Advanced Fiber Type-Specific Protein Profiles Derived from Adult Murine Skeletal Muscle

Abstract

Skeletal muscle is a heterogeneous tissue consisting of blood vessels, connective tissue, and muscle fibers. The last are highly adaptive and can change their molecular composition depending on external and internal factors, such as exercise, age, and disease. Thus, examination of the skeletal muscles at the fiber type level is essential to detect potential alterations. Therefore, we established a protocol in which myosin heavy chain isoform immunolabeled muscle fibers were laser microdissected and separately investigated by mass spectrometry to develop advanced proteomic profiles of all murine skeletal muscle fiber types. All data are available via ProteomeXchange with the identifier PXD025359. Our in-depth mass spectrometric analysis revealed unique fiber type protein profiles, confirming fiber type-specific metabolic properties and revealing a more versatile function of type IIx fibers. Furthermore, we found that multiple myopathy-associated proteins were enriched in type I and IIa fibers. To further optimize the assignment of fiber types based on the protein profile, we developed a hypothesis-free machine-learning approach, identified a discriminative peptide panel, and confirmed our panel using a public data set.

Keywords: fiber types; laser microdissection; neuromuscular disorders; proteomics; skeletal muscle.

Figure 1

Differentiation among the four fiber types. (a) Principal component analysis (PCA) of fiber types (n = 56). Significant separation of type IIb (purple) and type IIx fibers (green) can be observed in principle component (PC2), while type I (blue) and type IIa fibers (red) formed a combined, but significantly separated, cluster from type IIx and type IIb fibers in PC2 with moderate segregation into type I and type IIa fibers. (b) PCA loadings, visualizing the proteins separating the four groups. The four fiber type characteristic myosin heavy chain (MYH) isoforms are indicated in red and were identified as major separators.

Figure 2

Hierarchical clustering: high protein intensities are shown in red, and low protein intensities are shown in green. The profile plots indicate trends of protein expression intensity in the different fiber types. Cluster 1 displays high intensities in type IIx and type IIb fibers. Proteins of cluster 1 were annotated to glycolysis, calcium signaling, and the sarcoplasmic reticulum. High intensities in cluster 2 were annotated to type IIa and type IIx fibers and could be associated with mitochondrial processes such as the tricarboxylic acid (TCA) cycle the oxidative phosphorylation (OXPHOS) pathway and the mitochondrial respiratory chain complexes. Cluster 3 shows high-intensity values in type I, type IIa, and type IIb and is composed of ribosomal and proteasomal proteins. Cluster 4 can be annotated to structural components of the sarcomere as well as mitochondrial processes, such as the OXPHOS pathway, the TCA cycle, and the respiratory chain but also has a unique annotation to fatty acid beta oxidation and high intensities in GO terms and pathways associated with structural components of the sarcomere and the cytoskeleton.

Figure 3

(a) Principal component analysis (PCA) for all fiber types (n = 56) based on our complete proteomic data. Clear discrimination of type IIb and T2X fibers is possible, whereas type I and type IIa fibers cluster together. (b) PCA based on our alpha-actinin-3 and myosin-1 peptide panel. Discrimination of type I and type IIa fibers is especially enhanced. (c,d) Boxplots of the intensity value distributions on a log2-scale of the four fiber types, type I, type IIa, type IIb, and type IIx, for alpha-actinin-3 peptide (c) and the myosin-1 peptide (d), which were used in our panel. The alpha-actinin-3 peptide TINEVENQVLTR differentiates well between the type IIa, type IIb, and type IIx fibers, and the myosin-1-peptide IAEQELLDASER differentiates well between type I and all other fiber types, especially type IIa. Thus, only these two peptides are needed to differentiate all four fiber types.

Figure 4

(a,b) Quantitative Smart Protein Layers (SPL) Western blot analysis of whole murine soleus and tibialis anterior muscle. SPL Western blotting system (NH DyeAGNOSTICS GmbH) was used for the detection of quantitative differences between selected candidate proteins. Total protein was pre-labeled with a red fluorescent fluorophore (700 nm), and a 12.5 kDa green fluorescence-labeled standard protein (SMA S, 800 nm) was spiked in, enabling error correction of differing sample loading and data normalization between experiments. Primary antibodies against myotilin (a), myotilin (E-10), sc-393957), and desmin (b), DAKO, M0760 Clone D33) were visualized by infrared secondary antibodies (IRDye goat anti-mouse 800 nm). SPL system analysis resulted in quantitative protein volumes (SPL normalized volume), determining a higher concentration in the soleus muscle for both proteins.