Abstract 1688: Mitochondrial Localization and Cellular Function of Cytoglobin: A Novel Calcineurin-Dependent Stress-Responsive Globin in the Cardiovascular System
Cytoglobin (Cygb) is a novel tissue hemoprotein and its cellular function is unknown. We have demonstrated that Cygb is regulated by the calcineurin/NFAT pathway, is predominantly expressed in the heart and brain, and its expression is upregulated with stress (hypoxic heart/brain and ischemic cardiomyopathic heart). To define the functional role of cytoglobin, in-vitro studies were undertaken to investigate the subcellular localization and mechanistic role(s) of cytoglobin within the cell. Western blot analysis indicated that Cygb is found in both the cytoplasm and the nucleus of normoxic C2C12 myoblasts; however, upon exposure to hypoxia (1% O2 for 16 hours) Cygb translocates out of the nucleus. The translocation process was blocked when C2C12 cells were incubated with leptomycin B, a potent inhibitor of nuclear export. Western blot analysis and immunocytochemistry also revealed that Cygb co-localizes to the mitochondria. Transfection assays involving C2C12 cells demonstrated that overexpression of cytoglobin promotes cellular viability as compared to control cells upon exposure to menadione (an agent that generates free radicals) (67±1.6% vs. 48±1.6%, respectively; p<.05; n=3) or anoxic conditions (72±8.0% vs. 49±0.7%, respectively; p<0.05; n=3). Conversely, knockdown of Cygb using siRNA (Cygb KO) resulted in a significant reduction in cell survival vs. control cells upon menadione exposure (61±0.5% vs. 75±0.2%, respectively; p<0.05; n=3) or anoxic conditions (32.7±0.2% vs. 63±1.5%, respectively; p<0.05; n=3). FACS analysis demonstrated more anoxic Cygb KO cells were in a GoG1 cell cycle arrest (77±5% Cygb KO cells vs. 62±8% control cell; p<0.05; n=3) and TUNEL assays revealed an increase in apoptosis in this cell population. In addition, there was a 6.3 fold increase in oxidative stress as measured by glutathione levels and a 1.5 fold increase in NO levels in stressed Cygb KO cells as compared to control cells (n=3). Finally, microarray analysis of Cygb KO cells confirmed dysregulation of genes involved in apoptosis and cell cycle regulation. These new data collectively establish that Cygb is a novel stress-responsive globin that may play a key signaling role as well as a cytoprotective role in the hypoxic/ischemic heart.