Regulation of the CASA autophagy pathway in two muscle cell lines under altered gravity as a model for tunable mechanical loading

Abstract

Protein homeostasis is essential for cell function and survival. Cells have to mitigate extrinsic and intrinsic factors that influence the balance of the cell’s proteome. Autophagy is one crucial mechanism to preserve protein homeostasis. Notably, the importance of chaperones and their regulating co-chaperones in autophagic pathways is increasingly revealed. One of these pathways is chaperone-assisted selective autophagy (CASA), which is essential for the degradation of large cytoskeletal components damaged under tension/mechanical strain. Furthermore, CASA is crucial for mechanotransduction in mammalian cells, muscle maintenance and plays an important role in the response of human skeletal muscle to resistance exercise. However, it is completely unknown, whether CASA is involved in the muscle cell response to altered gravitational loading. Intriguingly, CASA might play a crucial role in the underlying mechanisms of muscle cell adaption to altered gravity and spaceflight-induced muscle atrophy. Therefore, the expression levels of the BAG family molecular chaperone regulator 3 (BAG3), the heat shock protein family B (small) member 8 (HSPB8), filaminC (FLNC), the serine/threonine kinase 38 (STK38) and synaptopodin 2 (SYNPO2), which exert key-functions in CASA were investigated in two different muscle cell lines following the exposure to altered gravity conditions. Hereto, A7r5 vascular smooth-muscle cells and differentiated C2C12 myotubes were exposed to altered gravity in the German Aerospace Center (DLR)’s Multi-Sample Incubator Centrifuge (MuSIC) and 2D-slideflask-clinostat. A significant induction of CASA in the response of differentiated C2C12 myotubes to the influence of hypergravity was identified in this thesis. Based on a significant reduction in the protein-turnover, the temporal induction of CASA is possibly dependent on the magnitude of the applied hypergravitational load. In contrast, a reduction of CASA was not induced by the exposure to simulated microgravity. Here, a significant increase of SYNPO2 expression indicates actin-cytoskeleton remodeling. In A7r5 VSMCs, hypergravitational load results in a significant up-regulation of SYNPO2. This indicates an injury-like response with potential remodeling of the actin-cytoskeleton. In these cells, SYNPO2 protein turnover as well as steady-state expression is significantly reduced. BAG3, HSPB8, STK38 and FLNC expression is not significantly affected in the response of A7r5 to hypergravitational loading. Contrarily, CASA appears to be reduced in A7r5 by the exposure to simulated microgravity. This reduction of CASA is potentially associated with remodeling of the actin-cytoskeleton, adherence to the cell culture surface or alteration of proliferation. Thereby, tension-sensing and intracellular contraction might be altered. Conclusively, CASA inherits a role in the response of muscle cells to altered gravity. However, the induction appears to be muscle cell type specific, dependent on the gravitational load and temporally regulated. The temporal regulation of CASA in muscle cells as well as the role of the cytoskeleton adapter protein SYNPO2 in the cellular response to altered gravity remain subject to further studies.