Protein profile of fiber types in human skeletal muscle: a single-fiber proteomics study

Abstract

Background: Human skeletal muscle is composed of three major fiber types, referred to as type 1, 2A, and 2X fibers. This heterogeneous cellular composition complicates the interpretation of studies based on whole skeletal muscle lysate. A single-fiber proteomics approach is required to obtain a fiber-type resolved quantitative information on skeletal muscle pathophysiology.

Methods: Single fibers were dissected from vastus lateralis muscle biopsies of young adult males and processed for mass spectrometry-based single-fiber proteomics. We provide and analyze a resource dataset based on relatively pure fibers, containing at least 80% of either MYH7 (marker of slow type 1 fibers), MYH2 (marker of fast 2A fibers), or MYH1 (marker of fast 2X fibers).

Results: In a dataset of more than 3800 proteins detected by single-fiber proteomics, we selected 404 proteins showing a statistically significant difference among fiber types. We identified numerous type 1 or 2X fiber type-specific protein markers, defined as proteins present at 3-fold or higher levels in these compared to other fiber types. In contrast, we could detect only two 2A-specific protein markers in addition to MYH2. We observed three other major patterns: proteins showing a differential distribution according to the sequence 1 > 2A > 2X or 2X > 2A > 1 and type 2-specific proteins expressed in 2A and 2X fibers at levels 3 times greater than in type 1 fibers. In addition to precisely quantifying known fiber type-specific protein patterns, our study revealed several novel features of fiber type specificity, including the selective enrichment of components of the dystrophin and integrin complexes, as well as microtubular proteins, in type 2X fibers. The fiber type-specific distribution of some selected proteins revealed by proteomics was validated by immunofluorescence analyses with specific antibodies.

Conclusion: We here show that numerous muscle proteins, including proteins whose function is unknown, are selectively enriched in specific fiber types, pointing to potential implications in muscle pathophysiology. This reinforces the notion that single-fiber proteomics, together with recently developed approaches to single-cell proteomics, will be instrumental to explore and quantify muscle cell heterogeneity.

Keywords: Human skeletal muscle; Mass spectrometry; Muscle fiber types; Single-fiber proteomics.

Fig. 1

Global analysis of proteome differences between fiber types in human skeletal muscle, as determined by single-fiber proteomics. A Principal component analysis (PCA) performed on pure slow type 1 and fast 2A and 2X fibers (n = 61). Components were derived from proteins expressed in at least 5 fibers for each fiber type. MYHs were not considered. B Volcano plot of statistical significance against fold change, highlighting the most significantly different proteins between type 1 and 2A fibers. C Volcano plot as in B for type 1 vs 2X fibers. D Volcano plot as in B for type 2A vs 2X fibers

Fig. 2

Major patterns of protein distribution according to fiber type in human skeletal muscle. The fiber type distribution of representative proteins (indicated by gene name) is shown for each pattern, with values expressed as per cent of the maximal value. ACTN3, α-actinin-3; CYCS, cytochrome c; MYL2, MLC-2 slow; MYLK2, myosin light chain kinase 2; RYR1, ryanodine receptor 1; TNNC2, fast troponin C

Fig. 3

Myofibrillar proteins: fiber type distribution of representative proteins. Proteins showing similar distribution in the different types are not shown in the figure. Values are expressed as per cent of the maximal value. ACTN2, α-actinin-2; ACTN3, α-actinin-3; CAPZA1, CapZ α1; LMOD2, leiomodin-2; LRRC39, Myomasp; MYL1, MLC1/3-fast; MYL2, MLC-2slow; MYL3, MLC-1 slow; MYLPF, MLC2-fast; MYL6B, MLC-1sa; MYBPC1, myosin-binding protein C1; MYBPC2, myosin-binding protein C2; MYBPH, myosin-binding protein H; MYOM2, myomesin 2; MYOM3, myomesin 3; MYOZ2, myozenin 2; MYOZ3, myozenin 3; TNNC1, slow troponin C; TNNC2, fast troponin C; TNNI1, slow troponin I; TNNI2, fast troponin I; TNNT1, slow troponin T; TNNT2, fast troponin T; TPM1, α-tropomyosin; TPM3, γ-tropomyosin

Fig. 4

Mitochondrial proteins: fiber type distribution of representative OXPHOS proteins. Representative subunits of the electron transport chain complexes (CI to CIV) and of the ATP synthase complex (CV) are shown. CI, NADH:ubiquinone oxidoreductase; CII, succinate dehydrogenase; CIII, ubiquinol-cytochrome C reductase; CIV, cytochrome C oxidase; CV, ATP synthase. Values are expressed as per cent of the maximal value

Fig. 5

Mitochondrial proteins: fiber type distribution of TCA cycle proteins. A scheme of the TCA cycle is shown in the upper panel and the fiber type distribution of the constituent enzymes in the lower panel. ACO2, aconitase; CS, citrate synthase; DLST, dihydrolipoamide S-succinyltransferase; FH, fumarate hydratase; IDH2, isocitrate dehydrogenase, NADPH-dependent; IDH3A, isocitrate dehydrogenase, NADH-dependent, subunit α; MDH2, malate dehydrogenase, mitochondrial; OGDH, oxoglutarate dehydrogenase; SDHA, succinate dehydrogenase subunit A; SDHB, succinate dehydrogenase subunit B; SUCLA2, succinate-CoA ligase ADP-forming subunit β; SUCLG1, succinate-CoA ligase GDP/ADP-forming subunit α

Fig. 6

T tubule and SR proteins: fiber type distribution of representative proteins. The electron micrograph in the central panel shows a face view of the SR, with T tubules labeled by asterisks. Values are expressed as per cent of the maximal value. ASPH, junctin/junctate; ATL2, atlastin 2; ATP2A1, SERCA 1 (sarco(endo)plasmic reticulum calcium-ATPase 1); ATP2A2, SERCA 2; BIN1, bridging integrator 1 (amphiphysin 2); CACNAG1, voltage-dependent calcium channel Ca_V1.1 (dihydropyridine receptor, DHPR) γ1 subunit; CASQ1, calsequestrin 1; CASQ2, calsequestrin 2; DNM2, dynamin 2; FKBP1A, FK binding protein 1A, prolyl isomerase 1A; HRC, histidine rich Ca²⁺ binding protein; JPH1, junctophilin 1; JPH2, junctophilin 2; JSRP1, JP-45; PLN, phospholamban; RTN2, reticulon 2; RTN4, reticulon 4; RYR1, ryanodine receptor 1; SRL, sarcalumenin; STAC3, SH3 and cysteine-rich domain-containing protein 3; SYPL2, Mg29 (mitsugumin 29); TMEM38A, TRIC-A (trimeric intracellular cation channel A); TMEM38B, TRIC-B; TRDN, triadin (EM picture: modified from ref [28])

Fig. 7

Glycolysis, glycogenolysis, and glycerol phosphate shuttle: fiber type distribution of representative proteins. Values are expressed as per cent of the maximal value. AGL, debranching enzyme; ALDOA, aldolase; ENO3, enolase 3 (β-enolase); GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GP1, glucose-6-phosphate isomerase; GPD1, glycerol-3-phosphate dehydrogenase 1 (cytosolic); GPD2, glycerol-3-phosphate dehydrogenase 2 (mitochondrial); LDHA, lactate dehydrogenase A; PFKM, phosphofructokinase, muscle isoform; PGAM2, phosphoglycerate mutase 2; PGK1, phosphoglycerate kinase 1; PGM1, phosphoglucomutase 1; PKM, pyruvate kinase, muscle isoform; PYGM, glycogen phosphorylase; TPI1, triosephosphate isomerase 1

Fig. 8

Fiber type–specific distribution of selected proteins revealed by immunofluorescence analysis. Fiber types are labeled as 1 (type 1), 2A (type 2A), or 2X (type 2X). Hybrid 2A–2X fibers are labeled by asterisks. A–D Type 1–specific proteins. Each panel shows on the left a section stained for PGM5/aciculin (A), PDLIM1 (B), MCU (C), or IDH2 (D) and on the right a serial section stained with MYH-specific antibodies to reveal the three fiber types. No pure type 2X fibers is present in A and B. E–F Type 2–specific proteins. Each panel shows on the left a section stained for ACTN3 (E) or XIRP2 (F), on the center a serial section stained for MYH-specific antibodies to reveal the three fiber types, and on the right a section stained with anti-MYH antibody BF-35, which reacts with MYH7 and MYH2 but not with MYH1, thus stains all fiber types, except pure type 2X fibers. Note that whereas ACTN3 is especially abundant in type 2X fibers, as well as in hybrid 2A–2X fibers, XIRP2 is present at higher levels in both 2A and 2X fibers. WGA counterstain was applied to all sections, except those processed with the three anti-MYHs antibodies