Studies On The Expression Pattern And Cardiomyocyte Protection Roles Of Novel Gene MIP2

WD40 repeats are short motifs with-40 amino acids, often terminating in a Trp-Asp (W-D) dipeptide. WD-containing proteins have 4 to 16 repeating units, all of which are thought to form a circularised beta-propeller structure. The best characterized WD repeat protein is theβ-subunit of the G proteins, which contains seven WD40 repeats. The WD40 family of proteins comprises a large number of proteins and participates in a wide variety of cellular functions including signal transduction, transcription, RNA processing, and the cell cycle.MIP2 is a novel gene cloned in our laboratory that is up-regulated in ischemic preconditioned rat heart and we named it myocardial ischemic preconditioning up-regulated protein 2 (GenBank accession number: AY221751). It has an open reading frame of 1497 bp, encoding a polypeptide of 498 amino acids with an N-terminal CTLH domain and 5 C-terminal WD40 repeats.Using bioinformatics techniques it was found that MIP2 is an acidic protein with high hydrophobicity, but with no signal peptide. It contains four protein kinase C phophorylation sites, four N-glycosylation sites, seven casein kinaseⅡphosphorylation sites, indicating that the activation of MIP2 may be controlled by phosphorlation. Majority of its motifs are alpha helixs, extended strands and random coils in its secondary structure. Three-dimensional structure analysis showed that five WD-repeats of MIP2 form a propeller structure. SAGE (Several Analysis of Gene Expression) analysis revealed that MIP2 was ubiquitously expressed in normal human tissues and broad kinds of tumors, with a high expression in the heart.We next observed the expression pattern of MIP2 in models of myocardial ischemia/reperfusion (I/R) and myocardial ischemic preconditioning (IP) in rats. The mRNA and protein expression levels were up-regulated after I/R, IP, IP+I/R and H2O2 exposure. A higher mRNA expression of the MIP2 was observed in ischemic myocardium than in non-ischemic myocardium. To observe the subcellular localization of MIP2, a pEGFP-MIP2 fusion vector was constructed. It was found that MIP2 was located mainly in the cytoplasm, with a part of MIP2 distributed on mitochondria.It was also found that stable overexpression of MIP2 in rat cardiomyoblast cell line H9c2 resulted in an enhanced growth of the cells in a dose-dependant manner as measured by cell number. Overexpression of MIP2 resulted in a shorter cell cycle, as measured by flow cytometry. Collectively, these data suggested that MIP2 promoted the proliferation of H9c2 cells.It was further found that oxidative stress induced by H2O2 led to concurrent increases in necrosis and apoptosis in cultured H9c2 rat cardiomyocytes. Overexpression of MIP2 decreased the cardiotoxicity induced by H2O2, which is associated with inhibition of the activation of caspase-9 and caspase-3, as well as inhibition of releasing of cytochrome C from mitochondrion to cytoplasm. MIP2 silencing increased oxidative stress-induced necrosis and apoptosis in H9C2 cells. These results indicated that MIP2 could protect cardiomyocytes against oxidative stress-induced injury.