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Reactive Oxygen Species (ROS) are generated as by-products of aerobic metabolism. The production of ROS increases during xenobiotic stress and under multiple pathological conditions. Although ROS are considered harmful historically, mounting evidence recently indicates a signaling function of ROS, preceding to and regulating transcriptional or post-transcriptional events, contributing to cell death or cell survival and adaptation. Among the cellular defense mechanisms activated by ROS is formation of stress granules (SGs). The stalled translational apparatus, together with mRNA, aggregates into microscopically detectable and molecularly dynamic granules. We found that with H2O2, the dose most potent for inducing SGs in HeLa cells is 400–600 μM. With 200 μM H2O2, 2 h treatment induced the highest percentage of cells containing SGs. Whether ROS signaling pathways regulate the formation of SGs was tested using pharmacological inhibitors. We probed the potential role of PI3K, MAPKs, PKC or histone deacetylation in SG formation. Using deferoxamine as a positive control, we found a lack of inhibitory effect of wortmannin, LY-294002, JNK-I, SB-202190, PD-98059, or H89 when the percentage of cells containing SGs was counted. About 35% inhibition was observed with HDAC6 inhibitor Tubastatin A, whereas general HDAC inhibitor Trichostatin A provided a complete inhibition of SG formation. Our data point to the need of investigating the role of HDACs in SG formation during oxidative stress.

Coronary artery reperfusion is essential for the management of symptoms in the patients with myocardial ischemia. However, the benefit of reperfusion often comes at an expense of paradoxical injury, which contributes to the adverse events, and sometimes heart failure. Reperfusion is known to increase the production of reactive oxygen species (ROS). We address whether N-acetylcysteine (NAC) reduces the ROS and alleviates reperfusion injury by improving the clinical outcomes. A literature search for the randomized controlled trials (RCTs) was carried out in the five biomedical databases for testing the effects of NAC in patients undergoing coronary artery reperfusion by percutaneous coronary intervention, thrombolysis, or coronary artery bypass graft. Of 787 publications reviewed, 28 RCTs were identified, with a summary of 2,174 patients. A meta-analysis using the random effects model indicated that NAC administration during or prior to the reperfusion procedures resulted in a trend toward a reduction in the level of serum cardiac troponin (cTn) [95% CI, standardized mean difference (SMD) −0.80 (−1.75; 0.15), p = 0.088, n = 262 for control, 277 for NAC group], and in the incidence of postoperative atrial fibrillation [95% CI, relative risk (RR) 0.57 (0.30; 1.06), p = 0.071, n = 484 for control, 490 for NAC group]. The left ventricular ejection fraction or the measures of length of stay in intensive care unit (ICU) or in hospital displayed a positive trend that was not statistically significant. Among the nine trials that measured ROS, seven showed a correlation between the reduction of lipid peroxidation and improved clinical outcomes. These lines of evidence support the potential benefit of NAC as an adjuvant therapy for cardiac protection against reperfusion injury.

Myocardial ischemia or reperfusion increases the generation of reactive oxygen species (ROS) from damaged mitochondria, NADPH oxidases, xanthine oxidase, and inflammation. ROS can be removed by eight endogenous antioxidant and redox systems, many components of which are expressed under the influence of the activated Nrf2 transcription factor. Transcriptomic profiling, sequencing of Nrf2-bound DNA, and Nrf2 gene knockout studies have revealed the power of Nrf2 beyond the antioxidant and detoxification response, from tissue recovery, repair, and remodeling, mitochondrial turnover, and metabolic reprogramming to the suppression of proinflammatory cytokines. Multifaceted regulatory mechanisms for Nrf2 protein levels or activity have been mapped to its functional domains, Nrf2-ECH homology (Neh)1–7. Oxidative stress activates Nrf2 via nuclear translocation, de novo protein translation, and increased protein stability due to removal of the Kelch-like ECH-associated protein 1 (Keap1) checkpoint, or the inactivation of β-transducin repeat-containing protein (β-TrCP), or Hmg-CoA reductase degradation protein 1 (Hrd1). The promise of small-molecule Nrf2 inducers from natural products or derivatives is discussed here. Experimental evidence is presented to support Nrf2 as a lead target for drug development to further improve the treatment outcome for myocardial infarction (MI).

Background Myocardial infarction with non-obstructive coronary arteries (MINOCA) remains an unresolved challenge. Many different diagnostic approaches are often required to diagnose, confirm, and evaluate MINOCA. The prevalence can be as high as 13 % of all acute MI patients indicating that this condition is not rare. At this time, there have been no completed randomized clinical trials involving MINOCA patients, and a better understanding of the mechanisms and management of these patients is important. This exploratory analysis seeks to find possible etiologic factors, the value of novel biomarkers, and the effect of different treatment strategies in patients with MINOCA. Methods This prospective randomized pilot trial will include 150 patients with MINOCA. A thorough clinical, laboratory and imaging evaluation will be performed including novel biomarkers and modern imaging techniques (heart magnetic resonance imaging and noninvasive testing). The duration of the enrollment is 18 months, duration of the follow up is 12 months from the enrollment of the first patient. Results The trial is registered under www.clinicaltrials.gov: NCT04538924. The study is currently recruiting participants. Conclusions Since MINOCA is not a benign disease, the results of the current investigation could inform future diagnostic and therapeutic strategies and enhance the understanding of MINOCA patients.

Percutaneous coronary intervention (PCI) is the preferred treatment for acute coronary syndrome (ACS) secondary to atherosclerotic coronary artery disease. This nonsurgical procedure is also used for selective patients with stable angina. Although the procedure is essential for restoring blood flow, reperfusion can increase oxidative stress as a side effect. We address whether intravenous infusion of vitamin C (VC) prior to PCI provides a benefit for cardioprotection. A total of eight randomized controlled trials (RCT) reported in the literature were selected from 371 publications through systematic literature searches in six electronic databases. The data of VC effect on cardiac injury biomarkers and cardiac function were extracted from these trials adding up to a total of 1185 patients. VC administration reduced cardiac injury as measured by troponin and CK-MB elevations, along with increased antioxidant reservoir, reduced reactive oxygen species (ROS) and decreased inflammatory markers. Improvement of the left ventricular ejection fraction (LVEF) and telediastolic left ventricular volume (TLVV) showed a trend but inconclusive association with VC. Intravenous infusion of VC before PCI may serve as an effective method for cardioprotection against reperfusion injury.

Statins are among the most commonly prescribed drugs for the treatment of high blood cholesterol. Myotoxicity of statins in certain individuals is often a severe side effect leading to withdrawal. Using C2C12 and H9c2 cells, both exhibiting characteristics of skeletal muscle cells, we addressed whether resveratrol (RSV) can prevent statin toxicity. Statins decreased cell viability in a dose and time-dependent manner. Among the five statins tested, atorvastatin, simvastatin, lovastatin, pravastatin, and fluvastatin, simvastatin is the most toxic one. Simvastatin at 10 µM caused about 65% loss of metabolic activity as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays in C2C12 cells or H9c2 cells. Inhibition of metabolic activity correlates with an increase in caspase activity. RSV was found to protect H9c2 cells from simvastatin-induced activation of caspase-3/7. However, such protection was not found in C2C12 cells. This cell type-dependent effect of RSV adds to the complexity in muscle cell toxicity of statins.

Nrf2 gene encodes a transcription factor regulating the expression of antioxidant and detoxification genes. We test here whether Nrf2 plays a role for cardiac protection during ischemic injury in an effort to establish Nrf2 as a target for cardiac protection therapies. Cardiac ischemia induced by the left anterior descending (LAD) coronary artery ligation results in myocardial infarction (MI). Young mice surviving MI show minimal signs of heart failure. Mice lacking Nrf2 experience an accelerated progression to heart failure that occurs within 10days following induction of MI. Nrf2 knockout (Nrf2 KO) mice have a survival rate similar to wild type (WT) mice at 24h after MI, but a significantly higher mortality rate within 10days after MI (50% vs 86%). Morphological examination revealed maladaptive remodeling, including cardiac hypertrophy and dilated left ventricle in Nrf2 KO mice, which were absent in WT mice. Measurements of cardiac function revealed increased left ventricular mass and decreases in cardiac output in Nrf2 KO mice. In addition, Nrf2 KO mice show biomarkers of heart failure, such as elevated levels of β-MHC, ANF, and BNP mRNA in the myocardium. These data support that Nrf2 plays an important role in protecting the myocardium from ischemic injury. Lack of Nrf2 leads to rapid development of heart failure.

INTRODUCTION: The objective of this study was to investigate the cardioprotective effects of a dodecafluoropentane (DDFP)-based perfluorocarbon emulsion (DDFPe) as an artificial carrier for oxygen delivery to ischemic myocardium, using (99m)Tc-duramycin SPECT imaging. METHODS: Rat hearts with Ischemia-reperfusion (I/R) was prepared by coronary ligation for 45-min followed by reperfusion. The feasibility of (99m)Tc-duramycin in detecting myocardial I/R injury and its kinetic profile were first verified in the ischemic hearts with 2-h reperfusion (n = 6). DDFPe (0.6 mL/kg) was intravenously administered at 10 min after coronary ligation in fifteen rats and saline was given in thirteen rats as controls. (99m)Tc-duramycin SPECT images were acquired in the DDFPe-treated hearts and saline controls at 2-h (DDFPe-2 h, n = 7 and Saline-2 h, n = 6) or 24-h (DDFPe-24 h, n = 8 and Saline-24 h, n = 7) of reperfusion. RESULTS: SPECT images, showing “hot-spot” (99m)Tc-duramycin uptake in the ischemic myocardium, exhibited significantly lower radioactive retention and smaller hot-spot size in the DDFPe-2 h and DDFPe-24 h hearts compared to controls. The infarcts in the Saline-24 h hearts extended significantly relative to measurements in the Saline-2 h. The extension of infarct size did not reach a statistical difference between the DDFPe-2 h and DDFPe-24 h hearts. Ex vivo measurement of (99m)Tc-duramycin activity (%ID/g) was lower in the ischemic area of DDFPe-2 h and DDFPe-24 h than that of the Saline-2 h and Saline-24 h hearts (P < 0.05). The area of injured myocardium, delineated by the uptake of (99m)Tc-duramycin, extended more substantially outside the infarct zone in the controls. CONCLUSIONS: Significant reduction in myocardial I/R injury, as assessed by (99m)Tc-duramycin cell death imaging and histopathological analysis, was induced by DDFPe treatment after acute myocardial ischemia. (99m)Tc-duramycin imaging can reveal myocardial cell death in ischemic hearts and may provide a tool for the non-invasive assessment of cardioprotective interventions.

Corticosterone (CT), progesterone (PG), and retinoic acid (RA) are capable of inhibiting Doxorubicin (Dox) from inducing apoptosis in rat cardiomyocytes. Mechanistically, CT, PG, and RA induce increases of Bcl-xL protein and mRNA, and activate a 3.2 kb bcl-x gene promoter. CT and RA, but not PG, induced the activity of a 0.9 kb bcl-x promoter, containing sequences for AP-1 and NF-kB binding. RA, but not CT or PG, induced NF-kB activation. CT, but not PG or RA, induced AP-1 activation, and induction of the 0.9 kb bcl-x reporter by CT was inhibited by dominant negative c-Jun TAM-67 or removal of AP-1 binding site. Therefore, although CT, PG, and RA all induce Bcl-xL mRNA and protein, three independent mechanisms are in operation: while CT induces Bcl-xL via AP-1 transcription factor, and RA induces NF-kB activation and bcl-x promoter activity, PG induces Bcl-xL via a mechanism independent of NF-kB or AP-1.

Sublethal levels of oxidative stress are commonly associated with various pathophysiological conditions. Cardiomyocytes have the highest content of mitochondria among all cell types, allowing the study of mitochondria in cells surviving oxidative stress and address whether nuclear factor–erythroid-derived 2–related factor 2 (Nrf2) can reverse these changes. Mitochondria normally exist in elaborated networks, which were replaced by predominately individual punctuate mitochondria 24 h after exposure to a nonlethal dose of H2O2. Electron microscopy revealed that cells surviving H2O2 show swelling of mitochondria with disorganized cristae and areas of condensation. Measurements of functional mitochondria showed a H2O2 dose-dependent decrease over a course of 5 d. At the protein and mRNA levels, cells surviving H2O2 treatment show a reduction of mitochondrial components, cytochrome c, and cytochrome b. Nrf2 overexpression prevented H2O2 from inducing mitochondria morphologic changes and reduction of cytochrome b/c. Although Nrf2 is known as a transcription factor regulating antioxidant and detoxification genes, Nrf2 overexpression did not significantly reduce the level of protein oxidation. Instead, Nrf2 was found to associate with the outer mitochondrial membrane. Mitochondria prepared from the myocardium of Nrf2 knockout mice are more sensitive to permeability transition. Our data suggest that Nrf2 protects mitochondria from oxidant injury likely through direct interaction with mitochondria.—Strom, J., Xu, B., Tian, X., Chen, Q. M. Nrf2 protects mitochondrial decay by oxidative stress.

Xiuqing Tian,1 Qing Wang,1 Rui Guo,2 Lingling Xu,3 Qin M Chen,4 Yinglong Hou1 1Department of Cardiology, Qianfoshan Hospital of Shandong University, Jinan City, 2Department of Physiology, Nanjing University of Chinese Medicine, Nanjing City, 3College of Medicine, Weifang Medical University, Weifang City, People’s Republic of China; 4Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ, USA Background: Paroxetine is a selective serotonin reuptake inhibitor utilized in the treatment of depression and anxiety disorders. Recent studies have identified paroxetine as a G protein-coupled receptor kinase-2 (GRK2) inhibitor capable of reversing cardiac dysfunction and remodeling in experimental models of acute myocardial infarction (AMI). We determine the clinical importance of paroxetine on cardiac functions in patients having AMI with depression (AMID) in comparison with fluoxetine, an unrelated selective serotonin reuptake inhibitor that does not inhibit GRK2.Methods: Diagnosis of depression was based on the 17-item Hamilton Depression Scale and Self-rating Depression Scale in AMI patients after hospital admission. AMID patients were randomly assigned to paroxetine or fluoxetine for treatment of depression. Heart rate variability and cardiac function were evaluated. GRK2 protein levels were measured using peripheral lymphocytes and Western blot.Results: GRK2 expression in AMID patients was significantly higher than that in AMI patients without depression. In AMID patients, GRK2 levels were positively correlated with the 17-item Hamilton Depression Scale and the Self-rating Depression Scale scores, and negatively correlated with heart rate variability. Treatment of AMID patients with paroxetine significantly reduced the expression of GRK2, normalized the autonomic nervous system function, and improved cardiac performance. In contrast, fluoxetine normalized the autonomic nervous system but did not reduce the expression of GRK2 nor improved cardiac performance.Conclusion: This study suggests that paroxetine is effective for improving cardiac function in patients with AMID and such effect correlates with GRK2 reduction. Keywords: selective serotonin reuptake inhibitors, heart rate variability, G protein-coupled receptor kinase-2, acute myocardial infarction, depression

Psychological or physical stress causes an elevation of glucocorticoids in the circulating system. Glucocorticoids regulate a variety of physiological functions, from energy metabolism and biochemical homeostasis to immune response. Synthetic steroids are among the most prescribed drugs for immune suppression and chemotherapy. While glucocorticoids are best known for inducing apoptosis in a number of cell types, we have found that corticosteroids at stress relevant levels protect cardiomyocytes from apoptosis. Current study addresses whether glucocorticoids inhibit cardiac injury in vivo. Adult male C57BL6 mice were administered with dexamethasone (20mg/kg, i.p.) or vehicle control 20 h prior to left anterior descending coronary artery occlusion surgery. Myocardial infarction was measured by triphenyl tetrazoliumchloride staining in tissue slices and by levels of cardiac Troponin (cTn I) in the blood. Treatment of dexamethasone markedly reduced infarct size (19.6 ± 4.3%, vs. 29.2 ± 4.9%, p

NAD(P)H: quinone oxidoreductase 1 (NQO1) is a ubiquitous flavoenzyme that catalyzes two-electron reduction of various quinones by utilizing NAD(P)H as an electron donor. Our previous study found that progesterone (PG) can protect cardiomyocytes from apoptosis induced by doxorubicin (Dox). Microarray analyses of genes induced by PG had led to the discovery of induction of NQO1 mRNA. We report here that PG induces NQO1 protein and its activity in a dose-dependent manner. Whereas NQO1 is well known as a target gene of Nrf2 transcription factor due to the presence of antioxidant response element (ARE) in the promoter, PG did not activate the ARE, suggesting Nrf2-independent induction of NQO1. To address the role of NQO1 induction in PG-induced cytoprotection, we tested the effect of NQO1 inducer β-naphthoflavone and inhibitor dicoumarol. Induction of NQO1 by β-naphthoflavone decreased Dox-induced apoptosis and potentiated the protective effect of PG as measured by caspase-3 activity. PG-induced NQO1 activity was inhibited with dicoumarol, which did not affect PG-induced cytoprotection. Dicoumarol treatment alone potentiated Dox-induced caspase-3 activity. These data suggest that while NQO1 plays a role in PG-induced cytoprotection, there are additional components contributing to PG-induced cytoprotection.

While gender-based differences in heart disease have raised the possibility that estrogen (ES) or progesterone (PG) may have cardioprotective effects, recent controversy regarding hormone replacement therapy has questioned the cardiac effects of these steroids. Using cardiomyocytes, we tested whether ES or PG has protective effects at the cellular level. We found that PG but not ES protects cardiomyocytes from apoptotic cell death induced by doxorubicin (Dox). PG inhibited apoptosis in a dose dependent manner, by 12 ± 4.0% at 1 μM and 60 ± 1.0 % at 10 μM. The anti-apoptotic effect of PG was also time dependent, causing 18 ± 5% or 62 + 2% decrease in caspase-3 activity within 1 or 72 hours of pretreatment. While PG causes nuclear translocation of its receptor within 20 mins, the cytoprotective effect of PG was cancelled by mifepristone (MF), a PG receptor antagonist. Analyses using Affymetrix high-density oligonucleotide array and RT-PCR found that PG induced Bcl-xL, metallothionine, NADPH quinone oxidoreductase 1, glutathione peroxidase-3, and 4 isoforms of glutathione S-transferase. Western blot analyses revealed that PG indeed induced an elevation of Bcl-xL protein in a dose and time dependent manner. Nuclear run-on assay indicated that PG induced Bcl-xL gene transcription. Inhibiting the expression of Bcl-xL using siRNA reduced the cytoprotective effect of PG. Our data suggests that PG induces a cytoprotective effect in cardiomyocytes in association with induction of Bcl-xL gene.

Glucocorticoids induce COX-2 expression in rat cardiomyocytes. While investigating whether phosphatidylinositol 3 kinase (PI3K) plays a role in corticosterone (CT)-induced COX-2, we found that LY294002 (LY29) but not wortmannin (WM) attenuates CT from inducing COX-2 gene expression. Expression of a dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K-independent mechanisms in regulating CT-induced COX-2 expression. LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), and DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca(2+) concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT-induced COX-2 expression in cardiomyocytes.

Psychological stress increases the level of glucocorticoids in the circulating system. We found that dexamethasone administration in adult mice elevates the expression of COX-2 in the myocardium. With isolated neonatal cardiomyocytes, corticosterone (CT) at physiologically relevant doses (0.01-1 microM) induces the expression of COX-2 gene. The induction first appeared at 4 h and remained for at least 24 h with 1 microM CT treatment. This response is likely cardiomyocyte cell type specific since CT did not induce COX-2 expression in cardiac fibroblasts and glucocorticoids are known to suppress the expression of COX-2 in lymphocytes and several organs. Corticosteroids, but not estrogen or progesterone, induce COX-2 expression. The glucocorticoid receptor (GR) antagonist mifepristone (MF) prevented CT from inducing COX-2 gene, suggesting a GR-dependent induction in cardiomyocytes. COX-2 gene promoter deletion and mutation studies indicate a role of CCAAT/enhancer binding protein-beta (C/EBP-beta) in CT-induced COX-2 gene expression. Chromatin immunoprecipitation assays revealed that CT caused the binding of both GR and C/EBP-beta to COX-2 promoter, while MF pretreatment blocked such binding. Coimmunoprecipitation experiments demonstrated that CT treatment induced the interaction of GR with C/EBP-beta. Small interfering RNA against C/EBP-beta prevented CT from activating COX-2 promoter or elevating COX-2 protein. Our data suggest that the interaction between GR and C/EBP-beta contributes to elevated COX-2 gene transcription by CT in cardiomyocytes.

Chronic exposure to arsenic has been linked to tumorigenesis, cardiovascular disease, hypertension, atherosclerosis, and peripheral vascular disease; however, the molecular mechanisms underlying its pathological effects remain elusive. In this study, we investigated arsenic-induced alteration of focal adhesion protein complexes in normal, primary vascular smooth muscle cells. We demonstrate that exposure to environmentally relevant concentrations of arsenic (50 ppb As 3+ ) can alter focal adhesion protein co-association leading to activation of downstream pathways. Co-associated proteins were identified and quantitated via co-immunoprecipitation, SDS-PAGE, and Western blot analysis followed by scanning densitometry. Activation of MAPK pathways in total cell lysates was evaluated using phosphor-specific antibodies. In our model, arsenic treatment caused a sustained increase in FAK–src association and activation, and induced the formation of unique signaling complexes (beginning after 3-hour As 3+ exposure and continuing throughout the 12-hour time course studied). The effects of these alterations were manifested as chronic stimulation of downstream PAK, ERK and JNK pathways. Past studies have demonstrated that these pathways are involved in cellular survival, growth, proliferation, and migration in VSMCs.

FP prostanoid receptors are G-protein-coupled receptors whose physiological activator is prostaglandin-F(2alpha) (PGF(2alpha)). PGF(2alpha) has been implicated in wound healing and cardiac hypertrophy, which are both known to involve the induction of the immediate-early response gene, early growth response factor-1 (EGR-1). We hypothesized that activation of the human FP receptor by PGF(2alpha) could induce the expression of EGR-1 and found that 1 muM PGF(2alpha) produced a time-dependent induction of both mRNA and protein expression for EGR-1. This FP receptor-mediated induction of EGR-1 expression involved activation of the small GTPase Ras followed by activation of C-Raf and the mitogen-activated protein (MAP) kinase kinases 1 and 2 (MEK1/2). Thus, induction of EGR-1 expression by PGF(2alpha) was blocked using dominant-negative constructs of Ras and C-Raf and the Raf kinase inhibitor 4-(4-(3-(4-chloro-3-trifluoromethylphenyl)ureido)phenoxy)-pyridine-2-carboxyllic acid methyamide-4-methylbenzenesulfonate (BAY43-9006). Likewise, the MEK1/2 inhibitor 2'-amino-3'-methoxyflavone (PD98059) blocked the induction of EGR-1 expression by PGF(2alpha). FP receptor stimulation by PGF(2alpha) induced the phosphorylation of C-Raf, MEK1/2, and extracellular signal-regulated kinases 1 and 2, consistent with the activation of a MAP kinase signaling cascade. PGF(2alpha) was also found to induce the expression of EGR-1 in rat cardiomyocytes through the activation of endogenous FP receptors. This induction of EGR-1 expression in cardiomyocytes also involved the activation of Raf and MAP kinase signaling and was dependent on the activation of protein kinase C. This is the first report to show the regulation of EGR-1 expression after PGF(2alpha) activation of FP receptors and suggests that this could be an early event involved in wound healing and cardiac hypertrophy.

Normal human diploid fibroblasts (HDFs) undergo replicative senescence inevitably in tissue culture after a certain number of cell divisions. A number of molecular changes observed in replicative senescent cells occur in somatic cells during the process of aging. Genetic studies on replicative senescence indicate the control of tumor suppression mechanisms. Despite the significance of replicative senescence in aging and cancer, little is known about the central cause of the complex changes observed in replicative senescent cells. The interest in the phenomenon has intensified in recent years, since damaging agents, certain oncogenes and tumor suppressor genes have been found to induce features of senescence in early passage young HDFs or in immortalized tumor cells. The reported features of senescence are summarized here in order to clarify the concept of replicative senescence or premature senescence. The experimental results of extending the replicative life span by reducing ambient oxygen tension or by N-tert-butyl-alpha-phenylnitrone (PBN) argue a role of oxidative damage in replicative senescence. By inducing premature senescence with a pulse treatment of H2O2, we can study the role of the cell cycle checkpoint proteins p53, p21, p16 and Rb in gaining each feature of senescence. Although p53 and Rb control G1 arrest and Rb appears to control cell enlargement, activation of the senescent associate beta-galactosidase, loss of cell replication and multiple molecular changes observed in premature senescent or replicative senescent cells are likely controlled by mechanisms beyond the cell cycle checkpoints.

Mild doses of oxidative stress in the heart correlate with the induction of apoptosis or hypertrophy in cardiomyocytes (CMCs) and fibrosis or proliferation of fibroblasts. Three branches of mitogen-activated protein kinases (MAPKs), i.e., c-Jun N-terminal kinases (JNKs), extracellular signal-related kinases 1 and 2 (ERK1/2), and p38, are activated by oxidants in a variety of cell types, including CMCs. However, the initiation process of these signaling pathways remains unsolved. We explored the role of the epidermal growth factor (EGF) receptor in H(2)O(2)-induced MAPK activation using two different cell types from the same organ: CMCs and heart fibroblasts (HFs). Pretreatment of each cell type with EGF revealed differences in how CMCs and HFs responded to subsequent treatment with H(2)O(2): in CMCs, the second treatment resulted in little further activation of JNKs and ERK1/2, whereas HFs retained the full response of JNKs and ERK1/2 activation by H(2)O(2) regardless of EGF pretreatment. AG-1478 [4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline], a pharmacologic inhibitor of the EGF receptor tyrosine kinase, inhibited JNK and ERK1/2 activations but not p38 in both cell types. The data using the Src inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] resemble those found when using AG-1478 in either cell type. Pharmacologic inhibitors of matrix metalloproteinases (MMPs) further illustrated the difference between the two cell types. In HFs, MMP inhibitors GM6001 [N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-l-tryptophan methylamide] and BB2516 [[2S-[N4(R(*)),2R(*),3S(*)]]-N4-[2,2-dimethyl-1-[(methylamino)carbonyl]propyl]-N1,2-dihydroxy-3-(2-methylpropyl)butanediamide, marimastat] inhibited JNKs and ERK1/2 activation without affecting p38 activation by H(2)O(2) inhibitors. In contrast, these MMP failed to significantly inhibit the activation of JNKs, ERKs, or p38 in CMCs. These data suggest the complexity of the cell type-dependent signaling web initiated by oxidants in the heart.

Cardiac hypertrophy is an adaptive response to a number of heart diseases including myocardial infarction. Although it can be compensatory at first, sustained hypertrophy is often a transition to heart failure. We have found that cardiomyocytes in culture can survive mild doses of H2O2 but develop hypertrophy involving activation of p70 S6 kinase 1 (p70S6K1). Here, the role of p42/p44(ERK) and p38 MAPK in oxidant-induced hypertrophy is tested. H2O2- induced phosphorylation (activation) of p42/p44(ERK) and p38 within 10 min of 200 microM H2O2 exposure. Although p42/p44(ERK) remained highly phosphorylated from 60 to 120 min, the level of p38 phosphorylation reached highest at 60 min and started to decline at 90 min. Inhibiting ERKs with PD98059 attenuated H2O2-induced AP-1 activation but did not affect H2O2-induced p70S6K1 activation or cardiomyocyte enlargement as measured by increases in cell volume and protein content. In contrast, the p38 inhibitor SB202190 has no inhibitory effect on AP-1 activation but partially prevented H2O2 from inducing p70S6K1 activation and cell enlargement. These data suggest that while p42/p44(ERK) participates in gene expression associated with hypertrophy, p38 may regulate cell size increase by p70S6K1 activation.

Genetics versus oxidative stress have been long-standing points of contention among theories seeking to explain the root of aging. Because aging is the highest risk factor for many diseases, it is to our advantage to better understand the biological mechanisms of this process. Caloric restriction has been the only reliable means of extending lifespan in mammalian models until recently. The discovery of mutant strains of mice with increased longevity could be a significant contributor to our understanding of the genetic and molecular basis of human aging. One genetic approach that increases the longevity of mice is the removal of the p66Shc gene, which encodes a protein belonging to a family of adaptors for signal transduction in mitogenic and apoptotic responses. Normally, p66Shc is tyrosine phosphorylated (activated) by various extracellular signals including EGF and insulin. However, serine phosphorylation of p66Shc can occur after oxidative stress either in association with or independently of tyrosine phosphorylation. p66Shc serine phosphorylation has been linked to inactivation of members of forkhead transcription factors, resulting in increased intracellular oxidant levels and increased sensitivity to apoptosis. Knocking out p66Shc allows moderately elevated activity of forkhead transcription factors and better-equipped antioxidant defenses at the cellular level. Recent reports have suggested that methylation of the p66Shc promoter has important implications in its expression regulation. This leads us to hypothesize that the methylation status of the p66Shc promoter may differ between individuals and therefore contribute to variations of longevity. We present evidence arguing that decreasing oxidative stress or increasing resistance to oxidative damage as a result of genetic variation or p66Shc knockout is likely contributing to individual differences in longevity.

Cardiomyocytes in culture can survive low or mild doses of oxidants but later increase cell volume and protein content. To understand the mechanism, we determined the early signaling events of oxidative stress. With 200 μM H 2 O 2 , the activity of p70 S6 kinase-1 (p70S6K1) increased at 30 min and reached a plateau at 90 min. Dose-response studies at the 60 min time point show that p70S6K1 activity reached its highest level with 150 μM H 2 O 2 . Increased p70S6K1 activity correlated with phosphorylation of Thr389 and Thr421/Ser424 residues, suggesting the involvement of an upstream kinase. Phosphoinositide 3-kinase (PI3K) activity was elevated by 5 min, reached a plateau at 10 min, and remained more than 6-fold induced for at least 60 min after 200 μM H 2 O 2 exposure. The dose-response studies at 10 min found that 150 μM H 2 O 2 induced the highest PI3K activity. Increased PI3K activity correlated with tyrosine phosphorylation of the 85-kDa regulatory subunit. Inactivating PI3K with wortmannin prevented H 2 O 2 from inducing Thr389 phosphorylation and p70S6K1 activation. Wortmannin and rapamycin prevented H 2 O 2 from inducing increases in cell volume and protein content. The antineoplastic drugs doxorubicin and daunorubicin also induced significant enlargement of cardiomyocytes at 10 to 100 nM dose range. Although the glutathione synthesis inhibitor buthionine sulfoximine potentiated the effect of doxorubicin and H 2 O 2 , the antioxidant N -acetylcysteine prevented induction of cell enlargement. Our data suggest that oxidative stress induces activation of PI3K, which leads to p70S6K1 activation and enlargement of cell size.

A large volume of experimental data supports the presence of apoptosis in failing hearts. Apoptosis in many types of cells results from exposure to cytotoxic cytokines or damaging agents. Cytotoxic cytokines such as tumor necrosis factor (TNF)-alpha or Fas ligand (FasL) bind to their receptors to activate caspase-8, while damaging agents can cause mitochondrial release of cytochrome c, which can initiate activation of caspase-9. Caspase-8 or -9 can activate a cascade of caspases. The p53 protein is often required for damaging agent-induced apoptosis. An imbalance of proapoptotic factors versus prosurvival factors in the bcl-2 family precedes the activation of caspases. Given these typical changes of apoptosis found in many cell types, the apoptotic pathway in cardiomyocytes is somewhat unconventional since in vivo experimental data reveal that apoptosis does not appear to be controlled by TNF-alpha, FasL, p53 or decrease of bcl-2. In vitro and in vivo studies suggest the importance of mitochondria and activation of caspases in cell death occurring in failing hearts. Oxidants, excessive nitric oxide, angiotensin II and catecholamines have been shown to trigger apoptotic death of cardiomyocytes. Eliminating these inducers reduces apoptosis and reverses the loss of contractile function in many cases, indicating the feasibility of the pharmacological application of antioxidants, nitric oxide synthetase inhibitors, ACE inhibitors, angiotensin II receptor antagonists and adrenergic receptor antagonists. Most inducers of apoptosis initiate a cascade of signaling events, including activation of the p38 mitogen-activated protein kinase. Small molecule inhibitors of p38 have been shown to be capable of preventing apoptosis and loss of contractile function associated with ischemia and reperfusion. Although further experimental work is needed, several studies have already indicated the beneficial effect of caspase inhibitors against cell loss and features of heart failure in vitro and in vivo. These studies indicate the importance of inhibiting apoptosis in therapeutic interventions against heart failure.

Nrf2 is a bZIP transcription factor regulating the expression of antioxidant and detoxification genes. We have found that Nrf2 knockout mice have an increased infarction size in response to regional ischemic reperfusion and have a reduced degree of cardiac protection by means of ischemic preconditioning. With cycles of brief ischemia and reperfusion (5′I/5′R) that induce cardiac protection in wild type mice, an elevated Nrf2 protein was observed without prior increases of Nrf2 mRNA. When an mRNA species is being translated into a protein, it is occupied by multiple ribosomes. The level of ribosome-associated Nrf2 mRNA increased following cycles of 5′I/5′R, supporting de novo Nrf2 protein translation. A dicistronic reporter assay indicated a role of the 5′ untranslated region (5′ UTR) of Nrf2 mRNA in oxidative stress induced Nrf2 protein translation in isolated cardiomyocytes. Western blot analyses after isolation of proteins binding to biotinylated Nrf2 5′ UTR from the myocardium or cultured cardiomyocytes demonstrated that cycles of 5′I/5′R or oxidants caused an increased association of La protein with Nrf2 5′ UTR. Ribonucleoprotein complex immunoprecipitation assays confirmed such association indeed occurring in vivo. Knocking down La using siRNA was able to prevent Nrf2 protein elevation by oxidants in cultured cardiomyocytes and by cycles of 5′I/5′R in the myocardium. Our data point out a novel mechanism of cardiac protection by de novo Nrf2 protein translation involving interaction of La protein with 5′ UTR of Nrf2 mRNA in cardiomyocytes.

Many biomarkers of replicative senescence appear in stress-induced premature senescence (SIPS) of human diploid fibroblasts (HDFs). The mRNA level of key cell cycle regulators was studied in H 2 O 2 -induced premature senescence of HDFs expressing or not the papillomavirus E6 and E7 proteins, which enhanced, respectively, the proteolysis of p53 and Rb. The CdKI's p21 waf-1 and p16 Ink-4a were found overexpressed in H 2 O 2 -induced premature senescence, while p19 Ink-4d and p27 Kip-1 were repressed. The results obtained in E6 HDFs suggest that p21 waf-1 and p16 Ink-4a overexpressions are p53-independent, while p27 Kip-1 and p19 Ink-4d down-regulations are p53-dependent. E6 regulated Rb, p130, p53 and p16 Ink-4a mRNA level in non-stressing conditions, and regulated p130, p107, p53, p19 Ink-4d , p27 Kip-1 mRNA level in SIPS. SIPS modified the E6-mediated regulatory control on p107, p16 Ink-4a , p19 Ink-4d and p27 Kip-1 mRNA level, when compared to normal conditions. E7 regulated the mRNA level of all the genes studied, in all conditions, suggesting that the Rb family or other E7-interacting proteins might modify the expression of these genes. SIPS modified strongly the E7-mediated regulatory control on p107, p16 Ink-4a , p19 Ink-4d , p27 Kip-1 , p21 Waf-1 and Rb mRNA level, when compared to normal conditions. Further work is ongoing to test whether this E7-mediated regulatory control takes place through interactions with Rb or other E7-interacting proteins.

Human diploid fibroblasts (HDFs) inevitably undergo replicative senescence in culture after serial passages. Recent work indicates that early passage HDFs undergo irreversible growth arrest and develop features of senescence after being treated with oxidants and other agents. Senescence is usually measured by a decrease in DNA synthesis index and an increase in the activity of senescence-associated β-galactosidase (SA β-gal). We compared these two measurements here and found that IMR-90 HDFs lost the ability to synthesize DNA immediately but did not activate SA β-gal until 4 days after the treatment with 75 µM or 0.75 pmol/cell H2O2. Expression of human papillomaviral E6 or/and E7 genes results in reduction of p53 or/and Rb protein levels and increases in ED50 for DNA synthesis inhibition or SA β-gal expression. A small fraction of wild type and E7 expressing cells could not synthesize DNA and did not express SA β-gal one week following the treatment with H2O2 at doses lower than 150 µM or 1.5 pmol/cell. The dose response curve of SA β-gal activation overlapped with that of DNA synthesis inhibition in E6 and E6E7 expressing cells. The results indicate that the expression of SA β-gal correlates with inability of DNA synthesis in the majority of wild type, E6, E7 or E6E7 expressing cells one week after H2O2 treatment.

Glucocorticoids (GCs) are frequently prescribed pharmacological agents most notably for their immunosuppressive effects. Endogenous GCs mediate biological processes such as energy metabolism and tissue development. At the cellular level, GCs bind to the Glucocorticoid Receptor (GR), a cytosolic protein that translocates to the nuclei and functions to alter transcription upon ligand binding. Amongst a long list of genes activated by GCs is the Glucocorticoid Induced Leucine Zipper (GILZ). GC induced GILZ expression has been well established in lymphocytes and mediates GC induced apoptosis. Unlike lymphocytes, cardiomyocytes respond to GCs by gaining resistance against apoptosis. We determined GILZ expression in cardiomyocytes in vivo and in vitro. Expression of GILZ in mouse hearts as a result of GC administration was confirmed by Western blot analyses. GCs induced dose and time dependent elevation of GILZ expression in primary cultured rat cardiomyocytes, with dexamethasone (Dex) as low as 0.1 M being effective. Time course analysis indicated that GILZ protein levels increased at 8 hr and peaked at 48 hr after exposure to 1 M Dex. H9c2(2-1) cell line showed a similar response of GILZ induction by Dex as primary cultured rat cardiomyocytes, providing a convenient model for studying the biological significance of GILZ expression. With corticosterone (CT), an endogenous form of corticosteroids in rodents, 0.1–2.5 M was found to induce GILZ in H9c2(2-1) cells. Time course analysis with 1 M CT indicated induction of GILZ at 6 hr with peak expression at 18 hr. Inhibition of the GR by mifepristone led to blunting of GILZ induction by GCs. Our data demonstrate GILZ induction in cardiomyocytes both in vivo and in vitro by GCs, pointing to H9c2(2-1) cells as a valid model for studying the biological function of GILZ in cardiomyocytes.

Nrf2 gene encodes a transcription factor that regulates the expression of a cluster of antioxidant and detoxification genes. Recent works from our laboratory indicate that oxidative stress causes rapid de novo synthesis of Nrf2 protein. We have found that 5' Untranslated Region (5'UTR) of Nrf2 allows the mRNA to undergo an Internal Ribosomal Entry Site (IRES) mediated protein translation. Using liquid chromatography tandem MS, we have discovered that La/SSB protein bound to Nrf2 5'UTR in response to oxidative stress. In vitro RNA binding and in vivo ribonucleoprotein immunoprecipitation showed H(2)O(2) dose and time dependent increases of La/SSB binding to Nrf2 5'UTR. La/SSB protein translocated from the nuclei to cytoplasm and distributed in the perinuclear space in cells treated with H(2)O(2). Isolation of ribosomal fractions indicated that oxidants caused an association of La/SSB with ribosomes. Physical interaction of La/SSB with representative proteins from the small or large subunits of ribosomes was found to increase in cells responding to H(2)O(2) treatment. Knocking down La/SSB gene with siRNA prevented Nrf2 protein elevation or Nrf2 5'UTR activation by oxidants. In contrast, overexpression of La/SSB gene was able to enhance Nrf2 5'UTR activation and Nrf2 protein increase. Our data suggest that oxidants cause nuclear export of La/SSB protein and subsequent association of La/SSB with Nrf2 5'UTR and ribosomes. These events contribute to de novo Nrf2 protein translation because of oxidative stress.

Doxorubicin (Dox) is an antineoplastic agent that can cause cardiomyopathy in humans and experimental animals. As an inducer of reactive oxygen species and a DNA damaging agent, Dox causes elevated expression of p21(WAF1/Cip1/Sdi1) (p21) gene. Elevated levels of p21 mRNA and p21 protein have been detected in the myocardium of mice following Dox treatment. With chronic treatment of Dox, wild type (WT) animals develop cardiomyopathy evidenced by elongated nuclei, mitochondrial swelling, myofilamental disarray, reduced cardiac output, reduced ejection fraction, reduced left ventricular contractility, and elevated expression of ANF gene. In contrast, p21 knockout (p21KO) mice did not show significant changes in the same parameters in response to Dox treatment. In an effort to understand the mechanism of the resistance against Dox induced cardiomyopathy, we measured levels of antioxidant enzymes and found that p21KO mice did not contain elevated basal or inducible levels of glutathione peroxidase and catalase. Measurements of 6 circulating cytokines indicated elevation of IL-6, IL-12, IFNγ and TNFα in Dox treated WT mice but not p21KO mice. Dox induced elevation of IL-6 mRNA was detected in the myocardium of WT mice but not p21KO mice. While the mechanism of the resistance against Dox induced cardiomyopathy remains unclear, lack of inflammatory response may contribute to the observed cardiac protection in p21KO mice.

Aims Numerous lines of evidence suggest a role of oxidative stress in initiation and progression of heart failure. We identify novel pathways of oxidative stress in cardiomyocytes using proteomic technology. Methods and results Cardiomyocytes and cardiac fibroblasts isolated from rat hearts were treated with sublethal doses of H2O2 for detection of secreted protein factors in the conditioned media by mass spectrometry-based proteomics. Comparison between the two cell types leads to the finding that H2O2 caused an elevated cystatin C protein in the conditioned medium from cardiomyocytes. When cardiomyopathy was induced in mice by chronic administration of doxorubicin, elevated cystatin C protein was detected in the plasma. Myocardial ischaemia by left anterior descending coronary artery occlusion causes an increase in the level of cystatin C protein in the plasma. In myocardial tissue from the ischaemic area, an increase in cystatin C correlates with the inhibition of cathepsin B activity and accumulation of fibronectin and collagen I/III. Overexpressing cystatin C gene or exposing fibroblasts to cystatin C protein results in an inhibition of cathepsin B and accumulation of fibronectin and collagen I/III. Conclusion Oxidants induce elevated cystatin C production from CMCs. Cystatin C plays a role in cardiac extracellular matrix remodelling.

Heart failure is an end point of many types of heart disease. Although symptoms or cardiac structure may differ among individuals or stages of heart failure, the most common clinical presentation of heart failure involves reduced exercise tolerance, elevated blood levels of B-type natriuretic peptide (BNP), and reduced cardiac output. Unmanaged hypertension, ischemia, valvular disease, myocarditis, and toxin-induced cardiomyopathy are the common causes of heart failure. Morphologically, failing hearts show enlarged cardiomyocytes, and apoptosis of a small fraction of cardiomyocytes has also been detected. In parallel with these changes of cardiomyocytes, fibrosis is evident as proliferation of fibroblasts and altered secretion of extracellular matrix protein. The progression of heart failure is often related to activation of neurohormonal and renin–angiotensin–aldosterone systems, resulting in increased levels of catecholamines and angiotensin II. Catecholamines bind to β-adrenergic receptors in the plasma membrane of cardiomyocytes to initiate a cascade of signaling events regulating contraction–relaxation coupling. Chronic activation of β-adrenergic or angiotensin II signaling contributes to cardiac remodeling, cardiomyocyte hypertrophy, and heart failure. While catecholamines can undergo autooxidation to generate free radicals, angiotensin II produces reactive oxygen species (ROS) following receptor binding and activation of a membrane-associated nicotinamide adenine dinucleotide phosphate (reduced form) (NAD(P)H) oxidase. Additional sources of oxidants include ischemia and reperfusion injury, drugs undergoing redox cycling, and inflammatory response. ROS are known to induce apoptosis and have been shown to play a role in fibrosis. Recent evidence suggests that ROS contribute to hypertrophy of cardiomyocytes. Measurements of biomarkers of oxidative stress have indicated an association with heart failure. Transgenic animals null for antioxidant enzymes show increased cardiac injury consequential to ischemia and reperfusion, while overexpression of antioxidant enzymes or proteins protects the heart from various types of heart failure inducers. Several pharmacological agents currently used for treatment of heart failure, such as angiotensin-converting enzyme (ACE) inhibitors captopril and ramipril, angiotensin receptor blocker telmisartan, aldosterone receptor antagonist eplerenone, and β-blockers nebivolol and carvedilol, exhibit antioxidant activity. However, clinical trials of antioxidant vitamins have failed to clearly show their role in the prevention of heart failure or reduction of the mortality rate associated with heart failure. Therefore, the role of ROS in heart failure remains to be investigated.

Recent work from our laboratory found that corticosteroids induce transcriptional activation of cyclooxygenase-2 (COX-2) gene in cardiomyocytes. Here we report that COX-2 gene promoter mutation studies indicate a role of cAMP response element-binding protein (CREB) in corticosterone-induced COX-2 gene expression. Corticosterone causes activation of p38 MAPK and subsequent CREB phosphorylation at serine 133 in cardiomyocytes. The inhibitors of p38 MAPK, SB202190 and SB203580, block corticosterone from inducing CREB phosphorylation and COX-2 gene expression while dominant-negative p38 MAPK or CREB prevents corticosterone from activating COX-2 promoter. Corticosterone does not induce p38 MAPK activation or COX2 expression in cardiac fibroblasts or HEK293 cells transfected with glucocorticoid receptor, suggesting that p38 MAPK activation is cell specific and necessary for corticosterone-induced COX-2 expression in cardiomyocytes. While glucocorticoid receptor antagonist mifepristone inhibits COX-2 gene induction by corticosterone, mifepristone fails to inhibit p38 MAPK activation or CREB phosphorylation. In contrast, inhibition of p38 MAPK does not prevent corticosterone from activating glucocorticoid receptor. Our data suggest that two parallel signaling pathways, glucocorticoid receptor and p38 MAPK, act in concert to regulate the expression of COX-2 gene in cardiomyocytes.

Fibroblasts are the most ubiquitous cell types within our body. They produce various factors to maintain the texture and structure of a particular organ or tissue. To identify protein factors secreted by fibroblasts and alteration of these protein factors upon oxidative stress, HCA3 human skin diploid fibroblasts were exposed to a sublethal dose of H2O2, which induces a prematurely senescent phenotype. Conditioned media from prematurely senescent cells versus control cells were analyzed for proteins using an LC-MS/MS-based proteomic technique. Collagen alpha1(VI), collagen alpha2(I), fibronectin, lumican, and matrix metalloproteinase 2 were among the proteins consistently detected from control and H2O2-treated cells. Insulin-like growth factor-binding protein-6 (IGFBP-6) consistently showed up in the conditioned medium of H2O2-treated cells but not from untreated cells. Increased IGFBP-6 production due to H2O2 treatment was confirmed by RT-PCR and Western blot analyses. While H2O2 induced a dose-dependent elevation of IGFBP-6 mRNA, Western blot analyses detected elevated levels of IGFBP-6 protein in the conditioned medium of H2O2-treated cells. In comparison, fibronectin or matrix metalloproteinase 2 did not show changes at the mRNA level in cell lysates or at the protein level in the conditioned medium by H2O2 treatment. Using several types of toxins at sublethal doses, including cis-platin, hydroxyurea, colchicine, L-mimosine, rhodamine, dithiothreitol, or N-ethylmaleimide, we found that these agents induced increases of IGFBP-6 at mRNA and protein levels. An increased level of IGFBP-6 protein was detected in the plasma of aging mice and of young mice treated with doxorubicin. These data suggest that IGFBP-6 may serve as a sensitive biomarker of cell degeneration or injury in vitro and in vivo.

Oxidants cause activation of the AP-1 transcription factor in cardiomyocytes. c-Fos, a component of the AP-1 transcription factor, is transiently induced by H2O2 and the induction is sensitive to the protein synthesis inhibitor cycloheximide. With high percentage gel electrophoresis, multiple c-Fos bands were resolved by Western blot analyses, indicating post-translational modification of newly synthesized c-Fos protein after H2O2 exposure. Treatment of immunoprecipitated c-Fos protein with the type 2 serine/threonine phosphatase A (PP2A) and immunoblotting of c-Fos protein with antibodies against phosphorylated serine or threonine demonstrated that c-Fos was phosphorylated at serine residues. A pharmacological inhibitor of JNKs inhibited the formation of multiple c-Fos bands without affecting c-fos transcription. The proteasomal inhibitor MG132 and Proteasome Inhibitor I extended the time course of c-Fos protein elevation. An increase in ubiquitin was detectable in c-Fos protein from H2O2-treated cells. Interestingly, treating the whole cell lysates with PP2A, but not calcineurin (i.e. PP2B), resulted in disappearance of c-Fos protein and MG132 was able to prevent this loss. H2O2 caused an elevation of PP2B and total phosphatase activity. The phosphatase inhibitor okadaic acid, but not PP2B inhibiter cypermethrin, extended the time course of c-Fos protein elevation after H2O2 exposure. These data suggest that JNK-mediated phosphorylation of newly synthesized c-Fos protects the protein from being degraded by the proteasome. PP2B independent dephosphorylation contributes to degradation of c-Fos protein during oxidative stress response of cardiomyocytes.

Aging is the highest risk factor for cancer. Although oxidants are thought to contribute to both aging and cancer, the interplay between oxidative stress, aging, and cancer has not been well studied. Human diploid fibroblasts (HDFs) undergo premature senescence in response to sublethal doses of H(2)O(2). To test the hypothesis that senescent or senescent-like HDFs function as a tumor promoter, we have employed an in vitro skin tumor promotion model, in which colony formation is measured using initiated mouse keratinocyte 308 cells seeded at clonal density. 308 cells form colonies when co-cultured with normal HDFs only in the presence of the tumor promoter phorbol 12-myristate 13-acetate (TPA), which induces an average of 5.75 colonies. When co-cultured with H(2)O(2)-treated HDFs, 308 cells form an average of 30.3 colonies. To understand the mechanism behind this phenomenon, we tested whether conditioned medium of HDFs, HDF extracellular matrix (ECM), density of HDFs, or the contact between keratinocytes and HDFs plays a role in 308 cell colony formation. The conditioned medium from prematurely senescent cells resulted in an average of eightfold more 308 cell colonies formed than the conditioned medium from normal HDFs, and the growth-promoting effect of the conditioned medium was trypsin sensitive. The ECM alone was not able to induce 308 cell colony formation. Increasing the density of normal HDFs or contact with normal HDFs but not senescent-like HDFs was inhibitory to the growth of 308 cells. Measurement of Connexin 43 indicated a decreased expression of the protein, which suggests an impaired gap junction communication in senescent-like HDFs. We conclude that H(2)O(2)-treated fibroblasts not only lose contact inhibition of the growth of initiated keratinocytes perhaps related to reduced gap junction communication but also increase production of secreted protein factors to enhance the growth of 308 keratinocytes.

The biology of aging has been mysterious for centuries. Removal of the p66(Shc) gene, which encodes an adaptor protein for cell signaling, extends lifespan by approximately 30% in mice and confers resistance to oxidative stress. The absence of p66(Shc) correlates with reduced levels of apoptosis. Oxidants induce phosphorylation of serine36 on p66(Shc), contributing to inactivation of members of the Forkhead transcription factor family, some of which appear to regulate the expression of antioxidant genes. The expression of p66(Shc) is regulated by the methylation status of its promoter. This leads us to hypothesize that increased methylation of the p66(Shc) promoter might contribute to the absence of its expression and therefore extended longevity in particular individuals.

Cardiac hypertrophy is an end point of chronic cardiac toxicity from a number of toxicants. Doxorubicin, cocaine, acetaldehyde, monocrotaline, and azide are examples of these toxicants, which may induce hypertrophy by increasing oxidants, circulating levels of catecholamines, and hemodynamic load or by inducing hypoxia. We summarize here the major signal transduction pathways and common changes in gene expression found with the classical hypertrophy inducers angiotensin II, endothelin 1, and catecholamines. Activation of G-proteins, calcium signaling, phosphoinositide 3-kinase (PI3K), certain family members of protein kinase Cs (PKCs), and three branches of mitogenactivated protein kinases (MAPKs), i.e. extracellular signal-regulated kinases (ERKs), p38, and c-Jun N-terminal kinases (JNKs), are important for developing a hypertrophic phenotype in cardiomyocytes. Characteristic changes of gene expression in hypertrophy include the elevated transcription of atrial natriuretic factor (ANF), beta-myosin heavy chain (beta MHC), skeletal alpha-actin (SkA), certain variants of integrins and perhaps tubulin genes, and reduced expression of the sarcoplasmic reticulum proteins phospholamban and sarco(endo)plasmic reticulum Ca2+-ATPase 2 alpha (SERCA2 alpha), and of the ryanodine receptors. Although which toxicants induce these molecular changes remains to be tested, increasing lines of evidence support that oxidants play a central role in cardiac hypertrophy. Oxidants activate small G-proteins, calcium signaling, PI3K, PKCs, and MAPKs. Oxidants cause cardiomyocytes to enlarge in vitro. Recent developments in transgenic, genomic, and proteomic technologies will provide needed tools to reveal the mechanism of chronic cardiac toxicity at the cellular and molecular levels.

The tumor suppressor p53 protein is known to play a critical role in apoptosis. In normal human diploid fibroblasts (HDFs), expression of the human papillomaviral (HPV) E6 gene results in a reduction of p53 protein and an inhibition of oxidant induced apoptosis within 24 h. In comparison, expression of the HPV E7 gene causes down-regulation of Rb protein without inhibiting apoptosis. Here we determine whether HDFs expressing E6 undergo cell death with a delayed time course following H2O2 exposure. Appearances of caspase-3 activity, cell detachment, trypan blue uptake and aberrant nuclei were all delayed in E6 cells compared to wild type (wt) or E7 cells. A mutant E6 gene that failed to reduce p53 could not delay cell death. Morphological examination revealed nuclear condensation in dying wt or E7 cells but nuclear fragmentation in E6 cells. Flow cytometry analysis indicated an S phase distribution of dying wt or E7 cells but a G2/M phase distribution of dying E6 cells. An elevation of cyclin B was observed in dying E6 cells but not in apoptotic E7 cells. Dying E6 cells also had elevated levels of cdc-2 protein and histone kinase activity, suggesting that the cells died at mitosis. Electron microscopy studies showed that E6 cells may die at prophase or prometaphase. Overexpression of bcl-2 resulted in an inhibition of both caspase-3 and death of E7 or E6 cells. Inactivating caspases with zVAD-fmk also reduced the death rate of E7 and E6 cells. Our data indicate that expression of HPV E6 causes a delay and morphological modification of cell death induced by oxidants. E6 cells die at mitosis, which can be inhibited by bcl-2 overexpression or caspase inhibition.

Early passage human diploid fibroblasts develop senescent morphology prematurely within a week after a 2-hour pulse treatment with low or mild dose H(2)O(2). We test here the role of cell cycle checkpoints, cytoskeletal proteins and de novo protein synthesis in senescent morphogenesis following H(2)O(2) treatment. H(2)O(2) treatment causes transient elevation of p53 protein and prolonged inhibition of Rb hyperphosphorylation. Expression of human papillomaviral E6 gene prevented elevation of p53 but did not affect senescent morphogenesis. Expression of human papillomaviral E7 gene reduced the level of Rb protein and prevented induction of senescent morphology by H(2)O(2). The mutants of the E7 gene, in which the Rb family protein binding site was destroyed, could not reduce Rb protein or prevent H(2)O(2) from inducing senescent morphology. Senescent-like cells showed enhanced actin stress fibers. In untreated cells, vinculin and paxillin preferentially distributed along the edge of the cells. In contrast, vinculin and paxillin distributed randomly and sporadically throughout senescent-like cells. E7 expression prevented enhancement of actin filament formation and redistribution of vinculin or paxillin. Neither wild-type nor E7 cells showed changes in the protein level of actin, vinculin or paxillin measured by western blot after H(2)O(2) treatment. Finally, depletion of methionine in the culture medium after H(2)O(2) treatment prevented senescent morphogenesis without affecting dephosphorylation of Rb protein. Our results suggest that senescent morphology likely develops by a program involving activated Rb family proteins, enhancement of actin stress fibers, redistribution of focal adhesion proteins and de novo protein synthesis.

Cardiomyocyte hypertrophy and cell death are often observed in the end stages of heart failure. The triggers of these two cellular processes are not known under most pathological conditions. Oxidants are by-products of aerobic metabolism. The level of oxidants increases as a result of ischemic reperfusion. Using H9C2 and primary cultured neonatal rat cardiomyocytes, we found that a 2-h pulse treatment with H(2)O(2) at 250 microM or lower caused activation of DEVD sequence specific caspases. The activity of DEVD-ase peaked with 200 microM H(2)O(2) at 24 h. While a fraction of the cells detached and showed nuclear condensation, the majority of the cells (>55%) survived the treatment and appeared to enlarge when cultured for 5 days. These cells showed increases in cell surface area, cell volume, and protein content. With 200 microM H(2)O(2), treated cells appeared to be six times bigger in volume and contained three times more protein per cell than untreated cells. The enlarged cells showed enhanced actin stress fibers and disrupted myofibrils. Our data indicate that while H(2)O(2) can cause cell death, the surviving cardiomyocytes undergo hypertrophy.

Purpose Dodecafluoropentane emulsion (DDFPe) is a perfluorocarbon with high oxygen dissolving, transport, and delivery capacity that may offer the potential to limit ischemic injury prior to clinical reperfusion. Here we investigated the cardiac protective potential of DDFPe in a mouse model of myocardial infarction. Methods Myocardial infarction was initiated by permanent ligation of the left anterior descending (LAD) coronary artery. Mice were administered vehicle or 5-hydroxydecanoate (5-HD) intravenously 10 min before LAD occlusion followed by a single intravenous administration of vehicle or DDFPe immediately after occlusion. Heart tissue and serum samples were collected 24 after LAD occlusion for measurement of infarct size and cardiac troponin I (cTnI) levels, respectively. Results DDFPe treatment reduced infarct size by approximately 72 % (36.9 ± 4.2 % for vehicle vs 10.4 ± 2.3 % for DDFPe; p