The human cardiac and skeletal muscle proteomes defined by transcriptomics and antibody-based profiling

Abstract

Background: To understand cardiac and skeletal muscle function, it is important to define and explore their molecular constituents and also to identify similarities and differences in the gene expression in these two different striated muscle tissues. Here, we have investigated the genes and proteins with elevated expression in cardiac and skeletal muscle in relation to all other major human tissues and organs using a global transcriptomics analysis complemented with antibody-based profiling to localize the corresponding proteins on a single cell level.

Results: Our study identified a comprehensive list of genes expressed in cardiac and skeletal muscle. The genes with elevated expression were further stratified according to their global expression pattern across the human body as well as their precise localization in the muscle tissues. The functions of the proteins encoded by the elevated genes are well in line with the physiological functions of cardiac and skeletal muscle, such as contraction, ion transport, regulation of membrane potential and actomyosin structure organization. A large fraction of the transcripts in both cardiac and skeletal muscle correspond to mitochondrial proteins involved in energy metabolism, which demonstrates the extreme specialization of these muscle tissues to provide energy for contraction.

Conclusions: Our results provide a comprehensive list of genes and proteins elevated in striated muscles. A number of proteins not previously characterized in cardiac and skeletal muscle were identified and localized to specific cellular subcompartments. These proteins represent an interesting starting point for further functional analysis of their role in muscle biology and disease.

Fig. 1

Hierarchical clustering of human tissues. The relationship between the 100 tissue samples corresponding to 28 different tissues, based on pairwise Spearman correlations

Fig. 2

Sample correlations and classification of all human protein coding genes. Scatter plots of FPKM values for all detected genes in a two cardiac muscle samples, b two skeletal muscle samples, c cardiac muscle and skeletal muscle d and cardiac muscle and adipose tissue. e Pie chart showing the classification of all genes in cardiac muscle, based on transcript abundance and number of tissues with expression. f Pie chart showing the distribution of the expressed mRNA molecules in cardiac muscle. g Pie chart showing the classification of all genes in skeletal muscle. h Pie chart showing the distribution of expressed mRNA molecules in skeletal muscle

Fig. 3

Network plot of the cardiac and skeletal muscle enriched and group enriched genes. Red circle nodes represent a group of expressed genes and are connected to the respective enriched tissues (grey circles). The size of each red node is related to the square root of the number of genes enriched in a particular combination of tissues

Fig. 4

Immunohistochemical staining of proteins elevated in cardiac muscle. a Examples of eight proteins related to muscle contraction (MYH6, MYL4, MYL7, ACTC1, TNNI3, TNNT2, MYBPC3 and CACNA1C). CACNA1C was distinctly stained in sarcolemma and endothelial cells. b Examples of four proteins related to homeostasis and regeneration/repair, with NPPA displaying granular cytoplasmic expression, AKAP6 observed in nuclear membranes of cardiomyocytes, NES restricted to endothelial cells and BVES distinctly stained in sarcolemma. c Examples of eight proteins specific for intercalated discs (ATP1A3, CDH2, MYZAP, PKP2, ART3, FILIP1, POPDC2 and RAB9B). d Examples of two proteins with unknown function in cardiac muscle (SBK2 and SHD)

Fig. 5

Immunohistochemical staining of proteins elevated in skeletal muscle. a Examples of four proteins related to muscle contraction (MYH2, TNNT1, MYBPC1 and MYOT). MYH2 and TNNT1 were differentially expressed between type I and type II muscle fibers, while MYOT concentrated to Z-lines of the muscle fibers. b Examples of four proteins related to calcium function (RYR1, CASQ1, CAPN3 and JPH1) (c) Examples of four proteins related to enzymatic activity (AMPD1, PYGM, CA3 and ENO3). AMPD1 and PYGM revealed heterogeneous cytoplasmic expression and ENO3 was differentially expressed between type I and type II muscle fibers. d Examples of four proteins with unknown function in skeletal muscle (FAM166B, KLHL40, OR10AB1P and PHKG1). Both OR10AB1P and PHKG1 being differentially expressed between type I and type II muscle fibers

Fig. 6

Immunohistochemical staining of proteins elevated in both cardiac and skeletal muscle. a Examples of four proteins related to muscular contraction (MYH7, MYL3, TNNC1 and TPM3), in skeletal muscle higher expressed in type I muscle fibers. b Examples of four proteins related to calcium transport and storage (CAMK2B, CASQ1, HRC and SRL). c Examples of four proteins related to metabolism and myofibrillar organization (CKM, PGAM2, LDB3 and MURC). LDB3 was restricted to Z-lines of cardiac muscle fibers while more homogeneous in skeletal muscle. MURC showed distinct expression of sarcolemma in both cardiac and skeletal muscle. d Examples of four proteins with unknown function in cardiac and skeletal muscle, with ADSSL1 showing distinct nuclear staining, HHATL and C1ORF170 displaying cytoplasmic positivity, and SPTB revealing distinct immunoreactivity in sarcolemma, accompanied with positivity in erythrocytes