CREG ameliorates the phenotypic switching of cardiac fibroblasts after myocardial infarction via modulation of CDC42

Fig. 1. CREG expression was decreased in the border zone of C57BL/6J mice after MI. A Real-time PCR of CREG and αSMA mRNA expression in the border zone of C57BL/6J mice on post-MI days 7 and 14 (N = 5 per group). B, C Western blotting of CREG and αSMA protein expression in the border zone of C57BL/6J mice on post-MI days 7 and 14 (N = 5 per group). D Immunofluorescence staining of CREG (red) and αSMA (green) expression in the border zone of myocardium in C57BL/6J mice on post-MI days 7 and 14 (N = 3 per group). The arrow represented the co-location of CREG with αSMA. E Real-time PCR for CREG mRNA in primary cardiac fibroblast of MI group and sham group (N = 5 per group). F, G Western blotting for CREG in primary cardiac fibroblast of MI group and sham group (N = 5 per group). **P < 0.01 vs. sham group.

Fig. 2. CREG deficiency exacerbated cardiac dysfunction and cardiac fibrosis on day 14 after MI. A, B EF% and FS% in CREG heterozygous mice (CREG+/−) mice and littermate control mice on post-MI day 14 (sham group, N = 10; MI group of littermate control mice, N = 22; and MI group of CREG+/− mice, N = 17). C, D The heart weight-to-tibial length (HW/TL) ratio and heart length-to-tibial length (HL/TL) ratio in CREG+/− mice and littermate control mice on post-MI day 14 (N = 10 per group). E, F Masson trichrome staining in CREG+/− mice and littermate control mice on post-MI day 14 (N = 5 per group). G, H Fibrosis in border zone tissue in CREG+/− mice and littermate control mice on post-MI day 14 (N = 5 per group). **P < 0.01 vs. sham group; ##P < 0.01 vs. littermate control-MI group.

Fig. 3. CREG protein alleviated cardiac dysfunction and cardiac fibrosis on day 14 after MI. A, B EF% and FS% in C57BL/6J mice with overexpression of exogenous CREG on post-MI day 14 (N = 10 in sham group; N = 22 in MI and MI + vehicle groups; N = 25 in MI + CREG group). C, D The heart weight-to-tibial length (HW/TL) ratio and heart length-to-tibial length (HL/TL) ratio in C57BL/6J mice with overexpression of exogenous CREG on post-MI day 14 (N = 10 per group). E, F Masson trichrome staining in C57BL/6J mice with exogenous CREG recombinant protein on post-MI day 14 (N = 5 per group). G, H Fibrosis in the border zone tissue in C57BL/6J mice with overexpression of exogenous CREG on post-MI day 14 (N = 5 per group). **P < 0.01 vs. sham group; ##P < 0.01 vs. MI + vehicle group.

Fig. 4. CREG recombinant protein inhibited cardiac myofibroblast activation in the border zone of the myocardium after MI. A, B Western blotting analysis of αSMA and collagen-1 expression in the border zone of the myocardium in CREG heterozygous mice (CREG+/−) on post-MI day 14 (N = 5 per group). C Immunohistochemical analysis of αSMA and collagen-1 expression in the border zone of the myocardium in CREG+/− mice on post-MI day 14 (N = 3 per group). D, E Western blotting of αSMA and collagen-1 expression in the border zone of the myocardium in C57BL/6J mice with exogenous CREG recombinant protein (rCREG) on post-MI day 14 (N = 5 per group). F Immunohistochemical analysis of αSMA and collagen-1 expression in the border zone of the myocardium in C57BL/6J mice with rCREG on post-MI day 14 (N = 3 per group). **P < 0.01 vs. sham group; #P < 0.05 vs. littermate control-MI, ##P < 0.01 vs. MI + vehicle group.

Fig. 5. Knockdown of CREG enhanced the phenotypic switching of cardiac fibroblasts in vitro. A, B The effects of CREG siRNA (si-CREG) on the expression of αSMA and collagen-1 induced by hypoxia as determined by western blotting (N = 5 per group). C, D The effects of si-CREG on the expression of collagen-1 and PCNA induced by hypoxia as determined by immunofluorescence staining (N = 3 per group). Green indicated collagen-1 expression, red indicated PCNA expression, and blue indicated nuclei. E The effects of si-CREG on cell proliferation were determined by CCK8 assays (N = 5 per group). F The effects of si-CREG on cell migration were determined by wound healing assays (N = 3 per group). **P < 0.01 vs. si-control group; ##P < 0.01, #P < 0.05 vs. si-control+hypoxia group.

Fig. 6. CREG protein inhibited the phenotypic switching of cardiac fibroblasts in vitro. A, B The effects of CREG recombinant protein (rCREG) on the expression of αSMA and collagen-1 induced by hypoxia as determined by western blotting (N = 5 per group). C, D The effects of rCREG on the expression of collagen-1 and PCNA induced by hypoxia as determined by immunofluorescence staining (N = 3 per group). Green indicated collagen-1 expression, red indicated PCNA expression, and blue indicated nuclei. E The effects of rCREG on cell proliferation were determined by CCK8 assays (N = 5 per group). F The effects of rCREG on cell migration was determined by wound healing assays (N = 3 per group). **P < 0.01 vs. control group; ##P < 0.01, #P < 0.05 vs. BSA + hypoxia group.

Fig. 7. CREG protein inhibited hypoxia-induced cardiac myofibroblast activation by regulating CDC42 expression. A Mass spectrometry in the myocardium of CREG+/− mice and littermate control mice (N = 3 per group). B, C Western blotting of CDC42, Rac1, and RhoA expression in the myocardium of CREG+/− mice (N = 5 per group). D, E The effects of CREG recombinant protein (rCREG) and CDC42 overexpressed-adenovirus (adCDC42) on the expression of αSMA and collagen-1 induced by hypoxia as determined by western blotting (N = 5 per group). F, G The effects of rCREG and adCDC42 on the expression of collagen-1 and PCNA induced by hypoxia as determined by immunofluorescence staining (N = 3 per group). Green indicated collagen-1 expression, red indicated PCNA expression, and blue indicated nuclei. H CCK8 assays were used to evaluate the effects of rCREG and adCDC42 on cell proliferation (N = 5 per group). I The effects of rCREG and adCDC42 on cell migration were determined by wound healing assays (N = 3 per group). **P < 0.01, *P < 0.05 vs. littermate control or control group; ##P < 0.01, #P < 0.05 vs. BSA + hypoxia group; &P < 0.05 vs. adGFP+hypoxia; $P < 0.05 vs. adCDC42+ hypoxia group.

Fig. 8. CREG protein inhibited CDC42 expression and activity in vitro. A Real-time PCR of CDC42 mRNA expression in cardiac fibroblasts with CREG recombinant protein (rCREG, N = 5 per group). B, C Western blotting of CDC42 protein expression in cardiac fibroblasts with rCREG (N = 5 per group). D Real-time PCR of CREG mRNA expression in cardiac fibroblasts with CDC42 overexpressed-adenovirus (adCDC42, N = 5 per group). E, F Western blotting of CREG protein expression in cardiac fibroblasts with adCDC42 (N = 5 per group). G Pull-down assay to determine if the effects of rCREG on the CDC42 activity in mouse cardiac fibroblasts under hypoxia (N = 3 per group). H, I Immunoprecipitation of CDC42 with CREG or CREG with CDC42 in mouse cardiac fibroblasts (N = 3 per group). J Immunofluorescence staining for CREG and CDC42 in cardiac fibroblasts (N = 3 per group). K, L Western blotting of CREG and CDC42 expression in chloroquine-treated or MG132-treated cardiac fibroblasts. Red indicated CREG expression, green indicated CDC42 expression, blue indicated nuclei, and yellow indicated the co-location of CREG with CDC42 (N = 5 per group). **P < 0.01 vs. control or adGFP group; ##P < 0.01, #P < 0.05 vs.…