Isoproterenol-induced Myocardial Fibrosis Research Articles

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.

Preclinical Investigations on Anti-fibrotic Potential of Long-Term Oral Therapy of Sodium Astragalosidate in Animal Models of Cardiac and Renal Fibrosis.

In traditional Chinese medicine, Radix Astragali has played a vital role in treating progressive fibrotic diseases. One of its main active components, astragaloside IV, is a promising anti-fibrotic treatment despite its extremely low bioavailability. Our study aimed to optimize sodium astragalosidate (SA) by salt formation to improve solubility and oral absorption for anti-fibrotic therapy in vivo. Isoproterenol-induced myocardial fibrosis rat models and obese BKS-db mice presenting diabetic kidney fibrosis were used in this study. Daily oral administration of SA (20 mg/kg) for 14 days ameliorated cardiac fibrosis by reducing collagen accumulation and fibrosis-related inflammatory signals, including TNF-α, IL-1β, and IL-6. In db/db mice, SA (5,10, and 20 mg/kg per day for 8 weeks) dose-dependently alleviated lipid metabolism impairment and renal dysfunction when administered orally. Furthermore, Western blot and immunohistochemistry analyses demonstrated that SA treatment inhibited renal fibrosis by suppressing TGF-β1/Smads signaling. Taken together, our findings provide the oral-route medication availability of SA, which thus might offer a novel lead compound in preclinical trial-enabling studies for developing a long-term therapy to treat and prevent fibrosis.

Cichoric acid improves isoproterenol-induced myocardial fibrosis via inhibition of HK1/NLRP3 inflammasome-mediated signaling pathways by reducing oxidative stress, inflammation, and apoptosis.

Cichoric acid (CA), a natural phenolic compound found in many plants, has been reported to have antioxidant, anti-inflammatory, hypoglycemic, and other effects. The aim of this study was to determine the potential role and underlying mechanisms of CA in isoproterenol (ISO)-induced myocardial fibrosis (MF). The MF model was induced by subcutaneous ISO injection in mice. Blood and heart tissue were collected for examination. Hematoxylin and eosin staining and Masson's trichrome staining were used to evaluate the histopathological changes and collagen deposition. The production of reactive oxygen species markers was observed by fluorescence microscopy, the degree of cardiomyocyte microstructure injury was observed by transmission electron microscope, and oxidative stress factors were detected by kit method, and the effect of CA on inflammatory factors was detected by ELISA. The expression levels of collagen proteins and signaling pathways were further investigated by western blotting. The results showed that CA inhibited the expression of ISO-induced proinflammatory factors (TNF-α, IL-1β, and IL-18) and proteins (HK1, NLRP3, caspase-1, cleaved-caspase-1, and ASC), and regulated the expression of apoptotic factors (caspase-3, cleaved-caspase-3, Bax, and Bcl-2). The results indicated that CA may regulate the HK1/NLRP3 inflammasome pathway by inhibiting HK1 expression and play a protective role in MF.

Insights into the Mechanism of Supramolecular Self-Assembly in the Astragalus membranaceus-Angelica sinensis Codecoction.

Astragalus membranaceus (Fisch.) Bge. (AM) and Angelica sinensis (Oliv.) Diels (AS) constitute a classic herb pair in prescriptions to treat myocardial fibrosis. To date, research on the AM-AS herb pair has mainly focused on the chemical compositions associated with therapeutic efficacy. However, supermolecules actually exist in herb codecoctions, and their self-assembly mechanism remains unclear. In this study, supermolecules originating from AM-AS codoping reactions (AA-NPs) were first reported. The chemical compositions of AA-NPs showed a dynamic self-assembly process. AA-NPs with different decoction times had similar surface groups and amorphous states; however, the size distributions of these nanoparticles might be different. Taking the interaction between Z-ligustilide and astragaloside IV as an example to understand the self-assembly mechanism of AA-NPs, it was found that the complex could be formed with a molar ratio of 2:1. Later, AA-NPs were proven to be effective in the treatment of myocardial fibrosis both in vivo and in vitro, the in-depth mechanisms of which were related to the recovery of cardiac function, reduced collagen deposition, and inhibition of the endothelial-to-mesenchymal transition that occurred in the process of myocardial fibrosis. Thus, AA-NPs may be the chemical material basis of the molecular mechanism of the AM-AS decoction in treating isoproterenol-induced myocardial fibrosis. Taken together, this work provides a supramolecular strategy for revealing the interaction between effective chemical components in herb-pair decoctions.

Inhibition of Mogroside IIIE on isoproterenol-induced myocardial fibrosis through the TLR4/MyD88/NF-κB signaling pathway.

To investigate the effect of mogroside IIIE (MGIIIE) on isoproterenol (ISO)-induced myocardial fibrosis and explore its possible mechanisms. Forty C57BL/6 male mice (6-8 weeks) were randomly divided into a control group (n=10), model group (n=10), low MGIIIE dose group (n=10), and high MGIIIE dose group (n=10). Myocardial fibrosis was established by subcutaneous ISO injection. After 2 weeks of continuous gastric administration of MGIIIE, the cardiac structure was evaluated by echocardiography. Myocardial inflammation and fibrosis were evaluated by histology examination. Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), p-IκBα, p-NF-κB, transforming growth factor β1 (TGF-β1), and α-smooth muscle actin (α-SMA) expression were detected by western blot. Inflammatory cytokines (IL-1β, IL-6, and TNF-α) in the serum were examined by ELISA. In the in vitro study, Ang II (1 μmol/l) was used to stimulate the fibroblasts, then inflammation and fibrosis index were detected. MGIIIE inhibited inflammation and fibrosis and down-regulated TLR4, MyD88, TGF-β1, and α-SMA expression in the myocardium. In the in vitro study, MGIIIE ameliorates the deposition of Col Ш and Col I and decreases the release of inflammatory cytokines. MGIIIE increased p-IκBα and reduced p-NF-κB expression both in vivo and in vitro. MGIIIE plays a role in anti-myocardial fibrosis, by inhibiting TLR4/MyD88/NF-κB signaling expression, and decreasing inflammatory cytokine release. MGIIIE may represent a novel therapeutic strategy for treating cardiac fibrosis.

The Severity of Isoproterenol-induced Myocardial Fibrosis and Related Dysfunction in Mice is Strain-dependent

The Severity of Isoproterenol-induced Myocardial Fibrosis and Related Dysfunction in Mice is Strain-dependent

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.

Apigenin inhibits isoproterenol-induced myocardial fibrosis and Smad pathway in mice by regulating oxidative stress and miR-122-5p/155-5p expressions.

Apigenin, a flavonoid isolated from Apium graveolens, is an effective natural active ingredient that inhibits transforming growth factor-β1 (TGF-β1)-induced cardiac fibroblasts (CFs) differentiation and collagen synthesis. However, its effects on isoproterenol-induced myocardial fibrosis in mice remain unknown. This study aimed to examine the effect of apigenin in the prevention of myocardial fibrosis. A mouse model of myocardial fibrosis induced by isoproterenol was established, and the mice were given apigenin 75-300 mg/kg orally for 40 days. The results showed that the heart weight coefficient, myocardial hydroxyproline, collagen accumulation, and malondialdehyde levels in the apigenin-treated groups were significantly reduced. In contrast, the activity of myocardial superoxide dismutase and glutathione peroxidase were significantly enhanced. The results of real-time quantitative polymerase chain reaction and western blot assays showed that apigenin could significantly upregulate the expressions of myocardial microRNA-122-5p (miR-122-5p), c-Ski, and Smad7 and downregulate the expressions of myocardial miR-155-5p, α-smooth muscle actin, collagen I/III, NF-κB, TGF-β1, hypoxia-inducible factor-1α (HIF-1α), Smad2/3, and p-Smad2/3. In vitro, the differentiation and extracellular matrix production, as well as TGF-β1/Smads axis, were further reduced after treatment of miR-122-5p mimic or miR-155-5p inhibitor-transfected and TGF-β1-stimulated CFs with apigenin. These results suggested that apigenin increased the expression of miR-122-5p and decreased the expression of miR-155-5p, which subsequently downregulated and upregulated the target genes HIF-1α and c-Ski, respectively. Furthermore, apigenin administration downregulated TGF-β1-induced Smad2/3 and upregulated Smad7. In addition, it reduced the NF-κB/TGF-β1 signaling pathway axis by increasing antioxidant ability to exert the antifibrotic effects.

Effects of maslinic acid on isoproterenol-induced myocardial fibrosis in mice

Objective: To investigate the effect of maslinic acid (MA) on isoproterenol (ISO)-induced myocardial fibrosis in mice. Methods: ISO was used to induce myocardial fibrosis in adult male C57BL/6 mice, and MA was administered for two weeks to detect the effects of MA on cardiac function and fibrosis. Molecular changes of fibrosis markers and signaling pathways were detected by RT-PCR and western blotting. Phosphate buffer saline (PBS), PBS+SB203580 (p38 MAPK inhibitor), PBS+MA, ISO, ISO+SB203580, ISO+MA were added to the primary cultured rat fibroblasts. Cells were collected after 48 h for subsequent detection. Results: In this study, the mouse model of myocardial fibrosis was successfully established. The left ventricular faction shortening (FS) and maximum rate of rise and maximum rate of fall of pressure in left ventricular chamber (±dp/dt) of the ISO+MA group were significantly higher than those of the ISO group ((35.1±1.8)% vs (28.5±2.6)%, (7 256±153) mmHg/s vs (6 402±240) mmHg/s, (7 156±163) mmHg/s vs (6 319±219) mmHg/s, all P<0.05). The levels of interstitial and perivascular collagen deposition in the ISO+MA group were higher than those in the ISO group (P<0.05), the relative mRNA levels of COL-1, COL-3 and TGF-β in the ISO+MA group were significantly lower than those in the ISO group, with the relative expression levels of 1.70±0.24 vs 3.69±0.34, 1.72±0.56 vs 4.84±0.82, 1.52±0.19 vs 2.64±0.29, respectively (all P<0.05). The phosphorylation levels of p38 MAPK, Smad3 and protein expression level of TGF-β1 in ISO+MA group were lower than those in ISO group (relative expression levels were 1.67±0.35 vs 2.61±0.58, 1.68±0.23 vs 2.52±0.19,1.56±0.15 vs 2.48±0.26, respectively, all P<0.05). The results of in vitro cell experiments showed that the mRNA levels of COL-1, COL-3 and TGF-β in the SB203580 and MA groups were significantly lower than those in the ISO group (relative expression levels were 2.25±0.51, 2.16±0.48 vs 5.29±1.21; 1.58±0.34, 1.69±0.29 vs 4.97±1.32; 1.41±0.31, 1.55±0.38 vs 3.53±0.56, respectively, all P<0.05). The phosphorylation levels of p38 MAPK and Smad3 in the SB203580 MA groups was significantly lower than those in the ISO group, and the protein expression level of TGF-β1 was lower than that in the ISO group (1.81±0.18, 1.77±0.16 vs 2.56±0.32; 1.85±0.21, 1.81±0.17 vs 2.48±0.37; 1.84±0.24, 1.72±0.17 vs 2.52±0.29, all P<0.05). Conclusion: Maslinic acid can inhibit the phosphorylation of p38 MAPK, thereby preventing the canonical TGF-β1/Smads fibrosis signaling pathway to achieve an anti-fibrosis role.

N-Propargyl caffeate amide (PACA) prevents cardiac fibrosis in experimental myocardial infarction by promoting pro-resolving macrophage polarization.

Macrophages control the initiation and resolution of cardiac fibrosis in post-infarction cardiac remodeling. The aim of the present study was to investigate whether N-propargyl caffeate amide (PACA) could suppress myocardial fibrosis via regulating macrophage polarization. By using rat model of isoproterenol-induced myocardial fibrosis, we discovered that PACA could reduce cardiac fibrosis in a dose-dependent manner. To elucidate the anti-fibrotic mechanisms, we examined whether PACA affected pro-inflammatory M1 and pro-resolving macrophage biomarkers in macrophage polarization. As result, PACA reduced the expression of pro-inflammatory M1 biomarkers (e.g., iNOS, TNF-α, CXCL10, IL-6, CCL2 and CD80) while increased the expression of pro-resolving M2a biomarkers (e.g., IL-10, arginase-1, FZZ1, YM-1 and CD163) in LPS-stimulated RAW264.7 macrophages. PACA also suppressed the elevation of M1 biomarker ED1 in the early phase but up-regulated the expression of pro-resolving biomarker ED2 in the later phase. Moreover, PACA reduced the expression of pro-fibrotic TGF-β1 and PDGF-α while maintained or even increased the production of pro-apoptotic MMP-13, MMP-9 and TRAIL. Importantly, mechanistic studies revealed that PACA might promote the switch of macrophage polarization towards a pro-resolving macrophage phenotype via activating PPAR-γ pathway. Taken together, this study suggested that PACA might be a drug candidate for preventing cardiac fibrosis in myocardial infarction.

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.

Ginsenoside Re Improves Isoproterenol-Induced Myocardial Fibrosis and Heart Failure in Rats.

Objective. Panax ginseng is used widely for treatment of cardiovascular disorders in China. Ginsenoside Re is the main chemical component of P. ginseng. We aimed to investigate the protective effect of ginsenoside Re on isoproterenol-induced myocardial fibrosis and heart failure in rats. Methods. A model of myocardial fibrosis and heart failure was established by once-daily subcutaneous injection of isoproterenol (5 mg/kg/day) to rats for 7 days. Simultaneously, rats were orally administrated ginsenoside Re (5 or 20 mg/kg) or vehicle daily for 4 weeks. Results. Isoproterenol enhanced the heart weight, myocardial fibrosis, and hydroxyproline content in rat hearts. Ginsenoside Re inhibited (at least in part) the isoproterenol-induced increase in heart weight, myocardial fibrosis, and hydroxyproline content. Compared with the isoproterenol group, treatment with ginsenoside Re ameliorated changes in left ventricular systolic pressure, left ventricular end diastolic pressure, and the positive and negative maximal values of the first derivative of left ventricular pressure. Ginsenoside Re administration also resulted in decreased expression of transforming growth factor (TGF)-β1 in serum and decreased expression of Smad3 and collagen I in heart tissue. Conclusion. Ginsenoside Re can improve isoproterenol-induced myocardial fibrosis and heart failure by regulation of the TGF-β1/Smad3 pathway.

Immunohistochemical characterization of myofibroblasts appearing in isoproterenol-induced rat myocardial fibrosis.

Fibrotic lesion is formed by myofibroblasts capable of producing collagens. The myofibroblasts are characterized by immunoexpressions of vimentin, desmin and α-smooth muscle actin (α-SMA) in varying degrees. The cellular characteristics remain investigated in myocardial fibrosis. We analyzed immunophenotypes of myofibroblasts appearing in isoproterenol-induced myocardial fibrosis in rats until 28 days after injection (10 mg/kg body weight); the lesions developed as interstitial edema and inflammatory cell reaction on 8 hr and days 1 and 3, and fibrosis occurred on days 1, 3, 7, 14, and 21 by gradual deposition of collagens, showing the greatest grade on day 14; the lesions gradually reduced with sporadic scar until day 28. Myofibroblasts expressing vimentin and α-SMA increased with a peak on day 3, and then, gradually decreased onwards. Interestingly, Thy-1 expressing cells appeared in the affected areas, apparently being corresponding to the grade similar to vimentin- and α-SMA-positive cells. Thy-1 is expressed in immature mesenchymal cells such as pericytes with pluripotent nature. The immunoreactivity for A3-antigen, a marker for immature mesenchymal cells, was seen in some surrounding cells. There were no cells reacting with antibodies to nestin or glial fibrillary acidic protein, although hepatic myofibroblats have been reported to react with these antibodies. Collectively, myofibroblasts appearing in rat myocardial fibrosis may have been derived from immature mesenchymal cells positive for Thy-1 or A3-antigen, with thereafter showing expressions of vimentin and α-SMA in differentiation.

Abstract 101: FHL2 -null Mice Showing Less Cardiac Remodeling

The four and a half Lin11, Isl-1 and Mec-3 (LIM) domain protein 2 (FHL2) is a member of the four and a half LIM domain-only (FHL) gene family, and has been shown to play an important role in cardiac remodeling. Here, we tested the hypothesis that isoproterenol-induced myocardial fibrosis in mice lacking FHL2 is related to resistant to myocardial fibrosis. We compared the cardiac phenotypes of FHL2 -null mice. Extra-cellular matrix remodeling and apoptosis are examined to determine the roles of FHL2 on cardiac fibrosis induced by isoproterenol. Compared to wild type littermates, FHL2 -null mice exhibited lower mortality. Masson’s Trichrome and silver stains showed significantly less severe ventricle fibrosis in FHL2-null mice. In addition, FHL2 -null mice exhibited lower levels of TGF-β, collagen I/III, but higher membrane-type matrix metalloproteinase-1 (MT1-MMP/MMP-14) during cardiac remodeling. This study shows that alternations of FHL2 might be implicated in the pathogenesis of cardiac fibrosis and suggests FHL2 as novel targets for cardiac fibrosis therapy. Further studies are required to define the mechanism by which FHL2 modulates cardiac remodeling.

Lumican-null mice are susceptible to aging and isoproterenol-induced myocardial fibrosis

With aging and stress, the myocardium undergoes structural remodeling, often leading to fibrosis. The purpose of this study is to examine whether lumican, one of the class II small leucine-rich proteoglycans, has a protective role in cardiac remodeling and fibrosis. In attempts to elucidate the hypothesis that lumican may have a protective role in cardiac remodeling and fibrosis, we compared the cardiac phenotypes of young (3-month-old) and elder (6-month- and 12-month-old) lumican-null (Lum−/−) mice. Extra-cellular matrix remodeling and apoptosis are examined to determine the roles of lumican on age-dependent cardiac fibrosis induced by isoproterenol. Compared to wild type littermates, Lum−/− mice exhibited higher mortality due to significantly impaired systolic function, which was associated with an increase of atrial natriuretic peptide (ANP) secreted by the ventricles in response to excessive stretching of myocytes. Masson's Trichrome and silver stains showed significantly more severe ventricle fibrosis in Lum−/− mice. Interestingly, rate of cell death mediated via apoptosis illustrated by the expression of caspase 3 and TUNEL assay was lower in Lum−/− mice after isoproterenol infusion. In addition, Lum−/− mice exhibited higher levels of TGF-β, collagen I/III, and membrane-type matrix metalloproteinase-1 (MT1-MMP/MMP-14) during cardiac remodeling. This study shows that alternations of lumican might be implicated in the pathogenesis of cardiac fibrosis and suggests lumican as novel targets for cardiac fibrosis therapy. Further studies are required to define the mechanism by which lumican modulates cardiac remodeling.

Transgenic overexpression of transient receptor potential vanilloid subtype 1 attenuates isoproterenol-induced myocardial fibrosis in mice.

Transient receptor potential vanilloid subtype1 (TRPV1) is a non-selective cation channel with high permeability to Ca2+. Intracellular Ca2+signaling is an essential regulator of endothelial nitric oxide(NO) synthase(eNOS) that plays a beneficial role in myocardial fibrosis. The aim of the present study was to determine the role of TRPV1 in isoproterenol-induced myocardial fibrosis. Transgenic mice overexpressing TRPV1 were generated on a C57BL/6J genetic background. An animal model of myocardial fibrosis was created by subcutaneously injecting the mice with isoproterenol. We found that the wild-type mice exhibited a significant increase in heart/body weight ratio, left ventricle/body weight ratio, left ventricular end-diastolic pressure(LVEDP), the cardiac fibrotic lesion area and collagen content, as well as a marked decrease in eNOS phosphorylation and NO/cyclic guanosine monophosphate(cGMP) levels at 2weeks after the administration of isoproterenol(all p<0.01). However, these changes were significantly attenuated in the TRPV1 transgenic mice(p<0.05 or p<0.01). Moreover, the beneficial effects on myocardial fibrosis exerted by the overexpression of TRPV1 were attenuated by the administration of the eNOS inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME)(all p<0.05). Similar anti-fibrotic effects were observed in invitro experiments with primary cultured cardiac fibroblasts. The findings of our study suggest that TRPV1 overexpression attenuates isoproterenol‑induced myocardial fibrosis.

Roles of heat shock factor 1 in isoproterenol‑induced myocardial fibrosis in mice.

Although it is well known that isoproterenol (ISO) causes myocardial hypertrophy and myocardial fibrosis in rats, it has remained elusive whether heat shock factor 1 (HSF1) has a role in this process. The present study aimed to investigate the possible roles of HSF1 in ISO-induced fibrosis in mice. It was found that after administration of ISO in Kunming and HSF1-/+ mice, there was a large number of fibers deposited around blood vessels and among cardiocytes, accompanied with an obvious increase in the protein expressions of type I or III collagen and heat shock protein 47 (HSP47), as indicated by western blot analysis. After intervention with insulin-like growth factor 1 (IGF-1), myocardial fibrosis was significantly attenuated, with a paralleled decrease in the expression of collagen and HSP47 in the mice. However, in HSF1-/- mice, fiber hyperplasy was not observed after injection of ISO, and the levels of type I or III collagen and HSP47 were not significantly increased at the protein and mRNA level. Furthermore, it was demonstrated that after subcutaneous injection of ISO into the back of Kunming and HSF1-/+ mice, large amounts of HSF1 protein were localized to the nucleus, and there was an increase in phosphorylated HSF1 as indicated by western blot and immunohistochemical analysis, respectively. Intervention with IGF-1 inhibited HSF1 activation mediated by ISO. These results suggested that HSF1 is required for myocardial fibrosis in ISO-treated mice, and the underlying molecular mechanism may involve the regulation of HSP47.

GW25-e4167 QiShen YiQi Pills ameliorates ventricular fibrosis and dysfunction via regulation the expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in the isoproterenol-induced myocardial fibrosis rats

GW25-e4167 QiShen YiQi Pills ameliorates ventricular fibrosis and dysfunction via regulation the expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in the isoproterenol-induced myocardial fibrosis rats

Withaferin-A Reduces Type I Collagen Expression In Vitro and Inhibits Development of Myocardial Fibrosis In Vivo

Type I collagen is the most abundant protein in the human body. Its excessive synthesis results in fibrosis of various organs. Fibrosis is a major medical problem without an existing cure. Excessive synthesis of type I collagen in fibrosis is primarily due to stabilization of collagen mRNAs. We recently reported that intermediate filaments composed of vimentin regulate collagen synthesis by stabilizing collagen mRNAs. Vimentin is a primary target of Withaferin-A (WF-A). Therefore, we hypothesized that WF-A may reduce type I collagen production by disrupting vimentin filaments and decreasing the stability of collagen mRNAs. This study is to determine if WF-A exhibits anti-fibrotic properties in vitro and in vivo and to elucidate the molecular mechanisms of its action. In lung, skin and heart fibroblasts WF-A disrupted vimentin filaments at concentrations of 0.5–1.5 µM and reduced 3 fold the half-lives of collagen α1(I) and α2(I) mRNAs and protein expression. In addition, WF-A inhibited TGF-β1 induced phosphorylation of TGF-β1 receptor I, Smad3 phosphorylation and transcription of collagen genes. WF-A also inhibited in vitro activation of primary hepatic stellate cells and decreased their type I collagen expression. In mice, administration of 4 mg/kg WF-A daily for 2 weeks reduced isoproterenol-induced myocardial fibrosis by 50%. Our findings provide strong evidence that Withaferin-A could act as an anti-fibrotic compound against fibroproliferative diseases, including, but not limited to, cardiac interstitial fibrosis.

Abstract 220: Induction of Heme Oxygenase-1 Enhances Matrix Metalloproteinase-9 Secretion and Myocardial Fibrosis

Background: Heme oxygenase (HO)-1 is a stress-inducible rate-limiting enzyme in heme catabolism into carbon monoxide, biliverdin and free iron. Whereas HO-1 exerts cardioprotective effect, such as anti-hypertrophic and anti-apoptotic effect in cardiomyocytes, the influence of HO-1 on cardiac fibroblasts has not been clarified. We examined the effect of HO-1 induced by cobalt protoporphyrin IX (CoPPIX) on matrix metalloproteinase (MMP)-9 secretion, a biomarker of cardiac remodeling, in cardiac fibroblasts. Methods and Results: Cardiac fibroblasts were isolated from male Wistar rats. Secreted MMP-9 protein into culture medium and HO-1 expression in cell lysates were measured by Western blotting. CoPPIX (0.1-1 μ M) increased HO-1 expression and enhanced interleukin (IL)-1β (4 ng/ml, 24 h)-induced MMP-9 secretion (n=4). Mn 2 (CO) 10 (0.3-10 μ M), a carbon monoxide donor, increased IL-1β-induced MMP-9 secretion (n=4), although bilirubin (0.3-10 μ M), a metabolite of biliverdin, and FeSO 4 (0.3-10 μ M), a free iron donor, had no effect. Mn 2 (CO) 10 increased reactive oxygen species (ROS) generation and the enhanced effect of Mn 2 (CO) 10 on IL-1β-induced MMP-9 secretion was abolished by an antioxidant EUK-134 (10 μ M, n=4). We also examined the effect of CoPPIX on isoproterenol (5 mg/kg/day, subcutaneous injection, for 1 or 7 days)-induced cardiac remodeling in male Wistar rats. CoPPIX (1 mg/kg) was administered intraperitoneally every 2 days and this treatment increased HO-1 expression. Hearts were excised at the day 2 or day 8. Myocardial fibrosis was evaluated by Azan staining. At the day 8, CoPPIX worsened isoproterenol-induced myocardial fibrosis significantly (n=6), but had no effect on the increase of heart weight. CoPPIX also enhanced isoproterenol-induced MMP-9 expression at the day 2 (n=10). Conclusion: Induction of HO-1 by CoPPIX enhances IL-1β-induced MMP-9 secretion via ROS generation through heme-derived carbon monoxide in cardiac fibroblasts and isoproterenol-induced myocardial fibrosis and MMP-9 expression. These findings suggest that HO-1 induction does not necessarily exert cardioprotective effects.