Carbon monoxide donors or heme oxygenase-1 (HO-1) overexpression blocks interleukin-18-mediated NF-kappaB-PTEN-dependent human cardiac endothelial cell death.

The objective of this study was to determine whether heme oxygenase-1 (HO-1) or heme metabolites exert cytoprotective effects on interleukin-18-mediated endothelial cell (EC) death. Treatment with interleukin (IL)-18 increased NF-kappaB activation and PTEN induction, suppressed Akt activation, and stimulated EC death. While ectopic expression of p65 enhanced PTEN transcription, adenoviral transduction of dnIkappaB-alpha, dnp65, or dnIKKbeta was inhibitory. Furthermore, IL-18 suppressed HO-1 mRNA expression via enhanced mRNA degradation. Overexpression of HO-1, treatment with HO-1 inducer hemin, or the CO donor cobalt (III) protoporphyrin IX all reversed IL-18-mediated NF-kappaB activation, PTEN induction, Akt suppression, and EC death. Furthermore, hemin induced HO-1 expression, and HO-1 knockdown, HO-1 inhibition, or CO scavengers all reversed the prosurvival effects of hemin. In addition, the CO donors CORM-1 and CORM-3 and the heme metabolites biliverdin and bilirubin attenuated IL-18-induced EC death via a similar signaling pathway. IL-18 induced p38alpha MAPK activation, and suppressed p38beta isoform expression. While p38alpha knockdown attenuated, p38beta knockdown potentiated IL-18-mediated EC death. Hemin and HO-1 reversed IL-18-mediated p38alpha induction and restored p38beta levels. These results demonstrate that IL-18 suppresses HO-1 expression and induces EC death. HO-1 overexpression, HO-1 induction, or treatment with heme metabolites all reverse IL-18-mediated p38alpha MAPK and NF-kappaB activation, PTEN induction, Akt suppression, and EC death. Thus, HO-1 inducers and CO donors may have the therapeutic potential to effectively block IL-18 signaling and reduce IL-18-dependent vascular injury and inflammation.

Interleukin-18 knockout mice display maladaptive cardiac hypertrophy in response to pressure overload.

Interleukin (IL)-18 is a cardiotropic proinflammatory cytokine chronically elevated in the serum of patients with cardiac hypertrophy (LVH). The purpose of this study was to examine the role of IL-18 in pressure-overload hypertrophy using wild type (WT) and IL-18 -/- (null) mice. Adult male C57Bl/6 mice underwent transaortic constriction (TAC) for 7days or sham surgery. Heart weight/body weight ratios showed blunted hypertrophy in IL-18 null TAC mice compared to WT TAC animals. Microarray analyses indicated differential expression of hypertrophy-related genes in WT versus IL-18 nulls. Northern, Western, and EMSA analyses showed Akt and GATA4 were increased in WT but unchanged in IL-18 null mice. Our results demonstrate blunted hypertrophy with reduced expression of contractile-, hypertrophy-, and remodeling-associated genes following pressure overload in IL-18 null mice, and suggest that IL-18 plays a critical role in the hypertrophic response.

Impact of physiological variables and genetic background on myocardial frequency-resistivity relations in the intact beating murine heart.

Conductance measurements for generation of an instantaneous left ventricular (LV) volume signal in the mouse are limited, because the volume signal is a combination of blood and LV muscle, and only the blood signal is desired. We have developed a conductance system that operates at two simultaneous frequencies to identify and remove the myocardial contribution to the instantaneous volume signal. This system is based on the observation that myocardial resistivity varies with frequency, whereas blood resistivity does not. For calculation of LV blood volume with the dual-frequency conductance system in mice, in vivo murine myocardial resistivity was measured and combined with an analytic approach. The goals of the present study were to identify and minimize the sources of error in the measurement of myocardial resistivity to enhance the accuracy of the dual-frequency conductance system. We extended these findings to a gene-altered mouse model to determine the impact of measured myocardial resistivity on the calculation of LV pressure-volume relations. We examined the impact of temperature, timing of the measurement during the cardiac cycle, breeding strain, anisotropy, and intrameasurement and interanimal variability on the measurement of intact murine myocardial resistivity. Applying this knowledge to diabetic and nondiabetic 11- and 20- to 24-wk-old mice, we demonstrated differences in myocardial resistivity at low frequencies, enhancement of LV systolic function at 11 wk and LV dilation at 20-24 wk, and histological and electron-microscopic studies demonstrating greater glycogen deposition in the diabetic mice. This study demonstrated the accurate technique of measuring myocardial resistivity and its impact on the determination of LV pressure-volume relations in gene-altered mice.

H2O2 activates Nox4 through PLA2-dependent arachidonic acid production in adult cardiac fibroblasts.

Stimulated production of reactive oxygen species (ROS) by plasma membrane-associated nicotinamide adenine dinucleotide phosphate oxidases (Nox) in non-phagocytic cells regulates a number of biological processes including growth, vessel tone, and oxygen sensing. The purpose of this study was to investigate H(2)O(2)-stimulated ROS production in primary adult cardiac fibroblasts (CF). Results demonstrate that CF express an H(2)O(2)-inducible oxidant generating system that is inhibitable by diphenylene iodonium (DPI) and sensitive to antioxidants. In addition to H(2)O(2), generation of ROS was stimulated potently by 1-oleoyl-2-acetyl-sn-glycerol (OAG) and arachidonic acid (AA) in a protein kinase C-independent manner. Pretreatment with arachidonyl trifluoromethyl ketone was nearly as effective as DPI at reducing H(2)O(2)- and OAG-stimulated oxidant generation indicating a central role for phospholipase A(2) (PLA(2)) in this signaling pathway. Co-stimulation with H(2)O(2) and OAG did not increase ROS generation as compared to OAG alone suggesting both agonists signal through a shared, rate-limited enzymatic pathway involving PLA(2). Co-stimulation with H(2)O(2) and AA had additive effects indicating these two agonists stimulate oxidant production through a parallel activation pathway. Reverse transcriptase-coupled polymerase chain reaction and Western blotting demonstrate primary cardiac fibroblasts express transcripts and protein for Nox4, p22, p47, and p67 phox. Transfections with Nox4 small inhibitory ribonucleic acid oligonucleotides or p22 phox antisense oligonucleotides significantly downregulated stimulated Nox activity. Inhibitors of nitric oxide synthases were without effect. We conclude adult CF express Nox4/p22 phox-containing oxidant generating complex activated by H(2)O(2), OAG, and AA through a pathway that requires activation of PLA(2).

Impact of brief oxidant stress on primary adult cardiac fibroblasts.

Reperfusion of ischemic myocardium (I/R) is associated with local release of a brief pulse of reactive oxygen species. The purpose of this study was to determine the effects of brief H2O2 stimulation on primary adult cardiac fibroblast phenotype. We demonstrate that brief H2O2 exposure results in transient phosphorylations of p38 and ERK which peaked by 15 min. Proliferation was minimally affected by either H2O2 or MAPK inhibition. Pretreatment with SB203580 or U0126 revealed that p38 enhances or maintains migration rates while ERK retarded migration. Peroxide exposure increased necrosis from 4% at baseline to >12% while reducing apoptosis by 3.5-fold. p38 inhibition resulted in increased necrosis and apoptosis while ERK inhibition had minimal effects. In conclusion, primary adult cardiac fibroblasts exposed to brief H2O2 exhibit an altered phenotype characterized by reduced migration and apoptosis and increased necrosis resulting, in part, from the differential effects of p38 and ERK signaling.

TNF-alpha and H2O2 induce IL-18 and IL-18R beta expression in cardiomyocytes via NF-kappa B activation.

Myocardial ischemia/reperfusion is characterized by oxidative stress and induction of proinflammatory cytokines. Interleukin (IL)-18, a member of the IL-1 family, acts as a proinflammatory cytokine, and is induced during various immune and inflammatory disorders. Therefore, in the present study we investigated whether IL-18 expression is regulated by cytokines and oxidative stress in cardiomyocytes. TNF-alpha induced rapid and sustained activation of NF-kappaB whereas H(2)O(2) induced delayed and transient activation. Both TNF-alpha and H(2)O(2) induced IL-18 mRNA and precursor protein in cardiomyocytes, and IL-18 release into culture supernatants. However, only TNF-alpha led to sustained expression. Expression of IL-18Rbeta, but not alpha, was induced by both agonists. TNF-alpha and H(2)O(2) induced delayed expression of IL-18 BP. Pretreatment with PDTC attenuated TNF-alpha and H(2)O(2) induced IL-18 and IL-18Rbeta, but not basal expression of IL-18Ralpha. These results indicate that adult cardiomyocytes express IL-18 and its receptors, and proinflammatory cytokines and oxidative stress regulate their expression via activation of NF-kappaB. Presence of both ligand and receptors suggests IL-18 impacts myocardial biology through an autocrine pathway.

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

Reperfusion of ischemic myocardium leads to a local burst of free radicals, increased [Ca(2+)](i), and the release of proinflammatory cytokines. The purpose of this study was to determine whether brief exposure of cardiac fibroblasts to H(2)O(2) is associated with transient changes in [Ca(2+)](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 microm H(2)O(2). In addition, low passage cultures were exposed to a pulse of H(2)O(2) and assayed for IL-6 expression. A brief exposure of H(2)O(2) led to a large intracellular Ca(2+) transient with a mean transient magnitude of 318 +/- 28 nm (mean +/- S.D., n = 12). Stimulation in the absence of [Ca(2+)](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 [Ca(2+)](o) did not exhibit a Ca(2+) 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 Ca(2+) by pretreatment with thapsigargin in the absence of [Ca(2+)](o) attenuated H(2)O(2)-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 H(2)O(2) resulted in a large Ca(2+) transient with intracellular and extracellular Ca(2+) contributions. Furthermore, brief H(2)O(2) exposure led to calcium-dependent IL-6 expression.