heart failure with reduced ejection fraction (HFrEF)

AimsAdiponectin has been shown to mediate cardioprotective effects and levels are typically reduced in patients with cardiometabolic disease. Hence, there has been intense interest in developing adiponectin-based therapeutics. The aim of this translational research study was to examine the functional significance of targeting adiponectin signaling with the adiponectin receptor agonist ALY688 in a mouse model of heart failure with reduced ejection fraction (HFrEF), and the mechanisms of cardiac remodeling leading to cardioprotection. Methods and resultsWild-type mice were subjected to transverse aortic constriction (TAC) to induce left ventricular pressure overload (PO), or sham surgery, with or without daily subcutaneous ALY688-SR administration. Temporal analysis of cardiac function was conducted via weekly echocardiography for 5 weeks and we observed that ALY688 attenuated the PO-induced dysfunction. ALY688 also reduced cardiac hypertrophic remodeling, assessed via LV mass, heart weight to body weight ratio, cardiomyocyte cross sectional area, ANP and BNP levels. ALY688 also attenuated PO-induced changes in myosin light and heavy chain expression. Collagen content and myofibroblast profile indicated that fibrosis was attenuated by ALY688 with TIMP1 and scleraxis/periostin identified as potential mechanistic contributors. ALY688 reduced PO-induced elevation in circulating cytokines including IL-5, IL-13 and IL-17, and the chemoattractants MCP-1, MIP-1β, MIP-1alpha and MIP-3α. Assessment of myocardial transcript levels indicated that ALY688 suppressed PO-induced elevations in IL-6, TLR-4 and IL-1β, collectively indicating anti-inflammatory effects. Targeted metabolomic profiling indicated that ALY688 increased fatty acid mobilization and oxidation, increased betaine and putrescine plus decreased sphingomyelin and lysophospholipids, a profile indicative of improved insulin sensitivity. ConclusionThese results indicate that the adiponectin mimetic peptide ALY688 reduced PO-induced fibrosis, hypertrophy, inflammation and metabolic dysfunction and represents a promising therapeutic approach for treating HFrEF in a clinical setting.

Renalase mediates macrophage-to-fibroblast crosstalk to attenuate pressure overload-induced pathological myocardial fibrosis.

A potential antifibrotic mechanism in pathological myocardial remodeling is the recruitment of beneficial functional subpopulations of macrophages or the transformation of their phenotype. Macrophages are required to activate molecular cascades that regulate fibroblast behavior. Identifying mediators that activate the antifibrotic macrophage phenotype is tantamount to identifying the button that retards pathological remodeling of the myocardium; however, relevant studies are inadequate. Circulating renalase (RNLS) is mainly of renal origin, and cardiac myocytes also secrete it autonomously. Our previous studies revealed that RNLS delivers cell signaling to exert multiple cardiovascular protective effects, including the improvement of myocardial ischemia, and heart failure. Here, we further investigated the potential mechanism by which macrophage phenotypic transformation is targeted by RNLS to mediate stress load-induced myocardial fibrosis. Mice subjected to transverse aortic constriction (TAC) were used as a model of myocardial fibrosis. The co-incubation of macrophages and cardiac fibroblasts was used to study intercellular signaling. The results showed that RNLS co-localized with macrophages and reduced protein expression after cardiac pressure overload. TAC mice exhibited improved cardiac function and alleviated left ventricular fibrosis when exogenous RNLS was administered. Flow sorting showed that RNLS is essential for macrophage polarization towards a restorative phenotype (M2-like), thereby inhibiting myofibroblast activation, as proven by both mouse RAW264.7 and bone marrow-derived macrophage models. Mechanistically, we found that activated protein kinase B is a major pathway by which RNLS promotes M2 polarization in macrophages. RNLS may serve as a prognostic biomarker and a potential clinical candidate for the treatment of myocardial fibrosis.

The crucial relationship between miRNA-27 and CSE/H2S, and the mechanism of action of GLP-1 in myocardial hypertrophy.

Cardiac hypertrophy is the most prevalent compensatory heart disease that ultimately leads to spontaneous heart failure. Mounting evidence suggests that microRNAs (miRs) and endogenous hydrogen sulfide (H2S) play a crucial role in the regulation of cardiac hypertrophy. In this study, we aimed to investigate whether inhibition of miR-27a could protect against cardiac hypertrophy by modulating H2S signaling. We established a model of cardiac hypertrophy by obtaining hypertrophic tissue from mice subjected to transverse aortic constriction (TAC) and from cells treated with angiotensin-II. Molecular alterations in the myocardium were quantified using quantitative real time PCR (qRT-PCR), Western blotting, and ELISA. Morphological changes were characterized by hematoxylin and eosin (HE) staining and Masson's trichrome staining. Functional myocardial changes were assessed using echocardiography. Our results demonstrated that miR-27a levels were elevated, while H2S levels were reduced in TAC mice and myocardial hypertrophy. Further luciferase and target scan assays confirmed that cystathionine-γ-lyase (CSE) was a direct target of miR-27a and was negatively regulated by it. Notably, enhancement of H2S expression in the heart was observed in mice injected with recombinant adeno-associated virus vector 9 (rAAV9)-anti-miR-27a and in cells transfected with a miR-27a inhibitor during cardiac hypertrophy. However, this effect was abolished by co-transfection with CSE siRNA and the miR-27a inhibitor. Conversely, injecting rAAV9-miR-27a yielded opposite results. Interestingly, our findings demonstrated that glucagon-like peptide-1 (GLP-1) agonists could mitigate myocardial damage by down-regulating miR-27a and up-regulating CSE. In summary, our study suggests that inhibition of miR-27a holds therapeutic promise for the treatment of cardiac hypertrophy by increasing H2S levels. Furthermore, our findings unveil a novel mechanism of GLP-1 agonists involving the miR-27a/H2S pathway in the management of cardiac hypertrophy.

Abstract 17336: A Hepato-Cardiac FGF21 Signaling Axis Promotes Cardiac Hypertrophy

FGF21 is expressed mainly in the liver and other organs, including the heart. The role of FGF21 is controversial, with potential benefits for obesity and certain heart conditions, but also associations with heart failure and increased mortality. We found higher serum FGF21 levels (2.5-fold) in left ventricular hypertrophy patients (N=31) compared to those without hypertrophy (N=45). Similarly, mice subjected to Transverse Aortic Constriction (TAC) had elevated plasma FGF21 levels and hepatic expression. Surprisingly, a time course experiment showed that liver FGF21 production peaked 3 days post-TAC (prior to the onset of hypertrophy), while an unexpected increase of cardiac FGF21 expression was observed 2 weeks post-TAC, coinciding with hypertrophy and dysfunction. FGF21 expression in skeletal muscle did not change. As in mice, hypertensive individuals (N=6) showed a 14-fold increase in cardiac FGF21 mRNA levels compared to normotensive (N=6). To delineate the role of hepatic and cardiac FGF21 in hypertrophy, TAC was performed on mice with hepatocyte- (HEP-FGF21-KO) or cardiomyocyte-specific (CM-FGF21-KO) FGF21 ablation. Both HEP-FGF21-KO and CM-FGF21-KO mice were protected from hypertrophy, pro-hypertrophic signaling, fibrosis, and cardiac dysfunction. Notably, hepatocyte FGF21 deletion prevented cardiac FGF21 upregulation with TAC, suggesting control of CM FGF21 expression by liver-derived FGF21 and highlighting the role of CM FGF21 in driving cardiac hypertrophy. This was confirmed by treatment of AC16 human cardiomyocytes with recombinant FGF21 that increased endogenous FGF21 expression and cell size. RNAseq analysis revealed attenuation of cardiac fibrosis- and hypertrophy-related transcriptome in both HEP-FGF21-KO and CM-FGF21-KO mice. To explore if FGF21 increase is a cause and not a consequence of cardiac hypertrophy, we developed anti-sense oligonucleotides against FGF21 and administered them in C57BL/6 mice with TAC. ASO-FGF21 treatment attenuated cardiac hypertrophy and fibrosis and improved cardiac function. Thus, FGF21 emerges as a therapeutic target for pathological cardiac hypertrophy.

Abstract 17132: IRE1α Protects Against Cardiac Fibrosis via Regulating Selective Transcript Degradation

Background: Upon cardiac injury, cardiac fibroblasts become activated and differentiate intomyofibroblasts to maintain cardiac structure and function through secretion of stabilizingextracellular matrix (ECM) components. However, if this persists, it leads to stiffening of themyocardium and heart failure. The unfolded protein response (UPR) responds to an increaseddemand for folding of nascent secreted ECM proteins trafficked through the endoplasmicreticulum such as collagen. IRE1α, the most evolutionarily conserved arm of the UPR, actsadaptively both by enhancing protein folding of secreted proteins and reducing prolongedincreases in protein synthesis as occurs during pathologic cardiac fibrosis. Hypothesis: IRE1α protects against cardiac fibrosis via inhibiting pathologic sustained fibroblastactivation. Methods: Primary cardiac fibroblasts (CFBs) were cultured with transforming growth factor beta(TGF-β) to promote activation, in vitro . Small molecule targeting approaches were utilized tospecifically inhibit or activate IRE1α. IRE1α-floxed mice were injected with AAV9 to knockoutIRE1α specifically in myofibroblasts (IRE1mfbKO) and were subjected to transverse aorticconstriction (TAC) as a model of heart failure to stimulate fibroblast activation, in vivo . Results: While small molecule activation of IRE1α significantly blunted CFB activation inresponse to TGF-β, specific inhibition of IRE1α’s endonuclease domain enhanced CFBresponsiveness to TGF-β. Importantly, this effect occurred independent of IRE1α’s canonicalmechanism of activating the transcription factor, XBP1, implicating regulated IRE1α-dependentdecay (RIDD) as a novel anti-fibrotic protective mechanism. Transcriptomics of CFBs identifieda novel “RIDD-ome” of pro-fibrotic mRNA targets of IRE1α-mediated degradation. Finally, inresponse to TAC, IRE1mfbKO mice exhibited an exacerbated decrease in cardiac function andmarked increase interstitial fibrosis. Conclusions: IRE1α protects against pathologic differentiation of CFBs in response to TGF-β or TACvia regulating the degradation of a novel subset of pro-fibrotic transcripts. Thus, small moleculetargeting of IRE1α represents a novel approach to mitigating cardiac fibrosis.

Corin deficiency impairs cardiac function in mouse models of heart failure.

Corin is a protease in the natriuretic peptide system. Deleterious CORIN variants are associated with hypertension and heart disease. It remains unclear if and to what extent corin deficiency may contribute to heart failure (HF). Corin knockout (KO) mice were used as a model. Cardiac function was assessed by echocardiography and tissue analysis in Corin KO mice at different ages or subjected to transverse aortic constriction (TAC), which increased pressure overload. Heart and lung tissues were analyzed for cardiac hypertrophy and lung edema using wheat germ agglutinin, Sirius red, Masson's trichrome, and Prussian blue staining. Recombinant corin was tested for its effect on cardiac function in the TAC-operated Corin KO mice. Selected gene expression in the heart was examined by RT-PCR. ELISA was used to analyze factors in plasma. Corin KO mice had progressive cardiac dysfunction with cardiac hypertrophy and fibrosis after 9 months of age, likely due to chronic hypertension. When Corin KO mice were subjected to TAC at 10-12 weeks of age, cardiac function decreased more rapidly than in similarly treated wild-type mice. When the TAC-operated Corin KO mice were treated with recombinant corin protein, cardiac dysfunction, hypertrophy, and fibrosis were ameliorated. The corin treatment also decreased the gene expression associated with cardiac hypertrophy and fibrosis, increased plasma cGMP levels, lowered plasma levels of N-terminal pro-atrial natriuretic peptide, angiotensin II, and aldosterone, and lessened lung edema in the Corin KO mice subjected to TAC. Corin deficiency impairs cardiac function and exacerbates HF development in mice. Corin protein may be used to reduce cardiac hypertrophy and fibrosis, suppress the renin-angiotensin-aldosterone system, and improve cardiac function in HF.

Abstract P2097: Ablation Of Fibroblast Human Antigen R (HuR) Mitigates Cardiac Fibrosis

Background: Cardiac fibrosis is a common pathology associated with various heart diseases and is attributed to the activation of resident fibroblasts. Limiting fibroblast-to-myofibroblast differentiation and proliferation could be a pragmatic approach to mitigate adverse cardiac outcomes. Recent studies have shown that RNA-binding proteins are critical in the post-transcriptional regulation of fibroblast-to-myofibroblast differentiation. Human antigen R (HuR), an RNA-binding protein, modulates post-transcriptional gene expression in various pathophysiology, including heart diseases. However, the direct role of cardiac myofibroblast HuR is largely unknown. Methods and Results: To investigate the role of myofibroblast-specific HuR in cardiac fibrosis, we generated a tamoxifen-inducible periostin-Cre-driven myofibroblast-specific HuR knockout (KO) mouse. Control and KO mice were subjected to transverse aortic constriction (TAC), and cardiac functions were assessed by echocardiography. Histological and morphometric analyses were used to evaluate cardiac fibrosis and remodeling eight weeks post-injury. Our results demonstrated that myofibroblast-specific deletion of HuR limited cardiac fibrosis and preserved cardiac functions in HuR knockout mice compared to the control group. Further, the knockdown of HuR in human cardiac fibroblasts suppressed myofibroblast differentiation after treatment with TGF-β1 (10 ng/ml), as observed by a decrease in cell migration and production of extracellular matrix-associated molecules. Interestingly, an in vivo BrdU labeling assay showed a reduction in myofibroblast proliferation in KO mice subjected to TAC compared to the control. Further investigation confirmed that HuR regulates cyclin D1 mRNA stability in TGF-β1-stimulated cardiac fibroblasts, and deletion of HuR restricts myofibroblast proliferation in a cyclin D1-dependent manner. Conclusion: Our findings indicate that HuR plays a central role in the post-transcriptional regulation of cardiac fibrosis and could be a potential therapeutic target for clinical applications.

Abstract P2047: Fast Skeletal Myosin Binding Protein-C Expression Exacerbates Dysfunction In Heart Failure

Background: During heart failure, gene and protein expression profiles undergo extensive compensatory and pathological remodeling. We have previously observed that fast skeletal myosin binding protein-C (fMyBP-C) is upregulated in diseased mouse hearts. While fMyBP-C shares significant homology with its cardiac paralog, cardiac myosin binding protein-C (cMyBP-C), there are key differences in the functional differences which may affect cardiac function. Also, it is unknown if the fMyBP-C expression in the heart is a pathological or compensatory response. Objectives: We aim to elucidate the consequence of increased fMyBP-C expression in the heart. Methods: To determine the sufficiency of fMyBP-C to cause cardiac dysfunction, we generated cardiac-specific fMyBP-C over-expression mice. These mice were further crossed into a cMyBP-C null model to assess the effect of fMyBP-C in the heart in the absence of cMyBP-C function. Finally, fMyBP-C knockout mice were subjected to transverse aortic constriction (TAC) to define the requirement of fMyBP-C during heart failure development. Results: We confirmed the upregulation of fMyBP-C in several models of cardiac disease. Genetic over-expression of low levels of fMyBP-C caused mild cardiac remodeling and sarcomere dysfunction. High expression of fMyBP-C in a heart failure model further exacerbated the cardiac disease, hypertrophy and fibrosis. On the other hand, following 8 weeks of TAC, fMyBP-C null mice demonstrated greater protection against heart failure development. Mechanistically, this may be due to the greater instability of the myosin super-relaxed state compared to wild-type littermates. Conclusions: These findings suggest that the elevated expression of fMyBP-C in diseased hearts is a pathological response. Targeted therapies to prevent upregulation of fMyBP-C may prove beneficial in the treatment of heart failure.

Cinnamic acid mitigates left ventricular hypertrophy and heart failure in part through modulating FTO-dependent N6-methyladenosine RNA modification in cardiomyocytes

Left ventricular hypertrophy leads to heart failure, a serious medical condition associated with high rates of hospitalization and mortality. Limited success with the existing pharmacological treatments necessitates the development of mechanisms-based new therapies to better control the progression from left ventricular hypertrophy to heart failure. The current work investigated the pharmacological potentials and mechanisms of naturally occurring cinnamic acid in the treatment of left ventricular hypertrophy and heart failure. The in vitro findings reveal that cinnamic acid attenuates the hypertrophic responses and mitochondrial dysfunction in the phenylephrine (PE)-stimulated cardiomyocytes. Furthermore, cinnamic acid offsets PE-induced increases in N6-methyladenosine (m6A) RNA modification and reductions in the expression of the key m6A demethylase FTO in cardiomyocytes. Most importantly, FTO knockdown abrogates anti-hypertrophic and mitochondrial protective effects of cinnamic acid in the PE-stimulated cardiomyocytes. The in vivo results further demonstrate that cinnamic acid mitigates left ventricular hypertrophy, left ventricular systolic dysfunction and ultrastructural impairment of cardiomyocyte mitochondria and myofibrils in the mice subjected to transverse aortic constriction (TAC)-induced pressure overload. Moreover, FTO knockdown abolishes these beneficial effects of cinnamic acid in the TAC mice. In conclusion, the work here demonstrates for the first time that cinnamic acid is effective at mitigating pressure overload-induced left ventricular hypertrophy and heart failure in part by modulating the expression of FTO and the level of FTO-dependent m6A RNA modification in cardiomyocytes. These novel findings warrant further evaluation of cinnamic acid as a pharmacological agent/component to complement the existing treatment of pressure overload-mediated left ventricular hypertrophy and heart failure.

Integrin beta-like 1 mediates fibroblast-cardiomyocyte crosstalk to promote cardiac fibrosis and hypertrophy.

Crosstalk between fibroblasts and cardiomyocytes (CMs) plays a critical role in cardiac remodelling during heart failure (HF); however, the underlying molecular mechanisms remain obscure. Recently, a secretory protein, Integrin beta-like 1 (ITGBL1) was revealed to have detrimental effects on several diseases, such as tumours, pulmonary fibrosis, and hepatic fibrosis; whereas the effect of ITGBL1 on HF is unclear. The purpose of this study was to evaluate its contribution to volume overload-induced remodelling. In this study, we identified ITGBL1 was highly expressed in varied heart diseases and validated in our TAC mice model, especially in fibroblasts. To investigate the role of ITGBL1 in in vitro cell experiments, neonatal rat fibroblasts (NRCFs) and cardiomyocytes (NRCMs) were performed for further study. We found that in comparison to NRCMs, NRCFs expressed high levels of ITGBL1. Meanwhile, ITGBL1 was upregulated in NRCFs, but not in NRCMs following angiotensin-II (AngII) or phenylephrine stimulation. Furthermore, ITGBL1 overexpression promoted NRCFs activation, whereas knockdown of ITGBL1 alleviated NRCFs activation under AngII treatment. Moreover, NRCFs-secreted ITGBL1 could induce NRCMs hypertrophy. Mechanically, ITGBL1-NME/NM23 nucleoside diphosphate kinase 1 (NME1)-TGF-β-Smad2/3 and Wnt signalling pathways were identified to mediate NRCFs activation and NRCMs hypertrophy, respectively. Finally, the knockdown of ITGBL1 in mice subjected to transverse aortic constriction (TAC) surgery recapitulated the in vitro findings, demonstrating blunted cardiac fibrosis, hypertrophy, and improved cardiac function. ITGBL1 is an important functional mediator between fibroblast-cardiomyocyte crosstalk and could be an effective target for cardiac remodelling in HF patients.

LCZ696 (sacubitril/valsartan) inhibits pulmonary hypertension induced right ventricular remodeling by targeting pyruvate dehydrogenase kinase 4

BackgroundRight ventricular (RV) function is a major prognostic factor in patients with cardiopulmonary disease. Effective medical therapies are available for left heart failure, but they are usually less effective or even ineffective in right heart failure. Here, we tested the hypothesis that LCZ696 (sacubitril/valsartan) can attenuate pressure overload-induced RV remodeling by inhibiting pyruvate dehydrogenase kinase 4 (PDK4). MethodsAdult male C57 mice were subjected to transverse aortic constriction (TAC), pulmonary artery constriction (PAC), or sham surgery. Bioinformatics analysis was used to screen for common differentially expressed genes (DEGs) between TAC and PAC. Chemical compounds targeting DEGs were predicted by molecular docking analysis. Effects of LCZ696 on PAC-induced RV remodeling and the associated PDK4-related mechanisms were investigated. ResultsWe found 60 common DEGs between PAC and TAC, and Pdk4 was one of the downregulated DEGs. From 47 chemical compounds with potential cardiovascular activity and PDK4 protein binding ability, we selected LCZ696 to treat PAC-induced RV remodeling because of its high docking score for binding PDK4. Compared with vehicle-treated PAC mice, LCZ696-treated mice had significantly smaller RV wall thickness and RV diameters, less myocardial fibrosis, lower expression of PDK4 protein, and less phosphorylation of glycogen synthase kinase-3β (p-GSK3β). In PAC mice, overexpression of Pdk4 blocked the inhibitory effect of LCZ696 on RV remodeling, whereas conditional knockout of Pdk4 attenuated PAC-induced RV remodeling. ConclusionsPdk4 is a common therapeutic target for pressure overload-induced left ventricular and RV remodeling, and LCZ696 attenuates RV remodeling by downregulating Pdk4 and inhibiting PDK4/p-GSK3β signal.

GSDMD-mediated pyroptosis promotes cardiac remodeling in pressure overload

ABSTRACT Background Gasdermin D (GSDMD) forms membrane pores to execute pyroptosis. But the mechanism of how cardiomyocyte pyroptosis induces cardiac remodeling in pressure overload remains unclear. We investigated the role of GSDMD-mediated pyroptosis in the pathogenesis of cardiac remodeling in pressure overload. Methods Wild-type (WT) and cardiomyocyte-specific GSDMD-deficient (GSDMD-CKO) mice were subjected to transverse aortic constriction (TAC) to induce pressure overload. Four weeks after surgery, left ventricular structure and function were evaluated by echocardiographic, invasive hemodynamic and histological analysis. Pertinent signaling pathways related to pyroptosis, hypertrophy and fibrosis were investigated by histochemistry, RT-PCR and western blotting. The serum levels of GSDMD and IL-18 collected from healthy volunteers or hypertensive patients were measured by ELISA. Results We found TAC induced cardiomyocyte pyroptosis and release of pro-inflammatory cytokines IL-18. The serum GSDMD level was significantly higher in hypertensive patients than in healthy volunteers, and induced more dramatic release of mature IL-18. GSDMD deletion remarkably mitigated TAC-induced cardiomyocyte pyroptosis. Furthermore, GSDMD deficiency in cardiomyocytes significantly reduced myocardial hypertrophy and fibrosis. The deterioration of cardiac remodeling by GSDMD-mediated pyroptosis was associated with activating JNK and p38 signaling pathways, but not ERK or Akt signaling pathway. Conclusion In conclusion, our results demonstrate that GSDMD serves as a key executioner of pyroptosis in cardiac remodeling induced by pressure overload. GSDMD-mediated pyroptosis activates JNK and p38 signaling pathways, and this may provide a new therapeutic target for cardiac remodeling induced by pressure overload.

Fibroblast growth factor 18 alleviates stress-induced pathological cardiac hypertrophy in male mice

Fibroblast growth factor-18 (FGF18) has diverse organ development and damage repair roles. However, its role in cardiac homeostasis following hypertrophic stimulation remains unknown. Here we investigate the regulation and function of the FGF18 in pressure overload (PO)-induced pathological cardiac hypertrophy. FGF18 heterozygous (Fgf18+/−) and inducible cardiomyocyte-specific FGF18 knockout (Fgf18-CKO) male mice exposed to transverse aortic constriction (TAC) demonstrate exacerbated pathological cardiac hypertrophy with increased oxidative stress, cardiomyocyte death, fibrosis, and dysfunction. In contrast, cardiac-specific overexpression of FGF18 alleviates hypertrophy, decreased oxidative stress, attenuates cardiomyocyte apoptosis, and ameliorates fibrosis and cardiac function. Tyrosine-protein kinase FYN (FYN), the downstream factor of FGF18, was identified by bioinformatics analysis, LC-MS/MS and experiment validation. Mechanistic studies indicate that FGF18/FGFR3 promote FYN activity and expression and negatively regulate NADPH oxidase 4 (NOX4), thereby inhibiting reactive oxygen species (ROS) generation and alleviating pathological cardiac hypertrophy. This study uncovered the previously unknown cardioprotective effect of FGF18 mediated by the maintenance of redox homeostasis through the FYN/NOX4 signaling axis in male mice, suggesting a promising therapeutic target for the treatment of cardiac hypertrophy.

Impairment of β-adrenergic regulation and exacerbation of pressure-induced heart failure in mice with mutations in phosphoregulatory sites in the cardiac CaV1.2 calcium channel.

The cardiac calcium channel CaV1.2 conducts L-type calcium currents that initiate excitation-contraction coupling and serves as a crucial mediator of β-adrenergic regulation of the heart. We evaluated the inotropic response of mice with mutations in C-terminal phosphoregulatory sites under physiological levels of β-adrenergic stimulation in vivo, and we assessed the impact of combining mutations of C-terminal phosphoregulatory sites with chronic pressure-overload stress. Mice with Ser1700Ala (S1700A), Ser1700Ala/Thr1704Ala (STAA), and Ser1928Ala (S1928A) mutations had impaired baseline regulation of ventricular contractility and exhibited decreased inotropic response to low doses of β-adrenergic agonist. In contrast, treatment with supraphysiogical doses of agonist revealed substantial inotropic reserve that compensated for these deficits. Hypertrophy and heart failure in response to transverse aortic constriction (TAC) were exacerbated in S1700A, STAA, and S1928A mice whose β-adrenergic regulation of CaV1.2 channels was blunted. These findings further elucidate the role of phosphorylation of CaV1.2 at regulatory sites in the C-terminal domain for maintaining normal cardiac homeostasis, responding to physiological levels of β-adrenergic stimulation in the fight-or-flight response, and adapting to pressure-overload stress.