On Marathons and Sprints: An Integrated Quantitative Proteomics and Transcriptomics Analysis of Differences Between Slow and Fast Muscle Fibers

Abstract

Skeletal muscle tissue contains slow as well as fast twitch muscle fibers that possess different metabolic and contractile properties. Although the distribution of individual proteins in fast and slow fibers has been investigated extensively, a comprehensive proteomic analysis, which is key for any systems biology approach to muscle tissues, is missing. Here, we compared the global protein levels and gene expression profiles of the predominantly slow soleus and fast extensor digitorum longus muscles using the principle of in vivo stable isotope labeling with amino acids based on a fully lysine-6 labeled SILAC-mouse. We identified 551 proteins with significant quantitative differences between slow soleus and fast extensor digitorum longus fibers out of >2000 quantified proteins, which greatly extends the repertoire of proteins differentially regulated between both muscle types. Most of the differentially regulated proteins mediate cellular contraction, ion homeostasis, glycolysis, and oxidation, which reflect the major functional differences between both muscle types. Comparison of proteomics and transcriptomics data uncovered the existence of fiber-type specific posttranscriptional regulatory mechanisms resulting in differential accumulation of Myosin-8 and ␣-protein kinase 3 proteins and mRNAs among others. Phosphoproteome analysis of soleus and extensor digitorum longus muscles identified 2573 phosphosites on 973 proteins including 1040 novel phosphosites. The in vivo stable isotope labeling with amino acids-mouse approach used in our study provides a comprehensive view into the protein networks that direct fiber-type specific functions and allows a detailed dissection of the molecular composition of slow and fast muscle tissues with unprecedented Skeletal muscles contain different types of fibers, which are responsible for specific biological properties and functions of individual muscles. Muscle fibers have been classified into slow type I and fast type II fibers mainly based on myofibrillar ATP staining and immunohistochemistry using specific antibodies (1).Slow type I fibers show a red tint, contain high numbers of mitochondria, and their energy supply is mainly based on oxidative metabolism. These features enable slow fibers to execute long lasting contractions, which are essential for the maintenance of body posture. The primary function of type II fibers is the rapid contraction of muscles. Fast fibers are divided into three additional subclasses: Type IIb and IIx (also known as IId) are glycolytic fibers, whereas type IIa fibers are more comparable to oxidative type I fibers (2). Type II fibers, which mainly derive their energy from glycolysis, are thus more susceptible to fatigue compared with Type I fibers.Muscle fibers have also been classified based on the expression of different isoforms of myosin heavy chain (MyHC) proteins. Myosins are the major contractile proteins and their activation by ATP and Ca 2ϩ ions results in shortening of muscle fibers. For example, slow type I fibers express MyHCI␤ a...

The transcriptional coactivator PGC-1α is dispensable for chronic overload-induced skeletal muscle hypertrophy and metabolic remodeling

“…Importantly, our findings imply that the reduction in peak force induced by SA is mainly related to MyHC-2B, SERCA1, MLCK2, and MLC2 expression. These proteins are highly expressed in fast-twitch muscles, and they play a pivotal role in muscle force potentiation (25,26). In accordance, we found that peak force, MyHC-2B mRNA/protein and MLCK2 mRNA levels were preserved in mKO OVL, whereas MLC2 protein levels were even increased.…”

“…The contractile properties of muscle cells are in part determined by the specific expression of genes involved in calcium handling and excitation-contraction coupling (24,25). Thus, mTg CON showed a shift toward a slow phenotype, mainly reflected by a lower mRNA level of calsequestrin 1 (CSQ1) and myosin heavy chain 2 B (MyHC-2B), in contrast to higher levels of the respective slow isoform of these genes, CSQ2 and MyHC-1 (Fig.…”

“…4 A-C). Importantly, the genes up-and down-regulated by SA are characteristic of slowand fast-twitch skeletal muscle, respectively (24,25). At the Values are mean ± SEM.…”

Identification of Disease Specific Pathways Using in Vivo SILAC Proteomics in Dystrophin Deficient mdx Mouse

“…These numbers are similar to those previously reported in other skeletal muscle proteome studies (29,30). However, these numbers are lower than those from a recent study in which a highly sensitive mass spectrometer was used on samples that were subjected to extensive fractionation (31). The identification of a low number of proteins in skeletal muscle can be attributed to the huge dynamic range between high and low abundant proteins in the skeletal muscle tissue (32).…”

The sirtuin SIRT6 regulates stress granules formation in C. elegans and in mammals

“…The resulting tryptic peptides were extracted with acetonitrile and desalted with reverse-phase C18 STAGE tips (Rappsilber et al, 2007). Mass spectrometric experiments were performed on a nano-flow HPLC system (Proxeon) connected to LTQorbitrap and Velos LTQOrbitrap-XL instruments (Thermo Fisher Scientific) equipped with a nanoelectrospray source (Proxeon) (Drexler et al, 2012). The RAW files were analyzed with the MaxQuant software tool (Version 14.10) (Cox and Mann, 2008).…”