Studies On Expression And Function Of Human Heart Developmental Candidate Gene Ahnakβ

The process of heart development is quite complicated, which is regulated by a series of genes. Although many of the heart developmental genes have been identified, there are still a lot of them to be identified.With the aim of identifying the genes involved in human heart development and cardiovascular diseases, we have isolated and characterized a heart developmental candidate gene AHNAKβfrom a human heart cDNA library. AHNAKβ(GenBank accession Number: NM₀24060) is mapped to chromosome 11q12.2. The full-length cDNA is long 1108 bp and contains an open reading frame (ORF) of 450 nucleotides, which encodes a deduced 16.0-kDa protein consisting of 149 amino acids. AHNAKβis the transcript variant 2 of AHNAK. The AHNAK transcript variant 1 AHNAKa is long 18,815 base pairs, encoding a huge protein with 680 KDa. By BLAST, we found that the N-terminals of AHNAKβand AHNAKa are the same, but their C-terminals are completely different. The difference between the two transcript variants has aroused our interest. The studies have shown that AHNAKa could control cardiac contraction, it suggesting that AHNAKβmay be associated with heart development.In order to study the AHNAKβgene expression patterns, we generated polyclonal anti-AHNAKβantibodies by injecting a GST fusion protein. This fusion protein corresponding to amino acids 106-149 from the C-terminals of AHNAKβwas selected as an immunogen.To examine the level of AHNAKβexpression quantitatively, Western blot analyses were performed using the protein from adult mouse. AHNAKβexpression was readily detectable in the heart, and a similar level of expression was maintained in intestine.In the present study, we show the temporal and spatial expression patterns of AHNAKβin mouse, by means of whole mount In Situ Hybridization and immunohystochemical experiments. The results of these experiments demonstrate, in both embryo and adult, AHNAKβhas expressed in the heart.We next set out to determine the intracellular localization of endogenous AHNAKβduring development by immunohistochemical staining. Primary cultured cardiomyocytes prepared from neonatal mice were immunostained with anti-AHNAKβantibody. The expression patterns suggest that AHNAKβis distributed in the cytoplasm. Further investigation by electron micrographs places AHNAKβin myofilament and mitochondria.From a screen using a GAL4 AD-fused human heart cDNA library and GAL4-BD fused human AHNAKβas bait in the yeast two-hybrid system, we tested eight candidate proteins that may interact with AHNAKβ, and one of the positive clones is TNNI3, which is a heart-specific gene, with related to Familial hypertrophic myocarditis. The interaction between TNNI3 and AHNAKβwas confirmed using immunoprecipitation (IP) in vitro.In conclusion, these results from above experimental procedures may suggest a role for AHNAKβin the regulation of heart development. These works offer a good foundation for further investigation.