Infarction was associated with increased RhoA/ROCK activation as shown by enhanced RhoA translocalization from the cytosol to the membrane. performance. Fasudil showed similarly increased expression of M2 macrophages as nicorandil. The beneficial effects of nicorandil on fibroblast differentiation were blocked by adding glibenclamide. However, glibenclamide P005091 cannot abolish the attenuated fibrosis of fasudil, implying that RhoA/RhoA\kinase is usually a downstream effector of KATP channel activation. Nicorandil polarized macrophages into M2 phenotype by inhibiting RhoA/RhoA\kinase pathway, which leads to attenuated myofibroblast\induced cardiac fibrosis after myocardial infarction. intraperitoneal injection to block IL\10 binding, once a day, for 3 days, starting at the first day after inducing infarction. At day 3 after infarction, all hearts (= 7 per group) were used for Western blot of \SMA and for immunohistology of myofibroblast infiltration at the border zone ( 2 mm outside the infarct). Experimental MI After anaesthesia with intraperitoneal dose of ketamine\xylazine (90C9 mg/kg), rats were intubated and the left anterior descending artery was ligated using a 6\0 silk as our previous description 12. Sham rats underwent the same procedure except the suture was exceeded under the coronary artery and then removed. Mortality in the animals with MI was ~30% within P005091 the first 24 hrs. None of the sham\operated animals died. Echocardiogram At day 28 after operation, echocardiographic measurements were done under lightly anaesthesia with intraperitoneal injection of ketamine\xylazine (25C4 mg/kg). For a detailed method, please refer to the Supplementary material online. Infarct size and haemodynamic measurements A section, taken from the equator of the LV, was fixed in 10% formalin Colec10 and embedded in paraffin for determination of infarct size. Haemodynamic parameters were measured at the end of the study as described in detail in the Supplementary material online. Real\time RT\PCR of IL\6, IL\1, iNOS, CD206, and IL\10 Real\time quantitative RT\PCR was performed from samples obtained from the border zone with the TaqMan system (Prism 7700 Sequence Detection System, PE Biosystems) at day 3 as previously described 12. We analysed the expression of gene markers for M1 (IL\1IL\10as a loading control. For a detailed method, please refer to the Supplementary material online. Western blot analysis of RhoA translocation, iNOS, IL\10, and \SMA Samples were obtained from either the border zone at day 3 or the remote zone ( 2 mm outside the infarct) at day 28. Experiments were replicated three times and results expressed as the mean value as described in detail in the Supplementary material online. Immunohistochemical analysis of CD68, iNOS, IL\10 and \SMA To confirm the downstream pathways of KATP channel, immunohistochemical staining for M1 and M2 markers was performed on LV muscle. Cryosections were performed at a thickness of 5 m and incubated with antibodies against CD68 (a marker for all those macrophages; Abcam, Cambridge, MA, USA), iNOS (a P005091 marker for M1; Cell Signalling Technology, Danvers, MA, USA), IL\10 (a marker for M2c; R& D systems, Abingdon, UK), and \SMA (a marker for myofibroblast; Sigma, St. Louis, MO, USA). The antibody had been tested for specificity in the rat. Isotype\identical directly conjugated antibodies P005091 served as a negative control. Ten random scans per section were analysed and averaged. Quantification was calculated as the percentage of positively stained area to total area at a magnification of 400. Morphology and morphometry of cardiac fibrosis The interstitial collagen.