A novel peroxide-induced calcium transient regulates interleukin-6 expression in cardiac-derived fibroblasts

Abstract

Reperfusion of ischemic myocardium leads to a local burst of free radicals, increased [Ca2+]i, and the release of proinflammatory cytokines. The purpose of this study was to determine whether brief exposure of cardiac fibroblasts to H2O2 is associated with transient changes in [Ca2+]i levels and whether this stimulus is sufficient to induce interleukin-6 (IL-6) expression. Cardiac derived fibroblasts were isolated from adult male rats and cultured under standard conditions. Individual coverslip-attached fibroblasts were loaded with the calcium probe Fura-2/AM and exposed to a single 3-min pulse of 100 μM H2O2. In addition, low passage cultures were exposed to a pulse of H2O2 and assayed for IL-6 expression. A brief exposure of H2O2 led to a large intracellular Ca2+ transient with a mean transient magnitude of 318 ± 28 nM (mean ± S.D., n = 12). Stimulation in the absence of [Ca2+]o led to a 59% reduction in mean transient magnitude (129 ± 23 nM, n = 10, p < 0.001), whereas pretreatment with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C resulted in a 37% reduction (199 ± 25 nM, n = 10, p < 0.01). Cells treated with xestospongin C and stimulated in the absence of [Ca2+]o did not exhibit a Ca2+ transient. Time-dependent IL-6 release was significantly elevated by 4 h (368 ± 64 pg/mg protein, p < 0.01) and increased further by 24 h (1030 ± 76 pg/mg protein). The depletion of cellular Ca2+ by pretreatment with thapsigargin in the absence of [Ca2+]o attenuated H2O2-induced IL-6 mRNA expression while blocking protein release. These data show that the exposure of cardiac fibroblasts to a brief pulse of physiological levels of H2O2 resulted in a large Ca2+ transient with intracellular and extracellular Ca2+ contributions. Furthermore, brief H2O2 exposure led to calcium-dependent IL-6 expression.