Objective: Skeletal muscle cells are continuously exposed to mechanical forces. Mechanical sensors such as filamin C (FLNc) and integrins mediate intracellular signaling pathways governed by protein kinases to maintain contractile functions even under extensive mechanical strain. Changes in protein folding and posttranslational modifications can directly regulate the function, turnover, and the interactions of key players within the contractile apparatus to maintain proteostasis in mechanically stressed myotubes and thereby enable muscle fibers to adapt and protect themselves. In this work, we designed a functional, quantitative phosphoproteomics approach to identify signaling mechanisms involved in acute mechanical stress protection in skeletal muscle cells.

Methods: To analyze the phospho-signaling network in C2 myotubes under mechanical stress, we applied electrical pulsed stimulation (EPS) and subsequently performed global quantitative phosphoproteomics using triple stable isotope dimethyl labeling in combination with high-pH RPLC for peptide fractionation and IMAC for phosphopeptide enrichment before LC-MS. Phosphoproteomics data analysis was performed to reveal stress-activated protein kinases. Functional proteomics and biochemical analyses were performed to identify those kinases specifically targeting the large signaling adaptor FLNc to regulate its protein interactions and functions at the myofibrillar Z-disc of contracting skeletal myotubes.

Results: We established a vast myotube phosphoproteome with 33,000 phosphosites localized in 7,226 proteins. 21,428 phosphosites were quantified in ≥ 3/6 biological replicates. Under acute mechanical stress, 2,243 phosphopeptides were significantly upregulated, while 700 were downregulated. Kinase-substrate enrichment analysis showed that mechanical stress markedly increased the activity of JNK1, p38α, ERK1 and PKCα. Most phosphorylation events on FLNc were found in its mechano-sensing domain 20 and its dimerization domain 24. Both domains are known to mediate binding to Z-disc proteins and proteostasis factors. We found that numerous proline-directed motifs in FLNc showed increased phosphorylation under mechanical stress. Inhibition of the MAP kinases in skeletal myotubes during mechanical stress revealed JNK1, p38α, and ERK1 as regulators of PxSP sites, which show different time-dependent activation profiles. Functional analysis of mechanical stress responsive phosphosites in d24 using FLNc mutants mimicking either the non- or the phosphorylated isoform(s) showed regulation of FLNc-HSPB7 binding, which affects FLNc homodimerization for actin-crosslinking at Z-discs. On a physiological level, both FLNc phosphomutants led to higher lesion areas in mechanically stressed skeletal myotubes. We concluded that dynamic phosphorylation/dephosphorylation cycles are crucial for the role of FLNc in stabilizing and maintaining sarcomeres as well as for FLNc homeostasis during mechanical stress.