Analysis of newly established EST databases reveals similarities between heart regeneration in newt and fish

Abstract

Background: The newt Notophthalmus viridescens possesses the remarkable ability to respond to cardiac damage by formation of new myocardial tissue. Surprisingly little is known about changes in gene activities that occur during the course of regeneration. To begin to decipher the molecular processes, that underlie restoration of functional cardiac tissue, we generated an EST database from regenerating newt hearts and compared the transcriptional profile of selected candidates with genes deregulated during zebrafish heart regeneration.

Results: A cDNA library of 100,000 cDNA clones was generated from newt hearts 14 days after ventricular injury. Sequencing of 11520 cDNA clones resulted in 2894 assembled contigs. BLAST searches revealed 1695 sequences with potential homology to sequences from the NCBI database. BLAST searches to TrEMBL and Swiss-Prot databases assigned 1116 proteins to Gene Ontology terms. We also identified a relatively large set of 174 ORFs, which are likely to be unique for urodele amphibians. Expression analysis of newt-zebrafish homologues confirmed the deregulation of selected genes during heart regeneration. Sequences, BLAST results and GO annotations were visualized in a relational web based database followed by grouping of identified proteins into clusters of GO Terms. Comparison of data from regenerating zebrafish hearts identified biological processes, which were uniformly overrepresented during cardiac regeneration in newt and zebrafish.

Conclusion: We concluded that heart regeneration in newts and zebrafish led to the activation of similar sets of genes, which suggests that heart regeneration in both species might follow similar principles. The design of the newly established newt EST database allows identification of molecular pathways important for heart regeneration.

Figure 1

Flowchart of EST sequence processing for contig analysis and GenBank submission. 11520 sequence reads were subtracted from bad trace data, failed sequence reads, empty vectors, and short transcripts to obtain 9696 high quality ESTs. Assembly of ESTs into 2894 contigs was followed by BLAST searches with 5 different algorithms to obtain 6669 best hits for 1695 contigs with scores ≤ 1e-05. Hits with smallest score were selected for contig analysis to evaluate weak and significant similarities to NCBI entries and to evaluate the distribution of organisms with closest similarity. Contigs were split into their respective ESTs for submission to GenBank. Best hits from all BLAST algorithms were attached to facilitate further analysis.

Figure 2

Determination of potential open reading frames from BLAST hits with no identifiable homology. 174 potential ORFs longer than 22 amino acids in length were identified by EST scan. 32% of all ORFs were longer than 150 amino acids.

Figure 3

Gene expression profile of selected newt-zebrafish homologues. Quantification of gene expression patterns by RT-PCR of potential newt-zebrafish homologues during newt heart regeneration in undamaged (day 0) and damaged newt hearts 4, 7, 14, and 21 days (day 4 to day 21) after injury (n = 3 for each time point). a) Expression of muscle creatin kinase, hsp90 alpha, and slc25a4. Potential zebrafish homologues showed a similar expression pattern although reduced mRNA levels were observed at 4 days to 14 days after injury. b) Expression of tubulin alpha 1, keratin 4 and tubulin beta. A delayed time course was apparent in regenerating newt hearts with increased levels only at 21 days after injury. c) Expression of collagen typeI alpha 1, similar to ferritin heavy chain, and similar to cathepsin K. Similar changes in expression levels were found both in newts and zebrafish at all time points after injury. Expression changes of more than two fold were statistically significant with the exception of tubulin 1 alpha at 21 days after injury (p < 0.05 by paired students t-test). Error bars are shown as ± STDEV.

Figure 4

Ratio of proteins annotated to selected ancestor GO terms relative to all proteins annotated to the GO term biological process for newt, deregulated zebrafish proteins, and protein entries from the complete GOA database. A more than 10 fold accumulation in newt and zebrafish EST datasets was detected within the GO term nodes wound healing, muscle contraction and circulation.

Figure 5

Accumulation of proteins annotated to GO terms containing more than 2 zebrafish proteins and more than 7 newt proteins. Fold enrichment was determined by comparing the ratio of proteins annotated to the complete GOA dataset relative to the ratio of annotated newt and zebrafish proteins. 12 GO term nodes display more than 10 fold enrichment in proteins for newt and zebrafish datasets.

Figure 6

Expression profiles of selected mRNAs during newt heart regeneration. Expression of RNF 7, SFRP 1, Thioredoxin-like protein 4B and TCTP 1 was analysed by RT-PCR at 4, 7, 14 and 21 days after mechanical injury of newt ventricles (n = 3 for each time point). Expression of ribosomal protein S21 was analyzed as a non-modulated control. A more than 2 fold change in expression level was detected for all 4 selected genes during the newt heart regeneration. Statistically significant changes in expression (p < 0.05 by paired students t-test) were detected for RNF 7 and SFRP 1 at 14 and 21 days after injury and 7 and 14 days after injury for Thioredoxin-like protein 4B. Error bars are shown as ± STDEV. Please note that selected newt genes were so far not identified in regenerating zebrafish hearts.