THE HUMAN TISSUE PROTEOMES

The skeletal muscle-specific proteome


The main function of the skeletal muscle is contraction, which provides stability and movement of the body. The skeletal muscle consists of striated muscle cells that are fused together into long muscle fibers. The transcriptome analysis shows that 53% of all human proteins (n=19628) are expressed in the skeletal muscle and 320 of these genes show an elevated expression in skeletal muscle compared to other tissue types. An analysis of genes with elevated expression in skeletal muscle reveals that a majority of the corresponding proteins are expressed in the cytoplasm, with various functions related to skeletal muscle physiology.

  • 93 skeletal muscle enriched genes
  • Most enriched genes encode proteins involved in muscle contraction
  • 320 genes defined as elevated in the skeletal muscle
  • Most group enriched genes share expression with heart

Figure 1. The distribution of all genes across the five categories based on transcript abundance in skeletal muscle as well as in all other tissues.


320 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. The function and cellular localization of known genes with tissue enriched expression in skeletal muscle (n=93), are well in-line with the function of the skeletal muscle.

Table 1. The genes with elevated expression in skeletal muscle

Category

Number of genes

Description

Tissue enriched 93 At least five-fold higher mRNA levels in a particular tissue as compared to all other tissues
Group enriched 118 At least five-fold higher mRNA levels in a group of 2-7 tissues
Tissue enhanced 109 At least five-fold higher mRNA levels in a particular tissue as compared to average levels in all tissues
Total 320 Total number of elevated genes in skeletal muscle


Table 2. The 12 genes with the highest level of enriched expression in skeletal muscle. "Predicted localization" shows the classification of each gene into three main classes: Secreted, Membrane, and Intracellular, where the latter consists of genes without any predicted membrane and secreted features. "mRNA (tissue)" shows the transcript level asáTPM values,áTS-score (Tissue Specificity score) corresponds to the score calculated as the fold change to the second highest tissue.

Gene

Description

Predicted localization

mRNA (tissue)

TS-score

MYH1 myosin, heavy chain 1, skeletal muscle, adult Intracellular 2754.7 735
ACTN3 actinin, alpha 3 (gene/pseudogene) Intracellular 556.7 119
NEB nebulin Intracellular 3274.3 110
MYH2 myosin, heavy chain 2, skeletal muscle, adult Intracellular 4307.0 99
ATP2A1 ATPase, Ca++ transporting, cardiac muscle, fast twitch 1 Intracellular,Membrane 2782.7 95
MYLPF myosin light chain, phosphorylatable, fast skeletal muscle Intracellular 6542.5 94
C8orf22 chromosome 8 open reading frame 22 Intracellular 362.2 88
TNNC2 troponin C type 2 (fast) Intracellular 9900.8 78
TNNT3 troponin T type 3 (skeletal, fast) Intracellular 9595.3 72
ANKRD23 ankyrin repeat domain 23 Intracellular 495.7 71
LBX1 ladybird homeobox 1 Intracellular 9.1 65
MYLK2 myosin light chain kinase 2 Intracellular 253.2 49

Some of the proteins predicted to be membrane-spanning are intracellular, e.g. in the Golgi or mitochondrial membranes, and some of the proteins predicted to be secreted can potentially be retained in a compartment belonging to the secretory pathway, such as the ER, or remain attached to the outer face of the cell membrane by a GPI anchor.

The skeletal muscle transcriptome


An analysis of the expression levels of each gene made it possible to calculate the relative mRNA pool for each of the categories. The analysis shows that 68% of the mRNA molecules derived from skeletal muscle tissue correspond to housekeeping genes and only 30% of the mRNA pool corresponds to genes categorized to be either skeletal muscle enriched, group enriched or, skeletal muscle enhanced. Thus, most of the transcriptional activity in the skeletal muscle relates to proteins with presumed housekeeping functions as they are found in all tissues and cells analyzed.

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 conformation differences 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) fibres. 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.



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 betwen cell surface and intracellular calcium release channels.



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.



Genes shared between the skeletal muscle and other tissues


There are 118 group enriched genes expressed in the skeletal muscle. Group enriched genes are defined as genes showing a 5-fold higher average level of mRNA expression in a group of 2-7 tissues, including skeletal muscle, compared to all other tissues.

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

Figure 2. An interactive network plot of theáskeletal muscleáenriched and group enriched genes connected to their respective enriched tissues (grey circles).áRedánodes represent the number ofáskeletal muscle enriched genes andáorangeánodes 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 toá5átissues, but the resulting lists show the complete set of group enriched genes in the particular tissue.



Skeletal muscle shares the largest number of genes with 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 fibres. 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 weight comes from 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 via impulses from the brain. Another important function of skeletal muscle is to elevate body temperature. The heat is generated when the muscles contract and causes blood vessels in the skin to dilate. In this manner, the skeletal muscles are also involved in regulation of the blood flow.

Skeletal muscle histology


The skeletal muscles together with the heart muscle are composed of striated muscle tissue that form 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 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.

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 the skeletal muscle are described and characterized, together with examples of immunohistochemically stained tissue sections that visualize protein expression patterns of proteins that correspond to genes with elevated expression in the skeletal muscle.

Transcript profiling and RNA-data analyses based on normal human tissues have been described previously (Fagerberg et al., 2013). Analyses of mRNA expression including over 99% of all human protein-coding genes was performed using deep RNA sequencing of 172 individual samples corresponding to 37 different human normal tissue types. RNA sequencing results of 5 fresh frozen tissues representing normal skeletal muscle was compared to 167 other tissue samples corresponding to 36 tissue types, in order to determine genes with elevated expression in skeletal muscle. A tissue-specific score, defined as the ratio between mRNA levels in skeletal muscle compared to the mRNA levels in all other tissues, was used to divide the genes into different categories of expression. These categories include: genes with elevated expression in skeletal muscle, genes expressed in all tissues, genes with a mixed expression pattern, genes not expressed in skeletal muscle, and genes not expressed in any tissue. Genes with elevated expression in skeletal muscle were further sub-categorized as i) genes with enriched expression in skeletal muscle, ii) genes with group enriched expression including skeletal muscle and iii) genes with enhanced expression in skeletal muscle.

Human tissue samples used for protein and mRNA expression analyses were collected and handled in accordance with Swedish laws and regulation and obtained from the Department of Pathology, Uppsala University Hospital, Uppsala, Sweden as part of the sample collection governed by the Uppsala Biobank. All human tissue samples used in the present study were anonymized in accordance with approval and advisory report from the Uppsala Ethical Review Board.


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Histology dictionary - the skeletal muscle