Raf kinase inhibitor protein mediates myocardial fibrosis under conditions of enhanced myocardial oxidative stress

Figures

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  Fig. 1. The extent of cardiac fibrosis and QTL analyses for cardiac and hepatic fibrosis in CCl4-treated recombinant inbred BxD mouse lines. a The extent of cardiac fibrosis, measured by picrosirius red staining, in CCl4-treated recombinant inbred lines descending from C57BL/6 J and DBA2/J strains (BxD) (n = 10–12 per group). b QTL analyses for cardiac (blue line) and hepatic (red line) fibrosis in the BxD reference population. Most QTL are organ-specific but few affect fibrosis across tissues. In particular on chromosome 5, the overlapping susceptibility loci indicated by the black box harbor the PEBP-I gene locus coding for RKIP. LRS-likelihood ratio statistics. c mRNA-expression of RKIP in the LV myocardium of CCl4-treated BxD mouse lines (n = 3–6 per group). d mean values of cardiac collagen content correlate positively with mean values of RKIP- mRNA in LV myocardium of BxD mouse lines (r = 0.4, p = 0.04, B x D n = 28)

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  Fig. 2. Systemic RKIP-deficiency reduces interstitial and replacement cardiac fibrosis, diminishes fibroblast proliferation, migration and production of intracellular fibronectin by cardiac fibroblasts in RKIP−/− N TAC mice. a Systemic RKIP-deficiency reduces cardiac fibrosis in CCl4-treated and TAC mice (n = 6–15 per group), b prevents an increase in fibroblast density in CCl4- treated N mice and reduces their number in N TAC mice (n = 6–15 per group). c Representative sections of the LV myocardium stained with picrosirius red and co-immunostaining for myocytic α-sarcomeric actin (red) and the fibroblast marker intracellular fibronectin (green). Nuclei are stained blue by DAPI. Bars = 30 µm. d RKIP-knockout in adult murine cardiac fibroblast reduces the number of migrating fibroblasts in a Boyden chamber with an insert size of 0.3 cm2 under basal conditions and after treatment with CCl4 and AngII. AngII but not CCl4 increased fibroblast migration. e shows representative migration assays. Migrating fibroblasts are immunostained for intracellular fibronectin (red). Bars = 100 µm. f RKIP-knockout in adult murine cardiac fibroblasts reduces AngII-stimulated protein expression of…

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  Fig. 3. Systemic RKIP-deficiency reduces the number of CXCR+ fibroblasts and circulating fibrocytes in the time course of cardiac fibrogenesis and ameliorates myocardial oxidative stress in RKIP−/− N TAC mice. a, b RKIP−/− N TAC mice demonstrate reduced number per mm2 (a) and reduced percentage (b) of CXCR4+ fibroblasts in the LV myocardium 1 week (n = 5–6 per group) and 5 weeks post TAC (n = 14–16 per group). Acute pressure overload does not influence both parameters (n = 4–8 per group). c CXCR4+ fibroblasts in a LV section from a WT N TAC mouse: intracellular fibronectin (green), CXCR4 (red), nuclei stained blue by DAPI and the overlay of the three stainings. Bars = 10 µm. d, e The numbers of CD45/collagen I-positive fibrocytes quantified by FACS were increased in the peripheral blood 1 day post TAC (n = 4–7 per group) and in the peripheral blood (d) and BM (n = 6–7 per group) (e) 5 weeks post TAC and was significantly reduced by RKIP-knockout. d, e show representative FACS blots for CD45 (X-axis) and collagen I (Y-axis) in the peripheral blood and in the BM respectively 5 weeks after surgery. The double positive cells in the upper right quadrant were counted as fibrocytes. One-way…

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  Fig. 4. Nuclear protein accumulation of Nrf2 is differently regulated by RKIP and enhanced by its knockout in the time course of pressure overload in N TAC mice. a, b Both the whole protein expression and phosphorylation of RKIP (Ser 153) were increased 1 day (a) (n = 13–15 per group) and 1 week (b) (n = 9–11 per group) post TAC in N mice. c There was no significant difference in protein expression and phosphorylation of RKIP (Ser 153) 5 week post TAC (n = 16 per group). d The nuclear protein accumulation of Nrf2 was not changed by 1 day-TAC (n = 3–6 per group) but was significantly reduced 1 week (n = 4–5 per group) post TAC in WT N mice. Systemic RKIP-deficiency slightly increased the nuclear protein accumulation of Nrf2 1 week post TAC and drastically increased it 5 weeks post TAC. e RKIP-knockout increases nuclear protein accumulation of Nrf2 in adult murine cardiac fibroblasts treated with Ang II (n = 6 per group). f Acute pressure overload significantly increased protein expression of Keap1 (n = 3–6 per group), which, however, was reduced 1 week (n = 5–6 per group) and 5 weeks (n = 7 per group) post TAC. RKIP-knockout enhanced protein expression of Keap1 1 week and 5 weeks post…

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  Fig. 5. Systemic RKIP-deficiency down-regulates Jak2/Fyn myocardial signaling in RKIP−/− N TAC mice. a Systemic RKIP-deficiency significantly reduced nuclear expression of the active form of Thr12 p-Fyn in both groups 5 weeks after surgery (n = 5–6 per group). b RKIP-knockout reduces nuclear expression of p-Fyn in adult murine cardiac fibroblasts treated with AngII (n = 6 per group). c Systemic RKIP-deficiency significantly increased myocardial expression of the active form of Tyr1007/1008 p-Jak2 in RKIP−/− SHAM, which was drastically reduced after 5 weeks of TAC (n = 6–8 per group). Heart tissue from WT and RKIP−/− was immunoprecipitated with anti-Jak2 antibody 5 weeks after surgery and then western blot for Tyr1007/1008 p-Jak2 was performed. RKIP−/−TAC N demonstrates diminished phosphorylation of Jak2. d Systemic RKIP-deficiency significantly enhanced myocardial expression of GRK2 after 5 weeks of TAC (n = 6 per group). Acute reduction of RKIP expression (e) in adult cardiac fibroblasts of C57BL/6N with siRNA (siR) significantly diminished the AngII-induced cytosolic protein expression of fibronectin (f) and increased the nuclear accumulation of Nrf2 (g) in cells treated with scrambled…

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  Fig. 6. Amelioration of cardiac fibrosis in RKIP−/−N versus RKIP−/−J mice after pressure overload. a, b Systemic RKIP deficiency (a) reduces cardiac fibrosis in RKIP−/− N TAC (n = 15 per group) (b) but increases in RKIP−/−J TAC mice (n = 5–9 per group). c Systemic RKIP-deficiency diminishes mRNA-expression of collagen Ia2 in RKIP−/−N TAC (n = 15 per group), d but enhances in RKIP−/−J TAC mice (n = 4–9 per group). e Representative sections of the LV myocardium stained with picrosirius red. Bars = 30 µm. Myocardial oxidative stress in cardiomyocytes and fibroblasts was evaluated by co-immunostaining for 8-dOHG and α–sarcomeric actin and intracellular fibronectin respectively. f Myocardial oxidative stress was significantly reduced in RKIP−/−N SHAM and RKIP−/−N TAC compared with control groups (n = 15–16 per group). g The percentage of 8-dOHG+ cardiomyocytes was significantly reduced in RKIP−/−J Control and was slightly increased in RKIP−/−J TAC compared with control groups (n = 4–9 per group). h The percentage of 8-dOHG+ fibroblasts in N mice was increased by TAC and diminished by RKIP-knockout (n = 15–16 per group). i RKIP−/−J Control mice demonstrated reduced basal ROS production in…

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  Fig. 7. Expression of RKIP and Nrf2 in the LV of N and J mice and myocardial samples from human non-failing and failing LV. a RKIP−/− N TAC (n = 8 per group) showed a twofold increase of nuclear accumulation of Nrf2 and protein expression of Keap1 (c). b RKIP−/− J Control mice (n = 4–7 per group) revealed enhanced the basal nuclear accumulation of Nrf2 and the protein expression of Keap1 (d), which was not further enhanced by TAC. e, f The enhanced nuclear accumulation of Nrf2 in RKIP−/− N TAC (e) and RKIP−/− J Control (f) was accompanied by an increased myocardial protein expression of the Nrf2-regulated antioxidative enzyme catalase and mitochondrial superoxide dismutase/SOD2 (g, h). i Both basal and TAC-induced mRNA expression of the gene coding for RKIP, phosphatidylethanolamine binding protein I (PEBP-I), was significantly lower in J than in N mice but was increased by TAC in J mice. The protein expression of p-RKIP, RKIP (j) and nuclear RKIP−/−N TAC (k) was similar in myocardial samples from human non-failing (n = 8) and failing (n = 15) LV myocardium. l Protein expression of RKIP correlated negatively with the nuclear expression of Nrf2 (r = − 0.38, p = 0.04). Mann–Whitney-test,…

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  Fig. 8. Schematic depiction of the results and proposed hypothesis. a In pressure-overloaded left-ventricular myocardium of WT N TAC mice Nnt is the dominant source of ROS and phospho-RKIP inhibits GRK2, preventing the down-regulation of AT1-receptor. TAC-induced production of AngII activates the AT1-Jak2-Fyn signaling cascade, leading to phosphorylation of Nrf2, its rapid translocation from the nucleus and degradation by proteasomes [36, 37]. The decreased nuclear content of Nrf2 diminishes the expression of antioxidant, cytoprotective ARE-genes. The enhanced myocardial ROS production leads to cell death and fibrosis. b Systemic RKIP-knockout in RKIP−/−N TAC leads to the activation of GRK2, which phosphorylates AT1-receptor, eliciting its internalization and degradation [34]. The downregulation of AT1-Jak2-Fyn signaling cascade inhibits nuclear export of Nrf2 [36]. The nuclear accumulation of Nrf2 enhances the expression of ARE-genes reducing myocardial ROS production, cardiac apoptosis, and fibrosis. c Due to the mutation of the Nnt gene, the inbred mouse strain C57BL/6 J is protected from oxidative stress and fibrosis in response to pressure overload [24]. In the pressure-overloaded…