Abstract
The teleost-specific whole genome duplication created multiple copies of genes allowing for subfunctionalization of isoforms. In this study, we show that the teleost cardiac Ca2+-binding troponin C (TnC) is the product of two distinct genes: cardiac TnC (cTnC, TnnC1a) and a fish-specific slow skeletal TnC (ssTnC, TnnC1b). The ssTnC gene is novel to teleosts as mammals have a single gene commonly referred as cTnC but which is also expressed in slow skeletal muscle. In teleosts, the data strongly indicate that these are two TnC genes are different paralogs. Because we determined that ssTnC exists across many teleosts but not in basal ray-finned fish (e.g., bichir), we propose that these paralogs are the result of an ancestral tandem gene duplication persisting only in teleosts. Quantification of mRNA levels was used to demonstrate distinct expression localization patterns of the paralogs within the chambers of the heart. In the adult zebrafish acclimated at 28°C, ssTnC mRNA levels are twofold greater than cTnC mRNA levels in the atrium, whereas cTnC mRNA was almost exclusively expressed in the ventricle. Meanwhile, rainbow trout acclimated at 5°C showed cTnC mRNA levels in both chambers significantly greater than ssTnC. Distinct responses to temperature acclimation were also quantified in both adult zebrafish and rainbow trout, with mRNA in both chambers shifting to express higher levels of cTnC in 18°C acclimated zebrafish and 5°C acclimated trout. Possible subfunctionalization of TnC isoforms may provide insight into how teleosts achieve physiological versatility in chamber-specific contractile properties.
Highlights
We show that the teleost cardiac Ca2ϩ-binding troponin C (TnC) is the product of two distinct genes: cardiac TnC and a fish-specific slow skeletal TnC
Because we determined that ssTnC exists across many teleosts but not in basal ray-finned fish, we propose that these paralogs are the result of an ancestral tandem gene duplication persisting only in teleosts
We show that the ssTnC and cTnC transcripts are expressed in a chamber-specific manner in both the zebrafish and rainbow trout hearts, indicating a possible involvement in functional differences in the atrium and ventricle like other chamber specific isoforms of contractile proteins [e.g., myosin heavy chain and myosin light chain [30, 46]]
Summary
All available TnC gene sequences homologous to zebrafish cTnC or ssTnC were found using the National Center for Biotechnology Information (Bethesda, MD) nonredundant protein database (http://www.ncbi.nlm.nih.gov/guide/ proteins/), the Ensembl Genome Browser (Wellcome Trust Genome Campus, Hinxton, Cambridge, UK) (http://uswest.ensembl.org/ index.html), and GenBank (http://www.ncbi.nlm.nih.gov/genbank/). BLAST searches [2] were set up for several species expressed sequence tag (EST) databases against zebrafish cTnC (TnnC1a) and ssTnC (TnnC1b) sequences (GI:28822162 and GI:50344823, respectively). Multiple amino acid sequence alignments were performed using Multiple Sequence Comparison by Log-Expectation [13] via MEGA5 (Molecular Evolutionary Genetics Analysis 5.0, Ref. 44). The evolutionary histories for these amino acid sequences were inferred using the maximum likelihood algorithm (MEGA5). The bootstrap consensus trees were inferred from 500 replicates. There were a total of 161 residues in the final dataset
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