The heart-specific proteome

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

201 genes show some level of elevated expression in the heart compared to other tissues. The three categories of genes with elevated expression in heart compared to other organs are shown in Table 1. The function and cellular localization of known genes with tissue enriched expression in heart (n=28), are well in line with the function of the heart.

Table 1. The genes with elevated expression in heart

Table 2. The 12 genes with the highest level of enriched expression in heart. "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 as TPM values, TS-score (Tissue Specificity score) corresponds to the score calculated as the fold change to the second highest tissue.

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 heart transcriptome

An analysis of the expression levels of each gene makes it possible to calculate the relative mRNA pool for each of the categories. The analysis shows that 82% of the mRNA molecules derived from heart tissue correspond to housekeeping genes and only 13% of the mRNA pool corresponds to genes categorized to be either heart enriched, group enriched or, heart enhanced. Thus, most of the transcriptional activity in the heart relates to proteins with presumed housekeeping functions as they are found in all tissues and cells analyzed.

Gene Ontology-based analysis of all the 201 genes identified as heart enriched (n=28), group enriched (n=92) or heart enhanced (n=81) indicates an overrepresentation of proteins associated with processes such as regulation of contraction, heart rate, ion transport, ATPase activity and heart development. The most common functions of these proteins are regulation of channel activity for e.g. ligand-gated and voltage-gated ion channels, and binding of proteins such as actin, myosin, tropomyosin, and troponin.

Protein expression of genes elevated in heart

In-depth analysis of the elevated genes in heart using antibody-based protein profiling allowed us to visualize the expression patterns of these proteins in different functional compartments including proteins related to contraction or homeostasis and regeneration, as well as proteins selectively expressed in intercalated discs.

Proteins specifically expressed in the heart related to contraction

To allow for the continuously beating and the long contraction period, the heart muscle is different from the skeletal muscle. As a result, several proteins related to contraction are only expressed in heart. The primary structural proteins in the heart 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, actin and troponin families solely expressed in heart muscle include MYH6, ACTC1 and TNNI3.

Proteins specifically expressed in the heart related to homeostasis and regeneration

In order to retain balanced levels of various substances in the body, heart plays an important role in homeostasis. One such example is the atrial natriuretic peptide NPPA, which controls extracellular fluid volume and electrolyte homeostasis. Mutations in NPPA are thought to be responsible for the development of atrial fibrillation (arrhythmia).

One example of a protein suggested to play a role in heart regeneration and repair is the neural stem cell protein NES, involved in brain and eye development but also implicated in reparative vascularization following myocardial infarction. NES is distinctly stained in endothelial cells.

Proteins specifically expressed in intercalated discs of the heart

One feature unique for heart muscle is the presence of intercalated discs, defined as the connections between two adjacent cardiomyocytes. Intercalated discs aid in contraction of multiple cardiomyocytes simultaneously as a unit, which is necessary for proper heart function. Examples of three proteins distinctly expressed in intercalated discs are ATP1A3, CDH2 and PKP2, involved in functions related to plasma membrane ion exchange, cell adhesion and cell junctions, respectively.

Genes shared between heart and other tissues

There are 92 group enriched genes expressed in the heart. 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 heart, compared to all other tissues.

In order to illustrate the relation of heart 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 the heart muscle enriched and group enriched genes connected to their respective enriched tissues (grey circles). Red nodes represent the number of heart 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 4 tissues, but the resulting lists show the complete set of group enriched genes in the particular tissue.

Heart shares the largest number of genes with skeletal muscle, which is expected as both heart and skeletal muscle are composed as striated muscles that share similar features. Two examples of proteins with shared expression in heart and skeletal muscle are MYH7 and LDB3. MYH7 is related to contraction and showed differential expression between slow (type I) and fast (type II) muscle fibers. LDB3 is involved in sarcomere organization and distinctly expressed in Z-discs.

Heart function

The heart is a coordinated muscle connected to the vascular system and specialized in pumping the blood through the body. The role of the cardiovascular system is to transport oxygen and nutrients to the cells, and to remove carbon dioxide and metabolic waste products from the body. The heart is made up of two atria and two ventricles. De-oxygenated blood from the body is pumped into the right atrium, passes through the tricuspid valve into the right ventricle, and then goes to the lungs where the blood is oxygenated and carbon dioxide is released. The oxygenated blood returns to the left atrium, and from there the blood moves to the left ventricle, through the bicuspid valve and is pumped out in the body via the aorta. The heart is hence separated in two parts, which are divided by the atrioventricular septum. The left and right sides are coordinated so that the two atria contract simultaneously, and the two ventricles contract simultaneously. This is controlled by the heart’s own electrical signaling system. The sinoatrial (SA) node generates electrical impulses that set the rate of the contraction. The signals from the SA node also pass through another node, the atrioventricular node, which delays the signals to ensure that the atria empty completely before the ventricles contract.

Heart histology

The heart muscle is highly vascularized and under nervous control to set the pace of heart beats. The cardiac and skeletal muscles are both composed of striated muscle tissue that forms parallel muscle fibers, but in contrast to skeletal muscle that consists of parallel linear fibers, the cardiac muscle cells (cardiomyocytes) are arranged in fibers that exhibit cross-striations formed by alternating segments of thick and thin protein filaments.

The major cell type in the heart is cardiomyocytes, which usually contain one or two nuclei and are rich in mitochondria. They are arranged in repeating units called sarcomeres, which contain myosin and actin proteins. In the microscope, this highly structured arrangement of sarcomeres appears as dark A-bands of thick filaments and light I-bands of thin filaments. Z-discs are located at the ends of each sarcomere, in between the I-bands, and appear as dark lines. Another typical feature of the cardiac muscle that is different from the skeletal muscle is the dark bands, known as intercalated discs, between myocytes where the membranes of adjacent cells are situated closely together. In addition to the muscle fibers, the myocardium also includes streaks of connective and cuffs of adipose tissue surrounding the smaller vessels. The cardiac muscle tissue is vascularized through the coronary arteries that branch into smaller vessels ending in a dense network of capillaries running between the fibers.

The histology of human heart 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 heart 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 heart 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 4 fresh frozen tissues representing normal heart muscle was compared to 168 other tissue samples corresponding to 36 tissue types, in order to determine genes with elevated expression in heart muscle. A tissue-specific score, defined as the ratio between mRNA levels in heart 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 heart muscle, genes expressed in all tissues, genes with a mixed expression pattern, genes not expressed in heart muscle, and genes not expressed in any tissue. Genes with elevated expression in heart muscle were further sub-categorized as i) genes with enriched expression in heart muscle, ii) genes with group enriched expression including heart muscle and iii) genes with enhanced expression in heart 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.

Relevant links and publications

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

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

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

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

Histology dictionary - the heart muscle