Cumene hydroperoxide induced changes in calcium homeostasis in cultured neonatal rat heart cells.

OBJECTIVE: The relationship between oxidative stress induced cell necrosis and perturbation of intracellular calcium homeostasis was investigated in cultured myocytes. METHODS: Cultured neonatal rat heart cells were loaded with fura-2 AM to measure cytosolic free calcium ([Ca2+]i). Probenecid, an inhibitor of organic anion transport, was present during the experiment to reduce efflux of fura-2 from the cytoplasm. Cells were exposed to cumene hydroperoxide, a toxic organic hydroperoxide that is known to induce oxidative stress in myocytes. The efficacy of the protective agents Trolox C (a vitamin E analogue) and chlorpromazine (a phospholipase inhibitor) on cumene hydroperoxide induced cell injury was determined. RESULTS: [Ca2+]i in control cells was constant (60 nM) during an incubation time of 45 min. Probenecid did not affect [Ca2+]i levels or cell viability under the experimental conditions. Cumene hydroperoxide caused a sustained rise in [Ca2+]i starting after 5-10 min, to a level of 1300 nM at 45 min. After 20-25 min the viability of the heart cells started to decline and after 45 min 44% of the cells were irreversibly injured. The loss of cell viability was expressed as percentage decrease of the fluorescence at 360 nm (the calcium independent wavelength), since the percent release of cellular alpha-hydroxybutyrate dehydrogenase (alpha-HBDH) activity equalled the percent decrease of the fluorescence at 360 nm. Trolox C and chlorpromazine almost completely prevented the cumene hydroperoxide induced alpha-HBDH release. The [Ca2+]i of myocytes incubated with cumene hydroperoxide in combination with Trolox C rose to 1000 nM without affecting cell viability. The cumene hydroperoxide induced rise in [Ca2+]i was markedly reduced by chlorpromazine (at t = 45 min, [Ca2+]i = 360 nM). Addition of Trolox C to untreated cells did not influence [Ca2+]i, whereas chlorpromazine alone induced a slight increase of [Ca2+]i up to 360 nM with complete preservation of cell viability. CONCLUSIONS: Trolox C and chlorpromazine are very effective inhibitors of cumene hydroperoxide induced perturbation of calcium homeostasis and subsequent cell death. A role for peroxidation of membrane phospholipids and activation of calcium dependent phospholipase in the cascade of events leading to irreversible injury is suggested.

Alpha-adrenergic agonist and endothelin-1 induced intracellular Ca2+ response in the presence of a Ca2+ entry blocker in cultured rat ventricular myocytes.

Previously we demonstrated that stimulation of cultured neonatal rat ventricular myocytes by either alpha 1-adrenergic agonist or endothelin-1 resulted in a rapid formation of total inositolphosphates, although the levels of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate did not rise significantly. The aim of this study was to examine whether stimulation by alpha 1-adrenergic agonist and endothelin-1 could still elicit phosphatidylinositol cycle mediated intracellular Ca2+ mobilization in these cells. The intracellular free Ca2+ concentration ([Ca2+]i) was measured by single cell imaging dual wavelength fluorescence microscopy in Fura-2-loaded cardiomyocytes. The interference of agonist induced [Ca2+]i responses by the beat to beat variation of [Ca2+]i was prevented by arresting the cells with the Ca2+ entry blocker diltiazem (10 microM). The [Ca2+]i response (expressed as % of baseline ratio of fluorescence intensities of Fura-2 at 340 nm and 380 nm excitation wavelength), induced by phenylephrine (10(-4) M) and endothelin-1 (10(-8) M) was small, up to 20% of baseline after 9-20 min. In contrast, Ca(2+)-influx induced by incubation in Na(+)-free buffer caused a steep increase of [Ca2+]i up to 150% of baseline after 30 s. Analysis of single cells following stimulation with phenylephrine or endothelin-1 showed heterogeneity with respect to a rise in [Ca2+]i. However, if rapid Ca(2+)-influx was induced by incubation in Na(+)-free buffer, [Ca2+]i responses in individual myocytes occurred homogeneously. It is concluded that the alpha 1-adrenergic agonist and endothelin-1 induced [Ca2+]i responses are delayed in time, small and quite heterogeneous among cells. The findings are in agreement with earlier observations which revealed no detectable overall increase of the Ca2+ releasing inositolphosphates under these conditions and suggest that other second messengers, such as 1,2-diacylglycerol, are involved in the agonist mediated Ca2+ signals.

Isoproterenol-induced cytotoxicity in neonatal rat heart cell cultures is mediated by free radical formation.

The cardiotoxic effect of the beta-adrenergic agonist isoproterenol was studied in cultured neonatal rat myocytes. A progressive increase in irreversible cell injury as determined by leakage of the cytoplasmic enzyme alpha-hydroxybutyrate dehydrogenase (alpha-HBDH) from the cells was noted at concentrations above 2.5 x 10(-4) M isoproterenol (exposure time 6 h). The isoproterenol-induced cell damage was reduced or prevented by several free radical scavengers: the application of Trolox C, a water-soluble vitamin E analogue, ICRF-159, a chelator of divalent cations, ascorbic acid, a potent antioxidant, as well as the enzymatic free radical scavengers superoxide dismutase and catalase reduced alpha-HBDH release. This study corroborates the hypothesis that oxidation products of isoproterenol, especially the formation of oxygen- and/or oxygen-derived free radicals, are responsible for the cytotoxicity observed after prolonged exposure to isoproterenol. In contrast to isoproterenol, exposure to 5 x 10(-4) M fenoterol, another beta-adrenergic agonist which is not oxidized, does not impair the viability of the myocytes. Moreover, application of the beta-blocker propranolol (10(-4) M, 10(-5)M) in combination with 5 x 10(-4) M isoproterenol does not prevent alpha-HBDH release. These findings suggest that isoproterenol-induced cardiotoxicity is not the result of excessive beta-adrenoceptor activation, but is mediated by the formation of free radicals.

Oxidative stress-induced perturbations of calcium homeostasis and cell death in cultured myocytes: role of extracellular calcium.

The role of extracellular calcium in the process of oxidative stress-induced calcium overload and cell death was investigated in cultured neonatal rat myocytes. Oxidative stress was induced by addition of cumene hydroperoxide (CHPO), a toxic organic hydroperoxide, in combination with varying extracellular calcium concentrations (1. normal calcium buffer: 2.5 mM Ca2+, 2. low calcium buffer: 5 microM Ca2+, 3. zero calcium buffer: 2.5 mM EGTA, no CaC12). Intracellular free calcium ion concentration ([Ca2+]i) was measured with fura-2 using a spectrofluorometer. To study the toxicant-induced changes in [Ca2+]i in more detail, single cell imaging was performed using digital imaging fluorescence microscopy (DIFM). In control experiments (in the absence of CHPO, but under different extracellular Ca2+ conditions) the [Ca2+]i remained at the basal level and cell viability was preserved. Administration of CHPO (50 microM) to the myocyte cultures generated a sustained increase in [Ca2+]i followed by loss of cell viability. A low extracellular calcium concentration (5 microM), in the absence or presence of diltiazem (10 microM), induced a delay in the rise in [Ca2+]i but was not able to prevent the CHPO-induced calcium overload and cell necrosis. Addition of EGTA (2.5 mM) to the low calcium buffer resulted also in CHPO-induced cell death, although no increase in [Ca2+]i was observed. In normal and low calcium buffers, DIFM revealed that CHPO produced a temporally and spatially heterogeneous distribution of [Ca2+]i in a group of myocytes. So, in the presence of normal or low extracellular Ca2+, CHPO intoxication of cultured myocytes leads to an increase of [Ca2+]i prior to the onset of cell death. If extracellular Ca2+ is chelated by EGTA, CHPO also induces cell death which is not preceded by cellular calcium overload. Apparently a disturbance in the calcium homeostasis is not causally related with oxidative stress-induced myocardial cell death.

Prevention of cumene hydroperoxide induced oxidative stress in cultured neonatal rat myocytes by scavengers and enzyme inhibitors.

Oxidative stress induced by cumene hydroperoxide was studied in cultured neonatal rat myocytes. A progressive increase of irreversible cell injury as determined by leakage of the cytoplastic enzyme alpha-hydroxybutyrate dehydrogenase (alpha-HBDH) from the cells was noted at concentrations ranging from 25-100 microM cumene hydroperoxide (incubation time 90 min). Cumene hydroperoxide-induced damage was reduced or prevented by several compounds: the application of Trolox C, a water-soluble vitamin E analogue, and of phospholipase A2 inhibitors chlorpromazine and (to a lesser extent) quinacrine prevented alpha-HBDH release. ICRF-159, a chelator of divalent cations, ascorbic acid, a potent antioxidant, and the cysteine protease inhibitor leupeptin did not reduce the cumene hydroperoxide-induced cytotoxicity. Detoxification of hydroperoxides by the glutathione peroxidase system results in an increased flux through the pentose phosphate shunt and loss of NADPH. Glucose inhibited the cumene hydroperoxide-induced alpha-HBDH release, probably by replenishing NADPH. These results indicate that cumene hydroperoxide, after exhaustion of the glutathione system, induces irreversible injury in cultured myocytes by a mechanism that depends to a large extent on deterioration of cellular membranes caused by lipid peroxidation and phospholipase activation.

Rapid gene transcription induced by stretch in cardiac myocytes and fibroblasts and their paracrine influence on stationary myocytes and fibroblasts.

Functional adaptation of cardiac cells in response to haemodynamic load requires dynamic alteration of gene expression. In this study, we examined early changes in gene expression following stretch in myocytes and fibroblasts isolated from neonatal rat hearts. In the first hour of biaxially applied static stretch, the changes in expression of immediate-early genes, such as c-fos, c-jun and fra-1, were quantified. The expression of the atrial natriuretic peptide (ANP) gene in myocytes was measured as an indication of the hypertrophic response. In stretched myocytes, expression of c-fos and ANP increased transiently to 227% and 176% respectively after 30 min stretch, whereas c-jun and fra-1 expression decreased in the 1st hour of stretch. In stretched fibroblasts the expression of c-fos and fra-1 increased transiently to maxima of 145% and 146% respectively after 30 min stretch, whereas c-jun expression did not change significantly. To study the indirect effects of stretch, as an indication of cross-talk between cardiac cells, stationary myocytes and fibroblasts were incubated with stretch-conditioned medium (CM) from stretched (0-60 min) myocytes and fibroblasts. CM from stretched myocytes reduced c-fos and induced c-jun expression in myocytes and fibroblasts, reduced fra-1 expression in myocytes but induced fra-1 expression in fibroblasts. CM from stretched fibroblasts induced c-fos expression and had little effect on c-jun expression in myocytes and fibroblasts, induced the fra-1 expression in myocytes but had little effect on fra-1 expression in fibroblasts. CM from myocytes and CM from fibroblasts induced ANP expression in myocytes to 206% and 120% respectively after 45 min stretch. Static stretch of myocytes and fibroblasts appears to stimulate, within 1 h, secretion of cell type-specific factors that participate in the regulation of proto-oncogene and ANP expression of stationary myocytes and fibroblasts. These early changes in gene transcription suggest that stretch of the myocardium initiates intracellular gene expression as well as cross-talk between the cell types.