Tachibana, Issei

ABSTRACT Dynamin proteins containing a GTPase domain, a pleckstrin homology motif and a proline-rich tail participate in receptor-mediated endocytosis in organisms ranging from insects to vertebrates. In addition, dynaminrelated GTPases, such as the yeast Golgi protein Vps1p, which lack both the pleckstrin homology motif and the proline-rich region, participate in vesicular transport within the secretory pathway in lower eukaryotes. However, no data is available on the existence of Vps1p-like proteins in mammalian cells. In this study, we report the identification and characterization of a novel gene encoding a human dynamin-related protein, DRP1, displaying high similarity to the Golgi dynamin-like protein Vps1p from yeast and to a Caenorhabditis elegans protein deposited in the databank. These proteins are highly conserved in their N-terminal tripartite GTPase domain but lack the pleckstrin homology motif and proline-rich region. Northern blot analysis reveals that the DRP1 mRNA is detected at high levels in human muscle, heart, kidney and brain. Immunolocalization studies in Chinese hamster ovary (CHO) cells using an epitope-tagged form of DRP1 and confocal microscopy show that this protein is concentrated in a perinuclear region that labels with the endoplasmic reticulum marker DiOC6(3) and the Golgi marker C5-DMB-Cer. In addition, the localization of DRP1 is highly similar to the localization of the endoplasmic reticulum and cis -Golgi GTPase Rab1A, but not to the staining for the trans -Golgi GTPase Rab6. Furthermore, overexpression of a cDNA encoding a GTP binding site mutant of DRP1 (DRP1K38E) in CHO cells decreases the amount of a secreted luciferase reporter protein, whereas the overexpression of wild-type DRP1 increases the secretion of this marker. Together, these results constitute the first structural and functional characterization of a mammalian protein similar to the yeast dynamin-related GTPase Vps1p and indicate that the participation of these proteins in secretion has been conserved throughout evolution.

Background —Cardiotrophin-1 (CT-1) is a potent hypertrophic factor discovered by coupling expression cloning in a mouse embryonic stem cell–based model of cardiogenesis. Methods and Results —The present study was designed to investigate the potential activation of atrial and ventricular CT-1 expression in pacing-induced experimental congestive heart failure (CHF) and its relationship to left ventricular hypertrophy by the method of Northern blot analysis and immunohistochemistry. We used a canine model of pacing-induced experimental CHF based on hemodynamic and neurohumoral characteristics that closely mimic human dilated cardiomyopathy. Northern blot analysis demonstrated that CT-1 gene expression was present in normal atrium and ventricle and was increased in CHF hearts. There was a positive correlation between ventricular CT-1 mRNA and left ventricular mass index. Immunohistochemistry revealed positive immunostaining in the atrial and ventricular cardiomyocytes from both normal and CHF hearts. CT-1 immunoreactivity was more intense in the atrium and ventricle from CHF hearts than in normal hearts. Conclusions —The present study demonstrates that both atrium and ventricle synthesize CT-1 and that cardiac production of CT-1 is augmented in a canine model of experimental CHF. This study also demonstrates that ventricular CT-1 mRNA correlates with left ventricular hypertrophy, suggesting that CT-1 plays an important role in the structural remodeling that characterizes CHF.