Background: Skeletal muscles are composed of a heterogeneous collection of fiber types with different physiological adaption in response to a stimulus and disease-related conditions. Each fiber has a specific molecular expression of myosin heavy chain molecules (MyHC). So far, MyHCs are currently the best marker proteins for characterization of individual fiber types, and several proteome profiling studies have helped to dissect the molecular signature of whole muscles and individual fibers. Methods: Herein, we describe a mass spectrometric workflow to measure skeletal muscle fiber type-specific proteomes. To bypass the limited quantities of protein in single fibers, we developed a Proteomics high-throughput fiber typing (ProFiT) approach enabling profiling of MyHC in single fibers. Aliquots of protein extracts from separated muscle fibers were subjected to capillary LC-MS gradients to profile MyHC isoforms in a 96-well format. Muscle fibers with the same MyHC protein expression were pooled and subjected to proteomic, pulsed-SILAC, and phosphoproteomic analysis. Results: Our fiber type-specific quantitative proteome analysis confirmed the distribution of fiber types in the soleus muscle, substantiates metabolic adaptions in oxidative and glycolytic fibers, and highlighted significant differences between the proteomes of type IIb fibers from different muscle groups, including a differential expression of desmin and actinin-3. A detailed map of the Lys-6 incorporation rates in muscle fibers showed an increased turnover of slow fibers compared to fast fibers. In addition, labeling of mitochondrial respiratory chain complexes revealed a broad range of Lys-6 incorporation rates, depending on the localization of the subunits within distinct complexes. Conclusion: Overall, the ProFiT approach provides a versatile tool to rapidly characterize muscle fibers and obtain fiberspecific proteomes for different muscle groups.

Aim Resistance exercise increases muscle mass over time. However, the early signalling events leading to muscle growth are not yet well‐defined. Here, we aim to identify new signalling pathways important for muscle remodelling after exercise. Methods We performed a phosphoproteomics screen after a single bout of exercise in mice. As an exercise model we used unilateral electrical stimulation in vivo and treadmill running. We analysed muscle biopsies from human subjects to verify if our findings in murine muscle also translate to exercise in humans. Results We identified a new phosphorylation site on Myocardin‐Related Transcription Factor B (MRTF‐B), a co‐activator of serum response factor (SRF). Phosphorylation of MRTF‐B is required for its nuclear translocation after exercise and is accompanied by the transcription of the SRF target gene Fos . In addition, high‐intensity exercise also remodels chromatin at specific SRF target gene loci through the phosphorylation of histone 3 on serine 10 in myonuclei of both mice and humans. Ablation of the MAP kinase member MSK1/2 is sufficient to prevent this histone phosphorylation, reduce induction of SRF‐target genes, and prevent increases in protein synthesis after exercise. Conclusion Our results identify a new exercise signalling fingerprint in vivo, instrumental for exercise‐induced protein synthesis and potentially muscle growth.

Heart development relies on PTMs that control cardiomyocyte proliferation, differentiation and cardiac morphogenesis. We generated a map of phosphorylation sites during the early stages of cardiac postnatal development in mice; we quantified over 10,000 phosphorylation sites and 5000 proteins that were assigned to different pathways. Analysis of mitochondrial proteins led to the identification of PGC-1-and ERR-induced regulator in muscle 1 (PERM1), which is specifically expressed in skeletal muscle and heart tissue and associates with the outer mitochondrial membrane. We demonstrate PERM1 is subject to rapid changes mediated by the UPS through phosphorylation of its PEST motif by casein kinase 2. Ablation of Perm1 in mice results in reduced protein expression of lipin-1 accompanied by accumulation of specific phospholipid species. Isolation of Perm1-deficient mitochondria revealed significant downregulation of mitochondrial transport proteins for amino acids and carnitines, including SLC25A12/13/29/34 and CPT2. Consistently, we observed altered levels of various lipid species, amino acids, and acylcarnitines in Perm1 − /− mitochondria. We conclude that the outer mitochondrial membrane protein PERM1 regulates homeostasis of lipid and amino acid metabolites in mitochondria.