The study identifies the importance of the Raf Kinase Inhibitor Protein (RKIP) for cardiac fibrogenesis. Importantly, we observed a differential regulation of cardiac fibrogenesis by RKIP depending on the myocardial redox state. C57BL/6-RKIP-deficient (RKIP−/−) N mice subjected to increased afterload (TAC) exhibit reduced myocardial fibrosis and oxidative stress. The mechanism relates to upregulation of the nuclear factor erythroid 2-related factor 2 (Nrf2), the main transcriptional activator of antioxidant proteins. In contrast, the Nnt-deficient RKIP−/− J TAC mice reveal diminished oxidative stress, increased LV fibrosis and enhanced nuclear Nrf2.
We identified RKIP as mediator of cardiac fibrosis associated with increased oxidative stress in the myocardium as result of the unbiased approach using genome wide QTL analyses in BxD lines. During our detailed research of the time course and the associated RKIP signalling, two investigations were published that revealed beneficial as well as detrimental myocardial effects of RKIP in mice [
11,
38]. Our data provide the explanation for these potentially discrepant observations showing that the differential effects can be caused by the different genetic backgrounds of the mouse strains and different expression level of RKIP. We have recently reported that C57BL/6 J but not C57BL/6 N mice are protected from myocardial oxidative stress, cardiomyocyte apoptosis and cardiac fibrosis in response to pressure overload due to a mutation of the nicotinamide nucleotide transhydrogenase (Nnt) gene [
29]. During cardiac pressure overload, Nnt functions in reverse-mode and, by depletion of antioxidant NADPH, facilitates mitochondrial ROS emission [
29]. As a result, profound differences between the N and the J strain with regard to the influence of RKIP on cardiac fibrogenesis are observed.
Effects of RKIP in C57BL/6-N mice
The analysis of the inbred BxD mouse lines revealed the association of RKIP with increased cardiac fibrosis and ROS production. Therefore we used
RKIP−/− N mice to investigate the influence of RKIP on myocardial fibrosis and oxidative stress. Systemic
RKIP-deficiency in the Nnt-positive N-strain significantly diminished both CCl
4- and TAC-induced interstitial and replacement cardiac fibrosis, fibroblast proliferation as well as the expression of fibrotic mediators such as
CTGF and
collagen. The activation of SDF-1/CXCR4 axis drastically increases fibroblast activity and migration during cardiac fibrogenesis [
16,
17]. Enhanced expression of SDF-1 in inflammatory cardiomyopathy is associated with increased fibrosis and mortality [
46]. Systemic
RKIP-deficiency significantly reduces the number and the percentage of CXCR4
+ fibroblasts ameliorating LV remodeling during pressure overload. Adult
RKIP−/− N cardiac fibroblasts show reduced migration capacity basal and after treatment with CCl
4 and angiotensin II, decreased angiotensin II-stimulated production of intracellular fibronectin. In agreement with recent reports in the literature, [
3,
11] TAC and CCl
4-treated RKIP
−/− N mice reveal decreased apoptosis and decreased proliferation of cardiomyocytes and non-cardiomyocytes. Thus, systemic
RKIP-deficiency in C57BL/6 N mice ameliorates interstitial and replacement cardiac fibrosis and cardiac cell turnover.
In addition to the resident cardiac fibroblasts, circulating bone marrow-derived fibroblasts (fibrocytes) contribute to cardiac fibrosis [
17,
42,
43]. Although recent experimental findings question the participation of circulating fibrocytes in pressure-overload induced fibrosis, clinical studies demonstrate an association between their elevated number in the peripheral blood and adverse clinical outcomes [
1,
19,
27]. Thus, the reduction of the fibrocyte numbers in the peripheral blood and bone marrow of pressure-overloaded C57BL/6 N is consistent with an ameliorative effect of systemic
RKIP-knockout.
Acute pressure overload increases ERK1/2 phosphorylation in WT which remains unchanged during the first week but is reduced below the basal level 5 weeks post TAC [
24]. Basal phosphorylation of ERK1/2 is enhanced by
RKIP-knockout and not changed by TAC. Recent reports demonstrate that the Raf-MEK-ERK signaling pathway plays a differential role during the time course of cardiac remodeling: increased phosphorylation and activation of ERK1/2 in early stage promotes cardiomyocyte hypertrophy and cardiac remodelling but in later stages decreased phosphorylation and downregulation of ERK1/2 increases myocyte apoptosis leading to LV dilatation and heart failure [
14,
24‐
26]. Thus, systemic
RKIP-deficiency ameliorates cardiac remodeling activating Raf-MEK-ERK signaling pathway during 5 weeks of pressure-overload.
TAC induced myocardial oxidative stress is a pivotal and well-characterized trigger of cardiac hypertrophy and fibrosis [
18,
23,
45]. Nrf2 is the main transcriptional activator of antioxidant proteins and enzymes protecting against cardiac hypertrophy and fibrosis during pressure overload [
7,
23,
45]. Immunostaining for 8-hydroxyguanosine revealed drastically increased oxidative stress both in pressure-overloaded cardiomyocytes and fibroblasts of WT N mice after TAC. Despite technical limitations of 8-hydroxyguanosine as oxidative damage marker [
32] clinical studies demonstrate an increased level in patients with cardiovascular disease [
8]. The finding of increased oxidative stress was confirmed by MDA concentration and decreased myocardial expressions of catalase and mitochondrial superoxide dismutase.
RKIP-knockout in N-mice markedly reduced oxidative stress and increased nuclear accumulation of Nrf2 [
2]. This increase was accompanied by an enhanced myocardial protein expression of Keap1. Keap1 functions as substrate adaptor protein for Cullin 3(CUL3)/Ring box protein 1 (RBX1)-dependent E3 ubiquitin ligase complex which promotes rapid degradation of Nrf2 in the absence of oxidative stress [
33]. Nrf2 regulates its protein level through an autoregulatory loop activating the expression of Keap1, CUL3 and RBX1 [
33]. Keap1 decreases apoptosis and inflammation by reduction of NF-κB activation through Keap1E3 ligase-mediated degradation of IKKβ [
6,
20]. The increased nuclear accumulation of Nrf2 is caused by down-regulated Jak2/Fyn myocardial signaling inhibiting export of Nrf2 from the nucleus (pathway depicted in the schematic (Fig.
8).
RKIP−/−N mice demonstrate increased myocardial expression of GRK2 in pressure-overload and abrogated angiotensin II response in isolated adult cardiac fibroblasts [
34]. We speculate that the ameliorative effect of Nrf2 in
RKIP−/− N TAC exceeds the deleterious one of GRK2 [
5,
9,
39]. Moreover, ERK1 phosphorylates GRK2 reducing its pro-fibrotic activity [
35]. Since RKIP is implicated in the regulation of different cell types we performed cell culture experiments to further confirm the influence of RKIP-knockout on the nuclear protein content of Nrf2 in adult cardiac fibroblasts. The fibroblasts from
RKIP−/− N mice demonstrate enhanced nuclear accumulation of Nrf2 after treatment with angiotensin II. Furthermore, small interfering RNA-mediated silencing of
RKIP expression in adult cardiac fibroblasts of C57BL/6 N mice significantly reduced angiotensin II-induced expression of intracellular fibronectin and increased the nuclear accumulation of Nrf2. Our findings are in agreement with recent data from cell culture experiments demonstrating ameliorative effects of
RKIP silencing: resistance to oxidative stress caused by increased nuclear accumulation of Nrf2 [
2] and retardation of cellular senescence elicited by enhanced activity of ERK [
22]. The retardation of aging can ameliorate cardiac fibrosis decreasing the number of cardiac myeloid and mesenchymal fibroblasts in age-dependent cardiac fibrosis [
43]. These findings suggest that reduction of Ang II signaling leading to nuclear accumulation of Nrf2 represents an important mechanism of the observed ameliorative effects of systemic
RKIP-deficiency in pressure-overloaded myocardium of C57BL/6 N mice.
Effects of RKIP in C57BL/6-J mice
The
Nnt-deficient C57BL/6 J strain is protected from myocardial oxidative stress in response to pressure overload [
29]. Thus, we performed the proof-of concept experiments on
RKIP−/− J mice to elucidate the role of the myocardial redox status in the observed effects of
RKIP-knockout. Overexpression
RKIP is associated with increased cardiac contractility that is mediated by the β1-adrenoceptor and with anti-apoptotic and anti-fibrotic effects mediated by the β2-adrenoceptor (AR) [
38]. The stimulation of β2-AR activates the cytoprotective antifibrotic and antiapoptotic phosphatidylinositol 3´kinase (PI3 K)-Akt signaling pathway [
38,
40]. Systemic
RKIP-deficiency in C57BL/6 J mice exaggerates pressure overload–induced cardiac failure [
38]. Systemic
RKIP deficiency has opposite effects on cardiac fibrosis in the pressure-overloaded myocardium of the two mouse strains: cardiac fibrosis and pro-fibrotic signaling were reduced in the
Nnt-positive N and increased in the
Nnt-negative J mice. Both basal and TAC-stimulated myocardial ROS production is markedly reduced in J mice. The expression of
PEBP-
I, the gene coding for RKIP, is higher in the N compared to the J mouse strain and TAC does not increase nuclear accumulation of Nrf2 in
RKIP−/− J mice. Thus, the fibrotic signaling of RKIP depends on the myocardial redox milieu. Under conditions of decreased myocardial ROS, detrimental effects of systemic
RKIP-deficiency override its antioxidant protection.
To elucidate a potential clinical relevance of our findings, we evaluated the protein expression of RKIP, its phosphorylation level and nuclear accumulation of Nrf2 in the myocardial samples from non-failing and failing human left ventricles. The data suggest that RKIP protein expression correlates negatively with the nuclear protein content of Nrf2 in the human hearts. These findings are consistent with the concept that myocardial ROS production that is typical for maladaptive cardiac remodeling switches the effects of RKIP expression to pro-fibrotic signaling [
4,
28]. Clearly, additional future studies are needed to confirm the role of RKIP during cardiac remodeling in humans.
Using immunohistochemistry to demonstrate the nuclear accumulation of Nrf2 has its limitations because of the potential non-specific cross-reactivity of some antibodies against Nrf2 [
30,
31]. The association between the nuclear accumulation of Nrf2 and the reduced ROS production was reported previously, e.g. using Nrf2-LacZ mice [
15] and GFP-Nrf2 fusion proteins [
44]. Furthermore, antibody-based and antibody-independent detection of nuclear localization of Nrf2 revealed shuttling of Nrf2 between the nucleus and the cytoplasm [
44].
In conclusion, the data show the important role of RKIP for the regulation of cardiac remodeling and fibrosis. Systemic RKIP deficiency ameliorates cardiac remodeling under conditions of increased myocardial production of reactive-oxygen species by activation of the Nrf2–Keap1 system. These findings may provide interesting perspectives to create novel strategies for the detection and prevention of myocardial fibrosis.