Clin Res Cardiol 107, Suppl 1, April 2018

Metformin modulates autophagic activity and differentially affects titin isoform switching in a cell cycle dependent manner

S. Sahin1, K. Bärthlein1, S. Bongardt1, M. Krüger1, S. Kötter1

1Institut für Herz- und Kreislaufphysiologie, Universitätsklinikum Düsseldorf, Düsseldorf;

Background: Titin is the third filament system in the sarcomere of cross striated muscle cells. In cardiac muscle two isoform types are co-expressed: the longer and more compliant N2BA (3.3-3.7 MDa) and the shorter and stiffer N2B (3.0 MDa). During perinatal cardiac development titin isoform expression switches from the foetal N2BA isoform to the adult N2BA and N2B isoforms. This process is mainly regulated by the splicing factor RNA-binding-protein 20 (RBM20) and modulated by hormones like T3 and insulin. Due to its gigantic size disintegration and degradation of the titin filament are a major challenge for cardiomyocytes, and the precise mechanisms involved in titin degradation are largely unknown. We have recently shown that titin is K48-polyubiquitinated, indicating titin degradation by the 26S-proteasome. Further studies also demonstrated connections of titin to the autolysosomal system. Here, we analysed the participation of the autolysosomal system and the autophagy modulator metformin in degradation of titin and the developmental isoform switch. Methods: Cell cultures of embryonic rat cardiomyocytes (ERCs) were treated in the absence or presence of the autophagy modulator metformin for up to 9 days. Isoform switch in cell culture for up to 9 days was analysed by 2.2 % SDS-PAGE. Autopagic flux was inhibited by 2-24 hours incubation with chloroquine. Protein levels of proteasomal and autophagy associated proteins were determined by Western blots. Immunofluorescent staining was performed using antibodies targeting proteasomal and autophagy associated proteins. Results: Inhibition of autophagy in ERCs for 24 hours by chloroquine between cultivation day 3 and 4 resulted in an increase in protein as well as specifically full length titin K63-dependent polyubiquitination. Autophagy inhibition resulted in accumulation of the autophagosomal markers LC3 and p62 protein. Metformin treatment for 24 hours reduced K63-dependent titin polyubiquitination and protein levels of LC3 and p62. Immunhistochemistry showed partial sarcomeric localisation of muscle specific E3 ligases (STUB1, Fbx32/Atrogin-1 and MuRF-3), which is supposed to be necessary for the ubiquitination of titin. We further observed that activation of autophagy by metformin had different effects on perinatal titin isoform switching. Within the first 5 days metformin accelerated titin isoform switching, accompanied by higher protein levels of the splicing factor RBM20. At later cultivation time points metformin treatment starting at day 6 of ERC culture reduced titin N2B and RBM20 levels compared to untreated control cells. Unc-51-like kinase 1 (ULK1), necessary for induction of the autophagosome formation, showed differential AMPK and mTOR-dependent phosphorylation on S555 and S757, indicating activation of autophagy in the first days and inhibition at later stages. Immunohistochemistry for the proliferation marker Ki67 indicated strongly reduced proliferation ability after 9 days compared to day 4. Conclusion: The titin filament becomes K63-polyubiquitinated suggesting at least a partial degradation by macroautophagy. Metformin as a modulator of autophagy differentially affect the titin isoform switch of ERCs. This is potentially based on metformin-induced activation of autophagy in proliferating and inhibition in non-proliferating ERCs. Under pathologic conditions altered autophagic activity could therefore affect titin degradation and isofom composition in cardiomyocytes.

http://www.abstractserver.de/dgk2018/jt/abstracts//P368.htm