Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Mar 7:7:43242.
doi: 10.1038/srep43242.

ZYZ-168 alleviates cardiac fibrosis after myocardial infarction through inhibition of ERK1/2-dependent ROCK1 activation

Shanshan Luo  1   2 , Tran Ba Hieu  2 , Fenfen Ma  3 , Ying Yu  2   4 , Zhonglian Cao  5 , Minjun Wang  1   2 , Weijun Wu  2 , Yicheng Mao  2 , Peter Rose  6 , Betty Yuen-Kwan Law  1 , Yi Zhun Zhu  1   2
Affiliations

ZYZ-168 alleviates cardiac fibrosis after myocardial infarction through inhibition of ERK1/2-dependent ROCK1 activation

Shanshan Luo et al. Sci Rep. .

Abstract

Selective treatments for myocardial infarction (MI) induced cardiac fibrosis are lacking. In this study, we focus on the therapeutic potential of a synthetic cardio-protective agent named ZYZ-168 towards MI-induced cardiac fibrosis and try to reveal the underlying mechanism. ZYZ-168 was administered to rats with coronary artery ligation over a period of six weeks. Ecocardiography and Masson staining showed that ZYZ-168 substantially improved cardiac function and reduced interstitial fibrosis. The expression of α-smooth muscle actin (α-SMA) and Collagen I were reduced as was the activity of matrix metalloproteinase 9 (MMP-9). These were related with decreased phosphorylation of ERK1/2 and expression of Rho-associated coiled-coil containing protein kinase 1 (ROCK1). In cardiac fibroblasts stimulated with TGF-β1, phenotypic switches of cardiac fibroblasts to myofibroblasts were observed. Inhibition of ERK1/2 phosphorylation or knockdown of ROCK1 expectedly reduced TGF-β1 induced fibrotic responses. ZYZ-168 appeared to inhibit the fibrotic responses in a concentration dependent manner, in part via a decrease in ROCK 1 expression through inhibition of the phosphorylation status of ERK1/2. For inhibition of ERK1/2 phosphorylation with a specific inhibitor reduced the activation of ROCK1. Considering its anti-apoptosis activity in MI, ZYZ-168 may be a potential drug candidate for treatment of MI-induced cardiac fibrosis.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Ratio of left ventricle/body weight and fibrotic area determined by Masson trichrome staining.
(a) Left ventricle/body weight ratio at 0 days, 15 days and 42 days of MI. Left ventricle wet weight increased in model group rats; ZYZ-168, LeoS and Cap treatment inhibited the increase of heart weight. (b) Representative images of Masson trichrome staining of hearts in different groups. (c) Fibrotic area was determined as the ratio of fibrotic scar (blue) average circumferences/left ventricle average inner circumferences. Data were expressed as mean ± SEM, six rats in each group were included. *P < 0.05 versus control group, **P < 0.01 versus control group, ***P < 0.001 versus control group, #P < 0.05 versus model group, ##P < 0.01 versus model group.
Figure 2
Figure 2
(a) Myocardial infarction caused abnormal expand of both cardiac systolic and diastolic volume, as well as reduction in ejection fraction over the 15 days and 42 days of infarction. (b) Statistical analysis of diastolic diameter, (c) systolic diameter and (d) ejection fraction after 15 days and 42 days infarction. ZYZ-168 treatment reduced systolic diameter and improved ejection function for 42 days treatment. ZYZ-168 treatment had no significant effect on diastolic diameter. n = 12 for each group. Data were analysed by one-way repeated measures ANOVA followed by post hoc analysis with Student–Newman–Keul’s test for multiple comparisons. *P < 0.05 versus control group, **P < 0.01 versus control group, #P < 0.05 versus model group.
Figure 3
Figure 3. Expression of α-SMA, Collagen I, MMP9 in peri-infarct tissue over 42 days of infarction and the activities of MMP2 and MMP-9 after 15-day and 42-day infarction.
Long-termed infarction significantly enhanced (a) α-SMA, P = 0.008, (b) Collagen I, P = 0.004 and (c) MMP9, P = 0.0078 expression in peri-infarct tissue, ZYZ-168 treatment attenuated increase of these proteins. (e) MMP-9 was expressed at the 92-kD band (pro-form) and the 84-kD band (active form); MMP-2 was evident at the 72-kD (pro-form) and the 62-kD band (active form). MMP9 activity increased obviously at 15 days of infarction compared with that of control group, and ZYZ-168 treatment inhibited MMP9 activity at the initial of 15 days. MMP2 activity was unchanged over the experiment period except that in control group, in which MMP9 activity was undetectable. n = 6 for each group (f) ZYZ-168 reduced serum TGF-β1 at 15 days of infarction (n = 8 for each group). Data were expressed as means ± SEM, *P < 0.05 versus Control group, **P < 0.01 versus control group, #P < 0.05 versus model group, ##P < 0.01 versus model group.
Figure 4
Figure 4. Levels of α-SMA, Collagen I, MMP9 in TGF-β stimulated cardiac fibroblasts.
(a) 48 hrs of TGF-β stimulation increased fibrotic related α-SMA, Collagen I and MMP9 expression in cardiac fibroblasts, ZYZ-168 pretreatment dose-dependently reduced the expression of these fibrotic related proteins. Values were means ± SEM of five independent tests. (b) MMP9 and MMP2 activity was determined by gelatin zymography. MMP9 was evident at 84-kD band (active form) and MMP2 was evident at 62-kD band (active form). Cardiac fibroblasts exposed to TGF-β for 48 hrs showed enhanced MMP9 activity rather than MMP2 activity. ZYZ-168 treatment dose-dependently inhibited MMP9 activity. Values were means ± SEM of five independent tests. (c) Representative immunofluorescent results for expression of α-SMA and MMP9 in TGF-β-stimulated cardiac fibroblasts. α-SMA: Green, MMP9: Red, magnification: 200×. *P < 0.05 versus Normal group, **P < 0.01 versus Normal group, #P < 0.05 versus TGF-β-treated group, ##P < 0.01 versus TGF-β-treated group.
Figure 5
Figure 5
Exposure of cardiac fibroblasts to 500 nM TGF-β led to a time-dependent (a) phosphorylation of ERK1/2 (p-ERK1/2) and (b) ZYZ-168 treatment dose-dependently rescued level of phosphor-ERK1/2. (c) Cardiac fibroblasts were pretreated with 10 μM PD98059 (PD) before TGF-β stimulation. PD98059 could inhibit MMP9 expression and (d) reduced MMP9 activity. (e) Phosphor-ERK1/2 expression in peri-infarct tissue was determined by western blotting, it enhanced in model group (M), and ZYZ-168 treatment reduced its level. In vitro data were means ± SEM of five independent samples. In vivo data were means ± SEM of six independent samples. (a–d): *P < 0.05, **P < 0.01 and ***P < 0.001 versus non-treated group, #P < 0.05 and ##P < 0.01 versus TGF-β-treated group. (e) **P < 0.01 versus control group, ##P < 0.01 versus model group.
Figure 6
Figure 6
Exposure of cardiac fibroblasts to 500 nM TGF-β led to a time-dependent (a) increase of ROCK1 expression and (b) ZYZ-168 dose-dependently reduced ROCK1 expression. (c) Cardiac fibroblasts were pretreated with 10 μM, 20 μM and 50 μM Y27632 for 24 hrs, followed by stimulated with TGF-β for 48 hrs. Y27632 dose-dependently reduced MMP9 expression. (d) Cardiac fibroblasts were transfected with ROCK1 siRNA1 (si1) or scramble siRNA (scra) for 48 hrs prior tostimulated with TGF-β, expression of ROCK1 and MMP9 (e) and activity of MMP9 were determined. (f) ROCK1 expression in peri-infarct tissue was determined by western blotting, ROCK1 expression enhanced in model group, and ZYZ-168 treatment reduced ROCK1 level. Data in vivo were represented as means ± SEM, and three independent samples were adopted. In vitro data were means ± SEM of five independent experiments. In vivo data were means ± SEM of three independent samples. *P < 0.05, **P < 0.01 versus non-treated group or control group, #P < 0.05, ##P < 0.01 versus TGF-β-treated group or model group.
Figure 7
Figure 7. Cardiac fibroblasts were pretreated with PD98059 (10 μM), followed by incubation with TGF-β, MRTF-A nuclear translocation and ROCK1 expression were determined.
(a) Inhibition of ERK1/2 phosphorylation effectively inhibited ROCK1 expression induced by TGF-β. Data represent means ± SEM of five independent experiments. **P < 0.01 versus non-treated or Control (Con) group, #P < 0.05 versus only TGF-β-treated group. (b) Immunofluorescent staining showed that incubation with TGF-β enhanced the nuclear translocation of MRTF-A (Green). And PD98059 seemed to inhibit MRTF-A nuclear translocation.
Figure 8
Figure 8. Activation of ROCK1 and ERK1/2 branches of the TGF-β1 pathway took part in the differenciation of cardiac fibroblasts to myofibroblasts.
In cardiac fibroblasts, TGF-β1 can phosphorylate ERK1/2 and activate ROCK 1, phosphorylated ERK1/2 can also increase expression of ROCK1. ZYZ-168 treatment inhibited TGF-β1-induced ERK1/2 phosphorylaton, resulting in hypoactivity of ROCK1. Black arrows indicate TGF-β1-induced myofibroblasts differenciation; red arrows indicate effects of ZYZ-168 treatment; dashed lines indicate the assumed mechanism which might be involved in ZYZ-168’s anti-fibrotic effects.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Cleland J. G., Coletta A. P. & Clark A. L. Clinical trials update from the American College of Cardiology 2007: Alpha, Everest, Fusion Ii, Validd, Parr-2, Remodel, Spice, Courage, Coach, Remadhe, pro-BNP for the evaluation of dyspnoea and THIS-diet. Eur J Heart Fail. 9, 740–745 (2007). - PubMed
    1. Leask A. Getting to the Heart of the Matter: New Insights Into Cardiac Fibrosis. Circ Res. 116, 1269–1276 (2015). - PubMed
    1. Moore M. T., Guimarães C. N., Yutzey K. E., Pucéat M. & Evans S. M. Cardiac fibroblasts: from development to heart failure. J Mol Med. 93, 823–830 (2015). - PMC - PubMed
    1. Frantz S. et al.. Transforming growth factor beta inhibition increases mortality and left ventricular dilatation after myocardial infarction. Basic Res Cardiol. 103, 485–492 (2008). - PubMed
    1. Klingberg F., Hinz B. & White E. S. The myofibroblast matrix: implications for tissue repair and fibrosis. J Pathol. 229, 298–309 (2013). - PMC - PubMed

Publication types