Isolation and Analysis of a transcriptional repressor to cause cardiac myopathy

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Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a type of transmembrane protein. HB-EGF is produced as a precursor (proHB-EGF) in cell membrane and metalloproteases cleave the ectodomain of proHB-EGF (ectodomain shedding) by various stimulations. A soluble form of HB-EGF (sHB-EGF) binds EGF receptors and activates them. It has been reported that a knock-in mouse with uncleavable mutant of proHB-EGF can not survive for a long term after birth because of onset of dilatation of a heart such as dilatation cardiomyopathy. However, Effects of ucproHB-EGF on adult mice remain unclear. In this report, ucproHB-EGF-induced H9c2 rat cardiomyoblasts significantly lead to cell death as compared with wild type-induced cells. And we also found that the cell death involved apoptosis by increasing TUNEL positive cells. Under hypoxia, ucproHB-EGF strongly induced cell death of H9c2. We have already shown that C-terminal fragment of proHB-EGF (HB-EGF-CTF) after shedding translocates to nuclear membrane and regulate transcription by releasing suppression of zinc finger transcriptional repressors such as premyeloid leukemia zinc finger protein (PLZF) and B-cell lymphoma protein (Bcl06). We hypothesized that uc proHB-EGF-induced cell death was due to failure of transcriptional response by insufficiency of HB-EGF-CTF production. Then, we performed DNA microarray assay to identify cell death-associated target genes, and found several genes not to response to hypoxia in uc proHB-EGF-induced H9c2 cells. In other hand, we investigated zinc finger transcriptional repressors in cardiac precursor cells differentiated from mouse ES cells with custom DNA microarray plates. As a result, several candidate genes for analysis were identified. These data suggest that insufficient shedding of proHB-EGF is associated with hypoxic cell death in cardiomyocytes. Further examinations could unveil a molecular mechanism of cardiomyopathy on set.