The skeletal muscle-specific proteome

The skeletal muscle transcriptome

Transcriptome analysis of the skeletal muscle can be visualized with regard to specificity and distribution of transcribed mRNA molecules (Figure 1). Specificity illustrates the number of genes with elevated or non-elevated expression in the skeletal muscle compared to other tissues. Elevated expression includes three subcategory types of elevated expression:

• Tissue enriched: At least four-fold higher mRNA level in skeletal muscle compared to any other tissues.
• Group enriched: At least four-fold higher average mRNA level in a group of 2-5 tissues compared to any other tissue.
• Tissue enhanced: At least four-fold higher mRNA level in skeletal muscle compared to the average level in all other tissues.

Distribution, on the other hand, visualizes how many genes that have, or do not have, detectable levels (NX≥1) of transcribed mRNA molecules in the skeletal muscle compared to other tissues. As evident in Table 1, all genes elevated in skeletal muscle are categorized as:

• Detected in single: Detected in a single tissue
• Detected in some: Detected in more than one but less than one third of tissues
• Detected in many: Detected in at least a third but not all tissues
• Detected in all: Detected in all tissues

Figure 1. (A) The distribution of all genes across the five categories based on transcript specificity in skeletal muscle as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in skeletal muscle as well as in all other tissues.

As shown in Figure 1, 907 genes show some level of elevated expression in the skeletal muscle compared to other tissues. The three categories of genes with elevated expression in skeletal muscle compared to other organs are shown in Table 1. In Table 2, the 12 genes with the highest enrichment in skeletal muscle are defined.

Table 1. Number of genes in the subdivided categories of elevated expression in skeletal muscle.

Table 2. The 12 genes with the highest level of enriched expression in skeletal muscle. "Tissue distribution" describes the transcript detection (NX≥1) in skeletal muscle as well as in all other tissues. "mRNA (tissue)" shows the transcript level in skeletal muscle as NX values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in skeletal muscle and the tissue with second highest expression level.

Protein expression of genes elevated in skeletal muscle

In-depth analysis of the elevated genes in skeletal muscle using antibody-based protein profiling allowed us to visualize the expression patterns of these proteins in different functional compartments including proteins related to i) contraction, ii) calcium function, and iii) enzymatic activity.

Proteins related to contraction expressed in the skeletal muscle

The primary structural proteins in the skeletal myocytes related to contraction are myosin and actin filaments, forming a striated pattern that can be observed in electron microscopy. Another protein family related to muscular contraction is the troponin family, regulating the binding of myosin to actin via conformational changes dependent on the calcium ion concentration in the cells. Examples of members of the myosin and troponin families solely expressed in skeletal muscle include MYH2 and TNNT1, with MYH2 being expressed in fast (type II) fibers and TNNT1 in slow (type I) fibers. Another example of a protein involved in skeletal muscle contraction is the myosin binding protein MYBPC1, which influences contraction by cross-bridging in the sarcomere.

MYH2
TNNT1
MYBPC1

Proteins related to calcium function expressed in the skeletal muscle

In both heart and skeletal muscle, contraction is dependent on the level of intracellular calcium. However, in contrast to cardiomyocytes, where calcium release is regulated via binding of calcium ions from the external environment to voltage-gated calcium channels, skeletal myocytes store calcium in the sarcoplasmic reticulum until a neuronal impulse triggers calcium influx along the myofilaments. Three examples related to calcium function with selective expression in skeletal muscle are RYR1, CASQ1 and JPH1. RYR1 is the ryanodine receptor acting as the calcium release channel, while CASQ1 is essential for calcium storage in the sarcoplasmic reticulum. JPH1 aids in the functional cross-talk between cell surface and intracellular calcium release channels.

RYR1
CASQ1
JPH1

Proteins related to enzymatic activity expressed in the skeletal muscle

Enzymatic activity is an important function in skeletal muscle physiology, which relates to various processes such as metabolism, glycogen storage and regeneration. Examples of three proteins implicated in enzymatic activities with selective expression in skeletal muscle include AMPD1, PYGM and ENO3. AMPD1 is an enzyme involved in the purine nucleotide cycle and plays a critical role in energy metabolism, while the enzyme PYGM is essential for carbohydrate metabolism and glycogenolysis. ENO3 is an isoenzyme suggested to play a role in muscle development and regeneration, with mutations associated with glycogen storage disease.

AMPD1
PYGM
ENO3

Gene expression shared between the skeletal muscle and other tissues

There are 202 group enriched genes expressed in skeletal muscle. Group enriched genes are defined as genes showing a 4-fold higher average level of mRNA expression in a group of 2-5 tissues, including skeletal muscle, compared to all other tissues.

In order to illustrate the relation of skeletal muscle tissue to other tissue types, a network plot was generated, displaying the number of genes with shared expression between different tissue types.

Figure 2. An interactive network plot of the skeletal muscle enriched and group enriched genes connected to their respective enriched tissues (grey circles). Red nodes represent the number of skeletal muscle enriched genes and orange nodes represent the number of genes that are group enriched. The sizes of the red and orange nodes are related to the number of genes displayed within the node. Each node is clickable and results in a list of all enriched genes connected to the highlighted edges. The network is limited to group enriched genes in combinations of up to 5 tissues, but the resulting lists show the complete set of group enriched genes in the particular tissue.

Skeletal muscle shares most group enriched gene expression with the heart, which is expected since both heart and skeletal muscles are striated muscles with many similarities. Two examples of proteins with shared expression in heart and skeletal muscle are MYH7 and LDB3. MYH7 is related to contraction and shows differential expression between slow (type I) and fast (type II) muscle fibers. LDB3 is involved in sarcomere organization and distinctly expressed in Z-discs of heart.

MYH7 - skeletal muscle
MYH7 - heart muscle

LDB3 - skeletal muscle
LDB3 - heart muscle

Skeletal muscle function

The skeletal muscle is one of the largest organs in the human body and up to 50% of the total body mass is skeletal muscle. The main function of skeletal muscle is contraction, which results in body movement but is also necessary for posture and stability of the body. In contrast to heart muscle, another striated muscle similar in structure, the contraction of skeletal muscles is under voluntary control and initiated by impulses from the brain. Another important function of skeletal muscle is to regulate body temperature. The heat is generated when the muscles contract and cause blood vessels in the skin to dilate. In this manner, the skeletal muscles are also involved in the regulation of blood flow.

Skeletal muscle histology

The skeletal muscles together with the heart muscle are composed of striated muscle tissue that forms parallel muscle fibers. Striated muscle tissue consists of myocytes arranged in long and thin multinucleated fibers that are crossed with a regular pattern of fine red and white lines, giving the muscle its distinctive appearance and its name. There are two types (fast and slow) of muscle fibers depending on the type of myosin present. These fiber types can not be distinguished in an ordinary hematoxylin-eosin (HE) staining.

Development and normal activity of skeletal muscle are dependent and closely integrated with the nervous system. Skeletal muscles are attached to the bone and contract voluntarily (via nerve stimulation) as opposed to the other common types of muscle, i.e. cardiac muscle and smooth muscle.

The major cell type in skeletal muscle is the myocyte. Myocytes are fused together during development to form large multinucleated cells called syncytia. The cells are rich in mitochondria and contain to a large extent actin and myosin proteins arranged in repeating units called sarcomeres. Histologically, this highly structured arrangement of sarcomeres appears as dark (A-bands) and light (I-bands) bands, which are clearly visible in the microscopic image. In addition to the muscle fibers, skeletal muscles also consist of adjacent streaks of connective and adipose tissue. Skeletal muscle tissue is highly vascularized with a fine network of capillaries running between the fibers.

Using light sheet microscopy and immunostaining we are able to study and visualize the complexity of skeletal muscle in detail. The video below visualizes skeletal muscle in red and its intricate network of nerves shown in teal. Full length version of the video is found here.

The histology of human skeletal muscle including detailed images and information about the different cell types can be viewed in the Protein Atlas Histology Dictionary.

Background

Here, the protein-coding genes expressed in skeletal muscle are described and characterized, together with examples of immunohistochemically stained tissue sections that visualize corresponding protein expression patterns of genes with elevated expression in skeletal muscle.

Transcript profiling was based on a combination of three transcriptomics datasets (HPA, GTEx and FANTOM5), corresponding to a total of 9332 samples from 113 different human normal tissue types. The final consensus normalized expression (NX) value for each tissue type was used for classification of all genes according to the tissue specific expression into two different categories, based on specificity or distribution.

Relevant links and publications

Uhlén M et al., Tissue-based map of the human proteome. Science (2015)
PubMed: 25613900 DOI: 10.1126/science.1260419

Yu NY et al., Complementing tissue characterization by integrating transcriptome profiling from the Human Protein Atlas and from the FANTOM5 consortium. Nucleic Acids Res. (2015)
PubMed: 26117540 DOI: 10.1093/nar/gkv608

Fagerberg L et al., Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteomics. (2014)
PubMed: 24309898 DOI: 10.1074/mcp.M113.035600

Lindskog C et al., The human cardiac and skeletal muscle proteomes defined by transcriptomics and antibody-based profiling. BMC Genomics. (2015)
PubMed: 26109061 DOI: 10.1186/s12864-015-1686-y

Histology dictionary - the skeletal muscle