Multikinase inhibitor sorafenib prevents pressure overload-induced left ventricular hypertrophy in rats by blocking the c-Raf/ERK1/2 signaling pathway

Abstract

Background: Left ventricular hypertrophy (LVH) is a potent risk factor for sudden death and congestive heart failure.

Methods: We tested the effect of sorafenib, a multikinase inhibitor (10 mg/kg, given orally, starting 2 days prior to banding, till sacrifice on day 14), on the development of LVH following aortic banding in rats.

Results: The latter resulted in significant LVH caused by both an increase in cardiomyocyte volume and interstitial collagen deposition. The observed LVH was entirely blocked by sorafenib downregulating both of these components. LVH was associated with PDGF-BB and TGFβ1 overexpression, as well as phosphorylation of c-raf and ERK1/2. Additionally, the transcription factors c-myc and c-fos leading to proliferation as well as the hypertrophy-inducing transcription factor GATA4 and its regulated gene ANP were all upregulated in response to aortic banding. All these overexpressions and upregulations were inhibited upon sorafenib treatment.

Conclusion: We show that sorafenib exhibits a regulatory role on the occurrence of LVH following AB in rats by blocking the rise in growth factors PDGF-BB and TGFβ1, activation of the corresponding c-Raf-ERK1/2 signaling pathway and effector mechanisms, including GATA4 and ANP. This effect of sorafenib may be of clinical importance in modulating the maladaptive hypertrophic response to pressure overload.

Figure 1

Sorafenib prevents the development of pressure overload-induced left ventricular hypertrophy (LVH) and cardiomyocyte volume. A. Following aortic banding (AB), LVH is anatomically evidenced by a significant rise in the ventricular weight to body weight (VW/BW) ratio (p < 0001, ***), which was blocked by sorafenib (p < 0001, ***). B. The observed LVH following AB is histologically accounted for by an increase in cardiomyocyte volume reflected by a significant increase in the cardiomyocyte cross-sectional area (CSA) expressed in μm2 (p < 0001, ***). This component of LVH was totally blocked by sorafenib treatment (p < 0.0001, ***). Cardiomyocyte CSA was measured in 10 different areas per section. Results were obtained from 2–4 sections per rat for a total of 6 rats selected at random from each group.

Figure 2

Sorafenib blocks interstitial collagen deposition following aortic banding. A. Interstitial collagen deposition following AB is evidenced by van Gieson’s staining and predominantly localizes to perivascular spaces. Sorafenib prevents interstitial collagen deposition after AB. B. Quantitative measurement of tissue collagen by SirCol assay normalized to total protein content (μg/mg). Sorafenib blocks this component of LVH (p < 0001, ***). All measurements were performed in duplicate.

Figure 3

Sorafenib inhibits the over expression of growth factors in myocardium. Tissue levels are determined by ELISA normalized to total protein content (pg/mg). A. Sorafenib abolishes the over expression of myocardial PDGF-BB levels in banded animals (p < 0001, ***). B. Sorafenib suppresses over expression of the pro-fibrotic growth factor TGFβ1 in myocardium in banded animals (p < 0001, ***). All measurements were performed in duplicate.

Figure 4

Sorafenib prevents phosphorylation of c-Raf and ERK1/2 in myocardium following AB. Left panel: representative Western blot for phosphorylated (upper band) and total (lower band) kinases from ventricle tissue. Right panel: ratios of phosphorylated to total kinases are presented as histograms. A. c-Raf phosphorylation is inhibited by sorafenib treatment in banded animals (p < 0001, ***). B. ERK1/2 phosphorylation is blocked by sorafenib treatment in banded animals (p < 0001, ***).

Figure 5

Sorafenib regulates the increased expression of nuclear transcription factors resulting from AB. mRNA of c-myc (A) and c-fos (B) and GATA4 (C) are measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR), and normalized to the reference gene GAPDH. Increased expression of pro-proliferative transcription factors c-myc (A) and c-fos (B) and pro-hypertrophic factor GATA4 (C) in AB as compared to sham-operated animals (p < 0.0001, *** for banding vehicle versus sham vehicle for all three factors) is inhibited by sorafenib (p < 0.0001, *** for banding vehicle versus banding sorafenib for all three factors). All measurements were performed in duplicate.

Figure 6

Sorafenib abolishes up-regulation of the pro-hypertrophic factor gene atrial natriuretic peptide ANP in banded animals (p < 0001, ***). ANP mRNA is measured by RT-qPCR and normalized to the reference gene GAPDH. All measurements were performed in duplicate.

Figure 7

Schematic representation of the blocking effect of sorafenib on development of left ventricle hypertrophy (LVH) induced by aortic banding. Mechanical stretch induced by pressure overload results in over-expression of growth factors, PDGF-BB and TGFβ1 in myocardium [5]. The original finding of this study shows sorafenib to inhibit the up-regulation of these growth factors following AB. Phosphorylation and hence activation of PDGFβR and TGFβ1R is known to activate the downstream signaling pathway of c-Raf/ERK1/2 [10]. Sorafenib is reported to block phosphorylation and activation of PDGFβR and c-Raf in cancer studies [23]. By blocking the c-Raf/ERK1/2 cascade, sorafenib down-regulates the expression of pro-proliferative (c-myc, c-fos) and pro-hypertrophic (GATA4) transcription factors as well. One of the pro-hypertrophic effector mechanisms regulated by GATA4 is ANP [14] which is in turn suppressed by sorafenib treatment. The sum of the inhibitory effects of sorafenib ends at the suppression of cardiomyocyte hypertrophy as well as of interstitial collagen deposition, the two main components of LVH. Blocking effect of sorafenib on expression of growth factor as demonstrated in this study. Blocking effect of sorafenib previously reported by Wilhelm et al. [23].