Fig 2: Down-regulated RhoA expression contributed to propofol-induced excessive relaxation in Ang II-pretreated HUASMCs. A, B western blot was used to test RhoA expression. GAPDH served as a loading control. n = 3 per group in (A) and n = 4 per group in (B). C “Parachute” dye coupling assay was applied to measure the function of GJs. In merged images, donor cells were stained in yellow by Calcein (green) and Dil (red); receiver cells were stained in green with Calcein. Each dot represents the ratio of “recipient cells” (green)/ “donor cells” (yellow) in one field. Three different fields were captured in every experiment. The experiment was repeated 4 times (n = 4 per group). D F-actin was stained with FITC-phalloidin (green) and cell nuclei was stained with DAPI (blue) in HUASMCs. Each dot represents the fluorescence intensity of one field. Three different fields were captured in every experiment. The experiment was repeated 4 times (n = 4 per group). E p-MLC2 and MLC2 were labeled by cellular immunofluorescence (red) and cell nuclei were stained by DAPI (blue) in HUASMCs. Each dot represents the fluorescence intensity of one field. Three different fields were captured in every experiment. The experiment was repeated 4 times (n = 4 per group). *p < 0.05, ** p < 0.01, *** p < 0.001. All values expressed as mean ± SEM

Fig 3: Enhancing Cx43-GJs function with retinoic acid reversed propofol-induced excessive relaxation in Ang II-pretreated HUASMCs. A F-actin was stained with FITC-phalloidin (green) and cell nuclei was stained with DAPI (blue) in HUASMCs. B p-MLC2 and MLC2 were labeled by cellular immunofluorescence (red) and cell nuclei were stained by DAPI (blue) in HUASMCs. C Bar plots showed the statistical results of F-actin level, p-MLC2 level and MLC2 level. Each dot represents the fluorescence intensity of one field. Three different fields were captured in every experiment. The experiment was repeated 3–5 times (n = 3–5 per group). ** p < 0.01, *** p < 0.001. All values expressed as mean ± SEM

Fig 4: Inhibition of Cx43-GJs function attenuated Ang II-increased [Ca2+]i, F-actin polymerization and MLC2 phosphorylation. A Detection of [Ca2+]i with fluo3-AM by flow cytometry on HUASMCs. B F-actin was stained with FITC-phalloidin (green) and cell nuclei was stained with DAPI (blue) in HUASMCs. Each dot represents the fluorescence intensity of one field. Three different fields were captured in every experiment. The experiment was repeated 3 times (n = 3 per group). C p-MLC2 and MLC2 were labeled by cellular immunofluorescence (red) and cell nuclei were stained by DAPI (blue) in HUASMCs. Each dot represents the fluorescence intensity of one field. Three different fields were captured in every experiment. The experiment was repeated 3–4 times (n = 3–4 per group). D Quantitation of fluorescence intensity (B, C) in each group. *p < 0.05, ** p < 0.01, *** p < 0.001. All values expressed as mean ± SEM

Fig 5: Propofol inhibited the activation of RhoA/LIMK2/cofilin and RhoA/MLCK signaling pathways. A Levels of P-LIMK2, LIMK2, P-cofilin and cofilin on HUASMCs were tested by western blot. GAPDH served as a loading control. n = 4 per group. B Levels of MLCK on HUASMCs were tested by western blot. GAPDH served as a loading control. n = 3 per group. C Levels of p-LIMK2, LIMK2, p-cofilin and cofilin on HUASMCs were tested by western blot. GAPDH served as a loading control. n = 3 per group. D Levels of MLCK on HUASMCs were tested by western blot. GAPDH served as a loading control. n = 3 per group. *p < 0.05, ** p < 0.01, *** p < 0.001. All values expressed as mean ± SEM. E The scheme of the mechanism that propofol induces excessive relaxation in Ang II-HUASMCs. (1) Comparison of the number and function of Cx43-GJs and [Ca2+]i in normal and Ang II-HUASMCs. (2) ? When HUASMCs are treated with propofol, propofol causes a prominent decrease in the function of Cx43-GJs in Ang II-HUASMCs compared with normal HUASMCs; ? This results in a more substantial decrease of intracellular Ca2+ mobility in Ang II-HUASMCs; ?-? Subsequently, propofol also leads to a stronger inhibition on the activation of RhoA protein and its downstream- RhoA/LIMK2/cofilin and RhoA/MLCK signaling pathways in Ang II-HUASMCs; ? Ultimately, compared to normal HUASMCs, the activation of F-actin polymerization and MLC2 phosphorylation were both mitigated more dramatically in Ang II-HUASMCs, which caused excessive relaxation