Osteopontin regulates hypoxic induction of NHE1 activity in H9C2 cells. Soumaya Bouchoucha, Fatima Mraiche Hypoxia, an important component of ischemia, is the result of an imbalance between oxygen supply and demand. Severe impairment of oxygen supply may result in a maladaptive cardiac phenotype. During myocardial ischemia, the Na+/H+ exchanger isoform 1 (NHE1), the main regulator of pHi, has been shown to be increased. It has been shown recently that NHE1 elevation increases Osteopontin (OPN) expression, a matricellular protein and proinflammatory cytokine that has been reported to have a protective role in ischemic injury. In this study we tested the hypothesis that hypoxic induction of NHE1 expression and subsequent alterations in H9c2 cells are mediated by OPN. H9c2 cardiomyoblasts were infected with GFP, NHE1 in the presence and absence of OPN adenoviruses following a hypoxic insult using cobalt chloride (CoCl2). Western blotting was performed to investigate the expression of OPN and NHE1. NHE1 activity was measured using BCECF-AM. H9c2 infected cells exposed to hypoxia were characterized by measuring cell viability. Inducing hypoxia for 6 hours caused a significant decrease in cell viability in H9c2 cells (GFP: 87±2.5% Vs 70.76±8.17% GFP + hypoxia; P<0.05). In NHE1 + hypoxia infected cells, viability was significantly lower than the control (NHE1 + hypoxia: 69.36±13.71% Vs 100.0 GFP + hypoxia; P<0.05). Interestingly, when overexpressing both OPN and NHE1+hypoxia, cell viability tended to increase. Compared to the control groups, CoCl2 increased the rate of recovery, which reflects the activity of NHE1, in all groups. These results were accompanied by an increase in OPN expression in the NHE1+ hypoxia infected cells (NHE1 + hypoxia: 167.36±10.01% Vs. 100.0 GFP + hypoxia; P<0.05), which validates that NHE1 activates the expression of OPN under hypoxic conditions. These results reveal a suspected role of osteopontin in protecting cell against ischemic injury owing to the increase of NHE1 expression and activity. Taken together, identified osteopontin may function as a survival factor against hypoxic induction of NHE1 cell death. Keywords: NHE1, Osteopontin, hypoxia.


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