Increased cell death in osteopontin-deficient cardiac fibroblasts occurs by a caspase-3-independent pathway

Reperfusion-induced oxidative injury to the myocardium promotes activation and proliferation of cardiac fibroblasts and repair by scar formation. Osteopontin (OPN) is a proinflammatory cytokine that is upregulated after reperfusion. To determine whether OPN enhances fibroblast survival after exposure to oxidants, cardiac fibroblasts from wild-type (WT) or OPN-null (OPN-/-) mice were treated in vitro with H2O2 to model reperfusion injury. Within 1 h, membrane permeability to propidium iodide ( PI) was increased from 5 to 60% in OPN-/- cells but was increased to only 20% in WT cells. In contrast, after 1 - 8 h of treatment with H2O2, the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling ( TUNEL)- stained cells was more than twofold higher in WT than OPN-/- cells. Electron microscopy of WT cells treated with H2O2 showed chromatin condensation, nuclear fragmentation, and cytoplasmic and nuclear shrinkage, which are consistent with apoptosis. In contrast, H2O2-treated OPN-/- cardiac fibroblasts exhibited cell and nuclear swelling and membrane disruption that are indicative of cell necrosis. Treatment of OPN-/- and WT cells with a cell-permeable caspase-3 inhibitor reduced the percentage of TUNEL staining by more than fourfold in WT cells but decreased staining in OPN-/- cells by similar to 30%. Although the percentage of PI-permeable WT cells was reduced threefold, the percent of PI-permeable OPN-/- cells was not altered. Restoration of OPN expression in OPN-/- fibroblasts reduced the percentage of PI-permeable cells but not TUNEL staining after H2O2 treatment. Thus H2O2-induced cell death in OPN-deficient cardiac fibroblasts is mediated by a caspase-3-independent, necrotic pathway. We suggest that the increased expression of OPN in the myocardium after reperfusion may promote fibrosis by protecting cardiac fibroblasts from cell death.