Nicorandil regulates the macrophage skewing and ameliorates myofibroblasts by inhibition of RhoA/Rho‐kinase signalling in infarcted rats

Figures

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  Figure 1. Western analysis of RhoA membrane fraction and cytosolic fraction from the border zone at day 3 after MI. Relative abundance was obtained by normalizing the protein density against that of β‐actin. Each column and bar represents mean ± SD. Each point is an average of 3 separate experiments. Sham (n = 10), Vehicle (Veh, n = 10), Nicorandil (Nic, n = 12), Glibenclamide (Glib, n = 11), Nic + Glib (n = 11), Fasudil (Fas, n = 9) and Fas + Glib (n = 9). *P < 0.05 compared with sham, Nic‐, Fas‐, and Fas + Glib‐treated groups. ANOVA with Bonferroni's correction.

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  Figure 2. Immunohistochemical staining of M1 (CD68+, iNOS +) and M2c (CD68+, IL‐10+) macrophage phenotype from the border zone at day 3 after MI. iNOS‐expressing CD68 (+) M1 macrophages were observed in infarcted myocardium treated with vehicle (Veh), but were significantly reduced by nicorandil (Nic) administration (upper panels). IL‐10‐expressing CD68 (+) M2 macrophages were predominant in nicorandil administered infarcted myocardium (lower panels). The iNOS‐expressing CD68 (+) or IL‐10‐expressing CD68 (+) macrophages were calculated and expressed as bar graphs. M, merged. The line length corresponds to 20 μm. Vehicle (Veh, n = 8), Nicorandil (Nic, n = 8). *P < 0.05 compared with vehicle.

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  Figure 3. Expression of gene markers and function for M1 and M2 macrophages from the border zone at day 3 after MI. (A) mRNA, (B) protein levels, (C) myocardial IL‐10 activity, (D) myocardial arginase activity and (E) serum plasmin activity. There was a significant increase in macrophage accumulation in the infarcted heart at day 3 after MI. There is an obvious shift towards M2 macrophage phenotype in infarcted groups treated with nicorandil, fasudil and fasudil+ glibenclamide as shown by reduced M1 (IL‐6,IL‐1β, iNOS) expression, increased M2 (CD206,IL‐10) mRNA and protein, and attenuated plasmin activity. Sham (n = 10), Vehicle (Veh, n = 10), Nicorandil (Nic, n = 12), Glibenclamide (Glib, n = 11), Nic + Glib (n = 11), Fasudil (Fas, n = 9) and Fas + Glib (n = 9). *P < 0.05 compared with sham, Nic‐, Fas‐, and Fas + Glib‐treated groups. ANOVA with Bonferroni's correction.

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  Figure 4. The role of IL‐10 on myofibroblast activation assessed by immunohistochemical staining and Western blot at day 3 after infarction. Positive staining for α‐SMA (brown color, a hallmark of myofibroblasts) in myocardium is significantly higher in the vehicle, which can be attenuated by administering nicorandil. After adding an IL‐10 receptor antibody (Ab), myofibroblast infiltration was significantly increased compared with nicorandil alone. Vehicle (Veh, n = 7), Nicorandil (Nic, n = 7), Nicorandil/IL‐10 receptor antibody (Nic/Ab, n = 7). *P < 0.05 compared with vehicle and Nic/Ab. ANOVA with Bonferroni's correction.

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  Figure 5. Representative M‐mode image reveals a hypokinetic‐to‐akinetic anterior wall and left ventricular (LV) dilation in the infarcted hearts (B–G) in contrast to normal anterior wall motion in sham‐operated heart (A). There are markedly dilated LV end‐diastolic diameter and LV end‐systolic diameter in groups treated with vehicle (B), Glibenclamide (D, Glib), Nicoradil (Nic) + Glib (E), and Fasudil (Fas) + Glib (G) compared with those in groups treated with Nic (C) and Fas (F). IVS, interventricular septum thickness.

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  Figure 6. Myofibroblast infiltration at day 28 after infarction. Representative immunohistochemical staining of α‐SMA, a hallmark of myofibroblasts (magnification 400×) at the remote zone in different treated rats. A, sham; B, vehicle; C, nicorandil; D, glibenclamide; E, nicorandil + glibenclamide; F, fasudil; G, fasudil + glibenclamide. The line length corresponds to 50 μm. To further confirm the protein levels of α‐SMA, Western blot was performed, showing similar results of immunohistochemical staining. Each column and bar represents mean ± SD. Sham (n = 10), Vehicle (Veh, n = 10), Nicorandil (Nic, n = 12), Glibenclamide (Glib, n = 11), Nic + Glib (n = 11), Fasudil (Fas, n = 9) and Fas + Glib (n = 9). *P < 0.05 compared with sham, Nic‐, Fas‐, and Fas + Glib‐treated groups. ANOVA with Bonferroni's correction.

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  Figure 7. Cardiac fibrosis at the remote zone at day 28. Representative sections from the remote zone with Sirius Red staining (red, magnification 400×) at 4 weeks after infarction. The line length corresponds to 50 μm. Hydroxyproline content was also shown to measure quantitative amount of fibrosis. Each column and bar represents mean ± SD. Sham (n = 10), Vehicle (Veh, n = 10), Nicorandil (Nic, n = 12), Glibenclamide (Glib, n = 11), Nic + Glib (n = 11), Fasudil (Fas, n = 9) and Fas + Glib (n = 9). *P < 0.05 compared with sham, Nic‐, Fas‐, and Fas + Glib‐treated groups. ANOVA with Bonferroni's correction.

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  Figure 8. Proposed schematic representation illustrates the involvement of nicorandil in macrophage‐ and myofibroblast‐related cardiac fibrosis in post‐infarcted rats. After MI, during the early stage, it is characterized by inflammatory M1 phenotype macrophages, however, at a late stage, M2 phenotype would become dominant. M1 and M2 phenotype macrophages can be converted into each other. IL‐10 activity in M2 macrophages inhibits myofibroblast differentiation, which antagonizes extracellular matrix production. Nicorandil administration exhibits potent anti‐fibrotic activity by suppressing collagen synthesis via increasing the activation of M2 macrophages and inhibiting myofibroblast differentiation.