Pin1 facilitates isoproterenol‑induced cardiac fibrosis and collagen deposition by promoting oxidative stress and activating the MEK1/2‑ERK1/2 signal transduction pathway in rats

Abstract

Peptidyl‑prolyl cis/trans isomerase, NIMA-interacting 1 (Pin1) is a member of a large superfamily of phosphorylation‑dependent peptidyl‑prolyl cis/trans isomerases, which not only regulates multiple targets at various stages of cellular processes, but is also involved in the pathogenesis of several diseases, including microbial infection, cancer, asthma and Alzheimer's disease. However, the role of Pin1 in cardiac fibrosis remains to be fully elucidated. The present study investigated the potential mechanism of Pin1 in isoprenaline (ISO)‑induced myocardial fibrosis in rats. The rats were randomly divided into three groups. Echocardiography was used to evaluate changes in the size, shape and function of the heart, and histological staining was performed to visualize inflammatory cell infiltration and fibrosis. Reverse transcription‑quantitative polymerase chain reaction analysis, immunohistochemistry and Picrosirius red staining were used to differentiate collagen subtypes. Additionally, cardiac‑specific phosphorylation of mitogen‑activated protein kinase kinase 1/2 (MEK1/2) and extracellular‑signal regulated protein kinase 1/2 (ERK1/2), and the activities of Pin1 and α‑smooth muscle actin (α‑SMA) and other oxidative stress parameters were estimated in the heart. The administration of ISO resulted in an increase in cardiac parameters and elevated the heart‑to‑body weight ratio. Histopathological examination of heart tissues revealed interstitial inflammatory cellular infiltrate and disorganized collagen fiber deposition. In addition, lipid peroxidation products and oxidative stress marker activity in plasma and tissues were significantly increased in the ISO‑treated rats. Western blot analysis showed significantly elevated protein levels of phosphorylated Pin1, MEK1/2, ERK1/2 and α‑SMA in remodeling hearts. Treatment with juglone following intraperitoneal injection of ISO significantly prevented inflammatory cell infiltration, improved cardiac function, and suppressed oxidative stresses and fibrotic alterations. In conclusion, the results of the present study suggested that the activation of Pin1 promoted cardiac extracellular matrix deposition and oxidative stress damage by regulating the phosphorylation of the MEK1/2‑ERK1/2 signaling pathway and the expression of α‑SMA. By contrast, the inhibition of Pin1 alleviated cardiac damage and fibrosis in the experimental models, suggesting that Pin1 contributed to the development of cardiac remodeling in ISO‑administered rats, and that the inactivation of Pin1 may be a novel therapeutic candidate for the treatment of cardiovascular disease and heart failure.

Figure 1

H&E and Masson's trichrome staining of myocardial tissues. (A) Histological analysis and pathological changes in cardiac tissue. Representative images of H&E-stained fields (magnification, ×40, top panels and ×200, bottom panels) are shown for the left ventricles of rats in the control group and rats treated with ISO+juglone or ISO alone. (B) Representative Masson's trichrome staining in the different groups. (C) Quantitative analysis of the myocardial interstitial collagen volume fraction (^*P<0.01 and ^**P<0.05 vs. control) and collagen volume fraction (^*P<0.0 vs. control) in the left ventricles of the experimental and control groups. Control, normal rat. ISO, rat intraperitoneally injected with ISO (5 mg/kg). ISO+J, ISO rat intraperitoneally injected with juglone (3 mg/kg). Data is presented as the mean ± standard error of the mean from at least three independent experiments. H&E, hematoxylin and eosin; ISO, isoprenaline; J, juglone.

Figure 2

Assessment of cardiac structure and function. (A) Representative M-mode echocardiograms in the control, ISO and ISO+J groups. (B) Bar charts from the left show the HW/BW ratio, EF%, IVS, FS%, and LVPW. ^*P<0.01 ISO group vs. control group; ^**P<0.05 ISO group vs. ISO+J group for EF%, IVS and FS%; ^*P<0.05 ISO group vs. control group for LVPW. Control, normal rat. ISO, rat intraperitoneally injected with ISO (5 mg/kg); ISO+J, ISO rat intraperitoneally injected with juglone (3 mg/kg). Data are presented as the mean ± standard error of the mean from at least three different independent experiments. ISO, isoprenaline; J, juglone; EF%, left ventricular ejection fraction; IVS, interventricular septal thickness; FS%, fractional shortening; LVPW, left ventricular posterior wall thickness.

Figure 3

Qualitative and quantitative analysis of collagen accumulation. (A) Picrosirius red staining of histological sections of the left ventricle to assess collagen deposition in different groups (magnification, ×40, top panels and ×200, bottom panels). (B) Immunofluorescent staining of collagen I, collagen III and total collagen proteins. Collagen I is stained red, collagen III is stained green, nuclei are stained blue with 4,6-diamidino-2-phenylindole (magnification, ×200). (C) Bar graphs shows the results of the quantitative analysis of the expression of collagen I, collagen III, total collagen proteins, collagen deposition, and mRNA levels of collagen type I and collagen type III, which were measured using an image analysis system. ^*P<0.01 ISO group vs. control group; ^**P<0.01 ISO group vs. ISO+J group control, normal rat. ISO, rat intraperitoneally injected with ISO (5 mg/kg). ISO+J: ISO rat intraperitoneally injected with juglone (3 mg/kg). Data are presented as the mean ± standard error of the mean from at least three independent experiments. ISO, isoprenaline; J, juglone.

Figure 4

Oxidative stress and measurements of MDA and SOD. (A) Oxidative stress was detected using the immunofluorescent probe-DHE (magnification, ×40 and ×200 above and below, respectively). Reactive oxygen species were stained red and nuclei were stained blue with 4,6-diamidino-2-phenylindole (magnification, ×200). (B) Expression of MDA in serum and SOD in heart tissue. ^*P<0.01 ISO group vs. control group; ^**P<0.01 ISO group vs. ISO+J group. Data are presented as the mean ± standard error of the mean from at least three independent experiments. Control, normal rat; ISO, rat intraperitone-ally injected with ISO (5 mg/kg); ISO+J, ISO rat intraperitoneally injected with juglone (3 mg/kg). ISO, isoprenaline; J, juglone; MDA, malondialdehvde; SOD, superoxide dismutase.

Figure 5

Expression of Pin1. (A) Representative immunostaining of Pin1. Pin1 was stained red and nuclei were stained blue with 4,6-diamidino-2-phenylindole. (B) Quantitative analysis of the expression of Pin1 via reverse transcription-quantitative polymerase chain reaction analysis and use of an associated image analysis system. GAPDH was used as the sample loading control. ^*P<0.01 ISO group vs. control group; ^**P<0.01 ISO group vs. ISO+J group. Control, normal rat; ISO, rat intraperitoneally injected with ISO (5 mg/kg); ISO+J, ISO rat intraperitoneally injected with juglone (3 mg/kg). Data are presented as the mean ± standard error of the mean derived from at least three independent experiments. ISO, isoprenaline; J, juglone; Pin1, peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1; GAPDH, glyceraldehyde phosphate dehydrogenase.

Figure 6

Expression of MEK1/2-ERK1/2 signal transduction pathway-related proteins. (A) Western blot analysis results showing the protein expression levels of α-SMA, Pin1, pERK1/2 and pMEK. (B) Ratio of α-SMA, Pin1, pMEK1/2 and pERK1/2 to GAPDH. ^*P<0.01. Control, normal rat; ISO, rat intraperitoneally injected with ISO (5 mg/kg); ISO+J, ISO rat intraperitoneally injected with juglone (3 mg/kg). Data are presented the mean ± standard error of the mean derived from at least three independent experiments. ISO, isoprenaline; J, juglone; MEK1/2, mitogen-activated protein kinase kinase 1/2; ERK1/2, extracellular-signal regulated protein kinase 1/2; pMEK1/2, phosphorylated MEK1/2; pERK1/2, phosphorylated ERK1/2; GAPDH, glyceraldehyde phosphate dehydrogenase.