Isoproterenol-induced Cardiac 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.

Malus toringoides (Rehd.) Hughes decoction alleviates isoproterenol-induced cardiac fibrosis by inhibiting cardiomyocyte inflammation and pyroptosis via the HK1/NLRP3 signaling pathway.

Malus toringoides (Rehd.) Hughes, called "Eseye (Ese)," is a traditional medicinal plant from the Tibet province of China that has proven effective in treating cardiac conditions due to its anti-inflammatory, antioxidative, and antiapoptotic properties. In this study, we explored the underlying protective mechanisms of Ese decoction in isoproterenol (ISO)-induced cardiac fibrosis (CF) and established the fact that treatment with an Ese decoction attenuated tissue injury, decreased the release of IL-1β, IL-18, TNF-α, and caspase-3, and elevated the Bax/Bcl-2 ratio in CF mice. We also found that with Ese treatment damage to the mitochondrial ultrastructure of myocardium was alleviated, and the level of reactive oxygen species was markedly diminished. Ese inhibited the expression of proteins associated with pyroptosis by the HK1/NLRP3 signaling pathway and also improved CF. Due to the anti-inflammatory, antioxidative, and antiapoptotic characteristics of Ese decoction, we found that Ese protected against ISO-induced CF, by inhibiting inflammation and pyroptosis as mediated by the HK1/NLRP3 signaling pathway.

Integrated miRNA-mRNA networks underlie attenuation of chronic β-adrenergic stimulation-induced cardiac remodeling by minocycline.

Adverse cardiac remodeling contributes to heart failure development and progression, partly due to inappropriate sympathetic nervous system activation. Although β-adrenergic receptor (β-AR) blockade is a common heart failure therapy, not all patients respond, prompting exploration of alternative treatments. Minocycline, an FDA-approved antibiotic, has pleiotropic properties beyond antimicrobial action. Recent evidence suggests it may alter gene expression via changes in miRNA expression. Thus, we hypothesized that minocycline could prevent adverse cardiac remodeling induced by the β-AR agonist isoproterenol, involving miRNA-mRNA transcriptome alterations. Male C57BL/6J mice received isoproterenol (30 mg/kg/day sc) or vehicle via osmotic minipump for 21 days, along with daily minocycline (50 mg/kg ip) or sterile saline. Isoproterenol induced cardiac hypertrophy without altering cardiac function, which minocycline prevented. Total mRNA sequencing revealed isoproterenol altering gene networks associated with inflammation and metabolism, with fibrosis activation predicted by integrated miRNA-mRNA sequencing, involving miR-21, miR-30a, miR-34a, miR-92a, and miR-150, among others. Conversely, the cardiac miRNA-mRNA transcriptome predicted fibrosis inhibition in minocycline-treated mice, involving antifibrotic shifts in Atf3 and Itgb6 gene expression associated with miR-194 upregulation. Picrosirius red staining confirmed isoproterenol-induced cardiac fibrosis, prevented by minocycline. These results demonstrate minocycline's therapeutic potential in attenuating adverse cardiac remodeling through miRNA-mRNA-dependent mechanisms, especially in reducing cardiac fibrosis. NEW & NOTEWORTHY We demonstrate that minocycline treatment prevents cardiac hypertrophy and fibrotic remodeling induced by chronic β-adrenergic stimulation by inducing antifibrotic shifts in the cardiac miRNA-mRNA transcriptome.

Irisin Treatment Prevents Isoproterenol-Induced Cardiac Fibrosis in Mice.

Cardiac fibrosis is a pathophysiological process that occurs as the end stage of cardiovascular diseases. Irisin is a myokine secreted mainly by skeletal muscle exerting pleiotropic effects. Previous studies found altered irisin levels in patients with cardiovascular diseases and irisin has been shown to preserve cardiac function after ischemia-reperfusion injury in mice. This study aimed to explore whether pretreatment with irisin prevents cardiac fibrosis induced in mice through a single injection of the beta-adrenergic agonist isoproterenol at a high dose. The cardiac fibrosis model was obtained through a single intraperitoneal administration of 160 mg/kg isoproterenol [ISO] in young C57BL/6J mice. Before ISO injection, mice were pretreated with irisin 100 μg/kg/week [irisin-ISO] or saline [veh-ISO] for 4 weeks. A third group of mice received saline for 4 weeks without ISO injection [CTRL]. The mice pretreated with irisin recovered faster than vehicle-treated mice after acute ISO stimulation, as measured by behavioral test. Twenty-four hours after ISO treatment, the serum levels of Troponin I were significantly lower in the group of mice pretreated with irisin compared with veh-ISO mice (p = 0.0117). Moreover, the expression of atrial natriuretic peptide (p = 0.0197) and alpha-smooth muscle actin (p = 0.0261) mRNAs in cardiac tissue of veh-ISO mice were 10- and 15-fold higher than CTRL mice, respectively, while pretreatment with irisin maintained their expression at control levels. Interestingly, 7 days after ISO, the expression of alpha-smooth muscle actin mRNA was still significantly lower in the irisin-ISO group than in the veh-ISO group (p = 0.0145). Moreover, we found increased cardiac hypertrophy, measured as heart-weight/tibia-length ratio, in veh-ISO mice versus CTRL mice (p = 0.0312) which was fully prevented in irisin-ISO mice (p = 0.0258). The cardiac fibrosis score assessed by Masson's trichrome staining was significantly lower in irisin-ISO mice versus veh-ISO mice (p = 0.0261). Notably, some mitochondrial genes, previously identified as controlled by irisin, were markedly increased in the early phase following ISO, whereas irisin maintained their expression similar to controls. Our results demonstrate the beneficial effect of irisin in preventing isoproterenol-induced cardiac hypertrophy and fibrosis.

Transcriptional changes during isoproterenol-induced cardiac fibrosis in mice.

Introduction: β-adrenergic stimulation using β-agonists such as isoproterenol has been routinely used to induce cardiac fibrosis in experimental animal models. Although transcriptome changes in surgical models of cardiac fibrosis such as transverse aortic constriction (TAC) and coronary artery ligation (CAL) are well-studied, transcriptional changes during isoproterenol-induced cardiac fibrosis are not well-explored. Methods: Cardiac fibrosis was induced in male C57BL6 mice by administration of isoproterenol for 4, 8, or 11days at 50mg/kg/day dose. Temporal changes in gene expression were studied by RNA sequencing. Results and discussion: We observed a significant alteration in the transcriptome profile across the different experimental groups compared to the saline group. Isoproterenol treatment caused upregulation of genes associated with ECM organization, cell-cell contact, three-dimensional structure, and cell growth, while genes associated with fatty acid oxidation, sarcoplasmic reticulum calcium ion transport, and cardiac muscle contraction are downregulated. A number of known long non-coding RNAs (lncRNAs) and putative novel lncRNAs exhibited differential regulation. In conclusion, our study shows that isoproterenol administration leads to the dysregulation of genes relevant to ECM deposition and cardiac contraction, and serves as an excellent alternate model to the surgical models of heart failure.

Paricalcitol Improved Cardiac Hypertrophy and Fibrosis through Upregulation of Fibroblast Growth Factor-23 and Downregulation of Transforming Growth Factor-beta in a Rat Model of Isoproterenol-Induced Cardiomyopathy

Acute cardiomyopathy is a significant global health concern and one of the leading causes of death in developed countries. Prior studies have shown an association between acute cardiomyopathy and low vitamin D levels. Although paricalcitol, a vitamin D receptor (VDR) activator, has demonstrated clinical benefits in patients with advanced kidney disease, its effect on cardiac remodeling in cardiomyopathy is unknown. This study aimed to investigate the relative effects of paricalcitol on cardiomyopathy in rats. Wistar–Kyoto rats were administered vehicle (sham control group) or isoproterenol to induce cardiomyopathy. Rats administered isoproterenol were subsequently treated with paricalcitol (experimental group) or vehicle (isoproterenol group). Picrosirius red and immunofluorescence staining were used to analyze cardiac fibrosis and hypertrophy. Immunohistochemistry staining was used to confirm the molecular mechanisms involved in isoproterenol-induced cardiomyopathy in rats. Injection of paricalcitol could reduce collagen and transforming growth factor-beta 1 (TGF-β1) levels while activating fibroblast growth factor receptor 1 (FGFR1) and fibroblast growth factor-23 (FGF23) without the help of Klotho, thereby reducing myocardial hypertrophy and fibrosis. As a VDR activator, paricalcitol reduces isoproterenol-induced cardiac fibrosis and hypertrophy by reducing the expression of TGF-β1 and enhancing the expression of VDR, FGFR1, and FGF23.

Application of recombinant TGF-β1 inhibitory peptide to alleviate isoproterenol-induced cardiac fibrosis.

Cardiac fibrosis is a remodeling process of the cardiac interstitium, characterized by abnormal metabolism of the extracellular matrix, excessive accumulation of collagen fibers, and scar tissue hyperplasia. Persistent activation and transdifferentiation into myofibroblasts of cardiac fibroblasts promote the progression of fibrosis. Transforming growth factor-β1 (TGF-β1) is a pivotal factor in cardiac fibrosis. Latency-associated peptide (LAP) is essential for activating TGF-β1 and its binding to the receptor. Thus, interference with TGF-β1 and the signaling pathways using LAP may attenuate cardiac fibrosis. Recombinant full-length and truncated LAP were previously constructed, expressed, and purified. Their effects on cardiac fibrosis were investigated in isoproterenol (ISO)-induced cardiac fibroblasts (CFs) and C57BL/6 mice. The study showed that LAP and tLAP inhibited ISO-induced CF activation, inflammation, and fibrosis, improved cardiac function, and alleviated myocardial injury in ISO-induced mice. LAP and tLAP alleviated the histopathological alterations and inhibited the elevated expression of inflammatory and fibrosis-related markers in cardiac tissue. In addition, LAP and tLAP decreased the ISO-induced elevated expression of TGF-β, αvβ3, αvβ5, p-Smad2, and p-Smad3. The study indicated that LAP and tLAP attenuated ISO-induced cardiac fibrosis via suppressing TGF-β/Smad pathway. This study may provide a potential approach to alleviate cardiac fibrosis. KEY POINTS: • LAP and tLAP inhibited ISO-induced CF activation, inflammation, and fibrosis. • LAP and tLAP improved cardiac function and alleviated myocardial injury, inflammation, and fibrosis in ISO-induced mice. • LAP and tLAP attenuated cardiac fibrosis via suppressing TGF-β/Smad pathway.

Exposure to Doxorubicin Modulates the Cardiac Response to Isoproterenol in Male and Female Mice.

Sex is a salient risk factor in the development of doxorubicin-induced cardiotoxicity. Sex differences in the heart's ability to respond to hypertrophic stimuli in doxorubicin-exposed animals have not been reported. We identified the sexual dimorphic effects of isoproterenol in mice pre-exposed to doxorubicin. Male and female intact or gonadectomized C57BL/6N mice underwent five weekly intraperitoneal injections of 4 mg/kg doxorubicin followed by a five-week recovery period. Fourteen days of subcutaneous isoproterenol injections (10 mg/kg/day) were administered after the recovery period. Echocardiography was used to assess heart function one and five weeks after the last doxorubicin injection and on the fourteenth day of isoproterenol treatment. Thereafter, mice were euthanized, and the hearts were weighed and processed for histopathology and gene expression analysis. Doxorubicin did not produce overt cardiac dysfunction in male or female mice before starting isoproterenol treatment. The chronotropic response to a single isoproterenol injection was blunted by doxorubicin, but the inotropic response was maintained in both males and females. Pre-exposure to doxorubicin caused cardiac atrophy in both control and isoproterenol-treated male mice but not in female mice. Counterintuitively, pre-exposure to doxorubicin abrogated isoproterenol-induced cardiac fibrosis. However, there were no sex differences in the expression of markers of pathological hypertrophy, fibrosis, or inflammation. Gonadectomy did not reverse the sexually dimorphic effects of doxorubicin. Additionally, pre-exposure to doxorubicin abrogated the hypertrophic response to isoproterenol in castrated male mice but not in ovariectomized female mice. Therefore, pre-exposure to doxorubicin caused male-specific cardiac atrophy that persisted after isoproterenol treatment, which could not be prevented by gonadectomy.

Set7 deletion attenuates isoproterenol-induced cardiac fibrosis and delays cardiac dysfunction.

Cardiovascular diseases are the main cause of death worldwide. Recent studies have revealed the influence of histone-modifying enzymes in cardiac remodeling and heart dysfunction. The Set7 methyltransferase regulates the expression of several genes through the methylation of histones and modulates the activity of non-histone proteins. However, the role of Set7 in cardiac remodeling and heart dysfunction remains unknown. To address this question, wild-type (WT) and Set7 knockout (KO) male mice were injected with isoproterenol or saline. WT mice injected with isoproterenol displayed a decrease in Set7 activity in the heart. In addition, WT and Set7 KO mice injected with isoproterenol exhibited cardiac hypertrophy. Interestingly, Set7 deletion exacerbated cardiac hypertrophy in response to isoproterenol but attenuated myocardial fibrosis. Echocardiograms revealed that WT mice injected with isoproterenol had lowered ejection fractions and fractional shortening, and increased E'-wave deceleration time and E/A ratio compared with their controls. Conversely, Set7 KO mice did not show alteration in these parameters in response to isoproterenol. However, prolonged exposure to isoproterenol induced cardiac dysfunction both in WT and Set7 KO mice. Both isoproterenol and Set7 deletion changed the transcriptional profile of the heart. Moreover, Set7 deletion increased the expression of Pgc1α and mitochondrial DNA content in the heart, and reduced the expression of cellular senescence and inflammation markers in response to isoproterenol. Taken together, our data suggest that Set7 deletion attenuates isoproterenol-induced myocardial fibrosis and delays heart dysfunction, suggesting that Set7 plays an important role in cardiac remodeling and dysfunction in response to stress.

MicroRNA-146a attenuates isoproterenol-induced cardiac fibrosis by inhibiting FGF2.

Cardiac fibrosis is a key factor of heart failure. Increasing evidence suggests that microRNAs (miRNAs/miRs) serve vital roles in the pathogenesis of cardiac fibrosis. The present study aimed to investigate the role of miR-146a-5p in isoproterenol (ISO)-induced cardiac fibrosis. Reverse transcription-quantitative PCR analysis demonstrated that miR-146a-5p expression was downregulated in ISO-treated rat heart tissue and ISO-induced cardiac fibroblasts (CFs). Conversely, the expression levels of basic fibroblast growth factor 2 (FGF2), collagen I and smooth muscle α-actin (α-SMA) were upregulated in ISO-treated rat cardiac tissue and CFs. Furthermore, viability and differentiation were inhibited in ISO-induced CFs transfected with miR-146a-5p mimics. Dual-luciferase reporter assay confirmed that miR-146a-5p targeted FGF2. Notably, FGF2 expression was suppressed following overexpression of miR-146a-5p, while FGF2 expression increased following miR-146a-5p knockdown. In addition, FGF2 knockdown suppressed the expression levels of FGF2, collagen I and α-SMA levels in CFs. Taken together, the results of the present study suggested that the miR-146a-5p/FGF2 pathway may be a novel therapy for cardiac fibrosis.

Stimulation of the atypical chemokine receptor 3 (ACKR3) by a small-molecule agonist attenuates fibrosis in a preclinical liver but not lung injury model

Atypical chemokine receptor 3 (ACKR3, formerly CXC chemokine receptor 7) is a G protein-coupled receptor that recruits β-arrestins, but is devoid of functional G protein signaling after receptor stimulation. In preclinical models of liver and lung fibrosis, ACKR3 was previously shown to be upregulated after acute injury in liver sinusoidal and pulmonary capillary endothelial cells, respectively. This upregulation was linked with a pro-regenerative and anti-fibrotic role for ACKR3. A recently described ACKR3-targeting small molecule agonist protected mice from isoproterenol-induced cardiac fibrosis. Here, we aimed to evaluate its protective role in preclinical models of liver and lung fibrosis. After confirming its in vitro pharmacological activity (i.e., ACKR3-mediated β-arrestin recruitment and receptor binding), in vivo administration of this ACKR3 agonist led to increased mouse CXCL12 plasma levels, indicating in vivo interaction of the agonist with ACKR3. Whereas twice daily in vivo administration of the ACKR3 agonist lacked inhibitory effect on bleomycin-induced lung fibrosis, it had a modest, but significant anti-fibrotic effect in the carbon tetrachloride (CCl4)-induced liver fibrosis model. In the latter model, ACKR3 stimulation affected the expression of several fibrosis-related genes and led to reduced collagen content as determined by picro-sirius red staining and hydroxyproline quantification. These data confirm that ACKR3 agonism, at least to some extent, attenuates fibrosis, although this effect is rather modest and heterogeneous across various tissue types. Stimulating ACKR3 alone without intervening in other signaling pathways involved in the multicellular crosstalk leading to fibrosis will, therefore, most likely not be sufficient to deliver a satisfactory clinical outcome.

The anticardiac fibrosis of total alkaloids of Plumula nelumbinis by regulating circulating lipidomic profile: In vivo study.

Plumula nelumbinis has great medicinal potential as a herbal tea and traditional drug in China. This study was aimed to evaluate the anticardiac fibrosis of the total alkaloids of P. nelumbinis (TAP). TAP at 50 mg/kg/day significantly ameliorated isoproterenol-induced cardiac fibrosis in mice (p < .05). The circulating lipidomics study revealed that TAP improved the lipid metabolism dysfunction in cardiac fibrosis. Meanwhile, TAP suppressed the lipid accumulation, decreased MDA level (p < .01) in heart, and increased FFA level (p < .01). Furthermore, integrating lipidomics, chemical profiles and pharmacology network analysis found that AMPK and PI3K/Akt signaling pathways were the potential targeted pathway by TAP to regulate lipid metabolism dysfunction including glycerophospholipid metabolism. Above all, TAP provided a potential anticardiac fibrosis effect partly through regulation of lipid profiles. PRACTICAL APPLICATIONS: The total alkaloids of Plumula nelumbinis (TAP) suppressed ISO-induced cardiac fibrosis in mice. Network pharmacology analysis and experiments revealed that TAP-regulated AMPK and PI3K/Akt signaling pathway to improve lipid metabolism disorder in cardiac fibrosis. This study provides evidence to the therapeutic potential of TAP in the treatment of ISO-induced cardiac fibrosis and could be a drug candidate for prevention and treatment of cardiac fibrosis.

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.

Nicotinamide mononucleotide attenuates isoproterenol-induced cardiac fibrosis by regulating oxidative stress and Smad3 acetylation

AimsCardiac fibrosis is a pathological hallmark of progressive heart diseases currently lacking effective treatment. Nicotinamide mononucleotide (NMN), a member of the vitamin B3 family, is a defined biosynthetic precursor of nicotinamide adenine dinucleotide (NAD+). Its beneficial effects on cardiac diseases are known, but its effects on cardiac fibrosis and the underlying mechanism remain unclear. We aimed to elucidate the protective effect of NMN against cardiac fibrosis and its underlying mechanisms of action. Materials and methodsCardiac fibrosis was induced by isoproterenol (ISO) in mice. NMN was administered by intraperitoneal injection. In vitro, cardiac fibroblasts (CFs) were stimulated by transforming growth factor-beta (TGF-β) with or without NMN and sirtinol, a SIRT1 inhibitor. Levels of cardiac fibrosis, NAD+/SIRT1 alteration, oxidative stress, and Smad3 acetylation were evaluated by real-time polymerase chain reaction, western blots, immunohistochemistry staining, immunoprecipitation, and assay kits. Key findingsISO treatment induced cardiac dysfunction, fibrosis, and hypertrophy in vivo, whereas NMN alleviated these changes. Additionally, NMN suppressed CFs activation stimulated by TGF-β in vitro. Mechanistically, NMN restored the NAD+/SIRT1 axis and inhibited the oxidative stress and Smad3 acetylation induced by ISO or TGF-β. However, the protective effects of NMN were partly antagonized by sirtinol in vitro. SignificanceNMN could attenuate cardiac fibrosis in vivo and fibroblast activation in vitro by suppressing oxidative stress and Smad3 acetylation in a NAD+/SIRT1-dependent manner.

Nucleotide-Binding Oligomerization Domain-Like Receptor 3 Deficiency Attenuated Isoproterenol-Induced Cardiac Fibrosis via Reactive Oxygen Species/High Mobility Group Box 1 Protein Axis.

Nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) is involved in fibrosis of multiple organs, such as kidney, liver, lung, and the like. However, the role of NLRP3 in cardiac fibrosis is still controversial and remains unclear. The study aims to investigate the role of NLRP3 on cardiac fibrosis induced by isoproterenol (ISO). In vivo, NLRP3 knockout and wild-type mice were subcutaneously injected with ISO to induce the cardiac fibrosis model. The results showed that NLRP3 deficiency alleviated the cardiac fibrosis and inflammation induced by ISO. In vitro, neonatal rat ventricular myocytes (NRVMs) and primary adult mouse cardiac fibroblasts of NLRP3 knockout and wild-type mice were isolated and challenged with ISO. Adenovirus (Ad-) NLRP3 and small interfering RNAs targeting NLRP3 were used to transfect NRVMs to overexpress or knockdown NLRP3. We found that NLRP3 could regulate high-mobility group box 1 protein (HMGB1) secretion via reactive oxygen species production in NRVMs and the HMGB1 secreted by NRVMs promoted the activation and proliferation of cardiac fibroblasts. Thus, we concluded that the NLRP3/reactive oxygen species/HMGB1 pathway could be the underlying mechanism of ISO-induced cardiac fibrosis.

Microsomal Prostaglandin E2 Synthase-1 Deletion Attenuates Isoproterenol-Induced Myocardial Fibrosis in Mice.

Deletion of microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibits inflammation and protects against atherosclerotic vascular diseases but displayed variable influence on pathologic cardiac remodeling. Overactivation of β-adrenergic receptors (β-ARs) causes heart dysfunction and cardiac remodeling, whereas the role of mPGES-1 in β-AR-induced cardiac remodeling is unknown. Here we addressed this question using mPGES-1 knockout mice, subjecting them to isoproterenol, a synthetic nonselective agonist for β-ARs, at 5 or 15 mg/kg per day to induce different degrees of cardiac remodeling in vivo. Cardiac structure and function were assessed by echocardiography 24 hours after the last of seven consecutive daily injections of isoproterenol, and cardiac fibrosis was examined by Masson trichrome stain in morphology and by real-time polymerase chain reaction for the expression of fibrosis-related genes. The results showed that deletion of mPGES-1 had no significant effect on isoproterenol-induced cardiac dysfunction or hypertrophy. However, the cardiac fibrosis was dramatically attenuated in the mPGES-1 knockout mice after either low-dose or high-dose isoproterenol exposure. Furthermore, in vitro study revealed that overexpression of mPGES-1 in cultured cardiac fibroblasts increased isoproterenol-induced fibrosis, whereas knocking down mPGES-1 in cardiac myocytes decreased the fibrogenesis of fibroblasts. In conclusion, mPGES-1 deletion protects against isoproterenol-induced cardiac fibrosis in mice, and targeting mPGES-1 may represent a novel strategy to attenuate pathologic cardiac fibrosis, induced by β-AR agonists. SIGNIFICANCE STATEMENT: Inhibitors of microsomal prostaglandin E2 synthase-1 (mPGES-1) are being developed as alternative analgesics that are less likely to elicit cardiovascular hazards than cyclooxygenase-2 selective nonsteroidal anti-inflammatory drugs. We have demonstrated that deletion of mPGES-1 protects inflammatory vascular diseases and promotes post-myocardial infarction survival. The role of mPGES-1 in β-adrenergic receptor-induced cardiomyopathy is unknown. Here we illustrated that deletion of mPGES-1 alleviated isoproterenol-induced cardiac fibrosis without deteriorating cardiac dysfunction. These results illustrated that targeting mPGES-1 may represent an efficacious approach to the treatment of inflammatory cardiovascular diseases.

Soluble Alpha-Klotho Alleviates Cardiac Fibrosis without Altering Cardiomyocytes Renewal

Heart disease is the leading cause of death worldwide. The major cause of heart failure is the death of the myocardium caused by myocardial infarction, detrimental cardiac remodeling, and cardiac fibrosis occurring after the injury. This study aimed at discovering the role of the anti-aging protein α-klotho (KL), which is the co-receptor of fibroblast growth factor-23 (FGF23), in cardiac regeneration, fibrosis, and repair. We found that the anti-apoptotic function of soluble KL in isoproterenol-treated H9c2 cardiomyocytes was independent of FGF23 in vitro. In vivo, isoproterenol-induced cardiac fibrosis and cardiomyocyte and endothelial cell apoptosis were reduced by KL treatment. Moreover, the number of Ki67-positive endothelial cells and microvessel density within the isoproterenol-injured myocardium were increased upon KL treatment. However, by using genetic fate-mapping models, no evident cardiomyocyte proliferation within the injured myocardium was detected with or without KL treatment. Collectively, the cardioprotective functions of KL could be predominantly attributed to its anti-apoptotic and pro-survival activities on endothelial cells and cardiomyocytes. KL could be a potential cardioprotective therapeutic agent with anti-apoptotic and pro-survival activities on cardiomyocytes and endothelial cells.

SIRT1 activation attenuates cardiac fibrosis by endothelial-to-mesenchymal transition

Endothelial-to-mesenchymal transition (EndMT) is closely related to the pathogenesis of various diseases, including cardiac fibrosis. Transforming growth factor (TGF)-β1 strongly induces EndMT, and sirtuin 1 (SIRT1) may play vital roles in TGF-β/Smad pathway inhibition. This study aimed to determine whether SIRT1 activation inhibits EndMT, thereby attenuating cardiac fibrosis. Cardiac fibrosis was induced in C57BL/6 mice by subcutaneously injecting isoproterenol. SIRT1 was activated and then suppressed by intraperitoneally injecting resveratrol (RSV) and EX527, respectively. EndMT was induced by adding TGF-β1 to H5V cells and measured by immunofluorescence and western blot. The role of SIRT1 in EndMT was determined by lentivirus-mediated overexpression of SIRT1. Interactions between SIRT1 and Smad2/3 in the TGF-β/Smad2/3 pathway were examined by immunoprecipitation. SIRT1 activation upregulated CD31 and vascular endothelial-cadherin, and downregulated α-smooth muscle actin, fibroblast-specific protein 1, and vimentin. SIRT1 upregulated and EX527 inhibited TGF-β receptor 1 (TGF-βR1) and P-Smad2/3 expression, respectively. SIRT1 activation and overexpression by RSV/SRT2104 and lentivirus transfection, respectively, reduced TGF-β1-induced EndMT. SIRT1 and Smad2/3 interaction was shown by immunoprecipitation in vivo and in vitro. TGF-βR1 and P-Smad2/3 expression was downregulated and Smad2/3 nuclear translocation was inhibited. In conclusion, SIRT1 activated by RSV attenuated isoproterenol-induced cardiac fibrosis by regulating EndMT via the TGF-β/Smad2/3 pathway.

Phosphocreatine Attenuates Isoproterenol-Induced Cardiac Fibrosis and Cardiomyocyte Apoptosis.

The present study was designed to further explore the role and the underlying molecular mechanism of phosphocreatine (PCr) for cardiac fibrosis in vivo. Isoproterenol (ISO) was used to induce cardiac fibrosis in rats. PCr administration ameliorated fibrosis by reducing collagen accumulation and fibrosis-related signals, including transforming growth factor beta 1 (TGF-β1), alpha smooth muscle actin (α-SMA), collagen type I, and collagen type III. Mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB) signaling pathways, including p38, extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p65, were highly activated by ISO and blocked by PCr. Moreover, PCr decreased ISO-induced matrix metalloproteinase-9 (MMP-9) and increased the tissue inhibitor of metalloproteinase-1 (TIMP-1) expression. Furthermore, PCr suppressed cardiomyocyte apoptosis induced by ISO, as shown by downregulated expression of the proapoptotic caspase-3, Bax, and upregulated expression of the antiapoptotic Bcl-2. Taken together, PCr can be an effective agent for preventing cardiac fibrosis and cardiomyocyte apoptosis.

Geniposide Alleviates Isoproterenol-Induced Cardiac Fibrosis Partially via SIRT1 Activation in vivo and in vitro.

Objective: Geniposide (GE) is a major component in the fruit of Gardenia jasminoides Ellis. Oxidative stress, endoplasmic reticulum (ER) stress, and canonical Smad3 pathway are implicated in the pathogenesis of cardiac fibrosis. We aim to investigate the protective roles of GE in isoproterenol (ISO)-induced cardiac fibrosis.Methods: ISO was used to induce cardiac fibrosis in male C57BL/6 mice. GE and the EX-527 were given for 2 weeks to detect the effects of GE on cardiac fibrosis. Levels of oxidative stress, ER stress, and Smad3 were evaluated by real time-PCR, Western blots, immunohistochemistry staining, immunofluorescence staining, and assay kits.Results: GE treatment alleviated cardiac dysfunction, fibrosis, and hypertrophy in mice response to ISO. Additionally, GE also suppressed the transformation of cardiac fibroblasts to myofibroblasts stimulated by transforming growth factor-β (TGF-β) in vitro. Mechanistically, GE inhibited the oxidative stress, ER stress, as well as Smad3 pathway activated by ISO or TGF-β. A selective antagonist of sirtuin 1 deacetylase (SIRT1), EX-527, partially counteracted the anti-fibrotic effect and weakened the inhibitory effect on the transformation of cardiac fibroblasts to myofibroblasts after the treatment of GE. Acetylated Smad3 (ac-Smad3), oxidative stress, as well as ER stress pathway were significantly enhanced after SIRT1 was blocked while phosphorylated Smad3 (P-Smad3) was not affected.Conclusion: GE could combat cardiac fibrosis in vivo and in vitro by inhibiting oxidative stress, ER stress, and ac-Smad3 in a SIRT1-dependent manner and suppressing P-Samd3 pathway independent of SIRT1 activation. GE is expected to be a promising agent against cardiac fibrosis.