294 Generation of endoplasmic reticulum protein 44 (ERP44) knockout/knockin mice to investigate the role of ERP44 in calcium signaling and ER stress in cardiomyocytes

Abstract

BACKGROUND: ERp44 is a novel unfolded protein response (UPR)-induced endoplasmic reticulum (ER) protein of the thioredoxin family. ERp44 transcripts are widely expressed in a variety of tissues and cell types and has been shown to be up regulated upon ER stress. ERp44 may be involved in multiple cellular functions including the inhibition of Ca release by inactivating inositol 1,4,5-trisphosphate receptor1 (IP3R1), and by supporting disulfide bond formation through reinforcing the Ero1 /oxidoreductase system. The role of ER stress in various cardiomyopathies is becoming clearer. Here, we hypothesize that ERp44 plays important roles in ER stress involved in heart disease by regulation of Ca signaling and activation of the ER stress response signaling. Dysregulation of Ca cycling in myocytes will activate ER stress pathway and lead to apoptotic cell death in cardiomyocytes. To study the role of ERp44 in Ca signaling and ER stress in vivo, we have generated ERp44 knockout/knockin mouse model systems. METHODS/RESULTS: We utilized the gene-trapped embryonic stem (ES) cell lines RST431 and XB599 (IGTC) to generate ERp44 (Txnd4) mutant mice through blastocyst injection. Analysis of the ERp44 (Txnd4) gene trapped lines RST431 and XB599 by RT-PCR and sequencing both mutant mice strains have the LacZ knockin allele inserted into intron 8 (RST431) and intron 1 (XB599). LacZ staining of tissue sections and immunoblot analysis of WT / ERp44-LacZ lines has confirmed LacZ protein expression in the heart, brain and other various tissues of these knockin ERp44 mutant mice. Mortality, in vivo echocardiography, isolated cardiomyocyte Ca2 transient assays, along with biochemical analysis of ER stress response pathways are reported. Ongoing work in our lab is focused on obtaining and analyzing ERp44-LacZ /ERp44LacZ double allele strains. CONCLUSION: We generated two ERp44 mutant mouse strains by utilizing gene trapped ES cell lines RST431 and XB599. RST431 mice have LacZ gene inserted in to downstream of ERp44 intron 8 and XB599 mice have LacZ inserted upstream of ERp44 intron 1. RST431 mouse strain represents a useful LacZ knockin ERp44 allele mouse model to study ER stress in the development of heart disease, as a reporter line, while the XB599 mouse strain represents the ERp44 knockout mouse model to study the role of ERp44 in development of ER stress involved heart disease. Heart and Stroke Foundation of Ontario, Canada Research Chair Program