Vitamins C and E attenuate apoptosis, beta-adrenergic receptor desensitization, and sarcoplasmic reticular Ca2+ ATPase downregulation after myocardial infarction

Oxidative stress plays an important role in mediating ventricular remodeling and dysfunction in heart failure (HF), but its mechanism of action has not been fully elucidated. In this study we determined whether a combination of antioxidant vitamins reduced myocyte apoptosis, beta-adrenergic receptor desensitization, and sarcoplasmic reticular (SR) Ca2+ ATPase downregulation in HF after myocardial infarction (MI) and whether these effects were associated with amelioration of left ventricular (LV) remodeling and dysfunction. Vitamins (vitamin C 300 mg and vitamin E 300 mg) were administered to rabbits 1 week after MI or sham operation for 11 weeks. The results showed that MI rabbits exhibited cardiac dilation and LV dysfunction measured by fractional shortening and the maximal rate of pressure rise (dP/dt), an index of contractility. These changes were associated with elevation of oxidative stress, decreases of mitochondrial Bcl-2 and cytochrome c proteins, increases of cytosolic Bax and cytochrome c proteins, caspase 9 and caspase 3 activities and myocyte apoptosis, and downregulation of beta-adrenergic receptor sensitivity and SR Ca2+ ATPase. Combined treatment with vitamins C and E diminished oxidative stress, increased mitochondrial Bcl-2 protein, decreased cytosolic Bax, prevented cytochrome c release from mitochondria to cytosol, reduced caspase 9 and caspase 3 activities and myocyte apoptosis, blocked beta-adrenergic receptor desensitization and SR Ca2+ ATPase downregulation, and attenuated LV dilation and dysfunction in HF after MI. The results suggest that antioxidant therapy may be beneficial in HF.     

Inhibition of NADPH oxidase reduces myocardial oxidative stress and apoptosis and improves cardiac function in heart failure after myocardial infarction

Increases in NADPH oxidase activity, oxidative stress, and myocyte apoptosis coexist in failing hearts. In cardiac myocytes in vitro inhibition of NADPH oxidase reduces apoptosis. In this study, we tested the hypothesis that NADPH oxidase inhibition reduces myocyte apoptosis and improves cardiac function in heart failure after myocardial infarction (MI). Rabbits with heart failure induced by MI and sham-operated animals were randomized to orally receive apocynin, an inhibitor of NADPH oxidase (15 mg per day) or placebo for 4 weeks. Left ventricular (LV) dimension and function were assessed by echocardiography and hemodynamics. Myocardial NADPH oxidase activity was measured by superoxide dismutase-inhibitable cytochrome c reduction assay, NADPH oxidase subunit p47phox expression by Western blot and immunofluorescence analysis, myocardial oxidative stress evaluated by 8-hydroxydeoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal (4-HNE) using immunohistochemistry, and myocyte apoptosis by TUNEL assay. MI rabbits exhibited LV dilatation and systolic dysfunction measured by LV fractional shortening and the maximal rate of LV pressure rise (dP/dt). These changes were associated with increases in NADPH oxidase activity, p47phox protein expression, 8-OHdG expression, 4-HNE expression, myocyte apoptosis, and Bax protein and a decrease in Bcl-2 protein. Apocynin reduced NADPH oxidase activity, p47phox protein, oxidative stress, myocyte apoptosis, and Bax protein, increased Bcl-2 protein, and ameliorated LV dilatation and dysfunction after MI. The results suggest that inhibition of NADPH oxidase may represent an attractive therapeutic approach to treat heart failure.     

Distinctive ERK and p38 signaling in remote and infarcted myocardium during post‐MI remodeling in the mouse

Global activation of MAP kinases has been reported in both human and experimental heart failure. Chronic remodeling of the surviving ventricular wall after myocardial infarction (MI) involves both myocyte loss and fibrosis; we hypothesized that this cardiomyopathy involves differential shifts in pro‐ and anti‐apoptotic MAP kinase signaling in cardiac myocyte (CM) and non‐myocyte. Cardiomyopathy after coronary artery ligation in mice was characterized by echocardiography, ex vivo Langendorff preparation, histologic analysis and measurements of apoptosis. Phosphorylation (activation) of signaling molecules was analyzed by Western blot, ELISA and immunohistochemistry. Post‐MI remodeling involved dramatic changes in the phosphorylation of both stress‐activated MAP (SAP) kinase p38 as well as ERK, a known mediator of cell survival, but not of SAP kinase JNK or the anti‐apoptotic mediator of PI3K, Akt. Phosphorylation of p38 rose early after MI in the infarct, whereas a more gradual rise in the remote myocardium accompanied a rise in apoptosis in that region. In both areas, ERK phosphorylation was lowest early after MI and rose steadily thereafter, though infarct phosphorylation was consistently higher. Immunostaining of p‐ERK localized to fibrotic areas populated primarily by non‐myocytes, whereas staining of p38 phosphorylation was stronger in areas of progressive CM apoptosis. Relative segregation of CMs and non‐myocytes in different regions of the post‐MI myocardium revealed signaling patterns that imply cell type‐specific changes in pro‐ and anti‐apoptotic MAP kinase signaling. Prevention of myocyte loss and of LV remodeling after MI may therefore require cell type‐specific manipulation of p38 and ERK activation. J. Cell. Biochem. 109: 1185–1191, 2010. © 2010 Wiley‐Liss, Inc.     

Antioxidant therapy attenuates JNK activation and apoptosis in the remote noninfarcted myocardium after large myocardial infarction

We hypothesized that the concomitant occurrence of increased oxidative stress, JNK activation, and myocyte apoptosis in the remote myocardium (RM) following a large myocardial infarction (MI) are causally related. Three days following coronary ligation, rats were randomized to treatment with probucol and PDTC (MI-T) or vehicle (MI). Control rats (C) underwent sham operation. At 7 weeks, TBARS assay showed increased level of lipid-peroxidation within the RM in the MI group vs C, which was completely inhibited in the MI-T group. Similarly, Western blot analysis showed a twofold increase in p-JNK in the MI group, vs C, which was attenuated in MI-T, a result confirmed by a JNK-kinase activity. Furthermore, apoptosis was increased within the RM in MI vs C, while this was inhibited in MI-T. We conclude that long-term antioxidant therapy with probucol and PDTC attenuates oxidative stress, JNK activation, and myocyte apoptosis within the RM after large MI.     

Antioxidants attenuate myocyte apoptosis and improve cardiac function in CHF: association with changes in MAPK pathways

Antioxidant vitamins reduce cardiac oxidative stress and cardiomyocyte apoptosis produced by exogenous norepinephrine (NE) and attenuate cardiac dysfunction in animals with pacing-induced congestive heart failure (CHF). This study was carried out to determine whether the mitogen-activated protein kinase (MAPK) signal transduction pathways are involved in oxidative stress-induced myocyte apoptosis. Rabbits with rapid pacing-induced CHF and sham operation were randomized to receive either a combination of antioxidant vitamins (beta-carotene, ascorbic acid, and alpha-tocopherol), alpha-tocopherol alone, or placebo for 8 wk. Compared with sham-operated animals, CHF animals exhibited increased oxidative stress as evidenced by decreased myocardial reduced-to-oxidized glutathione (GSH/GSSG) ratio (27 +/- 7 vs. 143 +/- 24, P < 0.05), myocyte apoptosis (77 +/- 18 vs. 17 +/- 4 apoptotic nuclei/10,000 cardiomyocytes, P < 0.05), increased total and phosphorylated c-Jun NH2-terminal protein kinase (p-JNK; 1.95 +/- 0.14 vs. 1.04 +/- 0.04 arbitrary units, P < 0.05) and phosphorylated p38 kinase (p-p38), and decreased phosphorylated extracellular signal-regulated kinase (p-ERK). Administration of antioxidant vitamins and alpha-tocopherol attenuated oxidative stress, myocyte apoptosis, and cardiac dysfunction, with reversal of the changes of total JNK, p-JNK, and p-ERK in CHF. Furthermore, because NE infusion produced changes of JNK, p-p38, and p-ERK similar to those in CHF, we conclude that NE may play an important role in the production of oxidative stress, MAPK activation, and myocyte apoptosis in CHF.     

Extracellular superoxide dismutase protects the heart against oxidative stress and hypertrophy after myocardial infarction

Extracellular superoxide dismutase (EC-SOD) contributes only a small fraction to total SOD activity in the heart but is strategically located to scavenge free radicals in the extracellular compartment. EC-SOD expression is decreased in myocardial-infarction (MI)-induced heart failure, but whether EC-SOD can abrogate oxidative stress or modify MI-induced ventricular remodeling has not been previously studied. Consequently, the effects of EC-SOD gene deficiency (EC-SOD KO) on left ventricular (LV) oxidative stress, hypertrophy, and fibrosis were studied in EC-SOD KO and wild-type mice under control conditions, and at 4 and 8 weeks after permanent coronary artery ligation. EC-SOD KO had no detectable effect on LV function in normal hearts but caused small but significant increases of LV fibrosis. At 8 weeks after MI, EC-SOD KO mice developed significantly more LV hypertrophy (LV mass increased 1.64-fold in KO mice compared to 1.35-fold in wild-type mice; p<0.01) and more fibrosis and myocyte hypertrophy which was more prominent in the peri-infarct region than in the remote myocardium. EC-SOD KO mice had greater increases of nitrotyrosine in the peri-infarct myocardium, and this was associated with a greater reduction of LV ejection fraction, a greater decrease of sarcoplasmic or endoplasmic reticulum calcium2+ ATPase, and a greater increase of atrial natriuretic peptide in the peri-infarct zone compared to wild-type mice. EC-SOD KO was associated with more increases of phosphorylated p38 (p-p38(Thr180/Tyr182)), p42/44 extracellular signal-regulated kinase (p-Erk(Thr202/Tyr204)), and c-Jun N-terminal kinase (p-JNK(Thr183/Tyr185)) both under control conditions and after MI, indicating that EC-SOD KO increases activation of mitogen-activated protein kinase signaling pathways. These findings demonstrate that EC-SOD plays an important role in protecting the heart against oxidative stress and infarction-induced ventricular hypertrophy.     

Progressive left ventricular remodeling and apoptosis late after myocardial infarction in mouse heart

We tested the hypothesis that left ventricular (LV) remodeling late after myocardial infarction (MI) is associated with myocyte apoptosis in myocardium remote from the infarcted area and is related temporally to LV dilation and contractile dysfunction. One, four, and six months after MI caused by coronary artery ligation, LV volume and contractile function were determined using an isovolumic balloon-in-LV Langendorff technique. Apoptosis and nuclear morphology were determined by terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) and Hoechst 33258 staining. Progressive LV dilation 1-6 mo post-MI was associated with reduced peak LV developed pressure (LVDP). In myocardium remote from the infarct, there was increased wall thickness and expression of atrial natriuretic peptide mRNA consistent with reactive hypertrophy. There was a progressive increase in the number of TUNEL-positive myocytes from 1 to 6 mo post-MI (2.9-fold increase at 6 mo; P < 0. 001 vs. sham). Thus LV remodeling late post-MI is associated with increased apoptosis in myocardium remote from the area of ischemic injury. The frequency of apoptosis is related to the severity of LV dysfunction.     

Human endothelial nitric oxide synthase gene delivery protects against cardiac remodeling and reduces oxidative stress after myocardial infarction

Nitric oxide (NO) has been shown to play a key role in the regulation of cardiac hypertrophy and fibrosis in response to myocardial ischemia in part by antagonizing the action of angiotensin II (Ang II). In this study, we investigated the potential protective role of human endothelial nitric oxide synthase (eNOS) in left ventricular (LV) remodeling after myocardial infarction (MI) by a somatic gene transfer approach. Male Wistar rats underwent coronary artery ligation to induce MI. One week after surgery, adenovirus encoding the human eNOS or luciferase gene under the control of the CMV promoter/enhancer was injected into rats via the tail vein, and animals were sacrificed at 1 and 5 weeks after gene transfer. Successful gene transfer was evaluated based on increased levels of NO and cGMP in the heart, measured at one week after eNOS gene delivery. Six weeks after MI, the LV end-diastolic pressure, heart weight, LV axis length and cardiomyocyte size were markedly increased compared to the Sham group, while eNOS gene delivery significantly reduced these parameters. Rats receiving control virus developed considerably more fibrotic lesions identified by Sirius Red staining and collagen I immunostaining compared to Sham rats, and eNOS gene delivery significantly reduced collagen accumulation. eNOS gene transfer also reduced TUNEL-positive apoptotic cells. The cardioprotective effect of NO was accompanied by reduced NADH and NADPH oxidase activities and superoxide formation, TGF-beta1 and p27 levels, JNK activation, NF-kappa B nuclear translocation, and caspase-3 activity. This study shows that NO may play an important role in attenuating cardiac remodeling and apoptosis after myocardial infarction via suppression of oxidative stress-mediated signaling pathways.     

Susceptibility to systolic dysfunction in the myocardium from chronically infarcted spontaneously hypertensive rats

We explored whether the hypertensive heart is susceptible to myocardial dysfunction in viable noninfarcted tissue post-myocardial infarction (MI), the potential mechanisms thereof, and the impact of these changes on pump function. Six to seven months after the ligation of the left anterior descending coronary artery, left ventricular (LV) myocardial systolic function, as assessed from the percent shortening of the noninfarcted lateral wall segmental length determined over a range of filling pressures (ultrasonic transducers placed in the lateral wall in anaesthetized, open-chest, ventilated rats) and the percent thickening of the posterior wall (echocardiography), was reduced in infarcted spontaneous hypertensive rats (SHR-MI) (P < 0.05) but not in normotensive Wistar-Kyoto (WKY-MI) animals compared with corresponding controls [SHR-sham operations (Sham) and WKY-Sham]. This change in the regional myocardial function in SHR-MI, but not in WKY-MI, occurred despite a similar degree of LV dilatation (increased LV end-diastolic dimensions and volume intercept of the LV end-diastolic pressure-volume relation) in SHR-MI and WKY-MI rats and a lack of difference in LV relative wall thinning, LV wall stress, apoptosis [terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL)], or necrosis (pathological score) between SHR-MI and WKY-MI rats. Although the change in regional myocardial function in the SHR-MI group was not associated with a greater reduction in baseline global LV chamber systolic function [end-systolic elastance (LV E(es)) and endocardial fractional shortening determined in the absence of an adrenergic stimulus], in the presence of an isoproterenol challenge, noninfarct-zone LV systolic myocardial dysfunction manifested in a significant reduction in LV E(es) in SHR-MI compared with WKY-MI and SHR and WKY-Sham rats (P < 0.04). In conclusion, these data suggest that with chronic MI, the hypertensive heart is susceptible to the development of myocardial dysfunction, a change that cannot be attributed to excessive chamber dilatation, apoptosis, or necrosis, but which in turn contributes toward a reduced cardiac adrenergic inotropic reserve.     

心梗后心肌重构过程中AT_(1A)、AT_2受体表达的变化

为探讨AT1、AT2 受体在心肌重构演变过程中的作用 ,本实验应用免疫组化、电镜技术和图像分析方法 ,观察了大鼠心梗后心肌重构过程中非梗塞区AT1、AT2 受体表达的动态变化。结果显示 ,心梗术后 3d ,电镜显示非梗塞区心肌细胞肌原纤维横纹消失 ,线粒体肿胀 ,成纤维细胞增多 ,免疫组化显示AT1A受体在非梗塞区心肌组织表达明显升高 (P <0 0 0 1) ,AT2 受体表达无明显变化 (P >0 0 5 ) ;心梗术后 14d ,可见心肌细胞肌原纤维横纹 ,心肌细胞间胶原纤维明显增多 ,同时AT1A受体在心肌的表达比心梗术后 3d时减弱 ,但仍高于对照组 (P <0 0 5 ) ,AT2 受体表达明显增加 (P <0 0 0 1)。结果提示 :心梗后非梗塞区心肌AT1A、AT2 受体表达先后上调 ,可能参与介导心肌重构过程     

In vivo TNF-alpha inhibition ameliorates cardiac mitochondrial dysfunction, oxidative stress, and apoptosis in experimental heart failure

Heart failure is associated with increased myocardial expression of TNF-alpha. However, the role of TNF-alpha in the development of heart failure is not fully understood. In the present study, we investigated the contribution of TNF-alpha to myocardial mitochondrial dysfunction, oxidative stress, and apoptosis in a unique dog model of heart failure characterized by an activation of all of these pathological processes. Male mongrel dogs were randomly assigned (n = 10 each) to 1) normal controls; 2) chronic pacing (250 beats/min for 4 wk) with concomitant administration of etanercept, a soluble p75 TNF receptor fusion protein, 0.5 mg/kg subcutaneously twice weekly; 3) chronic pacing with administration of saline vehicle. Mitochondrial function was assessed by left ventricular (LV) tissue mitochondrial respiratory enzyme activities. Oxidative stress was assessed with aldehyde levels, and apoptosis was quantified by photometric enzyme immunoassay for cytoplasmic histone-associated DNA fragments and terminal deoxynucleotide transferase-mediated nick-end labeling (TUNEL) assays. LV activity levels of mitochondrial respiratory chain enzyme complex III and V were reduced in the saline-treated dogs and restored either partially (complex III) or completely (complex V) in the etanercept-treated dogs. Aldehyde levels, DNA fragments, and TUNEL-positive cells were increased in the saline-treated dogs and normalized in etanercept-treated dogs. These changes were accompanied by an attenuation of LV dilatation and partial restoration of ejection fraction. Our data demonstrate that TNF-alpha contributes to progressive LV dysfunction in pacing-induced heart failure, mediated in part by a local impairment in mitochondrial function and increase in oxidative stress and myocyte apoptosis.     

Activating Nrf-2 Signaling Depresses Unilateral Ureteral Obstruction-Evoked Mitochondrial Stress-Related Autophagy,Apoptosis and Pyroptosis in Kidney

Exacerbated oxidative stress and inflammation may induce three types of programmed cell death, autophagy, apoptosis and pyroptosis in unilateral ureteral obstruction (UUO) kidney. Sulforaphane activating NF-E2-related nuclear factor erythroid-2 (Nrf-2) signaling may ameliorate UUO-induced renal damage. UUO was induced in the left kidney of female Wistar rats. The level of renal blood flow, cortical and medullary oxygen tension and reactive oxygen species (ROS) was evaluated. Fibrosis, ED-1 (macrophage/monocyte) infiltration, oxidative stress, autophagy, apoptosis and pyroptosis were evaluated by immunohistochemistry and Western blot in UUO kidneys. Effects of sulforaphane, an Nrf-2 activator, on Nrf-2- and mitochondrial stress-related proteins and renal injury were examined. UUO decreased renal blood flow and oxygen tension and increased renal ROS, 3-nitrotyrosine stain, ED-1 infiltration and fibrosis. Enhanced renal tubular Beclin-1 expression started at 4 h UUO and further enhanced at 3d UUO, whereas increased Atg-5-Atg12 and LC3-II expression were found at 3d UUO. Increased renal Bax/Bcl-2 ratio, caspase 3 and PARP fragments, apoptosis formation associated with increased caspase 1 and IL-1β expression for pyroptosis formation were started from 3d UUO. UUO reduced nuclear Nrf-2 translocation, increased cytosolic and inhibitory Nrf-2 expression, increased cytosolic Bax translocation to mitochondrial and enhanced mitochondrial Cytochrome c release into cytosol of the UUO kidneys. Sulforaphane significantly increased nuclear Nrf-2 translocation and decreased mitochondrial Bax translocation and Cytochrome c release into cytosol resulting in decreased renal injury. In conclusion, sulforaphane via activating Nrf-2 signaling preserved mitochondrial function and suppressed UUO-induced renal oxidative stress, inflammation, fibrosis, autophagy, apoptosis and pyroptosis.     

Temporal and spatial characteristics of apoptosis in the infarcted rat heart

Following myocardial infarction (MI), tissue repair/remodeling occurs in both the infarcted and noninfarcted myocardium. Apoptosis has been demonstrated to play an important role in these processes. In the present study, we sought to determine the temporal and spatial characteristics of apoptosis in the infarcted heart as well as to identify cells undergoing programmed cell death at different stages of repair/remodeling and their relationship to the expression of anti-/pro-apoptotic genes following MI. Our study has shown that apoptosis appears in both infarcted and noninfarcted myocardium, and cells undergoing apoptosis depend on the stage of healing. In the infarcted myocardium, apoptosis contributes to the loss of cardiomyocytes during the early stage of healing, elimination of inflammatory cells during the inflammatory phase of healing, and reduction of myofibroblasts with the fibrogenic phase of repair in the infarcted myocardium. In noninfarcted myocardium, cardiomyocyte apoptosis was observed from day 3 to 28 postMI. Cardiac apoptosis following MI is correlated with the increase of Bax expression.     

Cardiac myocyte apoptosis provokes adverse cardiac remodeling in transgenic mice with targeted TNF overexpression

Although cardiac myocyte apoptosis has been detected in explanted hearts from patients with end-stage dilated and ischemic cardiomyopathy, the relative contribution of apoptotic cell death to left ventricular (LV) remodeling and cardiac decompensation is not known. To determine whether progressive cardiac myocyte apoptosis contributes to the transition from a hypertrophic to a dilated cardiac phenotype that is observed in transgenic myosin heavy chain secreted TNF (MHCsTNF) mice with cardiac restricted overexpression of tumor necrosis factor (TNF), we assessed cardiac myocyte apoptosis (using a DNA ligase technique) in MHCsTNF mice and littermate control mice in relation to serial changes in LV structure, which was assessed using MRI. The prevalence of cardiac myocyte apoptosis increased progressively from 4 to 12 wk as the hearts of the MHCsTNF mice underwent the transition from a concentric hypertrophic to a dilated cardiac phenotype. Treatment of the MHCsTNF mice with the broad-based caspase inhibitor N-[(1,3-dimethylindole-2-carbonyl)-valinyl]-3-amino4-oxo-5-fluoropentanoic acid significantly decreased cardiac myocyte apoptosis and significantly attenuated LV wall thinning and adverse cardiac remodeling. Additional studies suggested that the TNF-induced decrease in Bcl-2 expression and activation of the intrinsic mitochondrial death pathway were responsible for the cardiac myocyte apoptosis observed in the MHCsTNF mice. These studies show that progressive cardiac myocyte apoptosis is sufficient to contribute to adverse cardiac remodeling in the adult mammalian heart through progressive LV wall thinning.     

Oleate prevents palmitate-induced cytotoxic stress in cardiac myocytes

The cytotoxicity of saturated fatty acids has been implicated in the pathophysiology of cardiovascular disease, though their effects on cardiac myocytes are incompletely understood. We examined the effects of palmitate and the mono-unsaturated fatty acid oleate on neonatal rat ventricular myocyte cell biology. Palmitate (0.5mM) increased oxidative stress, as well as activation of the stress-associated protein kinases (SAPK) p38, Erk1/2, and JNK, following 18h and induced apoptosis in approximately 20% of cells after 24h. Neither antioxidants nor SAPK inhibitors prevented palmitate-induced apoptosis. Low concentrations of oleate (0.1mM) completely inhibited palmitate-induced oxidative stress, SAPK activation, and apoptosis. Increasing mitochondrial uptake of palmitate with l-carnitine decreased apoptosis, while decreasing uptake with the carnitine palmitoyl transferase-1 inhibitor perhexiline nearly doubled palmitate-induced apoptosis. These results support a model for palmitate-induced apoptosis, activation of SAPKs, and protein oxidative stress in myocytes that involves cytosolic accumulation of saturated fatty acids.     

Osteopontin-stimulated apoptosis in cardiac myocytes involves oxidative stress and mitochondrial death pathway: role of a pro-apoptotic protein BIK

Increased osteopontin (OPN) expression in the heart, specifically in myocytes, associates with increased myocyte apoptosis and myocardial dysfunction. Recently, we provided evidence that OPN interacts with CD44 receptor, and induces myocyte apoptosis via the involvement of endoplasmic reticulum stress and mitochondrial death pathways. Here we tested the hypothesis that OPN induces oxidative stress in myocytes and the heart via the involvement of mitochondria and NADPH oxidase-4 (NOX-4). Treatment of adult rat ventricular myocytes (ARVMs) with OPN (20 nM) increased oxidative stress as analyzed by protein carbonylation, and intracellular reactive oxygen species (ROS) levels as analyzed by ROS detection kit and dichlorohydrofluorescein diacetate staining. Pretreatment with NAC (antioxidant), apocynin (NOX inhibitor), MnTBAP (superoxide dismutase mimetic), and mitochondrial K_ATP channel blockers (glibenclamide and 5-hydroxydecanoate) decreased OPN-stimulated ROS production, cytosolic cytochrome c levels, and apoptosis. OPN increased NOX-4 expression, while decreasing SOD-2 expression. OPN decreased mitochondrial membrane potential as measured by JC-1 staining, and induced mitochondrial abnormalities including swelling and reorganization of cristae as observed using transmission electron microscopy. OPN increased expression of BIK, a pro-apoptotic protein involved in reorganization of mitochondrial cristae. Expression of dominant-negative BIK decreased OPN-stimulated apoptosis. In vivo, OPN expression in cardiac myocyte-specific manner associated with increased protein carbonylation, and expression of NOX-4 and BIK. Thus, OPN induces oxidative stress via the involvement of mitochondria and NOX-4. It may affect mitochondrial morphology and integrity, at least in part, via the involvement of BIK.

     

Apoptosis induced by domoic acid in mouse cerebellar granule neurons involves activation of p38 and JNK MAP kinases

In mouse cerebellar granule neurons (CGNs) the marine neurotoxin domoic acid (DomA) induces neuronal cell death, either by apoptosis or by necrosis, depending on its concentration, with apoptotic damage predominating in response to low concentrations (100 nM). DomA-induced apoptosis is due to selective activation of AMPA/kainate receptors, and is mediated by DomA-induced oxidative stress, leading to mitochondrial dysfunction and activation of caspase-3. The p38 MAP kinase and the c-Jun NH2-terminal protein kinase (JNK) have been shown to be preferentially activated by oxidative stress. Here we report that DomA increases p38 MAP kinase and JNK phosphorylation, and that this effect is more pronounced in CGNs from Gclm (-/-) mice, which lack the modifier subunit of glutamate-cysteine ligase, have very low glutathione (GSH) levels, and are more sensitive to DomA-induced apoptosis than CGNs from wild-type mice. The increased phosphorylation of JNK and p38 kinase was paralleled by a decreased phosphorylation of Erk 1/2. The AMPA/kainate receptor antagonist NBQX, but not the NMDA receptor antagonist MK-801, prevents DomA-induced activation of p38 and JNK kinases. Several antioxidants (GSH ethyl ester, catalase and phenylbutylnitrone) also prevent DomA-induced phosphorylation of JNK and p38 MAP kinases. Inhibitors of p38 (SB203580) and of JNK (SP600125) antagonize DomA-induced apoptosis. These results indicate the importance of oxidative stress-activated JNK and p38 MAP kinase pathways in DomA-induced apoptosis in CGNs.     

Regulation of BAD protein by PKA, PKCdelta and phosphatases in adult rat cardiac myocytes subjected to oxidative stress

H2O2, as an example of oxidative stress, induces cardiac myocyte apoptosis. Bcl-2 family proteins are key regulators of the apoptotic response while their functions can be regulated by post-translational modifications including phosphorylation, dimerization or proteolytic cleavage. In this study, we examined the role of various protein kinases in regulating total BAD protein levels in adult rat cardiac myocytes undergoing apoptosis. Stimulation with 0.1 mM H2O2, which induces apoptosis, resulted in a marked down-regulation of BAD protein, which is attributed to cleavage by caspases since it can be restored in the presence of a general caspase inhibitor. Inhibition of PKC, p38-MAPK, ERK1/2 and PI-3-K did not influence the reduced BAD protein levels observed after stimulation with H2O2. On the contrary, inhibition of PKA or specifically PKCdelta resulted in up-regulation of BAD. Decreased caspase 3 activity was observed in H2O2 treated cells after inhibition of PKA or PKCdelta whereas inhibition of PKA also resulted in improved cell survival. Furthermore, addition of okadaic acid to inhibit selected phosphatases resulted in enhanced BAD cleavage. These data suggest that, during oxidative stress-induced cardiac myocyte apoptosis, there is a caspase-dependent down-regulation of BAD protein, which seems to be regulated by coordinated action of PKA, PKCdelta and phosphatases.