Hexavalent chromium-induced erythrocyte membrane phospholipid asymmetry

Hexavalent (VI) chromium is a global contaminant with cytotoxic activity. Chromium (VI) induces oxidative stress, inflammation, cell proliferation, malignant transformation and may trigger carcinogenesis and at the same time apoptosis. The toxic effects of chromium (VI) at least partially result from mitochondrial injury and DNA damage. Erythrocytes lack mitochondria and nuclei but may experience an apoptosis-like suicidal cell death, i.e. eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptosis may result from increase of cytosolic Ca²⁺ activity, ATP depletion and/or ceramide formation. The present study explored, whether chromium (VI) triggers eryptosis. Fluo-3-fluorescence was employed to determine cytosolic Ca²⁺-concentration, forward scatter to estimate cell volume, binding of fluorescent annexin V to detect phosphatidylserine exposure, hemoglobin concentration in the supernatant to quantify hemolysis, luciferin–luciferase to determine cytosolic ATP concentration and fluorescent anti-ceramide antibodies to uncover ceramide formation. A 48 h exposure to chromium (VI) (≥10 μM) significantly increased cytosolic Ca²⁺-concentration, decreased ATP concentration (20 μM), decreased forward scatter, increased annexin V-binding and increased (albeit to a much smaller extent) hemolysis. Chromium (VI) did not significantly modify ceramide formation. The effect of 20 μM chromium (VI) on annexin V binding was partially reversed in the nominal absence of Ca²⁺. The present observations disclose a novel effect of chromium (VI), i.e. Ca²⁺ entry and cytosolic ATP depletion in erythrocytes, effects resulting in eryptosis with cell shrinkage and cell membrane scrambling.     

The Role of Ca2+ on the DADS-induced Apoptosis in Mouse–Rat Hybrid Retina Ganglion Cells (N18)

Diallyl disulfide (DADS), a component of garlic, has been shown to induce growth inhibition and apoptosis in human cancer cell types. The present studies were designed to investigate the effects of DADS on mouse–rat hybrid retina ganglion cells (N18) to better understand its effect on apoptosis and apoptosis-related genes in vitro. Cell viability, cell cycle analysis, reactive oxygen species (ROS), Ca²⁺ production, mitochondria membrane potential, apoptosis induction, associated gene expression and caspases-3 activity were examined by flow cytometric assay and/or Western blot. After 24-h treatment with DADS, a dose- and time-dependent decrease in the viability of N18 cells was observed and the approximate IC₅₀ was 27.6 μM. The decreased percentage of viable cells are associated with the production of ROS then followed by the production of Ca²⁺ which is induced by DADS. DADS induced apoptosis in N18 cells via the activation of caspase-3. DADS increased the protein levels of p53, cytochrome c and phosphated JNK within 24 h of treatment and it decreased the levels of Bcl-2 and those factors may have led to the mitochondria depolarization of N18 cells. DADS induced apoptosis were accompanied by increased levels of Ca²⁺ and decreased mitochondrial membrane potential which then led to release the cytochrome c, cleavage of pro-caspase-3. Deleted levels of Ca²⁺ by BAPTA-AM 10 μM (intracellular calcium chelator) then led to decrease DADS-induced apoptosis. Inhibition of caspase-3 activation by inhibitor (z-VAD-fmk) completely blocked DADS-induced apoptosis on N18 cells. The results indicated that oxidative stress modulates cell proliferation and Ca²⁺ modulates the cell death induced by DADS.     

Early administration of nifedipine protects against angiotensin II‐induced cardiomyocyte hypertrophy through regulating CaMKII–SERCA2a pathway and apoptosis in rat cardiomyocytes

The calcium channel blocker (CCB), nifedipine, is a more effective treatment for early‐ than late‐stage cardiac hypertrophy. We investigated the effects of early‐ and late‐stage nifedipine administration on calcium homeostasis, CaMKII (Ca²⁺/calmodulin‐dependent protein kinase II) activity and apoptosis of cardiomyocytes under hypertrophic stimulation with angiotensin II (AngII). Primary rat cardiomyocytes were divided into five treatment groups: AK, AngII plus the CaMKII inhibitor, KN‐93; AN‐1 (early‐stage), AngII plus nifedipine × 48 h; AN‐2 (late‐stage), AngII × 48 h, then AngII plus nifedipine × 48 h; C, untreated; and A, AngII × 48 h. The t1/2β [time required for intracellular Ca²⁺ concentration ([Ca²⁺]i) to decline to one half of the peak value] decreased; however, CaMKII and SERCA2a (sarcoplasmic reticulum Ca²⁺‐ATPase 2a) activities increased in the AN‐1 group compared with the AK group. In the AN‐2 group compared with the AN‐1 group, CaMKII activity, t1/2α [time required for [Ca²⁺]i to increase from the bottom to one half of peak value], t1/2β, and apoptosis increased. These results indicate that the timing of CCB administration affects the calcium concentration and apoptosis of hypertrophic cardiomyocytes through the CaMKII–SERCA2a signalling pathway, thereby influencing the drug's protective activity against cardiomyocyte hypertrophy. Copyright © 2016 John Wiley & Sons, Ltd.     

Enhancement of hyperthermia-induced apoptosis by a new synthesized class of benzocycloalkene compounds

The aim of this study was to examine whether, a new synthesized class of benzocycloalkene derivatives (BCs), enhances apoptosis induced by hyperthermia. The combined effects of hyperthermia (44°C, 20 min) and BCs on apoptosis in human lymphoma U937 cells were investigated. Among the tested compounds (BC1 ∼ 9), the combined treatment of 10 μM BC2 or BC4 and hyperthermia showed the largest potency to induce DNA fragmentation at 6 h after hyperthermia. And enhancement of hyperthermia-induced apoptosis by BC2 or BC4 in a dose-dependent manner was observed. When the cells were treated first with BC2 or BC4 at a nontoxic concentration of 20 μM, and exposed to hyperthermia afterwards, a significant enhancement of heat-induced apoptosis was evidenced by DNA fragmentation, morphological changes and phosphatidylserine externalization. Flow cytometry revealed an increase of intracellular superoxide due to BC2 or BC4, which was further increased when hyperthermia was combined. Mitochondrial membrane potential was decreased and the activation of caspase-3 and caspase-8 was enhanced in the cells treated with the combination. The activation of Bid, but no change of Bax and Bcl-2 were observed after the combined treatment. The release of cytochrome c from mitochondria to cytosol, which was induced by hyperthermia, was enhanced by BC2 or BC4. An increase in the intracellular Ca²⁺ concentration [Ca²⁺]_i, externalization of Fas, and decrease in Hsp70 were observed following the combined treatment. These results indicate that the intracellular superoxide generated by BC2 or BC4 is involved in the enhancement of apoptosis through Fas-mitochondria caspase and [Ca²⁺]_i-dependent pathways, and a decrease in Hsp70 also contributed to the enhancement of apoptosis.     

Thapsigargin,a selective inhibitor of sarco-endoplasmic reticulum Ca<Superscript>2+</Superscript>-ATPases,modulates nitric oxide production and cell death of primary rat hepatocytes in culture

Increased cytosolic calcium ([Ca²⁺] _i ) and nitric oxide (NO) are suggested to be associated with apoptosis that is a main feature of many liver diseases and is characterized by biochemical and morphological features. We sought to investigate the events of increase in [Ca²⁺] _i and endoplasmic reticulum (ER) calcium depletion by thapsigargin (TG), a selective inhibitor of sarco-ER-Ca²⁺-ATPases, in relation to NO production and apoptotic and necrotic markers of cell death in primary rat hepatocyte culture. Cultured hepatocytes were treated with TG (1 and 5 μmol/L) for 0–24 or 24–48 h. NO production and inducible NO synthase (iNOS) expression were determined as nitrite levels and by iNOS-specific antibody, respectively. Hepatocyte apoptosis was estimated by caspase-3 activity, cytosolic cytochrome c content and DNA fragmentation, and morphologically using Annexin-V/propidium iodide staining. Hepatocyte viability and mitochondrial activity were evaluated by ALT leakage and MTT test. Increasing basal [Ca²⁺] _i by TG, NO production and apoptotic/necrotic parameters were altered in different ways, depending on TG concentration and incubation time. During 0–24 h, TG dose-dependently decreased iNOS-mediated spontaneous NO production and simultaneously enhanced hepatocyte apoptosis. In addition, TG 5 μmol/L produced secondary necrosis. During 24–48 h, TG dose-dependently enhanced basal NO production and rate of necrosis. TG 5 μmol/L further promoted mitochondrial damage as demonstrated by cytochrome c release. A selective iNOS inhibitor, aminoguanidine, suppressed TG-stimulated NO production and ALT leakage from hepatocytes after 24–48 h. Our data suggest that the extent of the [Ca²⁺] _i increase and the modulation of NO production due to TG treatment contribute to hepatocyte apoptotic and/or necrotic events.     

Klotho attenuates isoproterenol-induced hypertrophic response in H9C2 cells by activating Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase and inhibiting the reverse mode of Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript>-exchanger

Cardiac hypertrophy plays a major role in heart failure and is related to patient morbidity and mortality. Calcium overloading is a main risk for cardiac hypertrophy, and Na⁺/K⁺-ATPase (NKA) has been found that it could not only regulate intracellular Na⁺ levels but also control the intracellular Ca²⁺ ([Ca²⁺]_i) level through Na⁺/Ca²⁺-exchanger (NCX). Recent studies have reported that klotho could affect [Ca²⁺]_i level. In this study, we aimed at exploring the role of klotho in improving isoproterenol-induced hypertrophic response of H9C2 cells. The H9C2 cells were randomly divided into control and isoproterenol (ISO) (10 μM) groups. Klotho protein (10 μg/ml) or NKAα2 siRNA was used to determine the changes in isoproterenol-induced hypertrophic response. The alterations of [Ca²⁺]_i level were measured by spectrofluorometry. Our results showed that H9C2 cells which were treated with isoproterenol presented a higher level of [Ca²⁺]_i and hypertrophic gene expression at 24 and 48 h compared with the control group. Moreover, the expressions of NKAα1 and NKAα2 were both increased in control and ISO groups after treating with klotho protein; meanwhile, the NKA activity was increased and NCX activity was decreased after treatment. Consistently, the [Ca²⁺]_i level and hypertrophic gene expression were decreased in ISO group after klotho protein treatment. However, these effects were both prevented by transfecting with NKAα2 siRNA. In conclusion, these findings demonstrated that klotho inhibits isoproterenol-induced hypertrophic response in H9C2 cells by activating NKA and inhibiting the reverse mode of NCX and this effect may be associated with the upregulation of NKAα2 expression.     

Correlation between oxidative Stress and Alteration of Intracellular Calcium Handling in Isoproterenol-Induced Myocardial Infarction

Myocardial Ca²⁺ overload and oxidative stress are well documented effects associated to isoproterenol (ISO)-induced myocardial necrosis, but information correlating these two issues is scarce. Using an ISO-induced myocardial infarction model, 3 stages of myocardial damage were defined: pre-infarction (0–12 h), infarction (12–24 h) and post-infarction (24–96 h). Alterations in Ca²⁺ homeostasis and oxidative stress were studied in mitochondria, sarcoplasmic reticulum and plasmalemma by measuring the Ca²⁺ content, the activity of Ca²⁺ handling proteins, and by quantifying TBARs, nitric oxide (NO) and oxidative protein damage (changes in carbonyl and thiol groups). Free radicals generated system, antioxidant enzymes and oxidative stress (GSH/GSSG ratio) were also monitored at different times of ISO-induced cardiotoxicity. The Ca²⁺ overload induced by ISO was counterbalanced by a diminution in the ryanodine receptor activity and the Na⁺-Ca⁺² exchanger as well as by the increase in both calcium ATPases activities (vanadate- and thapsigargine-sensitive) and mitochondrial Ca²⁺ uptake during pre-infarction and infarction stages. Pro-oxidative reactions and antioxidant defences during the 3 stages of cardiotoxicity were observed, with maximal oxidative stress during the infarction. Significant correlations were found among pro-oxidative reactions with plasmalemma and sarcoplasmic reticulum Ca²⁺ ATPases, and ryanodine receptor activities at the onset and development of ISO-induced infarction. These findings could be helpful in the design of antioxidant therapies in this pathology.     

Endophilin A2-mediated alleviation of endoplasmic reticulum stress-induced cardiac injury involves the suppression of ERO1α/IP3R signaling pathway

Cardiac injury upon myocardial infarction (MI) is the leading cause of heart failure. The present study aims to investigate the role of EndoA2 in ischemia-induced cardiomyocyte apoptosis and cardiac injury. In vivo, we established an MI mouse model by ligating the left anterior descending (LAD) coronary artery, and intramyocardial injection of adenoviral EndoA2 (Ad-EndoA2) was used to overexpress EndoA2. In vitro, we used the siRNA and Ad-EndoA2 transfection strategies. Here, we reported that EndoA2 expression was remarkably elevated in the infarct border zone of MI mouse hearts and neonatal rat cardiomyocytes (NRCMs) stimulated with oxygen and glucose deprivation (OGD) which mimicked ischemia. We showed that intramyocardial injection of Ad-EndoA2 attenuated cardiomyocyte apoptosis and reduced endoplasmic reticulum (ER) stress in response to MI injury. Using siRNA for knockdown and Ad-EndoA2 for overexpression, we validated that knockdown of EndoA2 in NRCMs exacerbated OGD-induced NRCM apoptosis, whereas overexpression of EndoA2 attenuates OGD-induced cardiomyocyte apoptosis. Mechanistically, knockdown of EndoA2 activated ER stress response, which increases ER oxidoreductase 1α (ERO1α) and inositol 1, 4, 5-trisphosphate receptor (IP₃R) activity, thus led to increased intracellular Ca²⁺ accumulation, followed by elevated calcineurin activity and nuclear factor of activated T-cells (NFAT) dephosphorylation. Pretreatment with the IP₃R inhibitor 2-Aminoethoxydiphenylborate (2-APB) attenuated intracellular Ca²⁺ accumulation, and pretreatment with the Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) or the calcineurin inhibitor Cyclosporin A (CsA) inhibited EndoA2-knockdown-induced NRCM apoptosis. Overexpression of EndoA2 led to the opposite effects by suppressing ER-stress-mediated ERO1α/IP₃R signaling pathway. This study demonstrated that EndoA2 protected cardiac function in response to MI via attenuating ER-stress-mediated ERO1α/IP₃R signaling pathway. Targeting EndoA2 is a potential therapeutic strategy for the prevention of postinfarction-induced cardiac injury and heart failure.     

Activation of CaMKII via ER‐stress mediates coxsackievirus B3‐induced cardiomyocyte apoptosis

Cardiomyocyte apoptosis contributes to the development of coxsackievirus B3 (CVB3)‐induced myocarditis, but the mechanism for the apoptosis by CVB3 infection remains unclear. Here, we showed that CVB3‐induced endoplasmic reticulum (ER) stress response and apoptosis in cultured H9c2 cardiomyocytes. We found that Ca²⁺‐calmodulin‐dependent kinase II (CaMKII) was activated by ER stress‐dependent intracellular Ca²⁺ overload in the CVB3‐infected H9c2 cardiomyocytes. Treatment with an inhibitor of ER stress, 4‐phenylbutyric acid (4‐PBA), attenuated intracellular Ca²⁺ accumulation indirectly and reduced CaMKII activity. Inhibition of CaMKII with pharmacological inhibitor (KN‐93) or short hairpin RNA reduced CVB3‐induced H9c2 apoptosis and repressed cytochrome c release from mitochondria to cytoplasm; whereas overexpression of the activated mutant of CaMKII (CaMKII‐T287D) enhanced CVB3‐induced H9c2 apoptosis and mitochondrial cytochrome c release, which could be alleviated by blocking of mitochondrial Ca²⁺ uniporter or mitochondrial permeability transition pore. Further in vivo investigation revealed that blocking of CaMKII with KN‐93 prevented cardiomyocytes apoptosis and improved cardiac contractile function in CVB3‐infected mouse heart. Collectively, these findings provide a novel evidence that CaMKII plays a vital role in the promotion of CVB3‐induced cardiomyocyte apoptosis, which links ER stress and mitochondrial Ca²⁺ uptake.     

Antioxidant metallothionein alleviates endoplasmic reticulum stress-induced myocardial apoptosis and contractile dysfunction

Abstract

Aims. Endoplasmic reticulum (ER) stress exerts myocardial oxidative stress, apoptosis, and contractile anomalies, although the precise interplay between ER stress and apoptosis remains elusive. This study was designed to examine the impact of the cysteine-rich free radical scavenger metallothionein on ER stress-induced myocardial contractile defect and underlying mechanisms. Methods and results. Wild-type friendly virus B and transgenic mice with cardiac-specific overexpression of metallothionein were challenged with the ER stress inducer tunicamycin (1 mg/kg, intraperitoneal, 48 h) prior to the assessment of myocardial function, oxidative stress, and apoptosis. Our results revealed that tunicamycin promoted cardiac remodeling (enlarged left ventricular end systolic/diastolic diameters with little changes in left ventricular wall thickness), suppressed fractional shortening and cardiomyocyte contractile function, elevated resting Ca²⁺, decreased stimulated Ca²⁺ release, prolonged intracellular Ca²⁺ clearance, and downregulated sarco(endo)plasmic reticulum Ca²⁺-ATPase levels, the effects of which were negated by metallothionein. Treatment with tunicamycin caused cardiomyocyte mitochondrial injury, as evidenced by decreased mitochondrial membrane potential (??m, assessed by JC-1 staining), the effect of which was negated by the antioxidant. Moreover, tunicamycin challenge dramatically facilitated myocardial apoptosis as manifested by increased Bax, caspase 9, and caspase 12 protein levels, as well as elevated caspase 3 activity. Interestingly, metallothionein transgene significantly alleviated tunicamycin-induced myocardial apoptosis. Conclusion. Taken together, our data favor a beneficial effect of metallothionein against ER stress-induced cardiac dysfunction possibly associated with attenuation of myocardial apoptosis.     

Lanthanum chloride suppresses oxysterol-induced ECV-304 cell apoptosis via inhibition of intracellular Ca<Superscript>2+</Superscript> concentration elevation,oxidative stress,and activation of ERK and NF-κB signaling pathways

Experimental studies have demonstrated that oral administration of lanthanum chloride (LaCl₃) inhibits the development of atherosclerosis, but the related mechanism has not been fully elucidated. Oxysterols are toxic to the vascular endothelial cells which are important in preventing the formation and progression of atheromatous plaque. In this study, we examined the effect of LaCl₃ on oxysterol cholestane-3β,5α,6β-triol (Triol)-induced apoptosis and the related mechanisms in ECV-304 cells, a presumptive endothelial cell line. Incubation with Triol resulted in apoptosis of ECV-304 cells, as determined by Hoechst 33342 staining, fluorescein isothiocyanate labeled annexin V/propidium iodide double staining, and the loss of mitochondrial membrane potential. Triol activated extracellular-signal-regulated kinase (ERK) and nuclear factor κB (NF-κB), and inhibition of Triol-activated ERK and NF-κB signaling by specific inhibitors attenuated apoptosis induction by Triol in ECV-304 cells. Pretreatment with LaCl₃ (1 μM) for 12 h before exposure to Triol decreased Triol-mediated apoptosis as well as activation of ERK and NF-κB. In addition, Triol induced oxidative stress in ECV-304 cells, manifested by the increase of intracellular reactive oxygen species generation and malondialdehyde level, and the reduction of the content of total protein thiols and the activity of antioxidant glutathione peroxidases; LaCl₃ pretreatment significantly reversed these effects. Finally, LaCl₃ pretreatment significantly inhibited the increases of intracellular Ca²⁺ concentration induced by Triol. Our study suggests that Triol induced ECV-304 cell apoptosis, and LaCl₃ could suppress this effect probably by inhibiting intracellular Ca²⁺ concentration elevation, oxidative stress, as well as activation of ERK and NF-κB signaling pathways.     

Development of antithrombotic miniribozymes that target peripheral tryptophan hydroxylase

Transient receptor potential (TRP) proteins have been identified as cation channels that are activated by agonist–receptor coupling and mediate various cellular functions. TRPC7, a homologue of TRP channels, has been shown to act as a Ca²⁺ channel activated by G protein-coupled stimulation and to be abundantly expressed in the heart with an as-yet-unknown function. We studied the role of TRPC7 in G protein-activated signaling in HEK293 cells and cultured cardiomyocytes in vitro transfected with FLAG-tagged TRPC7 cDNA and in Dahl salt-sensitive rats with heart failure in vivo. TRPC7-transfected HEK293 cells showed an augmentation of carbachol-induced intracellular Ca²⁺ transient, which was attenuated under a Ca²⁺-free condition or in the presence of SK&F96365 (a Ca²⁺-permeable channel blocker). Upon stimulation with angiotensin II (Ang II), cultured neonatal rat cardiomyocytes transfected with TRPC7 exhibited a significant increase in apoptosis detected by TUNEL staining, accompanied with a decrease in the expression of atrial natriuretic factor and destruction of actin fibers, as compared with non-transfected cardiomyocytes. Ang II-induced apoptosis was inhibited by CV-11974 (Candesartan; Ang II type 1 [AT1] receptor blocker), SK&F96365, and FK506 (calcineurin inhibitor). In Dahl salt-sensitive rats, apoptosis and TRPC7 expression were increased in the failing myocardium, and a long-term treatment with temocapril, an angiotensin-converting enzyme inhibitor, suppressed both. Our findings suggest that TRPC7 could act as a Ca²⁺ channel activated by AT1 receptors, leading to myocardial apoptosis possibly via a calcineurin-dependent pathway. TRPC7 might be a key initiator linking AT1-activation to myocardial apoptosis, and thereby contributing to the process of heart failure.     

Oxidative stress as a component of chromium-induced cytotoxicity in rat calvarial osteoblasts

It has been documented that medical prosthetic alloys release metal ions into surrounding tissues and cause cytotoxicity, but the mechanisms remain undefined. In that regard the cellular oxidative stress may be a common pathway in cellular responses to metal ions. The objective of this study was to approach the hypothesis that oxidative stress mediates chromium-induced cytotoxicity in rat calvarial osteoblasts. Osteoblasts were exposed to different concentrations of Cr⁶⁺ or Cr³⁺ (5–20 μM) in the presence or absence of the antioxidant N-acetyl-cysteine (NAC; 1–5 mM). Cellular viability, differentiation, and intracellular ultrastructural alterations were evaluated by MTT assay, alkaline phosphatase (ALP) activity assay, and transmission electron microscopy. Cellular oxidative stress was evaluated by intracellular reactive oxygen species (ROS) production. ROS production was monitored by the oxidation-sensitive fluorescent probe 2′7′-dichlorofluorescin diacetate (DCFH-DA). A time- and concentration- dependent increased cytotoxicity, time-dependent increased intracellular ROS production were indicated on exposure to Cr⁶⁺. Pretreatment of osteoblasts with 1–5 mM NAC afforded dose-dependent cytoprotective effects against Cr⁶⁺-induced cytotoxicity in osteoblasts. NAC decreased the level of intracellular ROS induced by Cr⁶⁺, too. While Cr³⁺ and NAC did not have any significant effects on osteoblasts (5–20 μM). These results suggest that oxidative stress is involved in Cr⁶⁺-induced cytotoxicity in osteoblasts, and NAC can provide protection for osteoblasts against Cr⁶⁺-induced oxidative stress. Cr³⁺ (5–20 μM) have no significant cytotoxicity in osteoblasts based on the results of this study.     

Norepinephrine regulates the in vivo expression of the L-type calcium channel

The _1c subunit (DHP receptor) of the L-type Ca²⁺ channel is important for calcium homeostasis in cardiac muscle. The DHPr provides the primary mechanism for calcium influx during contraction. Published results demonstrate three in vitro signaling pathways that are important in the regulation of DHPr gene expression in neonatal cardiac myocytes, the protein kinase A (PKA), protein kinase C (PKC) pathways, and intracellular calcium. To determine whether these pathways are important in vivo, we treated adult rats with infusions of isoproterenol, or norepinephrine at 200 g/kg/h and assessed DHPr mRNA and protein levels. Following a 3-day infusion isoproterenol (ISO) and norepinephrine (NE) produced a small but insignificant reduction in DHPr mRNA levels. When the infusions were continued for 7 days isoproterenol increased DHPr mRNA accumulation to control levels while NE stimulated a 35% increase in DHPr mRNA levels and a 35% increase in protein abundance when compared to controls (p < 0.05). Furthermore, contractility and Ca²⁺ transient measurements of isolated cardiac myocytes from NE infused animals also display shortened duration of contraction/relaxation and increased intracellular free Ca²⁺ (DFFI) in response to electrical stimulation (p < 0.01). We conclude norepinephrine treatment alters DHPr mRNA and protein levels, and augments excitation-contraction coupling, and thus may be important for modulating cardiac calcium homeostasis in vivo.     

Nicotine mediates oxidative stress and apoptosis through cross talk between NOX1 and Bcl-2 in lung epithelial cells

Nicotine contributes to the onset and progression of several pulmonary diseases. Among the various pathophysiological mechanisms triggered by nicotine, oxidative stress and cell death are reported in several cell types. We found that chronic exposure to nicotine (48 h) induced NOX1-dependent oxidative stress and apoptosis in primary pulmonary cells. In murine (MLE-12) and human (BEAS-2B) lung epithelial cell lines, nicotine acted as a sensitizer to cell death and synergistically enhanced apoptosis when cells were concomitantly exposed to hyperoxia. The precise signaling pathway was investigated in MLE-12 cells in which NOX1 was abrogated by a specific inhibitor or stably silenced by shRNA. In the early phase of exposure (1 h), nicotine mediated intracellular Ca²⁺ fluxes and activation of protein kinase C, which in its turn activated NOX1, leading to cellular and mitochondrial oxidative stress. The latter triggered the intrinsic apoptotic machinery by modulating the expression of Bcl-2 and Bax. Overexpression of Bcl-2 completely prevented nicotine’s detrimental effects, suggesting Bcl-2 as a downstream key regulator in nicotine/NOX1-induced cell damage. These results suggest that NOX1 is a major contributor to the generation of intracellular oxidative stress induced by nicotine and might be an important molecule to target in nicotine-related lung pathologies.     

Penicillin V production by Penicillium chrysogenum in the presence of Fe3+ and in low-iron culture medium

Late-exponential-phasePenicillium chrysogenum mycelia grown in a complex medium possessed an intracellular iron concentration of 650 μmol/L (2.2±0.6 μmol per g mycelial dry mass). This iron reserve was sufficient to ensure growth and antibiotic production after transferring mycelia into a defined low-iron minimal medium. Although the addition of Fe³⁺ to the Fe-limited cultures increased significantly the intracellular iron levels the surplus iron did not influence the production of penicillin V. Supplements of purified majorP. chrysogenum siderophores (coprogen and ferrichrome) into the fermentation media did not affect the β-lactam production and intracellular iron level. Neither 150 nor 300 μmol/L extracellular Fe³⁺ concentrations disturbed the glutathione metabolism of the fungus, and increased the oxidative stress caused by 700 mmol/L H₂O₂. Nevertheless, when iron was applied in the Fe^II oxidation state the oxidative cell injuries caused by the peroxide were significantly enhanced.     

Calcineurin suppresses AMPK-dependent cytoprotective autophagy in cardiomyocytes under oxidative stress

Calcineurin signalling plays a critical role in the pathogenesis of many cardiovascular diseases. Calcineurin has been proven to affect a series of signalling pathways and to exert a proapoptotic effect in cardiomyocytes. However, whether it is able to regulate autophagy remains largely unknown. Here, we report that prolonged oxidative stress-induced activation of calcineurin contributes to the attenuation of adaptive AMP-activated protein kinase (AMPK) signalling and inhibits autophagy in cardiomyocytes. Primary cardiomyocytes exhibited rapid formation of autophagosomes, microtubule-associated protein 1 light chain 3 (LC3) expression and phosphorylation of AMPK in response to hydrogen peroxide (H₂O₂) treatment. However, prolonged (12 h) H₂O₂ treatment attenuated these effects and was accompanied by a significant increase in calcineurin activity and apoptosis. Inhibition of calcineurin by FK506 restored AMPK function and LC3 expression, and decreased the extent of apoptosis caused by prolonged oxidative stress. In contrast, overexpression of the constitutively active form of calcineurin markedly attenuated the increase in LC3 induced by short-term (3 h) H₂O₂ treatment and sensitised cells to apoptosis. In addition, FK506 failed to induce autophagy and alleviate apoptosis in cardiomyocytes expressing a kinase-dead K45R AMPK mutant. Furthermore, inhibition of autophagy by 3-methylanine (3-MA) or by knockdown of the essential autophagy-related gene ATG7 abrogated the protective effect of FK506. These findings suggest a novel role of calcineurin in suppressing adaptive autophagy during oxidative stress by downregulating the AMPK signalling pathway. The results also provide insight into how altered calcineurin and autophagic signalling is integrated to control cell survival during oxidative stress and may guide strategies to prevent cardiac oxidative damage.     

Insulin inhibits β-adrenergic action in ischemic/reperfused heart: a novel mechanism of insulin in cardioprotection

Objective Sympathetic overactivity is closely connected with cell injury and contractile dysfunction during myocardial ischemia/reperfusion (MI/R). Insulin exerts protection for the I/R heart and the underlying mechanisms remain unclear. This study aimed to investigate the ability of insulin to modulate β-adrenergic actions on myocardial contraction and post-ischemic injury in acute MI/R and the underlying mechanism. Methods Isolated hearts from adult SD rats were subjected to MI/R (30 min/2 h) and treated with isoproterenol (ISO) or/and insulin. Myocardial contraction, cardiomyocyte apoptosis, myocardial injury and infarction were assessed. In a separate study, isolated ventricular myocytes were subjected to simulated I/R (15/30 min) and myocyte shortening and intracellular Ca²⁺ transient in response to ISO during reperfusion were assessed with presence or absence of insulin. Results In isolated I/R hearts, insulin largely reversed the ISO-associated contractile functional impairment at 2 h after MI/R, inhibiting ISO-induced declines in heart rate and left ventricular systolic pressure by 34.0% and 23.0% and preventing ISO-induced elevation in left ventricular end-diastolic pressure by 28.7% respectively (all P < 0.05). In addition, ISO alone resulted in enlarged infarct size, elevated CK and LDH activity and increased apoptotic index in I/R hearts compared with vehicle, which were inhibited by treatment of insulin (all P < 0.05). Interestingly, in SI/R cardiomyocytes, insulin alone at 10⁻⁷ mol/l increased cell contraction whereas attenuated the positive inotropic response to ISO (10⁻⁹ mol/l) during R as evidenced by a 18.7% reduction in peak twitch amplitude and a 23.9% reduction in calcium transient amplitude (both P < 0.05). Moreover, insulin blunted ISO-mediated increase in PKA activity, enhanced the PKA-dependent phosphorylation of phospholamban (PLB), resulting in increased sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a) activity. Conclusion Insulin attenuated the contractile response to β-AR stimulation and suppressed ISO-elicited cardiac dysfunction and cell injury in MI/R. The inhibitory effect of insulin on the β-adrenergic action involved the inhibition of PKA-mediated Ca²⁺ transient and promotion of post-ischemic Ca²⁺ handling.     

Autonomous activation of CaMKII exacerbates diastolic calcium leak during beta-adrenergic stimulation in cardiomyocytes of metabolic syndrome rats

Autonomous Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation induces abnormal diastolic Ca²⁺ leak, which leads to triggered arrhythmias in a wide range of cardiovascular diseases, including diabetic cardiomyopathy. In hyperglycemia, Ca²⁺ handling alterations can be aggravated under stress conditions via the β-adrenergic signaling pathway, which also involves CaMKII activation. However, little is known about intracellular Ca²⁺ handling disturbances under β-adrenergic stimulation in cardiomyocytes of the prediabetic metabolic syndrome (MetS) model with obesity, and the participation of CaMKII in these alterations.MetS was induced in male Wistar rats by administering 30 % sucrose in drinking water for 16 weeks. Fluo 3-loaded MetS cardiomyocytes exhibited augmented diastolic Ca²⁺ leak (in the form of spontaneous Ca²⁺ waves) under basal conditions and that Ca²⁺ leakage was exacerbated by isoproterenol (ISO, 100 nM). At the molecular level, [³H]-ryanodine binding and basal phosphorylation of cardiac ryanodine receptor (RyR2) at Ser2814, a CaMKII site, were increased in heart homogenates of MetS rats with no changes in RyR2 expression. These alterations were not further augmented by Isoproterenol. SERCA pump activity was augmented 48 % in MetS hearts before β-adrenergic stimuli, which is associated to augmented PLN phosphorylation at T17, a target of CaMKII. In MetS hearts. CaMKII auto-phosphorylation (T287) was increased by 80 %. The augmented diastolic Ca²⁺ leak was prevented by CaMKII inhibition with AIP. In conclusion, CaMKII autonomous activation in cardiomyocytes of MetS rats with central obesity significantly contributes to abnormal diastolic Ca²⁺ leak, increasing the propensity for β-adrenergic receptor-driven lethal arrhythmias.