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

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

     

Protein kinase C and preconditioning: role of the sarcoplasmic reticulum

Activation of protein kinase C (PKC) is cardioprotective, but the mechanism(s) by which PKC mediates protection is not fully understood. Inasmuch as PKC has been well documented to modulate sarcoplasmic reticulum (SR) Ca2+ and because altered SR Ca2+ handling during ischemia is involved in cardioprotection, we examined the role of PKC-mediated alterations of SR Ca2+ in cardioprotection. Using isolated adult rat ventricular myocytes, we found that addition of 1,2-dioctanoyl-sn-glycerol (DOG), to activate PKC under conditions that reduced myocyte death associated with simulated ischemia and reperfusion, also reduced SR Ca2+. Cell death was 57.9 +/- 2.9% and 47.3 +/- 1.8% in untreated and DOG-treated myocytes, respectively (P < 0.05). Using fura 2 fluorescence to monitor Ca2+ transients and caffeine-releasable SR Ca2+, we examined the effect of DOG on SR Ca2+. Caffeine-releasable SR Ca2+ was significantly reduced (by approximately 65%) after 10 min of DOG treatment compared with untreated myocytes (P < 0.05). From our examination of the mechanism by which PKC alters SR Ca2+, we present the novel finding that DOG treatment reduced the phosphorylation of phospholamban (PLB) at Ser16. This effect is mediated by PKC-epsilon, because a PKC-epsilon-selective inhibitory peptide blocked the DOG-mediated decrease in phosphorylation of PLB and abolished the DOG-induced reduction in caffeine-releasable SR Ca2+. Using immunoprecipitation, we further demonstrated that DOG increased the association between protein phosphatase 1 and PLB. These data suggest that activated PKC-epsilon reduces SR Ca2+ content through PLB dephosphorylation and that reduced SR Ca2+ may be important in cardioprotection.     

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

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

Altered Spatiotemporal Dynamics of the Mitochondrial Membrane Potential in the Hypertrophied Heart

Chronically elevated levels of oxidative stress resulting from increased production and/or impaired scavenging of reactive oxygen species are a hallmark of mitochondrial dysfunction in left ventricular hypertrophy. Recently, oscillations of the mitochondrial membrane potential (ΔΨ_m) were mechanistically linked to changes in cellular excitability under conditions of acute oxidative stress produced by laser-induced photooxidation of cardiac myocytes in vitro. Here, we investigate the spatiotemporal dynamics of ΔΨ_m within the intact heart during ischemia-reperfusion injury. We hypothesize that altered metabolic properties in left ventricular hypertrophy modulate ΔΨ_m spatiotemporal properties and arrhythmia propensity.     

Pyridostigmine improves cardiac function and rhythmicity through RyR2 stabilization and inhibition of STIM1-mediated calcium entry in heart failure

Heart failure (HF) is characterized by asymmetrical autonomic balance. Treatments to restore parasympathetic activity in human heart failure trials have shown beneficial effects. However, mechanisms of parasympathetic-mediated improvement in cardiac function remain unclear. The present study examined the effects and underpinning mechanisms of chronic treatment with the cholinesterase inhibitor, pyridostigmine (PYR), in pressure overload HF induced by transverse aortic constriction (TAC) in mice. TAC mice exhibited characteristic adverse structural (left ventricular hypertrophy) and functional remodelling (reduced ejection fraction, altered myocyte calcium (Ca) handling, increased arrhythmogenesis) with enhanced predisposition to arrhythmogenic aberrant sarcoplasmic reticulum (SR) Ca release, cardiac ryanodine receptor (RyR2) hyper-phosphorylation and up-regulated store-operated Ca entry (SOCE). PYR treatment resulted in improved cardiac contractile performance and rhythmic activity relative to untreated TAC mice. Chronic PYR treatment inhibited altered intracellular Ca handling by alleviating aberrant Ca release and diminishing pathologically enhanced SOCE in TAC myocytes. At the molecular level, these PYR-induced changes in Ca handling were associated with reductions of pathologically enhanced phosphorylation of RyR2 serine-2814 and STIM1 expression in HF myocytes. These results suggest that chronic cholinergic augmentation alleviates HF via normalization of both canonical RyR2-mediated SR Ca release and non-canonical hypertrophic Ca signaling via STIM1-dependent SOCE.     

Increased expression of SERCA in the hearts of transgenic mice results in increased oxidation of glucose

While several transgenic mouse models exhibit improved contractile characteristics in the heart, less is known about how these changes influence energy metabolism, specifically the balance between carbohydrate and fatty acid oxidation. In the present study we examine glucose and fatty acid oxidation in transgenic mice, generated to overexpress sarco(endo)plasmic reticulum calcium-ATPase (SERCA), which have an enhanced contractile phenotype. Energy substrate metabolism was measured in isolated working hearts using radiolabeled glucose and palmitate. We also examined oxygen consumption to see whether SERCA overexpression is associated with increased oxygen utilization. Since SERCA is important in calcium handling within the cardiac myocyte, we examined cytosolic calcium transients in isolated myocytes using indo-1, and mitochondrial calcium levels using pericam, an adenovirally expressed, mitochondrially targeted ratiometric calcium indicator. Oxygen consumption did not differ between wild-type and SERCA groups; however, we were able to show an increased utilization of glucose for oxidative metabolism and a corresponding decreased utilization of fatty acids in the SERCA group. Cytosolic calcium transients were increased in myocytes isolated from SERCA mice, and they show a faster rate of decay of the calcium transient. With these observations we noted increased levels of mitochondrial calcium in the SERCA group, which was associated with an increase in the active form of the pyruvate dehydrogenase complex. Since an increase in mitochondrial calcium levels leads to activation of the pyruvate dehydrogenase complex (the rate-limiting step for carbohydrate oxidation), the increased glucose utilization observed in isolated perfused hearts in the SERCA group may reflect a higher level of mitochondrial calcium.     

The intracellular redox state is a core determinant of mitochondrial fusion

Mitochondrial hyperfusion has recently been shown to function as a cellular stress response, providing transient protection against apoptosis and mitophagy. However, the mechanisms that mediate this response remain poorly understood. In this study, we demonstrate that oxidized glutathione (GSSG), the core cellular stress indicator, strongly induces mitochondrial fusion. Biochemical and functional experiments show that GSSG induces the generation of disulphide‐mediated mitofusin oligomers, in a process that also requires GTP hydrolysis. Our data outline the molecular events that prime the fusion machinery, providing new insights into the coupling of mitochondrial fusion with the cellular stress response.     

Mitochondrial Safeguard: a stress response that offsets extreme fusion and protects respiratory function via flickering‐induced Oma1 activation

The connectivity of mitochondria is regulated by a balance between fusion and division. Many human diseases are associated with excessive mitochondrial connectivity due to impaired Drp1, a dynamin‐related GTPase that mediates division. Here, we report a mitochondrial stress response, named mitochondrial safeguard, that adjusts the balance of fusion and division in response to increased mitochondrial connectivity. In cells lacking Drp1, mitochondria undergo hyperfusion. However, hyperfusion does not completely connect mitochondria because Opa1 and mitofusin 1, two other dynamin‐related GTPases that mediate fusion, become proteolytically inactivated. Pharmacological and genetic experiments show that the activity of Oma1, a metalloprotease that cleaves Opa1, is regulated by short pulses of the membrane depolarization without affecting the overall membrane potential in Drp1‐knockout cells. Re‐activation of Opa1 and Mitofusin 1 in Drp1‐knockout cells further connects mitochondria beyond hyperfusion, termed extreme fusion, leading to bioenergetic deficits. These findings reveal an unforeseen safeguard mechanism that prevents extreme fusion of mitochondria, thereby maintaining mitochondrial function when the balance is shifted to excessive connectivity.     

Endoplasmic reticulum stress and mitochondrial dysfunction during aging: Role of sphingolipids

The endoplasmic reticulum (ER) plays a key role in the regulation of protein folding, lipid synthesis, calcium homeostasis, and serves as a primary site of sphingolipid biosynthesis. ER stress (ER dysfunction) participates in the development of mitochondrial dysfunction during aging. Mitochondria are in close contact with the ER through shared mitochondria associated membranes (MAM). Alteration of sphingolipids contributes to mitochondria-driven cell injury. Cardiolipin is a phospholipid that is critical to maintain enzyme activity in the electron transport chain. The aim of the current study was to characterize the changes in sphingolipids and cardiolipin in ER, MAM, and mitochondria during the progression of aging in young (3 mo.), middle (18 mo.), and aged (24 mo.) C57Bl/6 mouse hearts. ER stress increased in hearts from 18 mo. mice and mice exhibited mitochondrial dysfunction by 24 mo. Hearts were pooled to isolate ER, MAM, and subsarcolemmal mitochondria (SSM). LC-MS/MS quantification of lipid content showed that aging increased ceramide content in ER and MAM. In addition, the contents of sphingomyelin and monohexosylceramides are also increased in the ER from aged mice. Aging increased the total cardiolipin content in the ER. Aging did not alter the total cardiolipin content in mitochondria or MAM yet altered the composition of cardiolipin with aging in line with increased oxidative stress compared to young mice. These results indicate that alteration of sphingolipids can contribute to the ER stress and mitochondrial dysfunction that occurs during aging.     

Aerobic Interval Training Partly Reverse Contractile Dysfunction and Impaired Ca2+ Handling in Atrial Myocytes from Rats with Post Infarction Heart Failure

Background

There is limited knowledge about atrial myocyte Ca²⁺ handling in the failing hearts. The aim of this study was to examine atrial myocyte contractile function and Ca²⁺ handling in rats with post-infarction heart failure (HF) and to examine whether aerobic interval training could reverse a potential dysfunction.

Methods and results

Post-infarction HF was induced in Sprague Dawley rats by ligation of the left descending coronary artery. Atrial myocyte shortening was depressed (p<0.01) and time to relaxation was prolonged (p<0.01) in sedentary HF-rats compared to healthy controls. This was associated with decreased Ca²⁺ amplitude, decreased SR Ca²⁺ content, and slower Ca²⁺ transient decay. Atrial myocytes from HF-rats had reduced sarcoplasmic reticulum Ca²⁺ ATPase activity, increased Na⁺/Ca²⁺-exchanger activity and increased diastolic Ca²⁺ leak through ryanodine receptors. High intensity aerobic interval training in HF-rats restored atrial myocyte contractile function and reversed changes in atrial Ca²⁺ handling in HF.

Conclusion

Post infarction HF in rats causes profound impairment in atrial myocyte contractile function and Ca²⁺ handling. The observed dysfunction in atrial myocytes was partly reversed after aerobic interval training.     

A computational model of the human left-ventricular epicardial myocyte

A computational model of the human left-ventricular epicardial myocyte is presented. Models of each of the major ionic currents present in these cells are formulated and validated using experimental data obtained from studies of recombinant human ion channels and/or whole-cell recording from single myocytes isolated from human left-ventricular subepicardium. Continuous-time Markov chain models for the gating of the fast Na(+) current, transient outward current, rapid component of the delayed rectifier current, and the L-type calcium current are modified to represent human data at physiological temperature. A new model for the gating of the slow component of the delayed rectifier current is formulated and validated against experimental data. Properties of calcium handling and exchanger currents are altered to appropriately represent the dynamics of intracellular ion concentrations. The model is able to both reproduce and predict a wide range of behaviors observed experimentally including action potential morphology, ionic currents, intracellular calcium transients, frequency dependence of action-potential duration, Ca(2+)-frequency relations, and extrasystolic restitution/post-extrasystolic potentiation. The model therefore serves as a useful tool for investigating mechanisms of arrhythmia and consequences of drug-channel interactions in the human left-ventricular myocyte.     

Regulation of cardiac sarcoplasmic reticulum Ca release by luminal [Ca] and altered gating assessed with a mathematical model

Cardiac excitation-contraction coupling is initialized by the release of Ca from the sarcoplasmic reticulum (SR) in response to a sudden increase in local cytosolic [Ca] ([Ca]i) within the junctional cleft. We have tested the hypothesis that functional ryanodine receptor (RyR) regulation plays a major role in the regulation of myocyte Ca. A mathematical model with unique characteristics was used to simulate Ca homeostasis. Specifically, the model was designed to accurately represent the SR [Ca]-dependence of release from a variety of experimentally produced data sets. The simulated data for altered RyR Ca sensitivity demonstrated a regulatory feedback loop that resulted in the same release at lower [Ca]SR. This suggests that the primary role of myocyte RyR regulation may be to decrease SR [Ca] without decreasing the size of the [Ca]i transient. The model results suggest that this action moderates the increased SR [Ca] observed with adrenergic stimulation and may keep the [Ca]SR below the threshold for delayed afterdepolarizations and arrhythmia. However, increased Ca affinity of the RyR increased the probability of delayed afterdepolarizations when heart failure was simulated. We conclude that RyR regulation may play a role in preventing arrhythmias in healthy myocytes but that the same regulation may have the opposite effect in chronic heart failure.     

In Silico Screening of the Key Cellular Remodeling Targets in Chronic Atrial Fibrillation

Chronic atrial fibrillation (AF) is a complex disease with underlying changes in electrophysiology, calcium signaling and the structure of atrial myocytes. How these individual remodeling targets and their emergent interactions contribute to cell physiology in chronic AF is not well understood. To approach this problem, we performed in silico experiments in a computational model of the human atrial myocyte. The remodeled function of cellular components was based on a broad literature review of in vitro findings in chronic AF, and these were integrated into the model to define a cohort of virtual cells. Simulation results indicate that while the altered function of calcium and potassium ion channels alone causes a pronounced decrease in action potential duration, remodeling of intracellular calcium handling also has a substantial impact on the chronic AF phenotype. We additionally found that the reduction in amplitude of the calcium transient in chronic AF as compared to normal sinus rhythm is primarily due to the remodeling of calcium channel function, calcium handling and cellular geometry. Finally, we found that decreased electrical resistance of the membrane together with remodeled calcium handling synergistically decreased cellular excitability and the subsequent inducibility of repolarization abnormalities in the human atrial myocyte in chronic AF. We conclude that the presented results highlight the complexity of both intrinsic cellular interactions and emergent properties of human atrial myocytes in chronic AF. Therefore, reversing remodeling for a single remodeled component does little to restore the normal sinus rhythm phenotype. These findings may have important implications for developing novel therapeutic approaches for chronic AF.     

Regulation of the mitochondrial ATP-sensitive potassium channel in rat uterus cells by ROS

In previous study we demonstrated the presence of ATP-sensitive potassium current in the inner mitochondrial membrane, which was sensitive to diazoxide and glybenclamide, in mitochondria isolated from the rat uterus. This current was supposed to be operated by mitochondrial ATP-sensitive potassium channel (mitoK(ATP)). Regulation of the mitoK(ATP) in uterus cells is not studied well enough yet. It is well known that the reactive oxygen species (ROS) can play a dual role. They can damage cells in high concentrations, but they can also act as messengers in cellular signaling, mediating survival of cells under stress conditions. ROS are known to activate mitoK(ATP) during the oxidative stress in the brain and heart, conferring the protection of cells. The present study examined whether ROS mediate the mitoK(ATP) activation in myometrium cells. Oxidative stress was induced by rotenone. ROS generation was measured by 2',7'-dichlorofluorescin diacetate. The massive induction of ROS production was demonstrated in the presence of rotenone. Hyperpolarization of the mitochondrial membrane was also detected with the use of the potential-sensitive dye DiOC6 (3,3'-dihexyloxacarbocyanine iodide). Diazoxide, a selective activator of mitoK(ATP), depolarized mitochondrial membrane either under oxidative stress or under normal conditions, while mitoK(ATP) blocker glybenclamide effectively restored mitochondrial potential in rat myocytes. Estimated value for diazoxide to mitoK(ATP) under normoxia was four times higher than under oxidative stress conditions: 5.01 +/- 1.47-10(-6) M and 1.24 +/- 0.21 x 10(-6) M respectively. The ROS scavenger N-acetylcysteine (NAC) successfully eliminates depolarization of mitochondrial membrane by diazoxide under oxidative stress. These results suggest that elimination of ROS by NAC prevents the activation of mitoK(ATP) under oxidative stress. Taking into account the higher affinity of diazoxide to mitoK(ATP) under stress conditions than under normoxia, we conclude that the oxidative stress conditions are more favourable than normoxia for the activation of mitoK(ATP). Thus we hypothesize that the ROS regulate the activity of the mitoK(ATP) in myocytes.     

Anoxia-Reoxygenation Regulates Mitochondrial Dynamics through the Hypoxia Response Pathway,SKN-1/Nrf,and Stomatin-Like Protein STL-1/SLP-2

Many aerobic organisms encounter oxygen-deprived environments and thus must have adaptive mechanisms to survive such stress. It is important to understand how mitochondria respond to oxygen deprivation given the critical role they play in using oxygen to generate cellular energy. Here we examine mitochondrial stress response in C. elegans, which adapt to extreme oxygen deprivation (anoxia, less than 0.1% oxygen) by entering into a reversible suspended animation state of locomotory arrest. We show that neuronal mitochondria undergo DRP-1-dependent fission in response to anoxia and undergo refusion upon reoxygenation. The hypoxia response pathway, including EGL-9 and HIF-1, is not required for anoxia-induced fission, but does regulate mitochondrial reconstitution during reoxygenation. Mutants for egl-9 exhibit a rapid refusion of mitochondria and a rapid behavioral recovery from suspended animation during reoxygenation; both phenotypes require HIF-1. Mitochondria are significantly larger in egl-9 mutants after reoxygenation, a phenotype similar to stress-induced mitochondria hyperfusion (SIMH). Anoxia results in mitochondrial oxidative stress, and the oxidative response factor SKN-1/Nrf is required for both rapid mitochondrial refusion and rapid behavioral recovery during reoxygenation. In response to anoxia, SKN-1 promotes the expression of the mitochondrial resident protein Stomatin-like 1 (STL-1), which helps facilitate mitochondrial dynamics following anoxia. Our results suggest the existence of a conserved anoxic stress response involving changes in mitochondrial fission and fusion.     

Increased localization of APP‐C99 in mitochondria‐associated ER membranes causes mitochondrial dysfunction in Alzheimer disease

In the amyloidogenic pathway associated with Alzheimer disease (AD), the amyloid precursor protein (APP) is cleaved by β‐secretase to generate a 99‐aa C‐terminal fragment (C99) that is then cleaved by γ‐secretase to generate the β‐amyloid (Aβ) found in senile plaques. In previous reports, we and others have shown that γ‐secretase activity is enriched in mitochondria‐associated endoplasmic reticulum (ER) membranes (MAM) and that ER–mitochondrial connectivity and MAM function are upregulated in AD. We now show that C99, in addition to its localization in endosomes, can also be found in MAM, where it is normally processed rapidly by γ‐secretase. In cell models of AD, however, the concentration of unprocessed C99 increases in MAM regions, resulting in elevated sphingolipid turnover and an altered lipid composition of both MAM and mitochondrial membranes. In turn, this change in mitochondrial membrane composition interferes with the proper assembly and activity of mitochondrial respiratory supercomplexes, thereby likely contributing to the bioenergetic defects characteristic of AD.     

Calcium dysregulation increases right ventricular outflow tract arrhythmogenesis in rabbit model of chronic kidney disease

Chronic kidney disease (CKD) increases the risk of arrhythmia. The right ventricular outflow tract (RVOT) is a crucial site of ventricular tachycardia (VT) origination. We hypothesize that CKD increases RVOT arrhythmogenesis through its effects on calcium dysregulation. We analysed measurements obtained using conventional microelectrodes, patch clamp, confocal microscopy, western blotting, immunohistochemical examination and lipid peroxidation for both control and CKD (induced by 150 mg/kg neomycin and 500 mg/kg cefazolin daily) rabbit RVOT tissues or cardiomyocytes. The RVOT of CKD rabbits exhibited a short action potential duration, high incidence of tachypacing (20 Hz)-induced sustained VT, and long duration of isoproterenol and tachypacing-induced sustained and non-sustained VT. Tachypacing-induced sustained and non-sustained VT in isoproterenol-treated CKD RVOT tissues were attenuated by KB-R7943 and partially inhibited by KN93 and H89. The CKD RVOT myocytes had high levels of phosphorylated CaMKII and PKA, and an increased expression of tyrosine hydroxylase-positive neural density. The CKD RVOT myocytes exhibited large levels of I_to, I_Kr, NCX and L-type calcium currents, calcium leak and malondialdehyde but low sodium current, SERCA2a activity and SR calcium content. The RVOT in CKD with oxidative stress and autonomic neuron hyperactivity exhibited calcium handling abnormalities, which contributed to the induction of VT.     

Dantrolene prevents arrhythmogenic Ca2+ release in heart failure

In heart failure (HF), arrhythmogenic Ca(2+) release and chronic Ca(2+) depletion of the sarcoplasmic reticulum (SR) arise due to altered function of the ryanodine receptor (RyR) SR Ca(2+)-release channel. Dantrolene, a therapeutic agent used to treat malignant hyperthermia associated with mutations of the skeletal muscle type 1 RyR (RyR1), has recently been suggested to have effects on the cardiac type 2 RyR (RyR2). In this investigation, we tested the hypothesis that dantrolene exerts antiarrhythmic and inotropic effects on HF ventricular myocytes by examining multiple aspects of intracellular Ca(2+) handling. In normal rabbit myocytes, dantrolene (1 μM) had no effect on SR Ca(2+) load, postrest decay of SR Ca(2+) content, the threshold for spontaneous Ca(2+) wave initiation (i.e., the SR Ca(2+) content at which spontaneous waves initiate) and Ca(2+) spark frequency. In cardiomyocytes from failing rabbit hearts, SR Ca(2+) load and the wave initiation threshold were decreased compared with normal myocytes, Ca(2+) spark frequency was increased, and the postrest decay was potentiated. Using a novel approach of measuring cytosolic and intra-SR Ca(2+) concentration (using the low-affinity Ca(2+) indicator fluo-5N entrapped within the SR), we showed that treatment of HF cardiomyocytes with dantrolene rescued postrest decay and increased the wave initiation threshold. Additionally, dantrolene decreased Ca(2+) spark frequency while increasing the SR Ca(2+) content in HF myocytes. These data suggest that dantrolene exerts antiarrhythmic effects and preserves inotropy in HF cardiomyocytes by decreasing the incidence of diastolic Ca(2+) sparks, increasing the intra-SR Ca(2+) threshold at which spontaneous Ca(2+) waves occur, and decreasing the loss of Ca(2+) from the SR. Furthermore, the observation that dantrolene reduces arrhythmogenicity while at the same time preserves inotropy suggests that dantrolene is a potentially useful drug in the treatment of arrhythmia associated with HF.     

Implications of altered glutathione metabolism in aspirin-induced oxidative stress and mitochondrial dysfunction in HepG2 cells

We have previously reported that acetylsalicylic acid (aspirin, ASA) induces cell cycle arrest, oxidative stress and mitochondrial dysfunction in HepG2 cells. In the present study, we have further elucidated that altered glutathione (GSH)-redox metabolism in HepG2 cells play a critical role in ASA-induced cytotoxicity. Using selected doses and time point for ASA toxicity, we have demonstrated that when GSH synthesis is inhibited in HepG2 cells by buthionine sulfoximine (BSO), prior to ASA treatment, cytotoxicity of the drug is augmented. On the other hand, when GSH-depleted cells were treated with N-acetyl cysteine (NAC), cytotoxicity/apoptosis caused by ASA was attenuated with a significant recovery in oxidative stress, GSH homeostasis, DNA fragmentation and some of the mitochondrial functions. NAC treatment, however, had no significant effects on the drug-induced inhibition of mitochondrial aconitase activity and ATP synthesis in GSH-depleted cells. Our results have confirmed that aspirin increases apoptosis by increased reactive oxygen species production, loss of mitochondrial membrane potential and inhibition of mitochondrial respiratory functions. These effects were further amplified when GSH-depleted cells were treated with ASA. We have also shown that some of the effects of aspirin might be associated with reduced GSH homeostasis, as treatment of cells with NAC attenuated the effects of BSO and aspirin. Our results strongly suggest that GSH dependent redox homeostasis in HepG2 cells is critical in preserving mitochondrial functions and preventing oxidative stress associated complications caused by aspirin treatment.