Toxicity of airborne nanoparticles: Facts and challenges.

Air pollution is one of the most severe environmental healthhazards, and airborne nanoparticles (diameter <100 nm) are considered particularly hazardous to human health. They are produced by various sources such as internal combustion engines, wood and biomass burning, and fuel and natural gas combustion, and their origin, among other parameters, determines their intrinsic toxicity for reasons that are not yet fully understood. Many constituents of the nanoparticles are considered toxic or at least hazardous, including polycyclic aromatic hydrocarbons (PAHs) and heavy metal compounds, in addition to gaseous pollutants present in the aerosol fraction, such as NOx, SO2, and ozone. All these compounds can cause oxidative stress, mitochondrial damage, inflammation in the lungs and other tissues, and cellular organelles. Epidemiological investigations concluded that airborne pollution may affect the respiratory, cardiovascular, and nervous systems. Moreover, particulate matter has been linked to an increased risk of lung cancer, a carcinogenic effect not related to DNA damage, but to the cellular inflammatory response to the pollutants, in which the release of cytokines promotes the proliferation of pre-existing mutated cancer cells. The mechanisms behind toxicity can be investigated experimentally using cell cultures or animal models. Methods for gathering particulate matter have been explored, but standardized protocols are needed to ensure that the samples accurately represent chemical mixtures in the environment. Toxic constituents of nanoparticles can be studied in animal and cellular models, but designing realistic exposure settings is challenging. The air-liquid interface (ALI) system directly exposes cells, mimicking particle inhalation into the lungs. Continuous research and monitoring of nanoparticles and other airborne pollutants is essential for understanding their effects and developing active strategies to mitigate their risks to human and environmental health.

Is Intrinsic Cardioprotection a Laboratory Phenomenon or a Clinically Relevant Tool to Salvage the Failing Heart?

Cardiovascular diseases, especially ischemic heart disease, as a leading cause of heart failure (HF) and mortality, will not reduce over the coming decades despite the progress in pharmacotherapy, interventional cardiology, and surgery. Although patients surviving acute myocardial infarction live longer, alteration of heart function will later lead to HF. Its rising incidence represents a danger, especially among the elderly, with data showing more unfavorable results among females than among males. Experiments revealed an infarct-sparing effect of ischemic "preconditioning" (IPC) as the most robust form of innate cardioprotection based on the heart's adaptation to moderate stress, increasing its resistance to severe insults. However, translation to clinical practice is limited by technical requirements and limited time. Novel forms of adaptive interventions, such as "remote" IPC, have already been applied in patients, albeit with different effectiveness. Cardiac ischemic tolerance can also be increased by other noninvasive approaches, such as adaptation to hypoxia- or exercise-induced preconditioning. Although their molecular mechanisms are not yet fully understood, some noninvasive modalities appear to be promising novel strategies for fighting HF through targeting its numerous mechanisms. In this review, we will discuss the molecular mechanisms of heart injury and repair, as well as interventions that have potential to be used in the treatment of patients.

Krüppel-like factor (KLF)5: An emerging foe of cardiovascular health.

Krüppel-like factors (KLFs) are DNA-binding transcriptional factors, which regulate various pathways that pertain to development, metabolism and other cellular mechanisms. KLF5 was first cloned in 1993 and by 1999, it was reported as the intestinal-enriched KLF. Beyond findings that have associated KLF5 with normal development and cancer, it has been associated with various types of cardiovascular (CV) complications and regulation of metabolic pathways in the liver, heart, adipose tissue and skeletal muscle. Specifically, increased KLF5 expression has been linked with cardiomyopathy in diabetes, end-stage heart failure, and as well as in vascular atherosclerotic lesions. In this review article, we summarize research findings about transcriptional, post-transcriptional and post-translational regulation of KLF5, as well as the role of KLF5 in the biology of cells and organs that affect cardiovascular health either directly or indirectly. Finally, we propose KLF5 inhibition as an emerging approach for cardiovascular therapeutics.

Natural and synthetic antioxidants targeting cardiac oxidative stress and redox signaling in cardiometabolic diseases.

Cardiometabolic diseases (CMDs) are metabolic diseases (e.g., obesity, diabetes, atherosclerosis, rare genetic metabolic diseases, etc.) associated with cardiac pathologies. Pathophysiology of most CMDs involves increased production of reactive oxygen species and impaired antioxidant defense systems, resulting in cardiac oxidative stress (OxS). To alleviate OxS, various antioxidants have been investigated in several diseases with conflicting results. Here we review the effect of CMDs on cardiac redox homeostasis, the role of OxS in cardiac pathologies, as well as experimental and clinical data on the therapeutic potential of natural antioxidants (including resveratrol, quercetin, curcumin, vitamins A, C, and E, coenzyme Q10, etc.), synthetic antioxidants (including N-acetylcysteine, SOD mimetics, mitoTEMPO, SkQ1, etc.), and promoters of antioxidant enzymes in CMDs. As no antioxidant indicated for the prevention and/or treatment of CMDs has reached the market despite the large number of preclinical and clinical studies, a sizeable translational gap is evident in this field. Thus, we also highlight potential underlying factors that may contribute to the failure of translation of antioxidant therapies in CMDs.

Acute administration of the olive constituent, oleuropein, combined with ischemic postconditioning increases myocardial protection by modulating oxidative defense.

Oleuropein, one of the main polyphenolic constituents of olive, is cardioprotective against ischemia reperfusion injury (IRI). We aimed to assess the cardioprotection afforded by acute administration of oleuropein and to evaluate the underlying mechanism. Importantly, since antioxidant therapies have yielded inconclusive results in attenuating IRI-induced damage on top of conditioning strategies, we investigated whether oleuropein could enhance or imbed the cardioprotective manifestation of ischemic postconditioning (PostC). Oleuropein, given during ischemia as a single intravenous bolus dose reduced the infarct size compared to the control group both in rabbits and mice subjected to myocardial IRI. None of the inhibitors of the cardioprotective pathways, l-NAME, wortmannin and AG490, influence its infarct size limiting effects. Combined oleuropein and PostC cause further limitation of infarct size in comparison with PostC alone in both animal models. Oleuropein did not inhibit the calcium induced mitochondrial permeability transition pore opening in isolated mitochondria and did not increase cGMP production. To provide further insights to the different cardioprotective mechanism of oleuropein, we sought to characterize its anti-inflammatory potential in vivo. Oleuropein, PostC and their combination reduce inflammatory monocytes infiltration into the heart and the circulating monocyte cell population. Oleuropein's mechanism of action involves a direct protective effect on cardiomyocytes since it significantly increased their viability following simulated IRI as compared to non-treated cells. Οleuropein confers additive cardioprotection on top of PostC, via increasing the expression of the transcription factor Nrf-2 and its downstream targets in vivo. In conclusion, acute oleuropein administration during ischemia in combination with PostC provides robust and synergistic cardioprotection in experimental models of IRI by inducing antioxidant defense genes through Nrf-2 axis and independently of the classic cardioprotective signaling pathways (RISK, cGMP/PKG, SAFE).

The Molecular Mechanisms of Iron Metabolism and Its Role in Cardiac Dysfunction and Cardioprotection.

Iron is an essential mineral participating in different functions of the organism under physiological conditions. Numerous biological processes, such as oxygen and lipid metabolism, protein production, cellular respiration, and DNA synthesis, require the presence of iron, and mitochondria play an important role in the processes of iron metabolism. In addition to its physiological role, iron may be also involved in the adaptive processes of myocardial "conditioning". On the other hand, disorders of iron metabolism are involved in the pathological mechanisms of the most common human diseases and include a wide range of them, such as type 2 diabetes, obesity, and non-alcoholic fatty liver disease, and accelerate the development of atherosclerosis. Furthermore, iron also exerts potentially deleterious effects that may be manifested under conditions of ischemia/reperfusion (I/R) injury, myocardial infarction, heart failure, coronary artery angioplasty, or heart transplantation, due to its involvement in reactive oxygen species (ROS) production. Moreover, iron has been recently described to participate in the mechanisms of iron-dependent cell death defined as "ferroptosis". Ferroptosis is a form of regulated cell death that is distinct from apoptosis, necroptosis, and other types of cell death. Ferroptosis has been shown to be associated with I/R injury and several other cardiac diseases as a significant form of cell death in cardiomyocytes. In this review, we will discuss the role of iron in cardiovascular diseases, especially in myocardial I/R injury, and protective mechanisms stimulated by different forms of "conditioning" with a special emphasis on the novel targets for cardioprotection.

Potential markers and metabolic processes involved in the mechanism of radiation-induced heart injury.

Irradiation of normal tissues leads to acute increase in reactive oxygen/nitrogen species that serve as intra- and inter-cellular signaling to alter cell and tissue function. In the case of chest irradiation, it can affect the heart, blood vessels, and lungs, with consequent tissue remodelation and adverse side effects and symptoms. This complex process is orchestrated by a large number of interacting molecular signals, including cytokines, chemokines, and growth factors. Inflammation, endothelial cell dysfunction, thrombogenesis, organ dysfunction, and ultimate failing of the heart occur as a pathological entity - "radiation-induced heart disease" (RIHD) that is major source of morbidity and mortality. The purpose of this review is to bring insights into the basic mechanisms of RIHD that may lead to the identification of targets for intervention in the radiotherapy side effect. Studies of authors also provide knowledge about how to select targeted drugs or biological molecules to modify the progression of radiation damage in the heart. New prospective studies are needed to validate that assessed factors and changes are useful as early markers of cardiac damage.

Oxygen-Glucose Deprivation (OGD) Modulates the Unfolded Protein Response (UPR) and Inflicts Autophagy in a PC12 Hypoxia Cell Line Model.

Hypoxia is the lack of sufficient oxygenation of tissue, imposing severe stress upon cells. It is a major feature of many pathological conditions such as stroke, traumatic brain injury, cerebral hemorrhage, perinatal asphyxia and can lead to cell death due to energy depletion and increased free radical generation. The present study investigates the effect of hypoxia on the unfolded protein response of the cell (UPR), utilizing a 16-h oxygen-glucose deprivation protocol (OGD) in a PC12 cell line model. Expression of glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94), key players of the UPR, was studied along with the expression of glucose-regulated protein 75 (GRP75), heat shock cognate 70 (HSC70), and glyceraldehyde 3-phosphate dehydrogenase, all with respect to the cell death mechanism(s). Cells subjected to OGD displayed upregulation of GRP78 and GRP94 and concurrent downregulation of GRP75. These findings were accompanied with minimal apoptotic cell death and induction of autophagy. The above observation warrants further investigation to elucidate whether autophagy acts as a pro-survival mechanism that upon severe and prolonged hypoxia acts as a concerted cell response leading to cell death. In our OGD model, hypoxia modulates UPR and induces autophagy.

Low Dose Administration of Glutamate Triggers a Non-Apoptotic, Autophagic Response in PC12 Cells.

BACKGROUND/AIMS: Increasing amounts of the neurotransmitter glutamate are associated with excitotoxicity, a phenomenon related both to homeostatic processes and neurodegenerative diseases such as multiple sclerosis. METHODS: PC12 cells (rat pheochromocytoma) were treated with various concentrations of the non-essential amino acid glutamate for 0.5-24 hours. The effect of glutamate on cell morphology was monitored with electron microscopy and haematoxylin-eosin staining. Cell survival was calculated with the MTT assay. Expression analysis of chaperones associated with the observed phenotype was performed using either Western Blotting at the protein level or qRT-PCR at the mRNA level. RESULTS: Administration of glutamate in PC12 cells in doses as low as 10 µM causes an up-regulation of GRP78, GRP94 and HSC70 protein levels, while their mRNA levels show the opposite kinetics. At the same time, GAPDH and GRP75 show reduced protein levels, irrespective of their transcriptional rate. On a cellular level, low concentrations of glutamate induce an autophagy-mediated pro-survival phenotype, which is further supported by induction of the autophagic marker LC3. CONCLUSION: The findings in the present study underline a discrete effect of glutamate on neuronal cell fate depending on its concentration. It was also shown that a low dose of glutamate orchestrates a unique expression signature of various chaperones and induces cell autophagy, which acts in a neuroprotective fashion.

The PPARß/δ agonist GW0742 modulates signaling pathways associated with cardiac myocyte growth via a non-genomic redox mechanism.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily and appear to have beneficial effects in the cardiovascular system. PPARß/δ has been shown previously to exert an inhibitory effect on cardiac myocyte hypertrophy in vivo and in vitro although the exact mechanism is not fully clear yet. The principal signaling pathways that have been involved in triggering cardiac hypertrophic response are mitogen-activated protein kinases (MAPKs) and PI3K/Akt cascades. In this study, we sought to evaluate the potential effects evoked by PPARß/δ activation on signaling pathways that are implicated in cardiac myocyte growth responses. The selective PPARß/δ agonist GW0742 attenuated ERK1/2 and Akt phosphorylation that was stimulated by growth promoting agonists (phenylephrine, insulin or IGF-1). This effect was not reversed by the specific PPARß/δ antagonist, GSK0660, but was inhibited by vanadate, a potent protein tyrosine phosphatase inhibitor. In addition, GW0742 prevented the oxidation and inactivation of PTEN supporting further the notion that its inhibitory action on the agonist-induced kinase phosphorylation is mediated by the modulation of phosphatase activity. Furthermore, GW0742 abolished the agonist-induced intracellular generation of reactive oxygen species, independently of PPARß/δ activation. Our data reveals a new non-genomic mechanism of GW0742, which ameliorates the generation of reactive oxygen species and attenuates ERK1/2 and PI3K/Akt signaling, with implications in the regulation of cardiac hypertrophic response.

Activation of prosurvival signaling pathways during the memory phase of volatile anesthetic preconditioning in human myocardium: a pilot study.

According to a compelling body of evidence anesthetic preconditioning (APC) attenuates the deleterious consequences of ischemia-reperfusion and protects the heart through a mechanism similar to ischemic preconditioning. The present study was purported to investigate the intracellular signaling pathways activated in human myocardium in response to a preconditioning protocol with two different volatile anesthetics, namely isoflurane and sevoflurane. To this aim, phosphorylation of PKCα and -δ, ERK1/2, Akt, and GSK3ß was determined at the end of the APC protocol, in human atrial samples harvested from patients undergoing open-heart surgery. The results demonstrate that preconditioning with volatile anesthetics triggers the activation of PKCδ and -α isoforms and of prosurvival kinases, ERK1/2, and Akt, while inhibiting their downstream target GSK3ß during the memory phase.

Delayed cardioprotective effects of WY-14643 are associated with inhibition of MMP-2 and modulation of Bcl-2 family proteins through PPAR-α activation in rat hearts subjected to global ischaemia-reperfusion.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors regulating cardiac lipid metabolism and energy homeostasis. Although the activation of PPARs has been implicated in cardioprotection, the molecular mechanisms are largely unexplored. In this study, we aimed to investigate the effect of the PPAR-α agonist WY-14643 (WY), mimicking a delayed effect of preconditioning in rat hearts exposed to acute ischaemia-reperfusion (I/R) 24 h later, and to define whether antioxidative and antiapoptotic mechanisms are involved. Treatment with WY markedly attenuated post-ischaemic contractile dysfunction (as evidenced by the reduced infarct size), the higher left ventricular developed pressure (LVDP) recovery, and the decreased occurrence of arrhythmias. These effects were abolished in the presence of the PPAR-α antagonist MK886. Heme oxygenase-1, a key antioxidative enzyme implicated in cytoprotection, was upregulated in response to WY at baseline, but was markedly reduced after I/R, indicating reduced oxidative stress. WY treatment was also associated with decreased mRNA levels and enzymatic activity of matrix metalloproteinase-2, and increased ratios of Bcl-2:Bax proteins. These results indicate that PPAR-α activation by its selective ligand WY may confer delayed preconditioning-like protection in rat hearts subjected to I/R by modulating oxidative stress, activation of matrix metalloproteinase-2, and expression of Bcl-2 and Bax.

Silibinin protects H9c2 cardiac cells from oxidative stress and inhibits phenylephrine-induced hypertrophy: potential mechanisms.

Cardiac hypertrophy is the main response of the heart to various extrinsic and intrinsic stimuli, and it is characterized by specific molecular and phenotypic changes. Recent in vitro and in vivo studies indicate the involvement of reactive oxygen species in the hypertrophic response. In this study, silibinin, a plant flavonolignan extracted from milk thistle with potent antioxidant activity, was evaluated for its effects in (a) preventing hydrogen peroxide (H2O2)-induced cellular damage and (b) blocking the phenylephrine-induced hypertrophic response. Using the in vitro model of embryonic rat heart-derived H9c2 cells, we showed that silibinin has a rather safe profile as concentrations up to 200µM did not affect cell viability. Pretreatment of H9c2 cells with silibinin resulted in better protection of H9c2 cells under conditions of H2O2-induced cellular stress compared to untreated cells as indicated by cell viability and DNA fragmentation assays. Furthermore, silibinin attenuated the phenylephrine-induced hypertrophic response as evidenced by the measurement of cell surface, up-regulation of atrial natriuretic peptide and increase of cellular protein levels. Moreover, silibinin repressed the phenylephrine-induced phosphorylation of ERK1/2 kinases, while it appeared to inhibit the weakly activated by phenylephrine phosphorylation of Akt. Based on our results, silibinin may attenuate the phenylephrine-induced hypertrophic response of H9c2 cells via antioxidant mechanisms involving mainly the inhibition of the intracellular signaling pathways mediated by ERK1/2 MAPKs and Akt.

PPAR-alpha activation as a preconditioning-like intervention in rats in vivo confers myocardial protection against acute ischaemia-reperfusion injury: involvement of PI3K-Akt.

Peroxisome proliferator-activated receptors (PPAR) regulate the expression of genes involved in lipid metabolism, energy production, and inflammation. Their role in ischaemia-reperfusion (I/R) is less clear, although research indicates involvement of PPARs in some forms of preconditioning. This study aimed to explore the effects of PPAR-α activation on the I/R injury and potential cardioprotective downstream mechanisms involved. Langendorff-perfused hearts of rats pretreated with the selective PPAR-α agonist WY-14643 (WY, pirinixic acid; 3 mg·(kg body mass)·day(-1); 5 days) were subjected to 30 min ischaemia - 2 h reperfusion with or without the phosphatidylinositol 3-kinase (PI3K)-Akt inhibitor wortmannin for the evaluation of functional (left ventricular developed pressure, LVDP) recovery, infarct size (IS), and reperfusion-induced arrhythmias. A 2-fold increase in baseline PPAR-α mRNA levels (qPCR) in the WY-treated group and higher post-I/R PPAR-α levels compared with those in untreated controls were accompanied by similar changes in the expression of PPAR-α target genes PDK4 and mCPT-1, regulating glucose and fatty acid metabolism, and by enhanced Akt phosphorylation. Post-ischaemic LVDP restoration in WY-treated hearts reached 60% ± 9% of the pre-ischaemic values compared with 24% ± 3% in the control hearts (P < 0.05), coupled with reduced IS and incidence of ventricular fibrillation that was blunted by wortmannin. Results indicate that PPAR-α up-regulation may confer preconditioning-like protection via metabolic effects. Downstream mechanisms of PPAR-α-mediated cardioprotection may involve PI3K-Akt activation.

Signal transduction pathways through cytoprotective, apoptotic and hypertrophic stimuli: a comparative study in adult cardiac myocytes.

In response to pathophysiological stresses, cardiac myocytes undergo hypertrophic growth or apoptosis. Multiple signalling pathways have been implicated in these responses and among them, kinases such as mitogen-activated protein kinases (MAPKs) and Akt. However, the distinction between signalling pathways originally believed to be specific for either hypertrophy, apoptosis or cell survival is fading. The existing data, coming from different experimental systems, often are conflicting. In this study, we sought to compare aspects of intracellular signalling activated by diverse stimuli in a single experimental system, adult rat cardiac myocytes. Furthermore, we assessed the role of these stimuli in eliciting a particular cell phenotype, i.e. whether they promote hypertrophy, cell survival or apoptosis. The results demonstrate that the hypertrophic agonist phenylephrine is the most potent activator of MAPKs/mitogen and stress- activated kinase MSK1, although its effect on Akt phosphorylation is relatively minor. The pro-apoptotic concentration of H2O2 activates strongly both MAPKs and PI3K/Akt pathways. Insulin-like growth factor-1 has a minimal effect on phosphorylation of MAPKs/MSK1, but it is a potent activator of Akt. In conclusion, hypertrophic, pro-survival or apoptotic stimuli operate through the same signalling pathways with different time course and amplitude of kinase activation. Thus, to determine the effect of different stimuli on cell fate, it is important to assess signalling pathways as a network and not as a single pathway.

Multiple signalling pathways underlie the protective effect of levosimendan in cardiac myocytes.

Levosimendan is a cardiovascular drug for the treatment of acute and decompensated heart failure. The current weight of evidence on the cardioprotective effects of levosimendan originates from whole heart models and there is no information on the mechanism whereby signalling pathways are activated. In the present study, we investigated the effect of levosimendan on ischaemia/reperfusion injury and the underlying mechanism in cardiac myocytes. Pretreatment with levosimendan reversed the effects of ischaemia and ischaemia/reperfusion on cell viability and enhanced phosphorylation of Akt, p38-mitogen activated protein kinase (MAPK) and extracellular signal-regulated kinases 1/2 (ERK1/2). Inhibitors of these kinases and the blocker of the mitochondrial K(ATP) channels, 5-hydroxydecanoate, completely abolished the protection afforded by levosimendan. Levosimendan stimulated the phosphorylation of Akt, ERK1/2 and p38-MAPK with different kinetics and the activation of these pathways was dependent on the opening of the mitochondrial K(ATP) channels and the production of oxygen free radicals. The levosimendan-induced phosphorylation of ERK1/2 and Akt was reduced by inhibitors of epidermal growth factor receptor and Src. On the other hand, inhibition of the protein kinase A (PKA) pathway reduced phosphorylation of p38-MAPK. Furthermore, p38-MAPK was activated when a phosphodiesterase inhibitor or a selective PKA activator was used. Overall, our results suggest that levosimendan regulates the wiring of the natural salvaging pathways to execute the prosurvival signals. This network includes Akt, ERK1/2 and p38-MAPK. Opening of mitochondrial K(ATP) channels and the subsequent production of oxygen free radicals, the epidermal growth factor receptor/Src, and the cAMP/PKA pathways seem to mediate this response.

Non-genomic effects of thyroid hormone in adult cardiac myocytes: relevance to gene expression and cell growth.

Besides the well-characterized genomic action of thyroid hormone (TH), mediated by thyroid hormone receptors (TRs), accumulating data support the so-called non-genomic action of TH, which is often related to activation of signalling pathways. In this study, we sought to determine whether TH activates intracellular signalling pathways in the adult cardiac myocytes and whether such activation modulates cell growth and the expression of target proteins important in cardiac function. We demonstrate that TH promotes a rapid increase in the phosphorylation of several kinases, ERK1/2, PKCdelta, p38-MAPK and Akt. This activation is inhibited by triiodothyroacetic acid (triac), which is a TH analogue known to displace the hormone from membrane bound receptors, indicating that this TH effect is mediated through a cell membrane-initiated mechanism. Furthermore, using specific inhibitors of the TH-activated kinases, we show that the long-term effects of TH on the expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), alpha- and beta-myosin heavy chain (MHC) and cell growth are reverted, implying that what is initiated as a non-genomic action of the hormone interfaces with genomic effects. These data provide further insights into the underlying mechanisms of TH action in the heart with potentially important implications in the management of cardiac pathology.

Regulation of Bcl-2 phosphorylation in response to oxidative stress in cardiac myocytes.

Oxidative stress promotes cardiac myocyte death and has been implicated in the pathogenesis of many cardiovascular diseases. Bcl-2 family proteins are key regulators of the apoptotic response, while their functions can be regulated by post-transcriptional modifications including phosphorylation, dimerization or proteolytic cleavage. This study used adult cardiac myocytes to test the hypothesis that activation of specific kinase signalling pathways by oxidative stress may modulate either the expression or the phosphorylation of Bcl-2, with the resulting effect of a decrease or increase in its anti-apoptotic function. Stimulation of cardiac myocytes with 0.2 mM H(2)O(2), which induces apoptosis, resulted in a marked down-regulation of Bcl-2 protein simultaneously with an increase in its phosphorylation. Inhibition of p38-MAPK resulted in attenuation of Bcl-2 phosphorylation, whereas inhibition of ERK1/2, JNKs or PI-3-K had no effect. These data suggest that activation of p38 MAPK by oxidative stress results in the phosphorylation and degradation of Bcl-2 and the inactivation of its anti-apoptotic activity.

Differential roles of MAPKs and MSK1 signalling pathways in the regulation of c-Jun during phenylephrine-induced cardiac myocyte hypertrophy.

Gq-protein-coupled receptor (GqPCR) signalling is associated with the induction of cardiac myocyte hypertrophy, which is characterized by an increase in expression of immediate early genes via activation of pre-existing transcription factors. Here, we explore the role of MSK1 and MAPK signalling pathways in the regulation of the immediate early gene c-jun. The results provide further support for the role of MSK1 in cardiac myocyte hypertrophy and indicate that PE activates distinct signalling mechanisms which culminate with a complex activation of c-jun. ERK1/2 and JNKs are the principal kinases responsible for phosphorylation of c-Jun, whereas c-jun mRNA and protein up-regulation by PE is mediated by multiple signalling pathways that include MSK1, ERK1/2, p38-MAPK and JNKs. These signalling mechanisms seem to be critical to the phenotypic changes of cardiac myocytes in response to hypertrophic stimulation.

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.