TGF-beta(2)- and H2O2-Induced Biological Changes in Optic Nerve Head Astrocytes Are Reduced by the Antioxidant Alpha-Lipoic Acid

Background/Aims: The goal of the present study was to determine whether transforming growth factor-beta(2) (TGF-beta(2))- and oxidative stress-induced cellular changes in cultured human optic nerve head (ONH) astrocytes could be reduced by pretreatment with the antioxidant alpha-lipoic acid (LA). Methods: Cultured ONH astrocytes were treated with 1.0 ng/ml TGF-beta(2) for 24 h or 200 mu M hydrogen peroxide (H2O2) for 1 h. Lipid peroxidation was measured by a decrease in cis-pari-naric acid fluorescence. Additionally, cells were pretreated with different concentrations of LA before TGF-beta 2 or H2O2 exposure. Expressions of the heat shock protein (Hsp) alpha B-crystallin and Hsp27, the extracellular matrix (ECM) component fibronectin and the ECM-modulating protein connective tissue growth factor (CTGF) were examined with immunohistochemistry and real-time PCR analysis. Results: Both TGF-beta(2) and H2O2 increased lipid peroxidation. Treatment of astrocytes with TGF-beta(2) and H2O2 upregulated the expression of alpha B-crystallin, Hsp27, fibronectin and CTGF. Pretreatment with different concentrations of LA reduced the TGF-beta(2)- and H2O2-stimulated gene expressions. Conclusion: We showed that TGF-beta(2)- and H2O2-stimulated gene expressions could be prevented by pretreatment with the antioxidant LA in cultured human ONH astrocytes. Therefore, it is tempting to speculate that the use of antioxidants could have protective effects in glaucomatous optic neuropathy. Copyright (C) 2012 S. Karger AG, Base

Transient and sustained oxidative stress differentially activate the JNK1/2 pathway and apoptotic phenotype in H9c2 cells

The aim of this study was to investigate the activation of JNK1/2 signalling pathway and the respective cellular phenotype of H9c2 cardiac myoblasts during two distinct types of oxidative insult. We examined the dose- and time-dependent activation of JNK1/2 pathway by exogenous H2O2, both under transient and sustained stimulation. At 2 h of either sustained or transient treatment, maximal phosphorylation of c-Jun was observed, coincidently with the activation of nuclear JNK1/2; under sustained stress, these phosphorylation levels remained elevated above basal for up to 6 h, whereas under transient stress they declined to basal ones within 4 h of withdrawal. Furthermore, the JNK1/2 selective inhibitor SP600125 abolished the c-jun phosphorylation induced by oxidative stress. Our results using cell viability assays and light microscopy revealed that sustained H2O2 stimulation significantly and time-dependently decreased H9c2 viability, in contrast to transient stimulation; SP600125 (10 μM) abolished cell death induced by sustained as well as cell survival induced by transient oxidative stress. Hoechst staining showed an increase in DNA condensation during sustained, but not during transient stimulation. Moreover, from the antioxidants tested, catalase and superoxide dismutase prevented oxidative stress-induced cell death. Flow cytometry studies reconfirmed that sustained oxidative stress induced apoptosis, whereas transient resulted in the recovery of cardiac myoblasts within 24 h. We conclude that in H9c2 myoblasts, sustained activation of JNK1/2 signalling pathway during oxidative stimulation is followed by an apoptotic phenotype, while transient JNK1/2 activation correlates well with cell survival, suggesting a dual role of this signalling pathway in cell fate determination. © Springer Science+Business Media, LLC. 2007

Extracellular pH changes activate the p38-MAPK signalling pathway in the amphibian heart

We investigated the activation of the p38-MAPK signalling pathway during extracellular pH changes in the isolated perfused amphibian heart. Extracellular alkalosis (pH 8.5 or 9.5) maximally activated p38-MAPK within 2 min (4.17- and 3.20-fold, respectively) and this effect was reversible since the kinase phosphorylation levels decreased upon reperfusing the heart with normal Tris-Tyrode's buffer. Extracellular acidosis also activated p38-MAPK moderately, but persistently (1.65-fold, at 1 min and 1.91-fold, at 60 min). The alkalosis-induced p38-MAPK activation depended upon the Na+/H + exchanger (NHE) and Na+/K+-ATPase, because it was abolished when the NHE inhibitors amiloride and HOE642 and the Na +/K+-ATPase inhibitor, ouabain, were used. Our studies also showed that extracellular alkalosis (pH 8.5) induced MAPKAPK2 phosphorylation (2.59-fold, 2 min) and HSP27 phosphorylation (5.33-fold, 2 min) in a p38-MAPK-dependent manner, as it was inhibited with 1 μmol-1 SB203580. Furthermore, immunohistochemical studies of the phosphorylated forms of p38-MAPK and HSP27 revealed that these proteins were localised in the perinuclear region and dispersedly in the cytoplasm of ventricular cells during alkalosis. Finally, alkalosis induced the increase of HSP70 protein levels (1.52-fold, 5 min), but independently of p38-MAPK activation. These data indicate that the p38-MAPK signalling pathway is activated by extracellular pH changes and in the case of alkalosis this activation may have a protective role

Ventricular remodeling of single-chambered myh6 −/− adult zebrafish hearts occurs via a hyperplastic response and is accompanied by elastin deposition in the atrium

Zebrafish (Danio rerio) is widely used as an animal model to understand the pathophysiology of cardiovascular diseases. Here, we present the adult cardiac phenotype of weak atrium, myh6−/−, which carry mutations in the zebrafish atrial myosin heavy chain. Homozygous mutants survive to adulthood and are fertile despite their initial weak atrial beat. In adult mutants, the atrium remains hypoplastic and shows elastin deposition while mutant ventricles exhibit increased size. In mammals, hypertrophy is the most common mechanism resulting in cardiomegaly. Using immunohistochemistry and confocal microscopy to measure cardiomyocyte cell size, density and proliferation, we show that the enlargement of the myh6−/− ventricle is predominantly due to hyperplasia. However, we identified similar transcriptional profiles to the mammalian hypertrophy response via RT-PCR of the hyperplastic ventricles. Furthermore, we show activation of the ER-stress pathway by western blot analysis. In conclusion, we can assume, based on our model, that molecular signaling pathways associated with hypertrophy in mammals, in combination with ER-stress activation, result in hyperplasia in zebrafish. In addition, to our knowledge, this is the first time to report elastin deposition in the atrium. © 2019, Springer-Verlag GmbH Germany, part of Springer Nature

MAPK signaling pathways are needed for survival of H9c2 cardiac myoblasts under extracellular alkalosis

pH is one of the most important physiological parameters, with its changes affecting the function of vital organs like the heart. However, the effects of alkalosis on the regulation of cardiac myocyte function have not been extensively investigated. Therefore, we decided to study whether the mitogen-activated protein kinase (MAPK) signaling pathways [c-Jun NH 2-terminal kinases (JNKs), extracellular signal-regulated kinases (ERKs), and p38 MAPK] are activated by alkalosis induced with Tris-Tyrode buffer at two pH values, 8.5 and 9.5, in H9c2 rat cardiac myoblasts. These buffers also induced intracellular alkalinization comparable to that induced by 1 mM NH4Cl. The three MAPKs examined presented differential phosphorylation patterns that depended on the severity and the duration of the stimulus. Inhibition of Na+/H+ exchanger (NHE)1 by its inhibitor HOE-642 prevented alkalinization and partially attenuated the alkalosis (pH 8.5)-induced activation of these kinases. The same stimulus also promoted c-Jun phosphorylation and enhanced the binding at oligonucleotides bearing the activator protein-1 (AP-1) consensus sequence, all in a JNK-dependent manner. Additionally, mitogen- and stress-activated kinase 1 (MSK1) was transiently phosphorylated by alkalosis (pH 8.5), and this was abolished by the selective inhibitors of either p38 MAPK or ERK pathways. JNKs also mediated Bcl-2 phosphorylation in response to incubation with the alkaline medium (pH 8.5), while selective inhibitors of the three MAPKs diminished cell viability under these conditions. All these data suggest that alkalosis activates MAPKs in H9c2 cells and these kinases, in turn, modify proteins that regulate gene transcription and cell survival. Copyright © 2008 the American Physiological Society

The calpain-calpastatin system and the calcium paradox in the isolated perfused pigeon heart

To examine whether the calpain-callpastatin system is activated during the calcium paradox in the isolated perfused pigeon heart, we separated the protease from its inhibitor calpastatin and studied its kinetic properties. The protease exhibits kinetic properties similar to those of mammalian m-calpains. Ca2+ requirements for half and maximum activities are 220 muM and 2 mM, respectively. In the absence of Ca2+ the protease is strongly activated by Mn2+ or Sr2+. In the presence of Ca2+, Mn2+ and Sr2+ exhibit a synergistic effect; Mg2+ and Ba2+ have no effect, whereas Co2+, Ni2+ and Cd2+ completely inhibit its activation. Furthermore, we measured the activity of calpain and calpastatin under either conditions inducing a calcium paradox, or protecting the heart against this phenomenon. Although the calpain/calpastatin ratio is lowered during Ca2+ depletion, during Ca2+ repletion it is markedly inverted. Calpain activation during reperfusion is inhibited by the presence of 200 muM Mn2+ or Ba2+, in the Ca2+-free medium. Gel filtration of calpastatin, isolated from either untreated hearts or during Ca2+ depletion, produces two main peaks of similar to150 and 40 kDa of molecular mass, respectively, whereas calpastatin isolated during the 2(nd) min of reperfusion appears to be shifted to the 150 kDa form. All the above data suggest that this system may be involved in the induction of the calcium paradox in pigeon heart. Copyright (C) 2003 S. Karger AG, Basel