AMP-activated protein kinase protects cardiomyocytes against hypoxic injury through attenuation of endoplasmic reticulum stress

Oxygen deprivation leads to the accumulation of misfolded proteins in the endoplasmic reticulum (ER), causing ER stress. Under conditions of ER stress, inhibition of protein synthesis and up-regulation of ER chaperone expression reduce the misfolded proteins in the ER. AMP-activated protein kinase (AMPK) is a key regulatory enzyme involved in energy homeostasis during hypoxia. It has been shown that AMPK activation is associated with inhibition of protein synthesis via phosphorylation of elongation factor 2 (eEF2) in cardiomyocytes. We therefore examined whether AMPK attenuates hypoxia-induced ER stress in neonatal rat cardiomyocytes. We found that hypoxia induced ER stress, as assessed by the expression of CHOP and BiP and cleavage of caspase 12. Knockdown of CHOP or caspase 12 through small interfering RNA (siRNA) resulted in decreased expression of cleaved poly(ADP-ribose) polymerase following exposure to hypoxia. We also found that hypoxia-induced CHOP expression and cleavage of caspase 12 were significantly inhibited by pretreatment with 5-aminoimidazole-4-carboxyamide-1-beta-D-ribofuranoside (AICAR), a pharmacological activator of AMPK. In parallel, adenovirus expressing dominant-negative AMPK significantly attenuated the cardioprotective effects of AICAR. Knockdown of eEF2 phosphorylation using eEF2 kinase siRNA abolished these cardioprotective effects of AICAR. Taken together, these findings demonstrate that activation of AMPK contributes to protection of the heart against hypoxic injury through attenuation of ER stress and that attenuation of protein synthesis via eEF2 inactivation may be the mechanism of cardioprotection by AMPK.     

Role of adiponectin receptors in endothelin-induced cellular hypertrophy in cultured cardiomyocytes and their expression in infarcted heart

Adiponectin, an adipocyte-derived protein, has cardioprotective actions. We elucidated the role of the adiponectin receptors AdipoR1 and AdipoR2 in the effects of adiponectin on endothelin-1 (ET-1)-induced hypertrophy in cultured cardiomyocytes, and we examined the expression of adiponectin receptors in normal and infarcted mouse hearts. Recombinant full-length adiponectin suppressed the ET-1-induced increase in cell surface area and [(3)H]leucine incorporation into cultured cardiomyocytes compared with cells treated with ET-1 alone. Transfection of small interfering RNA (siRNA) specific for AdipoR1 or AdipoR2 reversed the suppressive effects of adiponectin on ET-1-induced cellular hypertrophy in cultured cardiomyocytes. Adiponectin induced phosphorylation of AMP-activated protein kinase (AMPK) and inhibited ET-1-induced ERK1/2 phosphorylation, which were also reversible by transfection of siRNA for AdipoR1 or AdipoR2 in cultured cardiomyocytes. Transfection of siRNA for alpha(2)-catalytic subunits of AMPK reduced the inhibitory effects of adiponectin on ET-1-induced cellular hypertrophy and ERK1/2 phosphorylation. Effects of globular adiponectin were similar to those of full-length adiponectin, and siRNA for AdipoR1 reversed the actions of globular adiponectin. Compared with normal left ventricle, expression levels of AdipoR1 mRNA and protein were decreased in the remote, as well as the infarcted, area after myocardial infarction in mouse hearts. In conclusion, AdipoR1 and AdipoR2 mediate the suppressive effects of full-length and globular adiponectin on ET-1-induced hypertrophy in cultured cardiomyocytes, and AMPK is involved in signal transduction through these receptors. AdipoR1 and AdipoR2 might play a role in the pathogenesis of ET-1-related cardiomyocyte hypertrophy after myocardial infarction.     

Endothelin-1- and isoproterenol-induced differential protein expression and signaling pathway in HL-1 cardiomyocytes

It is well known that the two chemical compounds endothelin-1 (ET-1) and isoproterenol (ISO) can individually induce cardiac hypertrophy through G protein-coupled receptors in cardiomyocytes. However, the cardiac hypertrophy signaling pathway activated by ET-1 and ISO is not well defined. Therefore, we investigated the protein expression profile and signaling transduction in HL-l cardiomyocyte cells treated with ET-1 and ISO. Following separation of the cell lysates by using 2-DE and silver staining, we identified 16 protein spots that were differentially expressed as compared to the controls. Of these 16 spots, three changed only after treatment with ET-1, whereas four changed only after treatment with ISO, suggesting that these two stimuli could induce different signaling pathways. In order to reveal the differences between ET-1- and ISO-induced signaling, we studied the different events that occur at each step of the signaling pathways, when selected biocomponents were blocked by inhibitors. Our results indicated that ET-1 and ISO used different pathways for phosphorylation of glycogen synthase kinase-3β (GSK3β). ET-1 mainly used the mitogen-activated protein kinase and phosphatidylinositol-3-kinase/AKT pathways to activate GSK3β, whereas under ISO stimulation, only the phosphatidylinositol-3-kinase/AKT pathway was required to trigger the GSK3β pathway. Furthermore, the strength of the GSK3β signal in ISO-induced cardiac hypertrophy was stronger than that in ET-1-induced cardiac hypertrophy. We found that these two agonists brought about different changes in the protein expression of HL-1 cardiomyocytes through distinct signaling pathways even though the destination of the two signaling pathways was the same.     

Decreased expression of calpain and calpastatin mRNA during development is highly correlated with muscle protein accumulation in neonatal pigs

It is well known that rapid gain of muscle mass in neonatal pigs is highly related to protein synthesis. However, the role of protein degradation in muscle gain of the neonatal period has not been well established. Calpains and their endogenous inhibitors, calpastatins, play a significant role in early-stage myofibrillar protein degradation. To investigate the role of calpain–calpastatin system in muscle protein accumulation, we studied the expressions of their mRNA in muscle tissue sampled at days 1, 4, 6, 12, 20 and 28 from a total of 36 neonatal pigs. The steady-state mRNA levels of calpains 1A, 2 and 3A, calpastatin types 1, 2 and 3, obtained by quantitative real-time PCR analysis, decreased by 2–4 folds at the age of 4 to 6 days compared to 1-day-old piglets. Then, the relatively low expression level was maintained through 28 days of age. Expressions of calpains 1A, 3A and calpastatin type 1 were significantly correlated with the measurements of muscle protein accumulations such as muscle protein content and RNA/protein ratio. Expressions of calpain 1A, calpastatin types 1 and 3 were negatively correlated with birth weight and fractional rate of growth. The levels of calpains 1A and 2 mRNA were correspondent to their protease activities. In conclusion, decreased levels of calpain and calpastatin expressions over development in neonatal pigs are associated with high protein accumulations, suggesting that dramatic muscle growth during the neonatal period may be partially controlled by down-regulated calpain–calpastatin system.     

Effect of hypotonic stress on ornithine decarboxylase mRNA expression in cultured cells.

This report examines the effect of hypotonic stress on ornithine decarboxylase (ODC) activity and ODC mRNA concentrations in LLC-PK1 cells. Earle's balanced salts solution minus glucose (EBSS-G) with decreasing concentrations of NaCl was utilized as the ODC induction medium. Hypotonic EBSS-G increased both the concentration of ODC mRNA and the specific activity of ODC in LLC-PK1 cells. Actinomycin D and cycloheximide prevented the increase in enzyme activity resulting from hypotonic stress. Actinomycin D was also a potent inhibitor of ODC mRNA expression resulting from hypotonic stress. Cycloheximide had very little effect on the induction of ODC mRNA in cells incubated in hypotonic EBSS-G. The magnitude of the increase in both ODC mRNA concentrations and enzyme activity was dependent on the incubation time in hypotonic media. The increase in ODC mRNA concentrations preceded the elevation in enzyme activity. ODC mRNA concentrations and the specific activity of ODC increased as a function of decreasing media osmolarity. The addition of putrescine, spermidine, and spermine to EBSS-G containing reduced NaCl suppressed the increase in LLC-PK1 ODC activity related to hypotonic stress. In contrast, these polyamines did not prevent the increase in ODC mRNA resulting from hypotonic shock. Furthermore, it was demonstrated that hypotonic stress increases ODC mRNA levels and enzyme activity in four additional cell lines from two different species. Based on these results it is suggested that one or more signal transducers associated with cell volume expansion enhance expression of the ODC gene.     

Role of protein phosphatases in hypoxic preconditioning

To find a protein kinase C (PKC)-independent preconditioning mechanism, hypoxic preconditioning (HP; i.e., 10-min anoxia and 10-min reoxygenation) was applied to isolated rat hearts before 60-min global ischemia. HP led to improved recovery of developed pressure and reduced end-diastolic pressure in the left ventricle during reperfusion. Protection was unaffected by the PKC inhibitor bisindolylmaleimide (BIM; 1 micromol/l). It was abolished by the inhibitor of protein phosphatases 1 and 2A cantharidin (20 or 5 micromol/l) and partially enhanced by the inhibitor of protein phosphatase 2A okadaic acid (5 nmol/l). In adult rat cardiomyocytes treated with BIM and exposed to 60-min simulated ischemia (anoxia, extracellular pH 6.4), HP led to attenuation of anoxic Na(+)/Ca(2+) overload and of hypercontracture, which developed on reoxygenation. This protection was prevented by treatment with cantharidin but not with okadaic acid. In conclusion, HP exerts PKC-independent protection on ischemic-reperfused rat hearts and cardiomyocytes. Protein phosphatase 1 seems a mediator of this protective mechanism.     

Antioxidant effects of American ginseng berry extract in cardiomyocytes exposed to acute oxidant stress

It is postulated that antioxidant properties of American ginseng root mediate its cardioprotective actions. The antioxidant capabilities of the American ginseng root have been demonstrated previously, however, the berry of the American ginseng has not yet been evaluated. In this study, we tested the American ginseng berry extract (AGBE) for its antioxidant effects in cell-free chemical systems using H(2)O(2)/FeSO(4) to generate hydroxyl radicals which were measured by a fluorescent probe, 2', 7'-dichlorofluorescin diacetate (DCFH/DA). Xanthine/xanthine oxidase was used to generate superoxide anion, which was measured by a fluorescent probe dihydroethidium (DHE). We found that AGBE decreased fluorescence significantly, suggesting that AGBE scavenges oxygen free radicals. We further tested whether AGBE (0.1-1 mg/ml) can protect cardiomyocytes from oxidative injury induced by exogenous or endogenous oxidants. Cells were exposed to either H(2)O(2) or antimycin A (a mitochondrial electron transport chain site III inhibitor that augments mitochondrial oxidant production). The resulting oxidant stress was measured using DCFH/DA and the cell death was assessed using propidium iodide staining. Pretreatment with AGBE (1 mg/ml) significantly attenuated DCF fluorescence by 49% or 85% and reduced cell death by 59% or 63% in cells exposed to H(2)O(2) or antimycin A, respectively. When the effects of extracts from berry and root of American ginseng were compared in cardiomyocytes exposed to antimycin A, we observed that AGBE conferred greater antioxidant protection at the same dose. We conclude that AGBE is a potent antioxidant that protects cardiomyocytes against oxidant-mediated injury and this protection is partly mediated by its free radical scavenging properties.     

Cardiac high molecular weight calmodulin-binding protein is homologous to calpastatin I and calpastatin II

Calpastatin is an endogenous inhibitor of calpain, which has been implicated in various physiological and pathological processes. In the present study we determined the molecular and inhibitory properties of HMWCaMBP, calpastatin I, and calpastatin II. Western blot analysis with antibodies raised against either full length HMWCaMBP or internal peptides that are common to all isoforms showed that all three homologs have common antigenic epitopes. However, additional Western blot analysis with N-terminal specific antibodies showed that all three proteins are different at the N-terminal end. HMWCaMBP is clearly different from two other homologues, calpastatin I and II, at the N-terminal end. In addition, HMWCaMBP also showed the same affinities for m-calpain as calpastatin I and calpastatin II. Our findings suggest that HMWCaMBP is the homolog of calpastatin and may be a CaM-binding form of calpastatin.     

Role of gamma-linolenic acid in counteracting doxorubicin-induced damage in cultured rat cardiomyocytes

The clinical usefulness of doxorubicin is limited by cardiotoxicity. We have demonstrated that doxorubicin has a dual negative effect on myocardial lipids, acting against highly unsaturated fatty acids (HUFAs) directly and desaturating/elongating enzymes required for their biosynthesis, thus decreasing linoleic and alpha -linolenic conversion to higher metabolites. Primary cultures of rat cardiomyocytes were challenged with different doxorubicin concentrations and doxorubicin exposure was followed by a 24-h recovery period in the absence or presence of serum, and of gamma -linolenic acid. Serum in the recovery medium did not appear to be essential for the restoration of the desaturating/elongating activities, and gamma -linolenic acid supplementation influenced only alpha -linolenic acid conversion. Serum, and particularly gamma-linolenic acid, were very important in increasing HUFA levels behind the pure biosynthesis. HUFA biosynthesis plays a role in counteracting doxorubicin toxicity, but it cannot completely overcome the depletion of these fatty acids; serum and exogenous gamma-linolenate are critical in filling the decreased HUFA pool.     

低氧预处理通过上调钙网蛋白表达减轻大鼠心肌细胞氧化应激损伤

本文旨在探讨钙网蛋(calreticulin,CRT)是否参与低氧预处理(hypoxic preconditioning,HPC)对心肌细胞氧化应激损伤的保护及其信号转导过程.将原代培养的Sprague.Dawley乳鼠心肌细胞随机分为8组:氧化应激(H2O2)组、短暂低氧(HPC)组、HPC H202组、SB203580(p38 MAPK特异性抑制剂) HPC H2O2组、干扰心肌细胞CRT表达的反义寡核苷酸(antiscnse oligodeoxynucleotides,AS)组、AS H2O2组、AS HPC H202组和对照组,以细胞存活率、乳酸脱氢酶(1actate dehydrogenase,LDH)漏出及流式细胞术检测细胞损伤情况;采用RT-PCR和Western blot分别检测CRT表达和p38MAPK磷酸化水平.结果表明:(1)HPC可减轻氧化应激损伤,与H202组比较,HPC H2O2组细胞存活率增高18.0%,细胞凋亡率和LDH漏出分别降低19.4%和53.0%(均P<0.05);HPC前以SB203580预孵育可消除HPC保护作用,与HPC H202组相比,SB203580 HPC H2O2组细胞凋亡率和LDH漏出分别增高13.1%和96.0%,存活率降低7.3%(均P<0.05);(2)氧化应激明显上调CRT表达(H202组较对照组高7.1倍,P<0.05);HPC也诱导CRT表达上调(HPC组较对照组高2.4倍,P<0.05),但上调程度较H2O2组低59%(P<0.05);即HPC可减轻氧化应激诱导的CRT过表达:(3)AS干扰CRT表达后,HPC保护作用降低,相关性分析显示HPC诱导的CRT适度表达与细胞存活率呈正相关(r=0.8023,P<0.05);(4)HPC前SB203580预孵育可抑制CRT表达上调(分别较HPC H2O2组和HPC组低75%和53%,均P<0.05).上述结果提示,HPC可能通过p38 MAPK信号途径诱导CRT表达上调,减轻心肌细胞氧化应激损伤.     

Influence of sustained mechanical stress on Egr-1 mRNA expression in cultured human endothelial cells

Restenosis after initially successful balloon angioplasty of coronary artery stenosis remains a major problem in clinical cardiology. Previous studies have identified pathogenetic factors which trigger cell proliferation and vascular remodeling ultimately leading to restenosis. Since there is evidence that endothelial cells adjacent to the angioplasty wound area synthesize factors which may initiate this process, we investigated the effects of mechanical stimulation on endothelial gene expression in vitro and focussed on the influence of sustained mechanical stress on expression of immediate early genes which have previously been shown to be induced in the vascular wall in vivo. Primary cultured human umbilical vein endothelial cells (HUVEC) and the human endothelial cell line EA.hy 926 were plated on collagen-coated silicone membranes and subjected to constant longitudinal stress of approximately 20% for 10 min to 6 h. Total RNA was isolated and the expression of the immediate early genes c-Fos and Egr-1 was studied by Northern blot analysis. We found a rapid upregulation c-Fos and Egr-1 mRNA which started at 10 min and reached its maxima at 30 min. HUVEC lost most of their stretch response after the third passage whereas immediate early gene expression was constantly in EA.hy 926 cells. Using specific inhibitors we investigated the contribution of several signal transduction pathways to stretch-activated Egr-1 mRNA expression. We found significant suppression of stretch-induced Egr-1 mRNA expression by protein kinase C (PKC) inhibition (p < 0.05) and by calcium depletion (EA.hy926, p < 0. 05; HUVEC, p = 0.063). No effect on stretch-activated Egr-1 mRNA expression was detected by inhibition of protein kinase A, blockade of stretch-activated cation channels or inhibition of microtubule synthesis. We conclude that sustained mechanical strain induces Egr-1 mRNA expression by PKC- and calcium-dependent mechanisms.     

The role of cell death and myofibrillar damage in contractile dysfunction of long-term cultured adult cardiomyocytes exposed to doxorubicin

In failing hearts cardiomyocytes undergo alterations in cytoskeleton structure, contractility and viability. It is not known presently, how stress-induced changes of myofibrils correlate with markers for cell death and contractile function in cardiomyocytes. Therefore, we have studied the progression of contractile dysfunction, myofibrillar damage and cell death in cultured adult cardiomyocytes exposed to the cancer therapy doxorubicin. We demonstrate, that long-term cultured adult cardiomyocytes, a well-established model for the study of myofibrillar structure and effects of growth factors, can also be used to assess contractility and calcium handling. Adult rat ventricular myocytes (ARVM) were isolated and cultured for a total of 14 days in serum containing medium. The organization of calcium-handling proteins and myofibrillar structure in freshly isolated and in long-term cultured adult cardiomyocytes was studied by immunofluorescence and electron microscopy. Excitation contraction-coupling was analyzed by fura 2 and video edge detection in electrically paced cardiomyocytes forming a monolayer, and cell death and viability was measured by TUNEL assay, LDH release, MTT assay, and Western blot for LC3. Adult cardiomyocytes treated with Doxo showed apoptosis and necrosis only at supraclinical concentrations. Treated cells displayed merely alterations in cytoskeleton organization and integrity concomitant with contractile dysfunction and up-regulation of autophagosome formation, but no change in total sarcomeric protein content. We propose, that myofibrillar damage contributes to contractile dysfunction prior to cell death in adult cardiomyocytes exposed to clinically relevant concentrations of anthracyclines.     

Cardioprotective effects of 3-phosphoinositide-dependent protein kinase-1 on hypoxic injury in cultured neonatal rat cardiomyocytes and myocardium in a rat myocardial infarct model

3-Phosphoinositide-dependent protein kinase-1 (PDK1) is involved in numerous cellular responses. In this study, we investigated the protective effects of PDK1 gene expression against hypoxic conditions in cultured rat CMCs (rCMCs) and in a rat myocardial infarction (MI) model using the lentiviral vector (LeV) system. LeV-PDK1 transfer effectively reduced the apoptotic cell death caused by hypoxic injury as compared to LeV-GFP transfer in rCMCs the expression of survival proteins increased in the LeV-PDK1 group, whereas apoptosis signaling decreased in the rCMCs and in infarcted hearts treated with LeV-PDK1. LeV-PDK1 transfer also reduced apoptosis and infarct size and attenuated myocardial wall thinning and ventricular remodeling in a rat MI model. These findings suggest that PDK1 has a protective role in the injured ischemic myocardium via overexpression of the cell survival pathway in CMCs. Hence PDK1 can be used as a treatment strategy for myocardial salvage inin hypoxic injury.     

Hyperoxia-mediated oxidative stress increases expression of UCP3 mRNA and protein in skeletal muscle

The uncoupling protein-3 (UCP3) is a mitochondrial protein expressed mainly in skeletal muscle. Among several hypotheses for its physiological function, UCP3 has been proposed to prevent excessive production of reactive oxygen species. In the present study, we evaluated the effect of an oxidative stress induced by hyperoxia on UCP3 expression in mouse skeletal muscle and C2C12 myotubes. We found that the hyperoxia-mediated oxidative stress was associated with a 5-fold and 3-fold increase of UCP3 mRNA and protein levels, respectively, in mouse muscle. Hyperoxia also enhanced reactive oxygen species production and UCP3 mRNA expression in C2C12 myotubes. Our findings support the view that both in vivo and in vitro UCP3 may modulate reactive oxygen species production in response to an oxidative stress.     

Delayed cytoprotection induced by hypoxic preconditioning in cultured neonatal rat cardiomyocytes: role of GRP78

Hypoxic preconditioning (HPC) has been well demonstrated to have potent protective effects in many cell types; however, the mechanisms responsible for this phenomenon are not fully understood. Recently, glucose-regulated protein 78 (GRP78), an inducible molecular chaperon, was indicated to be associated with ischemic preconditioning. We hypothesized that HPC protects cardiomyocytes against hypoxia by inducing GRP78 in cultured neonatal rat cardiomyocytes. HPC was induced by exposing cardiomyocytes to brief hypoxia (1% O(2), 30 min) followed by reoxygenation. GRP78 was expressed constitutively in cultured cardiomyocytes and its expression was enhanced at 12 h, peaked at 24 h (207.3+/-23.6% of the baseline), and was sustained for up to 72 h after HPC. Twenty-four hours after HPC, the myocytes were subjected to prolonged hypoxia (1% O(2), 12 h). The lactic dehydrogenase (LDH) release and malondialdehyde (MDA) content were reduced, while cell viability and superoxide dismutase (SOD) activity were increased in the preconditioned cells compared with the non-HPC cells. The GRP78 protein level was higher in cells exposed to both HPC and hypoxia than in the cells exposed to HPC alone or hypoxia alone. Heat shock protein 70 (HSP70) was induced in parallel by late HPC. Transfection of GRP78 antisense oligonucleotides blocked GRP78 expression but not HSP70, resulting in attenuated cardioprotection afforded by late HPC. Furthermore, inducing GRP78 by gene transfer protected cardiomyocytes from hypoxic injury. These findings demonstrate that the induction of GRP78 partially mediates the late HPC, suggesting that GRP78 is a novel mechanism responsible for the late cytoprotection of HPC.