Muscle tissue

The main functions of muscles are locomotion, maintenance of posture, as well as movement of inner organs. Muscle tissue is composed of specially differentiated muscle cells. Many muscle cells gather together and are surrounded by connective tissue to form a muscle bundle, which is rich in capillaries and fibers. In muscle cells, protein filaments of actin and myosin slide past one another, producing a contraction that alters both the shape and the length of muscle cells. There are three types of muscle tissue recognized in vertebrates, heart muscle, skeletal muscle and smooth muscle.

In the pig transcriptomic analysis, the heart, skeletal muscle and smooth muscle (from the intestine) are included and used for quantitative RNA measurements, representing muscle tissues. The heart is represented by three different sub parts of the heart (the ventricle wall, atrium and mitral valve) where the highest expression value among the three is used for representing the heart in the gene expression classification. The gene classification strategy highlights genes with an elevated level of expression in one or a group of tissues compared to all other tissues

700 genes are classified as heart elevated out of which 49 genes are highlighted as heart enriched. Based on the expression in skeletal muscle 583 genes are classified as elevated compared to other tissue types, out of which 78 genes are highlighted as tissue enriched in skeletal muscle. RNA expression levels in smooth muscle resulted in 185 genes classified as elevated in smooth muscle compared to other tissue types, out of which 4 genes are highlighted as smooth muscle enriched.

Histological image of the pig tissues used in the analysis can be found in the pig tissue dictionary.

Heart

Heart wall, heart atrium and heart mitral valve were all sampled and the highest expression value was used as representation of the heart. The mitral valve includes small pieces of the atrial muscle, explaining the high overlap between the two tissue samples. The main function of the heart is to pump blood and sustain the blood pressure needed for adequate circulation. The heart consists of a specialized form of striated muscle including cardiomyocytes as the main cell type.

Gene expression in heart is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 1 summarizes the number of genes in respective category. In total, 16026 genes are detected above cut of (1NX) in pig heart. The tissue distribution category highlights 18 genes only detected in heart while 700 genes are classified as heart elevated compared to other tissues. Table 1 shows the overlap for the heart elevated genes and tissue distribution category.

Figure 1, (A) The distribution of all genes across the five categories based on transcript specificity in heart as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in heart as well as in all other tissues. The combination of the two categories is shown in table 1.

Table 1, Number of genes in the subdivided categories of elevated expression and tissue distribution in heart

Heart enriched expression

To allow for the continuously beating and the long contraction period, the heart muscle is different from skeletal muscle. As a result, several proteins related to contraction are only expressed in the heart. Examples of members of the myosin, actin and troponin families solely expressed in heart muscle include ACTC1, TNNT2 and TNNI3. In order to retain balanced levels of various substances in the body, the heart plays an important role in homeostasis. Intracellular calcium homeostasis in the heart is achieved with calsequestrin (CASQ2) a calcium buffering protein.

Table 2, The 10 genes with the highest level of enriched expression in heart. "mRNA (tissue)" shows the transcript level in heart as NX values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in heart and the tissue with second highest expression level.

Expression comparison between different parts of the heart

The expression variation category enables further details related to expression heterogeneity within the tissues that include grouped samples. In this case, expression profile from heart wall is compared to expression profiles from heart atrium and mitral valve, resulting in 332 genes highlighted as variable between the three heart samples.

There are several genes with specific expression only in atrium and valve and lacking expression in ventricle wall, for example the atrial natriuretic peptide NPPA that controls extracellular fluid volume and electrolyte homeostasis as well as the atrial essential light chain protein MYL4.

Skeletal muscle

Gene expression in skeletal muscle is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 2 summarizes the number of genes in respective category. In total, 14549 genes are detected above cut of (1NX) in pig skeletal muscle. The tissue distribution category highlights 2 genes only detected in skeletal muscle while 583 genes are classified as skeletal muscle elevated compared to other tissues. Table 3 shows the overlap for the skeletal muscle elevated genes and tissue distribution category.

Figure 2, (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. The combination of the two categories is shown in table 3.

Table 3, Number of genes in the subdivided categories of elevated expression and tissue distribution in skeletal muscle

Skeletal muscle enriched expression

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

Skeletal muscle tissue is included in several other tissue types in the analysis, and not limited to the pure skeletal muscle sample. For example in several genes mentioned above expression levels can also be observed in mouth (lip, oral mucosa and tongue), urethral gland and synovial tissue, clearly indicating a fraction of skeletal muscle representation in these tissues. Indeed, when examining urethral gland or mouth histological sections in the pig tissue dictionary skeletal muscle fibers is observed.

Table 4, The 10 genes with the highest level of enriched expression in skeletal muscle. "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.

Smooth muscle

Smooth muscle fibers are found throughout the body in blood vessels and hollow organs. Through their ability to apply pressure by involuntary muscle contraction, they are able to regulate essential bodily functions, such as blood pressure and bowel movement. During contraction, dense bodies are used by smooth muscle cells as anchoring points for the actin and intermediate filaments to exert force upon. Smooth muscle fibers are built up of smooth muscle cells attached to each other using gap junctions to synchronize their response to stimuli.

Gene expression in smooth muscle is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 3 summarizes the number of genes in respective category. In total, 14807 genes are detected above cut of (1NX) in pig smooth muscle. The tissue distribution category highlights 1 genes only detected in smooth muscle while 185 genes are classified as smooth muscle elevated compared to other tissues. Table 5 shows the overlap for the smooth muscle elevated genes and tissue distribution category.

Figure 3, (A) The distribution of all genes across the five categories based on transcript specificity in smooth 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 smooth muscle as well as in all other tissues. The combination of the two categories is shown in table 5.

Table 5, Number of genes in the subdivided categories of elevated expression and tissue distribution in smooth muscle

Smooth muscle enriched expression

The primary structural proteins related to the contraction in smooth muscle cells are myosin and actin filament proteins. The typical smooth muscle actin (ACTG2) classified as smooth muscle enriched while the smooth muscle myosin (MYH11) is classified as enhanced due to the representation of smooth muscle in several other tissue types. This is further illustrated by the limited number of 4 genes classified as smooth muscle enriched.

TACR2 show smooth muscle elevated expression in both pig and human, while SYNM is also abundantly expressed in human skeletal muscle, which in pig is much lower compared to smooth muscle.

Table 6, The 10 genes with the highest level of enriched expression in smooth muscle. "mRNA (tissue)" shows the transcript level in smooth muscle as NX values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in smooth muscle and the tissue with second highest expression level.

Group enriched expression

Both heart and skeletal muscle are striated with actin and myosin arranged in sarcomeres. The differences are that heart muscle movment are involuntary and the myocytes (cardiac muscle) most often have a central (single) nucleus. The similarity between skeletal and heart muscle is clear when examining the list of genes classified as group enriched. PLN

Immunohistochemical detection of proteins with enriched expression in heart tissue, showing PLN detected in heart and skeletal muscle using antibody HPA026900.

Immunohistochemical detection of proteins with enriched expression in heart tissue, showing TNNC1 detected in heart and skeletal muscle using antibody HPA044848.

In order to illustrate the relation of muscle tissue to other tissue types, a network plot was generated, displaying the number of genes shared between different tissue types. Group enriched genes are defined as genes showing a 4-fold higher average level of mRNA expression in a group of 2-5 tissues, compared to all other tissues.

Figure 4. An interactive network plot of the muscle enriched and group enriched genes connected to their respective enriched tissues (grey circles). Black circles shows skeletal and heart muscle. Red nodes represent the number of tissue 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 3 tissues, but the resulting lists show the complete set of group enriched genes in the particular tissue. Due to the criteria of at least two genes shown for an individual node, smooth muscle is missing