ISO-induced Myocardial Fibrosis Research Articles

5-Demethylnobiletin Ameliorates Isoproterenol-Induced Cardiac Fibrosis and Apoptosis by Repressing the Sirt1/FOXO3a/NF-κB and Wnt/β-Catenin Pathways.

Apoptosis and fibrosis are two main factors leading to heart failure. 5-Demethylnobiletin (5-OH-Nob) is a natural polymethoxyflavone derived from the peel of citrus fruits that has many biological effects, such as antioxidative stress and anti-inflammatory effects. Here, we aimed to probe the function and mechanism of 5-OH-Nob in myocardial damage. Primary rat cardiac fibroblasts were exposed to isoproterenol (ISO, 10 µM) to establish an in vitro model of cardiac damage, and ISO (30 mg/kg/d) was used to induce myocardial fibrosis in mice. 5-OH-Nob was used for treatment in vivo and ex vivo. Functional assays revealed that 5-OH-Nob alleviated the apoptosis and fibrosis of cardiac fibroblasts treated with ISO and increased cell viability (p < 0.05). In vivo, 5-OH-Nob treatment ameliorated cardiac injury in ISO-treated mice (p < 0.05). Mechanistically, 5-OH-Nob treatment enhanced Sirt1 expression and suppressed ISO-mediated activation of the FOXO3a/nuclear transcription factor-κB (NF-κB) and Wnt/β-catenin pathways. Furthermore, Sirt1 inhibition attenuated the protective effect of 5-OH-Nob on ISO-induced cardiac apoptosis and fibrosis. Overall, 5-demethylnobiletin mediates the Sirt1/FOXO3a/NF-κB and Wnt/β-catenin pathways to mitigate ISO-induced myocardial fibrosis and apoptosis.

MiR-450a-2-3p targets ERK(1/2) to ameliorate ISO-induced cardiac fibrosis in mice

ObjectiveCardiac fibrosis is an important contributor to atrial fibrillation (AF). Our aim was to identify biomarkers for AF using bioinformatics methods and explore the regulatory mechanism of miR-450a-2-3p in cardiac fibrosis in mice.MethodsTwo datasets, GSE115574 and GSE79768, were obtained from the Gene Expression Omnibus (GEO) database and subsequently merged for further analysis. Differential gene expression analysis was performed to identify differentially expressed genes (DEGs) and miR-450a-2-3p-related differentially expressed genes (MRDEGs). To investigate the underlying mechanism of cardiac fibrosis, a mouse model was established by treating mice with isoproterenol (ISO) and the miR-450a-2-3p agomir.ResultsA total of 127 DEGs and 31 MRDEGs were identified and subjected to Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to determine the functions and pathways involved in AF. In the animal model, histological analysis using HE and Masson staining, as well as quantification of the collagen volume fraction (CVF), was performed. The increased expression of α-smooth muscle actin (α-SMA), collagen type I (COL1), collagen type III (COL3), and extracellular signal-regulated kinase 1/2 (ERK(1/2)) at both the transcriptional and translational levels indicated the significant development of myocardial fibrosis in mice induced with isoproterenol (ISO). In addition, the cross-sectional area of cardiomyocytes and the expression of atrial natriuretic peptide (NPPA) and brain natriuretic peptide (NPPB) were increased in the ISO group compared with the control group. However, after overexpression of the miR-450a-2-3p agomir through caudal vein injection, there was a notable improvement in cardiac morphology in the treated group. The expression levels of α-SMA, COL1, COL3, ERK(1/2), NPPA, and NPPB were also significantly decreased.ConclusionOur study reveals the mechanistic connection between ISO-induced myocardial fibrosis and the miR-450a-2-3p/ERK(1/2) signaling pathway, highlighting its role in the development of cardiac fibrosis. Modulating miR-450a-2-3p expression and inhibiting ERK(1/2) activation are promising approaches for therapeutic intervention in patients with AF.

Lnc PVT1 facilitates TGF-β1-induced human cardiac fibroblast activation in vitro and ISO-induced myocardial fibrosis in vivo through regulating MYC.

Cardiac fibrosis is a commonly seen pathophysiological process in various cardiovascular disorders, such as coronary heart disorder, hypertension, and cardiomyopathy. Cardiac fibroblast trans-differentiation into myofibroblasts (MFs) is a key link in myocardial fibrosis. LncRNA PVT1 participates in fibrotic diseases in multiple organs; however, its role and mechanism in cardiac fibrosis remain largely unknown. Human cardiac fibroblasts (HCFs) were stimulated with TGF-β1 to induce myofibroblast; Immunofluorescent staining, Immunoblotting, and fluorescence in situ hybridization were used to detect the myofibroblasts phenotypes and lnc PVT1 expression. Cell biological phenotypes induced by lnc PVT1 knockdown or overexpression were detected by CCK-8, flow cytometry, and Immunoblotting. A mouse model of myocardial fibrosis was induced using isoproterenol (ISO), and the cardiac functions were examined by echocardiography measurements, cardiac tissues by H&E, and Masson trichrome staining. In this study, TGF-β1 induced HCF transformation into myofibroblasts, as manifested as significantly increased levels of α-SMA, vimentin, collagen I, and collagen III; the expression level of lnc PVT1 expression showed to be significantly increased by TGF-β1 stimulation. The protein levels of TGF-β1, TGFBR1, and TGFBR2 were also decreased by lnc PVT1 knockdown. Under TGF-β1 stimulation, lnc PVT1 knockdown decreased FN1, α-SMA, collagen I, and collagen III protein contents, inhibited HCF cell viability and enhanced cell apoptosis, and inhibited Smad2/3 phosphorylation. Lnc PVT1 positively regulated MYC expression with or without TGF-β1 stimulation; MYC overexpression in TGF-β1-stimulated HCFs significantly attenuated the effects of lnc PVT1 knockdown on HCF proliferation and trans-differentiation to MFs. In the ISO-induced myocardial fibrosis model, lnc PVT1 knockdown partially reduced fibrotic area, improved cardiac functions, and decreased the levels of fibrotic markers. In addition, lnc PVT1 knockdown decreased MYC and CDK4 levels but increased E-cadherin in mice heart tissues. lnc PVT1 is up-regulated in cardiac fibrosis and TGF-β1-stimulated HCFs. Lnc PVT1 knockdown partially ameliorates TGF-β1-induced HCF activation and trans-differentiation into MFs in vitro and ISO-induced myocardial fibrosis in vivo, potentially through interacting with MYC and up-regulating MYC.

Zhen-wu-tang protects against myocardial fibrosis by inhibiting M1 macrophage polarization via the TLR4/NF-κB pathway

BackgroundMyocardial fibrosis is a risk factor that contributes to the increase in the incidence of cardiovascular disease and death, posing a significant threat to human health. Zhen-wu-tang (ZWT) is a classical Chinese medicinal recipe that has been extensively used to manage cardiovascular disorders throughout history. However, the fundamental processes involved in its effects were not clear. ObjectiveThis study examined the therapeutic effects of ZWT on myocardial fibrosis induced by isoproterenol (ISO) in mice, the effect of regulation and underlying mechanism on the polarization of M1 macrophage. MethodsIn vivo, a myocardial fibrosis mouse model was induced via intraperitoneal infusion of isoproterenol (ISO). ZWT or captopril (CAP) was administered intragastrically for 30 days. Cardiac function was evaluated by electrocardiogram (ECG) and echocardiography. By analysing myocardial fibrosis pathomorphologically and identifying fibrosis-related indicators, the protective effect of the ZWT on the heart was evaluated. A model of macrophage polarization was established in vitro by activating RAW264.7 cells with lipopolysaccharide (LPS). The regulatory effects of ZWT on macrophage polarization and the signalling pathways involved were examined by immunofluorescence staining, Western blotting (WB), quantitative real-time PCR (qRT-PCR) and siRNA transfection. ResultsZWT improved cardiac function; reduced fibrotic deposition in cardiac tissues; decreased α-SMA, collagen I, and collagen III levels; and inhibited myocardial fibrosis in mice with ISO-induced myocardial fibrosis. Furthermore, the results showed that ZWT could suppress M1 macrophage polarization by downregulating the expression of CD86 and iNOS in vitro and in vivo. Finally, the results confirmed that ZWT could significantly reduce TLR4/NF-κB signalling pathway activation. ConclusionZWT showed therapeutic effects on ISO-induced myocardial fibrosis mice, and reduced M1 macrophages polarization through inhibiting TLR4/NF-κB pathway, suggesting that ZWT is a promising drug for myocardial fibrosis treatment.

The isoproterenol-induced myocardial fibrosis: A biochemical and histological investigation

The isoproterenol (ISO)-induced myocardial fibrosis is considered a reliable and repeatable experimental model characterized by a relatively low mortality rate. Although is well-known that ISO stimulates the β1 adrenergic receptors at the myocardial level, a high degree of heterogeneity emerges around the doses and duration of the treatment generating unclear results. Therefore, we propose to gain insights into the progression of ISO-induced myocardial fibrosis, in order to critically analyze and optimize the experimental model. Male Wistar rats (12–14-week-old) were submitted to subcutaneous injection of ISO, in particular, two doses were selected: the commonly used dose of 5 mg/kg and a lower dose of 1 mg/kg, administered for 3 and 6 days. Biochemical and histological examinations were conducted either immediately after the last administration or after a recovering period of 7 or 14 days from the initial administration. Noteworthy, from our investigation emerged that even the lower dose of ISO was able to induce the maximal biochemical and histological alterations, suggesting that lower doses should be considered to control the progression of the damage more precisely and to identify a prodromic phase in which intervention with pharmacological or nutraceutical tools can be effectively attempted.

Engeletin mediates antiarrhythmic effects in mice with isoproterenol-induced cardiac remodeling

ObjectiveEngeletin is a potent natural compound with antioxidant and anti-inflammatory properties. However, its role in cardiac remodeling remains unclear. Herein, the aim of the present study was to explore the effects of engeletin on cardiac structural and electrical remodeling and its underlying mechanism. Methodsand results: A cardiac remodeling mice model using isoproterenol (ISO)-induced myocardial fibrosis was constructed and divided into the following four groups: control group; engeletin group; ISO group; engeletin + ISO group. Our results demonstrated that engeletin alleviated ISO-induced myocardial fibrosis and dysfunction. Moreover, engeletin significantly prolonged the QT and corrected QT (QTc) intervals, effective refractory period (ERP), and action potential duration (APD), and enhanced connexin protein 43 (Cx43) and ion channel expressions, thereby decreasing ventricular fibrillation (VF) susceptibility. Additionally, dihydroethidium staining illustrated that engeletin decreased reactive oxygen species (ROS) production. Of note, engeletin also increased the levels of superoxide dismutase and glutathione and decreased the activity of malondialdehyde and L-Glutathione oxidized. Moreover, engeletin significantly increased the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). Furthermore, in vitro administration of an Nrf2 inhibitor abolished the anti-oxidant properties of engeletin. ConclusionEngeletin ameliorated cardiac structural and electrical remodeling, ion channel remodeling, and oxidative stress induced by ISO in mice, thereby reducing VF susceptibility. These effects may be attributed to the anti-oxidant properties of engeletin associated with the Nrf2/HO-1 pathway.

Abstract P314: Loss Of Set7 Prevents Isoproterenol-induced Heart Dysfunction

Recent studies have revealed the influence of histone-modifying enzymes in cardiac remodeling and dysfunction. The Set7 methyltransferase regulates the expression of several genes through methylation of histones and modulates the activity of non-histone proteins. However, the role of Set7 in heart dysfunction remains unknown. To answer this question, wild type (WT) and Set7 knockout (KO) male mice were injected with isoproterenol (iso) or saline (s) subcutaneously for 14 days. The WTiso mice displayed decreased Set7 activity in the heart compared to WTs mice (Table 1). Both WTiso and KOiso mice exhibited increased heart weight to tibia length ratio (HW/TL) and cardiomyocyte area. However, KOiso mice had higher HW/TL and cardiomyocyte area compared to WTiso mice. Sirius Red staining revealed that both WT and KO mice injected with iso had increased myocardial fibrosis compared to their controls. Nonetheless, loss of Set7 attenuated iso-induced myocardial fibrosis. Echocardiogram showed that WTiso mice had lower ejection fraction (EF) and fractional shortening (FS), and higher E/A ratio compared to WTs mice. Conversely, KOiso mice did not show alteration on these parameters compared to their controls. RNA sequencing analysis revealed that biological processes related to oxidant detoxication, cellular respiration, and anti-inflammatory response were enriched in the heart of KOiso mice compared to WTiso mice. On the other hand, biological processes related to cell aging, interferon production, and immune response were downregulated in the heart of KOiso mice compared to WTiso mice. Collectively, our data suggest that Set7 deletion prevents iso-induced heart dysfunction.

MED1 Deficiency in Macrophages Aggravates Isoproterenol-Induced Cardiac Fibrosis in Mice

Mediator 1 (MED1), a key subunit of the mediator complex, interacts with various nuclear receptors and functions in lipid metabolism and energy homeostasis. Dilated cardiomyopathy-related ventricular dilatation and heart failure have been reported in mice with cardiomyocyte-specific Med1 deficiency. However, the contribution of macrophage-specific MED1 in cardiac remodeling remains unclear. In this study, macrophage-specific Med1 knockout (Med1ΔMac) mice were generated and exposed to isoproterenol (ISO) to induce cardiac fibrosis; these mice showed aggravated cardiac fibrosis compared with Med1fl/fl mice. The levels of expression of marker genes for myofibroblast transdifferentiation [α-smooth muscle actin (SMA)] and of profibrotic genes, including Col1a1, Col3a1, Postn, Mmp2, Timp1, and Fn1, were significantly increased in the cardiac tissues of Med1ΔMac mice with ISO-induced myocardial fibrosis. In particular, the transforming growth factor (TGF)-β-Smad2/3 signaling pathway was activated. In bone marrow-derived and peritoneal macrophages, Med1 deficiency was also associated with elevated levels of expression of proinflammatory genes, including Il6, Tnfa, and Il1b. These findings indicate that macrophage-specific MED1 deficiency may aggravate ISO-induced cardiac fibrosis via the regulation of the TGF-β-SMAD2/3 pathway, and the underlying mechanism may involve MED1 deficiency triggering the activation of inflammatory cytokines in macrophages, which in turn may stimulate phenotypic switch of cardiac fibroblasts and accelerate cardiac fibrosis. Thus, MED1 is a potential therapeutic target for cardiac fibrosis.

Huangqi Shengmai Yin Ameliorates Myocardial Fibrosis by Activating Sirtuin3 and Inhibiting TGF-β/Smad Pathway.

Myocardial fibrosis (MF) is an important pathological process in which a variety of cardiovascular diseases transform into heart failure. The main manifestation of MF is the excessive deposition of collagen in the myocardium. Here, we explored whether Huangqi Shengmai Yin (HSY) can inhibit isoprenaline (ISO)-induced myocardial collagen deposition in rats, thereby reducing the cardiac dysfunction caused by MF. The results of echocardiography showed that HSY upregulated fractional shortening and ejection fraction, and reduced the left ventricular systolic dysfunction in the rats with MF. Pathological results showed that HSY protected myocardium, inhibited apoptosis, and effectively reduced collagen deposition. HSY also inhibited the expression of collagen I and III and α-smooth muscle actin (α-SMA) in the heart tissue. HSY increased the expression of Sirtuin 3 (Sirt3) and inhibited the protein levels of the components in the transforming growth factor-β (TGF-β)/Smad pathway. At the same time, it also regulated the expression of related proteins in the matrix metalloproteinases family. In summary, HSY played a therapeutic role in rats with ISO-induced MF by protecting myocardium and inhibiting collagen deposition. Therefore, HSY is a potential therapeutic agent for ameliorating MF.

Liquiritigenin attenuates isoprenaline‑induced myocardial fibrosis in mice through the TGF‑β1/Smad2 and AKT/ERK signaling pathways.

Myocardial fibrosis is a pathological process characterized by excessive accumulation of extracellular matrix in myocardial interstitial spaces. Myocardial fibrosis is a fundamental process in ventricular remodeling and a primary contributor to the progression of heart failure. Liquiritigenin (LQ) is a flavanone compound with anti-oxidative, anti-carcinogenic, anti-inflammatory and estrogenic properties. The present study aimed to investigate the regulatory potential of LQ treatment in a mouse model of isoprenaline (ISO)-induced cardiac fibrosis and in cultured H9C2 cardiomyocytes stimulated with angiotensin II (Ang II). The treatment of ISO-induced mice with LQ significantly decreased the levels of cardiac injury-related proteins in the serum and ECM accumulation in mouse heart tissues. LQ treatment also effectively alleviated cardiac dysfunction in ISO-treated mice. Further analyses revealed that LQ inhibited ISO-induced collagen formation and activation of the transforming growth factor-β1 (TGF-β1)/Smad2 and protein kinase B (AKT)/extracellular signal-regulated kinase (ERK) signaling pathways. As a major pathological event in myocardial fibrosis, the apoptosis of cardiomyocytes has been considered a key mechanism contributing to impaired left ventricle performance. The pretreatment of rat cardiomyocytes with LQ significantly reduced the apoptosis of H9C2 cells, and inhibited Ang II-induced activation of the TGF-β1/Smad2 and AKT/ERK pathways. In conclusion, the present study revealed that LQ ameliorated ISO-induced myocardial fibrosis in mice and inhibited the apoptosis of cardiomyocytes in vitro by inhibiting the TGF-β1/Smad2 and AKT/ERK signaling pathways. These results suggested the anti-fibrotic and cardioprotective potential of LQ in fibrosis, thus supporting the use of LQ for the management of cardiomyocyte injury and myocardial fibrosis in patients with cardiac diseases.

8-Gingerol Ameliorates Myocardial Fibrosis by Attenuating Reactive Oxygen Species, Apoptosis, and Autophagy via the PI3K/Akt/mTOR Signaling Pathway.

8-gingerol (8-Gin) is the series of phenolic substance that is extracted from ginger. Although many studies have revealed that 8-Gin has multiple pharmacological properties, the possible underlying mechanisms of 8-Gin against myocardial fibrosis (MF) remains unclear. The study examined the exact role and potential mechanisms of 8-Gin against isoproterenol (ISO)-induced MF. Male mice were intraperitoneally injected with 8-Gin (10 and 20 mg/kg/d) and concurrently subcutaneously injected with ISO (10 mg/kg/d) for 2 weeks. Electrocardiography, pathological heart morphology, myocardial enzymes, reactive oxygen species (ROS) generation, degree of apoptosis, and autophagy pathway-related proteins were measured. Our study observed 8-Gin significantly reduced J-point elevation and heart rate. Besides, 8-Gin caused a marked decrease in cardiac weight index and left ventricle weight index, serum levels of creatine kinase and lactate dehydrogenase (CK and LDH, respectively), ROS generation, and attenuated ISO-induced pathological heart damage. Moreover, treatment with 8-Gin resulted in a marked decrease in the levels of collagen types I and III and TGF-β in the heart tissue. Our results showed 8-Gin exposure significantly suppressed ISO-induced autophagosome formation. 8-Gin also could lead to down-regulation of the activities of matrix metalloproteinases-9 (MMP-9), Caspase-9, and Bax protein, up-regulation of the activity of Bcl-2 protein, and alleviation of cardiomyocyte apoptosis. Furthermore, 8-Gin produced an obvious increase in the expressions of the PI3K/Akt/mTOR signaling pathway-related proteins. Our data showed that 8-Gin exerted cardioprotective effects on ISO-induced MF, which possibly occurred in connection with inhibition of ROS generation, apoptosis, and autophagy via modulation of the PI3K/Akt/mTOR signaling pathway.

Chronic peripheral ghrelin injection exerts antifibrotic effects by increasing growth differentiation factor 15 in rat hearts with myocardial fibrosis induced by isoproterenol.

This study aimed to investigate the anti-fibrotic effects of ghrelin in isoproterenol (ISO)-induced myocardial fibrosis and the underlying mechanism. Sprague-Dawley rats were randomized to control, ISO, and ISO + ghrelin groups. ISO (2 mg/kg per day, subcutaneous) or vehicle was administered once daily for 7 days, then ghrelin (100 microg/kg per day, subcutaneous) was administered once daily for the next 3 weeks. Ghrelin treatment greatly improved the cardiac function of ISO-treated rats. Ghrelin also decreased plasma brain natriuretic peptide level and ratios of heart weight to body weight and left ventricular weight to body weight. Ghrelin significantly reduced myocardial collagen area and hydroxyproline content, accompanied by decreased mRNA levels of collagen type I and III. Furthermore, ghrelin increased plasma level of growth differentiation factor 15 (GDF15) and GDF15 mRNA and protein levels in heart tissues, which were significantly decreased with ISO alone. The phosphorylation of Akt at Ser473 and GSK-3beta at Ser9 was decreased with ISO, and ghrelin significantly reversed the downregulation of p-Akt and p-GSK-3beta. Mediated by GDF15, ghrelin could attenuate ISO-induced myocardial fibrosis via Akt-GSK-3beta signaling.

6]-Gingerol Ameliorates ISO-Induced Myocardial Fibrosis by Reducing Oxidative Stress, Inflammation, and Apoptosis through Inhibition of TLR4/MAPKs/NF-κB Pathway.

[6]-Gingerol is one of the primary pungent constituents of ginger. While [6]-gingerol has many pharmacological effects, its benefits for myocardial fibrosis, including its exact role and underlying mechanisms, remain largely unexplored. The present study is designed to characterize the cardio-protective effects of [6]-gingerol in myocardial fibrosis mice and possible underlying mechanisms. Mice are subcutaneously injected with isoproterenol (ISO, 10mg kg-1 ) and gavaged with [6]-gingerol (10, 20mg kg-1 day-1 ) for 14 days. Pathological alterations, fibrosis, oxidative stress, inflammation response, and apoptosis are examined. In ISO-induced myocardial fibrosis, [6]-gingerol treatment decreases the J-point, heart rate, cardiac weight index, left ventricle weight index, creatine kinase (CK), and lactate dehydrogenase serum levels, calcium concentration, reactive oxygen species, malondialdehyde, and glutathione disulfide (GSSG), and increases levels of superoxide dismutase, catalase, glutathione, and GSH/GSSG. Further, [6]-gingerol improved ISO-induced morphological pathologies, inhibited inflammation and apoptosis, and suppressed the toll-like receptor-4 (TLR4)/mitogen-activated protein kinases (MAPKs)/nuclear factor κB (NF-κB) signaling pathways. The protective effect of [6]-gingerol in mice with ISO-induced myocardial fibrosis may be related to the inhibition of oxidative stress, inflammation, and apoptosis, potentially through the TLR4/MAPKs/NF-κB signaling pathway.

The Mechanism Underlying the Protective Effects of Tannic Acid Against Isoproterenol-Induced Myocardial Fibrosis in Mice.

Tannic acid (TA) belongs to a class of complex water-soluble polyphenolic derivatives that show anticarcinogenic, antiinflammatory, antioxidant, and scavenging activities. Here, we investigate the protective effects of TA against isoproterenol (ISO)-induced myocardial fibrosis (MF) in mice. Mice received TA and ISO dosing and were sacrificed 48 h later. The activities of creatine kinase (CK), creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), and mitochondria enzymes were measured. Cardiac histopathology was done using H&E, Sirius red, and Masson’s Trichrome staining. Immunohistochemical staining was applied to indicate changes in B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and basic fibroblast growth factor (bFGF) protein expressions in cardiac tissue. RT-PCR was used to measure the expression of atrial and brain natriuretic peptides (ANP and BNP, respectively), c-fos, and c-jun. Western blotting was used to measure the expression of nuclear factor-κB (NF-κB) p65, phosphorylated NF-κB p65), toll-like receptor 4 (TLR4), p38, phosphorylated p38, Bax, Bcl-2, and caspase-3. Compared to the ISO group, the TA group had reduced levels of TLR4, p38, p-p38, NF-κB (p65), p-NF-κB (p-p65), caspase-3, Bax, and Bcl-2, as well as CK, CK-MB, and LDH. These results indicate that TA protects against ISO-induced MF, possibly through its ability to suppress the TLR4-mediated NF-κB signaling pathway.

GHSR deficiency exacerbates cardiac fibrosis: role in macrophage inflammasome activation and myofibroblast differentiation.

Sustained activation of β-adrenergic signalling induces cardiac fibrosis, which marks progression to heart failure. GHSR (growth hormone secretagogue receptor) is the receptor for ghrelin, which is an orexigenic gastric hormone with newly defined cardiovascular effects. The present study determined the effects of GHSR deficiency in a mouse model of isoproterenol (ISO)-induced cardiac fibrosis and examined the underlying mechanism. Histochemical studies showed that GHSR deficiency exacerbated cardiac fibrosis. Quantitative RT-PCR, western blotting, and immunofluorescence staining demonstrated that cardiac fibroblasts isolated from GHSR-/- mice exhibited increased expression of marker genes for myofibroblast trans-differentiation (α-SMA, SM22, and calponin) upon transforming growth factor-β treatment compared to wild-type mice. RNA-sequencing of heart transcriptomes revealed that differentially expressed genes in GHSR-/- hearts were enriched in such biological processes as extracellular matrix organization, inflammatory response, lipid metabolism, cell cycle, migration, and adhesion. Particularly, GHSR deficiency increased Wnt/β-catenin pathway activation in ISO-induced myocardial fibrosis. In addition, loss of GHSR in macrophages instigated inflammasome activation with increased cleavage and release of interleukin-18. These results for the first time demonstrated that GHSR deficiency aggravated ISO-induced cardiac fibrosis, suggesting that GHSR was a potential target for the intervention of cardiac fibrosis.

Protective Effect of Hydroxysafflor Yellow A on Isoproterenol-Induced Myocardial Fibrosis in Rats

Aim: Present research was to explore the effects and mechanisms of hydroxysafflor yellow A in treatment of isoproterenol-induced myocardial fibrosis in rats. Methods: Dividing 50 SD rats as the normal control, ISO and HSYA treated groups, 10 rats in each group. Administering Rats by ISO subcutaneously (2 mg/kg · d) for 10 days induce myocardial fibrosis. HSYA treated groups were treated with 10, 20 and 40 mg/kg · d HSYA for 4 weeks (ig). The normal control and the model groups were treated with saline at equivalent volume. Measurement of hemodynamics index to evaluate the changes of cardiac function. And evaluating the relative index of difference groups. Results: Treatment of HSYA significantly inhibited heart rate (HR) and LVEDP, meanwhile improving LVSP compared with model group with dose-dependent. HE staining and Masson staining showed that diltiazem improve heart structure disorder, excessive accumulation of collagen fibers, meanwhile decreasing the value of collagen volume fraction (CVF) (P &lt; 0.05). The result showed diltiazem inhibited Collagen I and Collagen II which were up-regulation (P &lt; 0.001). HSYA could significantly increase SOD, GSH-Px levels (P &lt; 0.001), meanwhile reducing the MDA levels in ISO-induced myocardial fibrosis model (P &lt; 0.001) with dose-dependent. In addition, the elevated NF-κB, TGF-β1, CTGF expressions were effectively restored by HSYA(P &lt; 0.001). Conclusion: HSYA alleviated ISO induced myocardial fibrosis in rat, which might be correlation with the improvement of reduction of oxidative stress and inhibition of NF-κB signal pathway.

Crocin attenuates isoprenaline-induced myocardial fibrosis by targeting TLR4/NF-κB signaling: connecting oxidative stress, inflammation, and apoptosis.

Crocin is isolated from saffron and has multiple activities. There are many reports on its beneficial effects for cardiovascular disease, but crocin's effects on anti-myocardial fibrosis have not yet been reported. This study investigated crocin's effects and potential mechanisms on isoproterenol (ISO)-induced myocardial fibrosis (MF) in mice. Mice were infused intraperitoneally with crocin with concurrent ISO subcutaneous injections over 2weeks. Electrocardiography, cardiac weight index (CWI), hydroxyproline content, and heart morphology changes were observed. Administration of crocin markedly decreased heart rate, J-point elevation, QRS interval, CWI, and hydroxyproline content in the myocardial tissues, and improved heart pathologic morphology. Versus the control group, the ISO group showed an increase in lactate dehydrogenase and creatine kinase activities and malondialdehyde content. Meanwhile, superoxide dismutase, catalase, and glutathione contents decreased in the ISO group; crocin caused a significant reduction in oxidative stress levels in ISO-induced MF. ISO led to a significant increase in interleukin-1 and -6 and tumor necrosis factor-α in addition to nuclear factor kappa B (NF-κB) (p65) and toll-like receptor (TLR) 4 expressions. Crocin treatment suppressed these inflammatory cytokine expressions. Moreover, crocin treatment caused a significant decrease in connective tissue growth factor and transforming growth factor-β1 mRNA levels in addition to a decrease in B cell lymphoma-2, Bcl-2-associated X protein, caspase-3, and cleaved caspase-3 expressions. Crocin has a protective effect on ISO-induced MF, which may be associated with the TLR4/NF-κB (p65) signal transduction pathway.

Luteolin-7-diglucuronide attenuates isoproterenol-induced myocardial injury and fibrosis in mice.

Myocardial injury and ensuing fibrotic alterations impair normal heart architecture and cause cardiac dysfunction. Oxidative stress has been recognized as a key player in the pathogenesis of cardiac injury and progression of cardiac dysfunction, and promoting fibrosis. In the current study we investigated whether luteolin-7-diglucuronide (L7DG), a naturally occurring antioxidant found in edible plants, could attenuate isoproterenol (ISO)-induced myocardial injury and fibrosis in mice and the underlying mechanisms. Myocardial injury and fibrosis were induced in mice via injection of ISO (5 mg·kg-1·d-1, ip) for 5 or 10 d. Two treatment regimens (pretreatment and posttreatment) were employed to administer L7DG (5-40 mg·kg-1·d-1, ip) into the mice. After the mice were euthanized, morphological examinations of heart sections revealed that both L7DG pretreatment and posttreatment regimens significantly attenuated ISO-induced myocardial injury and fibrosis. But the pretreatment regimen caused better protection against ISO-induced myocardial fibrosis than the posttreatment regimen. Furthermore, L7DG pretreatment blocked ISO-stimulated expression of the genes (Cyba, Cybb, Ncf1, Ncf4 and Rac2) encoding the enzymatic subunits of NADPH oxidase, which was the primary source of oxidant production in mammalian cells. Moreover, L7DG pretreatment significantly suppressed ISO-stimulated expression of collagen genes Col1a1, Col1a2, Col3a1, and Col12a1 and non-collagen extracellular matrix genes fibrillin-1, elastin, collagen triple helix repeat containing 1 and connective tissue growth factor. In addition, L7DG pretreatment almost reversed ISO-altered expression of microRNAs that were crosstalking with TGFβ-mediated fibrosis, including miR-29c-3p, miR-29c-5p, miR-30c-3p, miR-30c-5p and miR-21. The current study demonstrated for the first time that L7DG is pharmacologically effective in protecting the heart against developing ISO-induced injury and fibrosis, justifying further evaluation of L7DG as a cardioprotective agent to treat related cardiovascular diseases.

Effect of U50,488H, a κ-opioid receptor agonist on myocardial α-and β-myosin heavy chain expression and oxidative stress associated with isoproterenol-induced cardiac hypertrophy in rat

Both oxidative stress and β-MHC expression are associated with pathological cardiac hypertrophy. β-adrenergic receptor stimulation plays an important role in cardiac hypertrophy. Recent studies have reported a negative interplay between opioid receptors and adrenoceptors in heart. This study investigated the effect of U50,488H (a selective κ-opioid receptor agonist) on myocardial oxidative stress and α- and β-MHC expression in isoproterenol-induced cardiac hypertrophy. Male Wistar rats were administered normal saline (control), isoproterenol (ISO) (5mg/kg BW s.c. OD), and isoproterenol with U50,488H (0.4 and 0.6mg/kg BW, i.p. OD) for 14days. In a separate group, nor-binaltorphimine (nor-BNI) (0.5mg/kg, BW, i.p.) (κ-receptor antagonist) was administered along with ISO and U50,488H. ISO administration caused significant increase in left ventricular (LV) wall thicknesses, LV mass in echocardiography, heart weight to body weight ratio, and myocyte size as compared to control. Both the doses of U50,488H offered significant protection against these changes. The higher dose of U50,488H significantly prevented ISO-induced increase in myocardial lipid peroxidation and depletion of myocardial antioxidants (glutathione, superoxide dismutase, and catalase), while a similar trend (although not significant) was observed with the lower dose also. ISO-induced myocardial fibrosis was also significantly attenuated by both the doses of U50,488H. Isoproterenol-induced β-MHC expression in the hypertrophied heart was not altered by either doses of U50,488H, however, the latter prevented the loss of myocardial α-MHC expression. All these effects of U50,488H were blocked by nor-BNI. This study provides the evidence that U50,488H reduced oxidative stress and preserved expression of α-MHC in isoproterenol-induced cardiac hypertrophy.