Characterization of zebrafish (Danio rerio) muscle ankyrin repeat proteins reveals their conserved response to endurance exercise

Abstract

Muscle proteins with ankyrin repeats (MARPs) ANKRD1 and ANKRD2 are titin-associated proteins with a putative role as transcriptional co-regulators in striated muscle, involved in the cellular response to mechanical, oxidative and metabolic stress. Since many aspects of the biology of MARPs, particularly exact mechanisms of their action, in striated muscle are still elusive, research in this field will benefit from novel animal model system. Here we investigated the MARPs found in zebrafish for protein structure, evolutionary conservation, spatiotemporal expression profiles and response to increased muscle activity. Ankrd1 and Ankrd2 show overall moderate conservation at the protein level, more pronounced in the region of ankyrin repeats, motifs indispensable for their function. The two zebrafish genes, ankrd1a and ankrd1b, counterparts of mammalian ANKRD1/Ankrd1, have different expression profiles during first seven days of development. Mild increase of ankrd1a transcript levels was detected at 72 hpf (1.74±0.24 fold increase relative to 24 hpf time point), while ankrd1b expression was markedly upregulated from 24 hpf onward and peaked at 72 hpf (92.18±36.95 fold increase relative to 24 hpf time point). Spatially, they exhibited non-overlapping expression patterns during skeletal muscle development in trunk (ankrd1a) and tail (ankrd1b) somites. Expression of ankrd2 was barely detectable. Zebrafish MARPs, expressed at a relatively low level in adult striated muscle, were found to be responsive to endurance exercise training consisting of two bouts of 3 hours of forced swimming daily, for five consecutive days. Three hours after the last exercise bout, ankrd1a expression increased in cardiac muscle (6.19±5.05 fold change), while ankrd1b and ankrd2 were upregulated in skeletal muscle (1.97±1.05 and 1.84±0.58 fold change, respectively). This study provides the foundation to establish zebrafish as a novel in vivo model for further investigation of MARPs function in striated muscle.

Fig 1. Gene organization, protein sequence alignment and domain structure of MARP family members from different species.

(a) Exon-intron structure of human (Hs) ANKRD1 and ANKRD2 and their counterparts in mouse (Mm) and zebrafish (Dr). Exons (boxes) and introns (lines) are drawn to scale. White boxes indicate 5’ and 3’ UTRs. The numbers on the right indicate the length of the genomic region. (b) Amino acid sequence alignment of human, mouse and zebrafish proteins. (c) Schematic representation of the structural domains of human, mouse and zebrafish proteins. The predicted domains are indicated by colored boxes. Domain positions are listed in S2 Table.

Fig 2. Phylogenetic tree of MARP homologs generated by PhyML algorithm.

The GeneBank accession numbers of the sequences are listed in S1 Table. Species abbreviations: Hs, Homo sapiens; Mm, Mus musculus; Gg, Gallus gallus; Xt, Xenopus tropicalis; Dr, Danio rerio; Am, Astyanax mexicanus. Confidence of nodes is indicated by numbers on individual branches.

Fig 3. Synteny comparisons between human (Hs) and zebrafish (Dr) gene loci.

ANKRD1/ankrd1a/ankrd1b (a) and ANKRD2/ankrd2 (b) genes, depicted in orange polygons, are presented with nearest neighbors (colored polygons, orthologs have the same color). The transcriptional orientation of the gene is indicated by the angled end of each polygon corresponding to the 3´ end. Image style was adapted from Genomicus.

Fig 4. Quantification of zebrafish MARP transcripts during development.

Expression levels of ankrd1a, ankrd1b and ankrd2 at indicated time points after fertilization were obtained using qPCR. The housekeeping gene rlp13a served as internal reference. Data (mean ± SD) are combined from four biological replicates and normalized to the 24 hpf time point. * denotes P<0.05 in comparison to control group (by one-way ANOVA/Tukey’s multiple comparison test). Agarose gels showing qPCR products for ankrd1a, ankrd1b, ankrd2 and rlp13a are also presented. Average Ct±SD values for MARP and reference (rpl13a) genes during zebrafish development at indicated time points are given in S3 Table.

Fig 5. Spatiotemporal expression of zebrafish ankrd1a and ankrd1b during development, from 24 to 120 hpf.

Representative images of whole mount ISH using probes detecting ankrd1a (a-f) and ankrd1b (h-m) transcripts at designated time points. Control staining for ankrd1a (g) and ankrd1b (n) was performed in 48 hpf embryos. Lateral and one dorsal (c) views are shown, anterior to the left.

Fig 6. Expression of the zebrafish MARP genes in adult heart and skeletal muscle.

Quantification of ankrd1a, ankrd1b and ankrd2 expression was done by qPCR, using mRNA isolated from the hearts of 8 fish, pooled in 4 groups and the skeletal muscles of 4 fish. Relative level of transcripts in adult heart is normalized to the transcript level in the skeletal muscle, set as 1. Bars represent the mean ± SD. * denotes P<0.05 in comparison to control group (by t-test). Average Ct±SD values for MARP and reference (rpl13a) genes in adult zebrafish heart and skeletal muscle are given in S4 Table.

Fig 7. Effects of swimming exercise on heart and skeletal muscle genes expression.

mRNA expression levels of chymotrypsinogen B1 (ctrb1), glycogen synthase 1 (gys1), lipoprotein lipase (lpl) and type I collagen, alpha 1a (col1a1a) in heart, and apelin receptor a (aplnra), apelin receptor b (aplnrb), peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (ppargc1a) and insulin-like growth factor 1 (igf1) in skeletal muscle of exercised fish expressed as a fold change over non-exercised controls set to 1. Bars represent the mean ± SD. * denotes P<0.05 and ** denotes P<0.005 in comparison to control group (by t-test). Fold change ± SD values for all tested cardiac and skeletal muscle genes of trained and control adult zebrafish are given in S5 Table.

Fig 8. Zebrafish MARP genes are differentially upregulated in adult heart and in skeletal muscle after endurance exercise.

Adult fish were subjected to endurance exercise for one week. Relative expression of MARP genes in zebrafish heart and skeletal muscle is expressed as fold change to non-exercised controls. Bars represent the mean ± SD. * denotes P<0.05 in comparison to control group (by t-test).