Einfluss des Wachstums-Differenzierungs-Faktors-15 (GDF-15) auf die Gen- und Proteinexpression des Musculus gastrocnemius bei hypercholesterinämischen (knockout) Mäusen.

Introduction: Growth differentiation factor-15 (GDF-15) is a distant member of the TGF-β superfamily and is induced under inflammatory and stress conditions particularly in macrophages, smooth muscle cells, cardiomyocytes, adipocytes and endothelial cells (Schlittenhardt et al., 2004; Bauskin et al., 2005; Bermúdez et al., 2008; Ding et al., 2009; Adela and Banerjee, 2015). In former studies we showed an induction of GDF-15 protein and mRNA expression in human arteriosclerotic vessel walls and a localisation in macrophages (Schlittenhardt et al., 2004; Schlittenhardt et al., 2005). GDF-15 is a new biomarker for cardiovascular stress and is associated with heart failure and cardiac remodeling (Andersson et al., 2016; Meijers et al., 2016). Recently, we have shown that GDF-15 deficiency inhibits atherosclerosis development and progression (de Jager et al., 2011; Bonaterra et al., 2012). The aim of the present study was to investigate the influence of GDF-15 on the expression of inflammatory, angiogenic, apoptotic and atrophy genes/proteins in the skeletal muscle of mice fed a cholesterol-enriched diet in a mouse model for experimental atherosclerosis (ApoE-/-). Methods: GDF-15 deficient (GDF-15-/-) mice were generated using a LacZknockin construct (Strelau et al., 2009). These GDF-15-/- mice were cross-bred with ApoE-/- mice, yielding the following genotypes: GDF-15+/+/ApoE+/+ (wild type; WT), GDF-15-/-/ApoE+/+, GDF-15+/+/ApoE-/- and GDF-15-/-/ApoE-/-. 10 weeks old mice received a cholesterol-enriched diet (CED) for 12 or 20 weeks. The weight and the blood lipid concentrations of the animals were measured before CED and after feeding a cholesterol-enriched diet for 12 or 20 weeks. Afterwards the gastrocnemius muscles were dissected and shock-frozen. RNA and proteins were isolated, relevant genes for inflammation, apoptosis, angiogenesis, atrophy and nicotinic acetylcholine receptor (AChR) were analysed by quantitative Real-Time RT-PCR (qRT-PCR). Proteins in skeletal muscle were quantified for pro-/anti-inflammatory cytokines and pro-/anti-angiogenic chemokines using a “Proteome Profiler™ Mouse Cytokine Array Kit” system. Results: The weight was increased in GDF-15-/-/ApoE+/+ mice after 20 weeks CED (10 %; p≤0.01) and in GDF-15-/-/ApoE-/- mice before CED (6 %; p≤0.01) and after 12 (16 %; p≤0.01) or 20 weeks CED (34 %; p≤0.001) compared with WT or GDF-15+/+/ApoE-/- mice (Kubo, 2013). The serum cholesterol level was increased in GDF-15-/-/ApoE-/- mice before CED (22 %; p≤0.05) and after 12 (34 %; p≤0,001) or 20 weeks CED (14 %; p≤0,05) compared with GDF-15+/+/ApoE-/- animals (Kubo, 2013). Further, the serum triglyceride level was increased in GDF-15-/-/ApoE+/+ mice after 12 (54 %; p≤0.05) or 20 weeks CED (35 %; p≤0.05) and in GDF-15-/-/ApoE-/- mice after 20 weeks CED (95 %; p≤0.01) in comparison with WT or GDF-15+/+/ApoE-/- mice (Kubo, 2013). After 12 weeks CED, qRT-PCR showed in gastrocnemius muscle of GDF-15-/-/ApoE+/+ mice a 21- and 6-fold (p≤0.05) IL-6 and IL-1β expression compared with WT. After 20 weeks CED, a 69 % and 98 % (p≤0.05) decrease of IL-6 and IL-1β expression in this group was measured. Pro-apoptotic Bax mRNA expression was 31-fold increased (p≤0.05) in gastrocnemius muscle of GDF-15-/-/ApoE+/+ mice after 12 weeks CED compared with WT. Moreover, we found in gastrocnemius muscle of GDF-15-/-/ApoE-/- mice a 86 % (p≤0.05) decrease of Bax expression compared with GDF-15+/+/ApoE-/- mice after 20 weeks CED. Expression of pro-angiogenic Notch1 gene was 97 % decreased (p≤0.05) in gastrocnemius muscle of GDF-15-/-/ApoE-/- compared with GDF-15+/+/ApoE-/- mice after 20 weeks CED. Expression of atrophy gene Fbxo32 was 23-fold increased (p≤0.05) in GDF-15-/-/ApoE+/+ mice after 12 weeks CED compared with WT mice. Furthermore, the analysis with membrane-based sandwich immunoassay (Proteome Profiler™ Mouse Cytokine Array Kit) showed in gastrocnemius muscle of GDF-15-/-/ApoE+/+ mice after 20 weeks CED a 1.5-3.1-fold increased protein expression of pro-inflammatory (e.g. IL-1β, IL-6, IL-17, IFN-γ and TNF-α) and a 1.2-2.5-fold increased protein expression of anti-inflammatory (e.g. IL-4, IL-10 and IL-13) cytokines compared with WT mice. In gastrocnemius muscle of GDF-15-/-/ApoE-/- mice after 20 weeks CED, a 39-85 % decrease of pro- and anti-inflammatory protein expression was measured compared with GDF-15+/+/ApoE-/- mice. The protein expression of inflammatory and pro-/anti-angiogenic chemokines (e.g. CCL2, CXCL1 and CXCL9) in gastrocnemius muscle of GDF-15-/-/ApoE+/+ mice after 20 weeks was 1.1-2.7-fold increased compared with WT mice. Protein expression of inflammatory and angiogenic chemokines in gastrocnemius muscle of GDF-15-/-/ApoE-/- mice after 20 weeks CED was 45-73 % decreased compared with GDF-15+/+/ApoE-/- mice. Conclusion: The lack of GDF-15 seems to modulate the expression of pro-/anti-inflammatory, pro-/anti-angiogenic, apoptotic and atrophy genes/proteins in gastrocnemius muscle dependent from the duration of CED application. Furthermore, GDF-15 influences the weight and the blood lipid concentrations.