|Abstract||The Na+/H+ exchanger-1 (NHE1) is a ubiquitously expressed housekeeping glycoprotein that functions to regulate intracellular pH. Enhanced expression/activity of NHE1 has been implicated in cardiac hypertrophy (CH). Recently, Transgenic mice expressing active-NHE1 demonstrated a>1500-fold increase in osteopontin (OPN) gene expression. OPN, a component of the extracellular matrix, has also been implicated in CH. In our study, upregulation of NHE1 in cardiomyocytes resulted in a significant increase in OPN protein expression (342.7%±69.22%). To determine whether OPN contributes to the hypertrophic effects of NHE1 during CH, cardiomyocytes were infected with active NHE1 in the presence and absence of OPN, or a silencing RNA (siRNA). CH was assessed by measuring cell area, protein content and atrial natriuretic peptide (ANP) mRNA. Overexpression of NHE1 and OPN in cardiomyocytes significantly increased cell area (158.4±3.59% of control); this was significantly reduced in the presence of OPN-siRNA (68.5±0.24% vs. 190.9±8.66% NHE1-infected). Protein content and ANP mRNA expression were also significantly increased in NHE1-infected cardiomyocytes, however they were significantly abrogated in the presence of OPN-siRNA (87.8±12.58% vs. 136.8±11% NHE1-infected) and (64.6±19.9% vs. 247.7±30.81% NHE1-infected). OPN appeared to contribute to NHE1-induced CH independent of extracellular-signal-regulated kinases, p90-ribosomal-S6 kinase and protein kinase B (Akt). Interestingly, NHE1 expression and activity in cardiomyocytes infected with NHE1 and OPN were significantly enhanced (636.5±128.75% of control) and (163.1±19.49% vs. 114.7±9.48% NHE1-infected). This was significantly abolished in the presence of siRNA-OPN (20.2 fold decrease±0.12 of control) and (68.5±0.24% vs. 586.5±103.54% NHE1-infected). Moreover, cleavage of OPN was enhanced in cardiomyocytes infected with both NHE1 and OPN, which was significantly reduced in the presence of siRNA OPN. Our study is the first to demonstrate a two-way cross talk between NHE1 and OPN in CH in vitro. Our findings demonstrate that NHE1 upregulates OPN in cardiomyocytes, which in turn regulates NHE1 expression/activity and contributes to the NHE1-induced hypertrophic response. Our study highlights OPN as a potential therapeutic target to reverse NHE1-induced CH and activity, one which maybe more beneficial than directly inhibiting NHE1.