IRX2 regulates angiotensin II-induced cardiac fibrosis by transcriptionally activating EGR1 in male mice.

Cardiac fibrosis is a common feature of chronic heart failure. Iroquois homeobox (IRX) family of transcription factors plays important roles in heart development; however, the role of IRX2 in cardiac fibrosis has not been clarified. Here we report that IRX2 expression is significantly upregulated in the fibrotic hearts. Increased IRX2 expression is mainly derived from cardiac fibroblast (CF) during the angiotensin II (Ang II)-induced fibrotic response. Using two CF-specific Irx2-knockout mouse models, we show that deletion of Irx2 in CFs protect against pathological fibrotic remodelling and improve cardiac function in male mice. In contrast, Irx2 gain of function in CFs exaggerate fibrotic remodelling. Mechanistically, we find that IRX2 directly binds to the promoter of the early growth response factor 1 (EGR1) and subsequently initiates the transcription of several fibrosis-related genes. Our study provides evidence that IRX2 regulates the EGR1 pathway upon Ang II stimulation and drives cardiac fibrosis.

Matrine attenuates pathological cardiac fibrosis via RPS5/p38 in mice.

Pathological cardiac fibrosis is a common feature in multiple cardiovascular diseases that contributes to the occurrence of heart failure and life-threatening arrhythmias. Our previous study demonstrated that matrine could attenuate doxorubicin-induced oxidative stress and cardiomyocyte apoptosis. In this study, we investigated the effect of matrine on cardiac fibrosis. Mice received aortic banding (AB) operation or continuous injection of isoprenaline (ISO) to generate pathological cardiac fibrosis and then were exposed to matrine lavage (200 mg·kg-1·d-1) or an equal volume of vehicle as the control. We found that matrine lavage significantly attenuated AB or ISO-induced fibrotic remodeling and cardiac dysfunction. We also showed that matrine (200 µmol/L) significantly inhibited the proliferation, migration, collagen production, and phenotypic transdifferentiation of cardiac fibroblasts. Mechanistically, matrine suppressed p38 activation in vivo and in vitro, and overexpression of constitutively active p38 completely abolished the protective effects of matrine. We also demonstrated that ribosomal protein S5 (RPS5) upregulation was responsible for matrine-mediated inhibition on p38 and fibrogenesis. More importantly, matrine was capable of ameliorating preexisting cardiac fibrosis in mice. In conclusion, matrine treatment attenuates cardiac fibrosis by regulating RPS5/p38 signaling in mice, and it might be a promising therapeutic agent for treating pathological cardiac fibrosis.

Secreted frizzled-related protein 2 prevents pressure-overload-induced cardiac hypertrophy by targeting the Wnt/ß-catenin pathway.

BACKGROUND AND AIM: Secreted frizzled-related protein 2 (sFRP2) has been reported to be involved in cardiovascular diseases. However, its role in cardiac hypertrophy induced by pressure overload is still elusive. We aimed to examine the role of sFRP2 in the development of cardiac hypertrophy in vivo and in vitro. METHODS AND RESULTS: Following cardiac hypertrophy stimulated by aortic banding (AB), the expression of sFRP2 was downregulated in the hypertrophic ventricle. Adeno-associated virus 9 (AAV9) was injected through the tail vein to overexpress sFRP2 in the mouse myocardium. Overexpression of sFRP2 alleviated cardiomyocyte hypertrophy and interstitial fibrosis, as identified by the reduced cardiomyocyte cross-sectional area, heart weight/body weight ratio, and left ventricular (LV) collagen ratio. Additionally, sFRP2 decreased cardiomyocyte apoptosis induced by pressure overload. Western blot showed that sFRP2 prevented the expression of active ß-catenin. The Wnt/ß-catenin agonist LiCl (1 mmol/kg) abolished the inhibitory effects of sFRP2 on cardiac hypertrophy and apoptosis, as evidenced by the increased cross-sectional area and LV collagen ratio and the deterioration of echocardiographic data. CONCLUSION: Our study indicated that decreased sFRP2 levels were observed in failing mouse hearts. Overexpression of sFRP2 attenuated myocyte hypertrophy and interstitial fibrosis induced by hypertrophic stimuli by inhibiting the Wnt/ß-catenin pathway. We revealed that sFRP2 may be a promising therapeutic target for the development of cardiac remodeling.

Osteocrin attenuates inflammation, oxidative stress, apoptosis, and cardiac dysfunction in doxorubicin-induced cardiotoxicity.

BACKGROUND: Inflammation, oxidative stress, and apoptosis contribute to the evolution of doxorubicin (DOX)-induced cardiotoxicity. Osteocrin (OSTN) is a novel secretory peptide mainly derived from the bone and skeletal muscle, and plays critical roles in regulating bone growth and physical endurance. Inspiringly, OSTN was also reported to be abundant in the myocardium that functioned as a therapeutic agent against cardiac rupture and congestive heart failure in mice after myocardial infarction. Herein, we investigated the role and potential mechanism of OSTN in DOX-induced cardiotoxicity. METHODS: Cardiac-restrict OSTN overexpression was performed by the intravenous injection of a cardiotropic AAV9 vector, and subsequently the mice received 15 mg/kg DOX injection (i.p., once) to induce acute cardiac injury. Besides, H9C2 cell lines were used to assess the possible role of OSTN in vitro by incubating with recombinant human OSTN or small interfering RNA against Ostn (siOstn). To clarify the involvement of protein kinase G (PKG), KT5823 and siPkg were used in vivo and in vitro. Mice were also administrated intraperitoneally with 5 mg/kg DOX weekly for consecutive 3 weeks at a cumulative dose of 15 mg/kg to mimic the cardiotoxic effects upon chronic DOX exposure. RESULTS: OSTN treatment notably attenuated, whereas OSTN silence exacerbated inflammation, oxidative stress, and cardiomyocyte apoptosis in DOX-treated H9C2 cells. Besides, cardiac-restrict OSTN-overexpressed mice showed an alleviated cardiac injury and malfunction upon DOX injection. Mechanistically, we found that OSTN activated PKG, while PKG inhibition abrogated the beneficial effect of OSTN in vivo and in vitro. As expected, OSTN overexpression also improved cardiac function and survival rate in mice after chronic DOX treatment. CONCLUSIONS: OSTN protects against DOX-elicited inflammation, oxidative stress, apoptosis, and cardiac dysfunction via activating PKG, and cardiac gene therapy with OSTN provides a novel therapeutic strategy against DOX-induced cardiotoxicity.

Levosimendan Protects against Doxorubicin-Induced Cardiotoxicity by Regulating the PTEN/Akt Pathway.

BACKGROUND AND AIMS: Myocyte apoptosis plays a critical role in the development of doxorubicin- (DOX-) induced cardiotoxicity. In addition to its cardiotonic effect, laboratory evidence indicates that levosimendan can inhibit apoptosis, but its role in DOX-induced cardiac injury remains unclear. Therefore, the present study is aimed at exploring whether levosimendan could attenuate DOX-induced cardiotoxicity. METHODS: Levosimendan (1 mg/kg) was administered to mice through oral gavage once daily for 4 weeks, and the mice were also subjected to an intraperitoneal injection of DOX (5 mg/kg) or saline, once a week for 4 weeks, to create a chronic model of DOX-induced cardiotoxicity. A morphological examination and biochemical analysis were used to evaluate the effects of levosimendan. H9C2 cells were used to verify the protective role of levosimendan in vitro. And an Akt inhibitor was utilized to verify the cardioprotection of levosimendan. RESULTS: Levosimendan reduced the cardiac dysfunction and attenuated the myocardial apoptosis induced by DOX in vivo and in vitro. Levosimendan also inhibited the activation of phosphatase and tensin homolog (PTEN) and upregulated P-Akt expression both in vivo and in vitro. And inhibition of Akt abolished the cardioprotection of levosimendan in vitro. CONCLUSION: Levosimendan may protect against DOX-induced cardiotoxicity via modulation of the PTEN/Akt signaling pathway.

AdipoRon, an adiponectin receptor agonist, attenuates cardiac remodeling induced by pressure overload.

AdipoRon, a small-molecule adiponectin receptor (AdipoR) agonist, has been reported to be implicated in cardiovascular diseases. However, its role in pressure-overload-induced cardiac remodeling is still elusive. To elucidate the role of AdipoRon in the pathogenesis of cardiac remodeling in vivo and vitro, in the left ventricle of human end-stage heart failure, the expression of AdipoR2 is upregulated. Meanwhile, increased expression of AdipoR2 was also observed in mice failing hearts. Oral administration of AdipoRon alleviated cardiac hypertrophy and fibrosis induced by pressure overload, as evidenced by the beneficial change of cross-sectional area of cardiomyocytes, heart weight-to-body weight ratio, gene expression of hypertrophic markers, ventricle collagen ratio, and cardiac function. The AMPKα activation mediated by AdipoRon significantly inhibited AngII-induced TGF-ß1 expression and cardiac fibroblast differentiation, and these inhibitory effects were abrogated by treatment with the AMPK inhibitor Compound C. Consistent with the above results, AdipoRon abolished the ability to retard AngII-induced TGF-ß1 expression in AMPKα2-/- cardiac fibroblasts. In AMPKα2-/- mice subjected to aortic banding, AdipoRon abolished the protective effect, as indicated by increased cross-sectional area, cardiac collagen ratio, and cardiac dysfunction. Our results demonstrated that AdipoR2 expression was markedly increased in the failing hearts. AdipoRon inhibited TGF-ß1 expression and myofibroblast differentiation in AMPKα-dependent manner in vitro. In line with the vitro results, AMPKα2-/- mice markedly abrogated the inhibitory effects of AdipoRon in cardiac remodeling. These results indicated AdipoRon may hold promise of an effective therapy against pressure-overload-induced cardiac remodeling. KEY MESSAGES: • The increased expression of AdipoR2 is observed in human and mice failing hearts, the changeable expression of AdipoR suggests the possible role of AdipoR in cardiac remodeling. • Oral administration of AdipoRon alleviates cardiac hypertrophy and fibrosis induced by pressure overload, and AMPKα activation mediated by AdipoRon significantly inhibited AngII-induced TGF-ß1 expression and cardiac fibroblast differentiation. • These findings provide new mechanistic insight and open new therapeutic pathways for heart failure.

Overexpression of CTRP3 protects against sepsis-induced myocardial dysfunction in mice.

C1q/tumor necrosis factor-related protein-3 (CTRP3) shows striking homologies of genomic structure to the adiponectin. In this study, we aimed to investigate the protective role of CTRP3 against sepsis-induced cardiomyopathy. Here, we overexpressed CTRP3 in myocardium by direct intramyocardial injection and constructed a model of lipopolysaccharide (LPS)-induced sepsis in mice. Our results demonstrated that cardiac-specific overexpression of CTRP3 remarkably attenuated myocardial dysfunction and increased the phosphorylation level of AMPKα during LPS-induced sepsis. The anti-inflammatory effects of CTRP3, as determined by decreased mRNA levels of TNF-α, IL-6 and a lower protein expression of phosphorylated NF-κB p65 and IκBα, was detected in mice following LPS treatment. Additionally, CTRP3 suppressed cardiac apoptosis induced by LPS in mice as indicated by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) staining and western blot for Cleaved-caspase3, Bax and Bcl-2. In conclusion, CTRP3 could protect against sepsis-induced myocardial dysfunction in mice. The cardioprotective effects of CTRP3 might be mediated by activating AMPKα signaling pathway and blunting inflammatory response and apoptosis.

Therapeutic Potential of Polyphenols in Cardiac Fibrosis.

Cardiac fibrosis, in response to injury and stress, is central to a broad constellation of cardiovascular diseases. Fibrosis decreases myocardial wall compliance due to extracellular matrix (ECM) accumulation, leading to impaired systolic and diastolic function and causing arrhythmogenesis. Although some conventional drugs, such as ß-blockers and renin-angiotensin-aldosterone system (RAAS) inhibitors, have been shown to alleviate cardiac fibrosis in clinical trials, these traditional therapies do not tend to target all the fibrosis-associated mechanisms, and do not hamper the progression of cardiac fibrosis in patients with heart failure. Polyphenols are present in vegetables, fruits, and beverages and had been proposed as attenuators of cardiac fibrosis in different models of cardiovascular diseases. Together with results found in the literature, we can show that some polyphenols exert anti-fibrotic and myocardial protective effects by mediating inflammation, oxidative stress, and fibrotic molecular signals. This review considers an overview of the mechanisms of cardiac fibrosis, illustrates their involvement in different animal models of cardiac fibrosis treated with some polyphenols and projects the future direction and therapeutic potential of polyphenols on cardiac fibrosis.

Pioglitazone Alleviates Cardiac Fibrosis and Inhibits Endothelial to Mesenchymal Transition Induced by Pressure Overload.

BACKGROUND/AIMS: Cardiac fibrosis, characterized by an unbalanced production and degradation of extracellular matrix components, is a common pathophysiology of multiple cardiovascular diseases. Recent studies suggested that endothelial to mesenchymal transition (EndMT) could be a source of activated fibroblasts and contribute to cardiac fibrosis. Here, the role of pioglitazone (PIO) in cardiac fibrosis and EndMT was elaborated. METHODS: Male C57BL/6 mice were subjected to aortic banding (AB), which was used to construct a model of pressure overload-induced cardiac hypertrophy. PIO and GW9662 was given for 4 weeks to detect the effects of PIO on EndMT. RESULTS: Our results showed PIO treatment attenuated cardiac hypertrophy, dysfunction and fibrosis response to pressure overload. Mechanistically, PIO suppressed the TGF-ß/Smad signaling pathway activated by 4-week AB surgery. Moreover, PIO dramatically inhibited EndMT in vivo and in vitro stimulated by pressure overload or TGF-ß. A selective antagonist of PPAR-γ, GW9662, neutralized the anti-fibrotic effect and abolished the inhibitory effect of EndMT during the treatment of PIO. CONCLUSION: Our data implied that PIO exerts an alleviative effect on cardiac fibrosis via inhibition of the TGF-ß/Smad signaling pathway and EndMT by activating PPAR-γ.

Rosmarinic acid attenuates cardiac fibrosis following long-term pressure overload via AMPKα/Smad3 signaling.

Agonists of peroxisome proliferator-activated receptor gamma (PPAR-γ) can activate 5' AMP-activated protein kinase alpha (AMPKα) and exert cardioprotective effects. A previous study has demonstrated that rosmarinic acid (RA) can activate PPAR-γ, but its effect on cardiac remodeling remains largely unknown. Our study aimed to investigate the effect of RA on cardiac remodeling and to clarify the underlying mechanism. Mice were subjected to aortic banding to generate pressure overload induced cardiac remodeling and then were orally administered RA (100 mg/kg/day) for 7 weeks beginning 1 week after surgery. The morphological examination, echocardiography, and molecular markers were used to evaluate the effects of RA. To ascertain whether the beneficial effect of RA on cardiac fibrosis was mediated by AMPKα, AMPKα2 knockout mice were used. Neonatal rat cardiomyocytes and fibroblasts were separated and cultured to validate the protective effect of RA in vitro. RA-treated mice exhibited a similar hypertrophic response as mice without RA treatment, but had an attenuated fibrotic response and improved cardiac function after pressure overload. Activated AMPKα was essential for the anti-fibrotic effect of RA via inhibiting the phosphorylation and nuclear translocation of Smad3 in vivo and in vitro, and AMPKα deficiency abolished RA-mediated protective effects. Small interfering RNA against Ppar-γ (siPpar-γ) and GW9662, a specific antagonist of PPAR-γ, abolished RA-mediated AMPKα phosphorylation and alleviation of fibrotic response in vitro. RA attenuated cardiac fibrosis following long-term pressure overload via AMPKα/Smad3 signaling and PPAR-γ was required for the activation of AMPKα. RA might be a promising therapeutic agent against cardiac fibrosis.

Nobiletin, a Polymethoxy Flavonoid, Protects Against Cardiac Hypertrophy Induced by Pressure-Overload via Inhibition of NAPDH Oxidases and Endoplasmic Reticulum Stress.

BACKGROUND/AIMS: An increase in oxidative stress has been implicated in the pathophysiology of pressure-overload induced cardiac hypertrophy. Nobiletin (NOB), extracted from the fruit peel of citrus, possesses anti-oxidative property. Our study aimed to investigate the protective role of NOB in the progression of cardiac hypertrophy in vivo and in vitro. METHODS: Mice received aortic banding (AB) operation to induce cardiac hypertrophy. Experimental groups were as follows: sham+vehicle (VEH/SH), sham+NOB (NOB/SH), AB+vehicle (VEH/AB), and AB+ NOB (NOB/AB). Animals (n = 15 per group) were treated with vehicle or NOB (50 mg/kg) for 4 weeks after disease onset. RESULTS: NOB prevented cardiac hypertrophy induced by aortic banding (AB), as assessed by the cross-sectional area of cardiomyocytes, heart weight-to-body weight ratio, gene expression of hypertrophic markers and cardiac function. In addition, NOB supplementation blunted the increased expression of NAPDH oxidase (NOX) 2 and NOX4 and mitigated endoplasmic reticulum (ER) stress and myocyte apoptosis in cardiac hypertrophy. Furthermore, NOB treatment attenuated the neonatal rat cardiomyocyte (NRCM) hypertrophic response stimulated by phenylephrine (PE) and alleviated ER stress. However, our data showed that NOB dramatically inhibited NOX2 expression but not NOX4 in vitro. Finally, we found that knockdown of NOX2 attenuated ER stress in NRCMs stimulated by PE. CONCLUSIONS: Inhibition of oxidative and ER stress by NOB in the myocardium may represent a potential therapy for cardiac hypertrophy. Moreover, there is a direct role of NOX2 in regulating ER stress stimulated by PE.

CTRP3 attenuates cardiac dysfunction, inflammation, oxidative stress and cell death in diabetic cardiomyopathy in rats.

AIMS/HYPOTHESIS: Oxidative stress, inflammation and cell death are closely involved in the development of diabetic cardiomyopathy (DCM). C1q/tumour necrosis factor-related protein-3 (CTRP3) has anti-inflammatory properties but its role in DCM remains largely unknown. The aims of this study were to determine whether CTRP3 could attenuate DCM and to clarify the underlying mechanisms. METHODS: Streptozotocin (STZ) was injected intraperitoneally to induce diabetes in Sprague-Dawley rats. Cardiomyocyte-specific CTRP3 overexpression was achieved using an adeno-associated virus system 12 weeks after STZ injection. RESULTS: CTRP3 expression was significantly decreased in diabetic rat hearts. Knockdown of CTRP3 in cardiomyocytes at baseline resulted in increased oxidative injury, inflammation and apoptosis in vitro. Cardiomyocyte-specific overexpression of CTRP3 decreased oxidative stress and inflammation, attenuated myocyte death and improved cardiac function in rats treated with STZ. CTRP3 significantly activated AMP-activated protein kinase α (AMPKα) and Akt (protein kinase B) in H9c2 cells. CTRP3 protected against high-glucose-induced oxidative stress, inflammation and apoptosis in vitro. AMPKα deficiency abolished the protective effects of CTRP3 in vitro and in vivo. Furthermore, we found that CTRP3 activated AMPKα via the cAMP-exchange protein directly activated by cAMP (EPAC)-mitogen-activated protein kinase kinase (MEK) pathway. CONCLUSIONS/INTERPRETATION: CTRP3 protected against DCM via activation of the AMPKα pathway. CTRP3 has therapeutic potential for the treatment of DCM.

Bezafibrate Attenuates Pressure Overload-Induced Cardiac Hypertrophy and Fibrosis.

Background. Peroxisome proliferator-activated receptor-α (PPAR-α) is closely associated with the development of cardiac hypertrophy. Previous studies have indicated that bezafibrate (BZA), a PPAR-α agonist, could attenuate insulin resistance and obesity. This study was designed to determine whether BZA could protect against pressure overload-induced cardiac hypertrophy. Methods. Mice were orally given BZA (100 mg/kg) for 7 weeks beginning 1 week after aortic banding (AB) surgery. Cardiac hypertrophy was assessed based on echocardiographic, histological, and molecular aspects. Moreover, neonatal rat ventricular cardiomyocytes (NRVMs) were used to investigate the effects of BZA on the cardiomyocyte hypertrophic response in vitro. Results. Our study demonstrated that BZA could alleviate cardiac hypertrophy and fibrosis in mice subjected to AB surgery. BZA treatment also reduced the phosphorylation of protein kinase B (AKT)/glycogen synthase kinase-3ß (GSK3ß) and mitogen-activated protein kinases (MAPKs). BZA suppressed phenylephrine- (PE-) induced hypertrophy of cardiomyocyte in vitro. The protective effects of BZA were abolished by the treatment of the PPAR-α antagonist in vitro. Conclusions. BZA could attenuate pressure overload-induced cardiac hypertrophy and fibrosis.

Asiatic Acid Protects against Cardiac Hypertrophy through Activating AMPKα Signalling Pathway.

BACKGROUND: AMPactivated protein kinase α (AMPKα) is closely involved in the process of cardiac hypertrophy. Asiatic acid (AA), a pentacyclic triterpene, was found to activate AMPKα in our preliminary experiment. However, its effects on the development of cardiac hypertrophy remain unclear. The present study was to determine whether AA could protect against cardiac hypertrophy. METHODS: Mice subjected to aortic banding were orally given AA (10 or 30mg/kg) for 7 weeks. In the inhibitory experiment, Compound C was intraperitoneally injected for 3 weeks after surgery. RESULTS: Our results showed that AA markedly inhibited hypertrophic responses induced by pressure overload or angiotensin II. AA also suppressed cardiac fibrosis in vivo and accumulation of collagen in vitro. The protective effects of AA were mediated by activation of AMPKα and inhibition of the mammalian target of rapamycin (mTOR) pathway and extracellular signal-regulated kinase (ERK) in vivo and in vitro. However, AA lost the protective effects after AMPKα inhibition or gene deficiency. CONCLUSIONS: AA protects against cardiac hypertrophy by activating AMPKα, and has the potential to be used for the treatment of heart failure.

Nobiletin attenuates cardiac dysfunction, oxidative stress, and inflammatory in streptozotocin: induced diabetic cardiomyopathy.

Diabetic cardiomyopathy, characterized by the presence of diastolic and/or systolic myocardial dysfunction, is one of the major causes of heart failure. Nobiletin, which is extracted from the fruit peel of citrus, is reported to possess anti-inflammatory, anti-oxidative, and hypolipidemic properties. The purpose of this study was to investigate whether nobiletin exerts the therapeutic effect on streptozotocin-induced diabetic cardiomyopathy (DCM) in mice. 80 experimental male C57BL mice were randomly assigned into four groups: sham + vehicle (VEH/SH), sham + nobiletin (NOB/SH), DCM + vehicle (VEH/DM), and DCM + nobiletin (NOB/DM). Nobiletin treatment ameliorated cardiac dysfunction in the DCM group, as shown by the result of echocardiography and hemodynamic measurements. Nobiletin treatment also blunted the mRNA expression of NADPH oxidase isoforms p67(phox), p22(phox), and p91(phox), and abated oxidative stress. Although administration of diabetic mice with nobiletin did not significantly effect the level of blood glucose, it decreased the TGF-ß1, CTGF, fibronectin, and collagen Iα expressions and blunted cardiac fibrosis. In addition, nobiletin inhibited the activation of c-Jun NH2-terminal kinase (JNK), P38, and NF-κB in the cardiac tissue of diabetic mice. Collectively, our study indicates that treatment with nobiletin mitigates cardiac dysfunction and interstitial fibrosis, and these beneficial of nobiletin may belong to the suppression of JNK, P38, and NF-κB signaling pathways.

Pioglitazone Protected against Cardiac Hypertrophy via Inhibiting AKT/GSK3ß and MAPK Signaling Pathways.

Peroxisome proliferator activated receptor γ (PPARγ) has been closely involved in the process of cardiovascular diseases. This study was to investigate whether pioglitazone (PIO), a PPARγ agonist, could protect against pressure overload-induced cardiac hypertrophy. Mice were orally given PIO (2.5 mg/kg) from 1 week after aortic banding and continuing for 7 weeks. The morphological examination and biochemical analysis were used to evaluate the effects of PIO. Neonatal rat ventricular cardiomyocytes were also used to verify the protection of PIO against hypertrophy in vitro. The results in our study demonstrated that PIO remarkably inhibited hypertrophic response induced by aortic banding in vivo. Besides, PIO also suppressed cardiac fibrosis in vivo. PIO treatment also inhibited the activation of protein kinase B (AKT)/glycogen synthase kinase-3ß (GSK3ß) and mitogen-activated protein kinase (MAPK) in the heart. In addition, PIO alleviated angiotensin II-induced hypertrophic response in vitro. In conclusion, PIO could inhibit cardiac hypertrophy via attenuation of AKT/GSK3ß and MAPK pathways.