Effects of heat shock protein 70 activation by metabolic inhibition preconditioning or kappa-opioid receptor stimulation on Ca2+ homeostasis in rat ventricular myocytes subjected to ischemic insults

Heat shock protein 70 (HSP70) mediates delayed cardioprotection of preconditioning. Cytosolic calcium ([Ca(2+)])(i) overload precipitates injury, whereas attenuation of [Ca(2+)](i) overload is believed to be responsible for cardioprotection. There is evidence suggesting a link between HSP70 and [Ca(2+)](i) homeostasis. We hypothesize that activation of HSP70 by preconditioning may restore [Ca(2+)](i) homeostasis altered by ischemic insults. To test the hypothesis, we determined the effects of preconditioning with metabolic inhibition or pretreating with U50,488H [trans-(+)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide (a kappa-opioid receptor agonist)] on viability and injury, HSP70 expression, and [Ca(2+)](i) in ventricular myocytes subjected to metabolic inhibition and anoxia (MI/A), with blockade of HSP70 synthesis. In myocytes with vehicle pretreatment, the percentage of dead cells determined by trypan blue exclusion, the injury reflected by release of lactate dehydrogenase, and the resting [Ca(2+)](i) measured by spectrofluorometry significantly increased, whereas the amplitude of electrically induced [Ca(2+)](i) transient decreased, after 10 min with 10 mM 2-deoxy-d-glucose and 10 mM sodium dithionite, known to cause MI/A. However, when myocytes were subjected for 30 min to either 20 mM lactate and 10 mM 2-deoxy-d-glucose (MIP) or 30 microM U50,488H (UP) 20 h before MI/A, the changes in viability and injury, and [Ca(2+)](i) responses were significantly attenuated. These were accompanied by a significantly increased HSP70 expression. Furthermore, blockade of HSP70 synthesis with selective antisense oligonucleotides abolished the beneficial effects of MIP or UP. This study provides first evidence that activation of HSP70 induced by preconditioning, which conferred delayed cardioprotection, restored partially the [Ca(2+)](i) homeostasis altered by ischemic insults.     

Calcium-activated potassium channel triggers cardioprotection of ischemic preconditioning

We tested the hypothesis that the high-conductance calciumactivated potassium (K(Ca)) channel is involved in the cardioprotection of preconditioning with ischemic insults. In the isolated perfused rat heart subjected to ischemia/reperfusion, effects of ischemic preconditioning (IPC) on infarct size and lactate dehydrogenase (LDH) release were abolished by 1 microM paxilline (Pax), an inhibitor of the K(Ca) channel, administered 30 min before, but not during, ischemia. In isolated ventricular myocytes subjected to metabolic inhibition and anoxia (MI/A), preconditioning with MI/A increased their viability, and the effect was abolished by administering Pax before MI/A. Like IPC, 10 microM NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-trifluoromethyl-2Hbenzimidazol-2-one; NS), an opener of K(Ca) channels, reduced infarct size and LDH release, effects attenuated by Pax. The harmful and protective effects of blockade and activation of the K(Ca) channel were accompanied by impaired and improved left ventricular contractile functions, respectively. In addition, the effect of NS was not altered by 100 microM 5-hydroxydecanoate, an inhibitor of the K(ATP) channel. Neither was the effect of 100 microM diazoxide, an activator of the K(ATP) channel, altered by Pax. Furthermore, opening of the mitochondrial permeability transition pore (mPTP) with 20 microM atractyloside abolished the beneficial effects of IPC or NS in the isolated rat heart and myocyte. Inhibition of mPTP opening with 0.2 microM cyclosporin A decreased the infarct size and LDH release and improved the contractile function, effects not attenuated by Pax. In conclusion, the study provides evidence that the K(Ca) channel triggers cardioprotection of IPC, which involves mPTP.     

钙离子、蛋白激酶C与大鼠心肌预适应相关性研究

目的 :观察钙离子、蛋白激酶C在大鼠心肌预适应保护机制中的作用。方法 :64只雄性SD大鼠随机分为 8组 ,A组正常灌流 2 0min缺血 40min再灌 3 0min(I/R) ;B组短暂缺血复灌 +I/R ;C组短暂缺血复灌 +L- 型钙通道阻滞剂 +I/R ;D组短暂缺血复灌 +PKC阻滞剂 +I/R ;E组钙通道激动剂 +I/R ;F组钙通道激动剂 +PKC阻滞剂 +I/R ;G组L- 型钙通道阻滞剂 +I/R ;H组PKC阻滞剂 +I/R。结果 :实验中观察到B、E组与A、E、C组相比 ,各项指标均提示心肌保护作用较好。结论 :短暂钙离子内流增加通过蛋白激酶C诱导出心肌内源性保护作用。     

The cardioprotective effects of preconditioning with endotoxin, but not ischemia, are abolished by a peroxisome proliferator-activated receptor-gamma antagonist

We investigated whether endogenous ligands of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) protect the heart against ischemia-reperfusion (I/R) injury. The selective PPAR-gamma antagonist GW9662 (2-chloro-5-nitrobenzanilide) was used in rat models of 1) regional myocardial I/R, 2) ischemic preconditioning, and 3) delayed cardioprotection by endotoxin. We also investigated the effects of the selective cyclooxygenase-2 inhibitor, parecoxib, on ischemic preconditioning and delayed cardioprotective effects of endotoxin. Male Wistar rats were anesthetized with sodium thiopentone. Animals were subjected to either 15 or 25 min of regional myocardial I/R and pretreated with the PPAR-gamma agonist ciglitazone (0.3 mg/kg), the PPAR-gamma antagonist GW9662 (1 mg/kg), or GW9662 and ciglitazone. Animals were also subjected to either 1) ischemic preconditioning alone, ischemic preconditioning, and pretreated with either GW9662 or parecoxib (20 mg/kg) or 2) lipopolysaccharide (LPS) (1 mg/kg) alone, LPS, and pretreated with ciglitazone, GW9662, or parecoxib (20 mg/kg). Myocardial infarct size was determined by p-nitroblue tetrazolium staining. The PPAR-gamma antagonist GW9662 (1 mg/kg) abolished the cardioprotection afforded by the potent PPAR-gamma agonist ciglitazone (0.3 mg/kg). Neither GW9662 nor parecoxib affected the cardioprotective effects of ischemic preconditioning. Pretreatment with ciglitazone did not provide additional cardioprotection to LPS-treated animals. Both GW9662 and parecoxib abolished the delayed cardioprotective effects of endotoxin. Thus, we propose that 1) endogenous ligands of PPAR-gamma are being generated by myocardial ischemia in sufficient amounts to attenuate myocardial I/R injury, and 2) that cyclooxygenase-2 metabolites contribute to (or even account for) the cardioprotective effects of endotoxin (second window of protection) by acting as endogenous PPAR-gamma ligands.     

The role of phosphodiesterase in mediating the effect of protein kinase C on cyclic AMP accumulation upon kappa-opioid receptor stimulation in the rat heart

This study determined whether phosphodiesterase (PDE) was activated by protein kinase C (PKC) upon kappa-receptor stimulation, and if so, to identify the isozyme. We first studied the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulphonate (U50,488H), a selective kappa-opioid receptor (OR) agonist, and phorbol-12-myristate-13-acetate (PMA), a PKC activator, on cAMP accumulation and PDE activity in rat ventricular myocytes when PKC and PDE were inhibited by respective inhibitors. Like PMA, U50,488H decreased the forskolin-stimulated cAMP accumulation and dose-dependently stimulated the PDE activity, which were antagonized by 10(-6) M chelerythrine and bisindolylmaleimide I, selective PKC antagonists. In addition, 3-isobutyl-1-methylxanthine, a PDE inhibitor, dose-dependently attenuated the inhibition on forskolin-stimulated cAMP accumulation and abolished the stimulation on PDE activity by U50,488H and PMA. The observations suggest that PKC may enhance cAMP degradation through activating PDE upon kappa-OR stimulation. To identify the isozyme(s) mediating the effect of PKC upon kappa-OR stimulation, selective inhibitors were used. We found that 10(-5) M Ro-20-1724, a selective cAMP-specific PDE (PDE-IV) inhibitor, abolished the inhibitory effects of U50,488H and PMA, whereas 8-methoxymethyl-3-isobutyl-1-methylxanthine, erythro-9-(2-hydroxy-3-nonyl) adenine, cilostamide, and zaprinast, selective inhibitors of Ca(2+)/calmodulin-dependent PDE (PDE-I), cGMP-stimulated PDE (PDE-II), cGMP-inhibited PDE (PDE-III), and cGMP-specific PDE (PDE-V), respectively, had no effect. Moreover, rolipram, another selective PDE-IV inhibitor, also dose-dependently attenuated the inhibition on forskolin-stimulated cAMP accumulation and stimulation on PDE activity by U50,488H and PMA. In conclusion, this study has provided evidence for the first time that PKC and PDE-IV mediate the action of kappa-OR.     

Protein kinase C mediates lipopolysaccharide- and phorbol-induced nitric-oxide synthase activity and cellular injury in the rat colon

The role of protein kinase C (PKC) in lipopolysaccharide (LPS)- and phorbol ester-induced changes in rat colonic cellular integrity and Ca(2+)-independent inducible nitric-oxide synthase (iNOS) activity was investigated. LPS treatment (3 mg kg(-1) i.p.) increased colonic cellular PKC activity within 1 h after administration. The percentage of nonviable cells and iNOS activity in response to LPS were reduced by pretreatment with the selective PKC antagonist GF 109203X (25 ng kg(-1) i.v.). Pretreatment with the selective iNOS inhibitor 1400W (5 mg kg(-1) s.c.) reduced the extent of cellular injury and iNOS activity but did not affect the increase in LPS-mediated PKC activation. Reduction of circulating neutrophils with anti-neutrophil serum reduced cell damage as well as the increases in PKC and iNOS activities in response to LPS. Intracolonic administration of the phorbol ester phorbol-12-myristate-13-acetate (PMA; 3 mg kg(-1)) increased colonic cellular PKC activity within 2 h after instillation. Cellular iNOS activity did not increase until 6 h after PMA administration. The colonic responses to PMA were eliminated by GF 109203X. The selective iNOS inhibitor 1400W reduced the increase in cell injury but did not affect the PKC activation in response to PMA. LPS treatment also increased in the proteins for PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta. PMA treatment resulted in PKC-delta and PKC-epsilon translocation from cytosol to membrane. These data suggest that PKC mediates iNOS activation and subsequent colonic cell injury in response to LPS administration. The delta- and epsilon-isozymes appear to be most closely associated with these responses.     

Direct preconditioning of cultured chick ventricular myocytes. Novel functions of cardiac adenosine A2a and A3 receptors.

Preconditioning with brief ischemia before a sustained period of ischemia reduces infarct size in the perfused heart. A cultured chick ventricular myocyte model was developed to investigate the role of adenosine receptor subtypes in cardiac preconditioning. Brief hypoxic exposure, termed preconditioning hypoxia, prior to prolonged hypoxia, protected myocytes against injury induced by the prolonged hypoxia. Activation of the adenosine A1 receptor with CCPA or the A3 receptor with C1-IB-MECA can replace preconditioning hypoxia and simulate preconditioning, with a maximal effect at 100 nM. While activation of the A2a receptor by 1 microM CGS21680 could not mimic preconditioning, its stimulation during preconditioning hypoxia, however, attenuated the protection against hypoxia-induced injury. Blockade of A2a receptors with the selective antagonist CSC (1 microM) during preconditioning hypoxia enhanced the protective effect of preconditioning. Nifedipine, which blocked the A2a receptor-mediated calcium entry, abolished the A2a agonist-induced attenuation of preconditioning. Isoproterenol, forskolin, and BayK 8644, which stimulated calcium entry, also attenuated preconditioning. Nifedipine blocked the increase in calcium uptake by these agents as well as their attenuating effect on preconditioning. The present study provides the first evidence that the adenosine A3 receptor is present on ventricular myocytes and can mediate simulation of preconditioning. The data demonstrate, for the first time, that activation of the A2a receptor antagonizes the preconditioning effect of adenosine, with increased calcium entry during the preconditioning stimuli as a novel mechanism.     

Cardioprotection of interleukin-2 is mediated via kappa-opioid receptors

We examined whether interleukin-2 (IL-2) protects the myocardium against injury induced by ischemia and reperfusion via the kappa-opioid receptor (OR). The cardioprotective effect of IL-2 was evaluated by measuring infarct size and lactate dehydrogenase (LDH) release in response to ischemia and reperfusion in the isolated rat heart. IL-2 at an optimal dose of 50 U/ml mimicked the effect of ischemic preconditioning by reducing infarct size and LDH release. The infarct and LDH-reducing effects of IL-2 were blocked by nor-binaltorphimine (5 microM), a kappa-OR antagonist, but not naltrindole (5 microM), a delta-OR antagonist known to block the action of its stimulation. Moreover, blockade of the mitochondrial ATP-sensitive potassium (mito-K(ATP)) channel with a selective antagonist, 5-hydroxydecanoate (100 microM), or a nonselective antagonist of K(ATP) channels, glybenclamide (100 microM), or blockade of protein kinase C (PKC) with its inhibitors chelerythrine (5 microM) or GF 109203X (10 microM) [3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride] abolished the protective effect of IL-2. Administration of free radical scavengers N-acetylcysteine (4 mM) or N-(2-mercaptopropionyl)-glycine (1 mM) also abolished the protective effects of IL-2 and U50,488H [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide], a selective kappa-OR agonist. This study provides the first evidence that IL-2 confers cardioprotection against injury induced by ischemia/reperfusion. The effect of IL-2 is mediated via kappa-OR as evidenced by kappa-OR antagonism and similar signaling mechanisms, mito-K(ATP), PKC, and reactive oxygen species involved in the cardioprotective effects of both IL-2 and kappa-OR stimulation.     

Role of nitric-oxide synthase,free radicals,and protein kinase C delta in opioid-induced cardioprotection

Opioids generate free radicals that mediate protection in isolated cultured cardiomyocytes. We hypothesize that the nature of these radicals is nitric oxide, and that nitric oxide activates the protein kinase C (PKC) delta isoform. Through this signal transduction pathway, opiates protect cardiomyocytes during hypoxia and reoxygenation. Cell viability was quantified in chick embryonic ventricular myocytes with propidium iodide. Oxygen radicals were quantified using a molecular probe, 2',7'-dichlorofluorescin diacetate (DCFH-DA). After a 10-min infusion of the opioid delta receptor agonist BW373U86 (BW; 2 or 20 pM) and a 10-min drug-free period, cells were subjected to hypoxia for 1 h followed by reoxygenation for 3 h. BW produced a concentration-dependent reduction in cardiomyocyte death (2 pM, 35.3 +/- 3.9%, n = 5; 20 pM, 21.5 +/- 4.0%, n = 8, p < 0.05 versus controls) and attenuated oxidant stress compared with controls (43.3 +/- 4.2%, n = 8). The increase in DCFH-DA oxidation with BW before hypoxia was abolished by the specific nitric-oxide synthase inhibitors nitro-L-arginine methyl ester (L-NAME) or N(G)-monomethyl-L-arginine (L-NMMA) (100 microM each). L-NAME or L-NMMA blocked the protective effects of BW. BW selectively increased the activity of PKC delta isoform in the particulate fraction, and its protection was abolished by the selective PKC delta inhibitor rottlerin (1 microM). Similar to BW, infusion with 5 microM of the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) reduced cardiomyocyte death (24.6 +/- 3.7, n = 8), and this protection was blocked by chelerythrine or rottlerin. Chelerythrine and rottlerin had no effect on BW-generated oxygen radicals before hypoxia, but they abolished the protection of SNAP. The nature of DCFH oxidation produced by opioid delta receptor stimulation is nitric oxide. Nitric oxide mediates cardioprotection via activating PKC delta in isolated myocytes.     

N-Benzyladriamycin-14-valerate (AD 198): a non-cardiotoxic anthracycline that is cardioprotective through PKC-epsilon activation

N-Benzyladriamycin-14-valerate (AD 198) is one of several novel anthracycline protein kinase C (PKC)-activating agents developed in our laboratories that demonstrates cytotoxic superiority over doxorubicin (Adriamycin; DOX) through its circumvention of multiple mechanisms of drug resistance. This characteristic is attributed at least partly to the principal cellular action of AD 198: PKC activation through binding to the C1b (diacylglycerol binding) regulatory domain. A significant dose-limiting effect of DOX is chronic, dose-dependent, and often irreversible cardiotoxicity ascribed to the generation of reactive oxygen species (ROS) from the semiquinone ring structure of DOX. Despite the incorporation of the same ring structure in AD 198, we hypothesized that AD 198 might also be cardioprotective through its ability to activate PKC-epsilon, a key component of protective ischemic preconditioning in cardiomyocytes. Chronic administration of fractional LD(50) doses of DOX and AD 198 to mice results in histological evidence of dose-dependent ventricular damage by DOX but is largely absent from AD 198-treated mice. The absence of significant cardiotoxicity with AD 198 occurs despite the equal ability of DOX and AD 198 to generate ROS in primary mouse cardiomyocytes. Excised rodent hearts perfused with AD 198 prior to hypoxia induced by vascular occlusion are protected from functional impairment to an extent comparable to preconditioning ischemia. AD 198-mediated cardioprotection correlates with increased PKC-epsilon activation and is inhibited in hearts from PKC-epsilon knockout mice. These results suggest that, despite ROS production, the net cardiac effect of AD 198 is protection through activation of PKC-epsilon.     

Hypercholesterolemia abrogates sevoflurane-induced delayed preconditioning against myocardial infarct in rats by alteration of nitric oxide synthase signaling

The aim of the current study was to determine whether hypercholesterolemia affects the delayed sevoflurane preconditioning against myocardial ischemia-reperfusion (IR) injury and, if so, the underlying mechanism. Male Sprague-Dawley rats fed 2% cholesterol-enriched chow for 8 weeks were subjected to sevoflurane preconditioning (2.4% vol/vol, 1 h) 24 h before myocardial ischemia was induced by occluding the left anterior descending coronary artery for 30 min followed by reperfusion for 120 min. The hemodynamic parameters left ventricular developed pressure, left ventricular end-diastolic pressure, and maximal rise/fall rate of left ventricular pressure were continuously monitored, and myocardial infarct size was determined at the end of reperfusion. The protein expression of myocardial nitric oxide synthase (NOS), Bcl-2, and Bad was assessed before ischemia. We found that the left ventricular hemodynamic parameters during the whole IR procedure and the myocardial infarct size did not significantly differ between the normocholesterolemic and hypercholesterolemic control groups. The hemodynamic parameters were all markedly improved during the reperfusion period, and the myocardial infarct size was significantly reduced by delayed sevoflurane preconditioning in normocholesterolemic rats, but all of these improvements were reversed by N-(3-(aminomethyl)benzyl) acetamidine (1400W, 1 mg/kg; i.v., 10 min before ischemia), a selective inducible NOS (iNOS) inhibitor, and 5-hydroxy decanoate sodium (5 mg/kg, i.v., 10 min before ischemia), a mitochondrial ATP-dependent K? channel blocker. Such cardiac improvement induced by delayed sevoflurane preconditioning did not occur in hypercholesterolemic rats and was not exacerbated by 1400W or 5-hydroxy decanoate sodium. The expression of myocardial iNOS was markedly enhanced by delayed sevoflurane preconditioning in normocholesterolemic, but not in hypercholesterolemic rats. The expression of endothelial NOS and Bad did not differ among all groups. The expression of myocardial phosphorylated endothelial NOS, Bcl-2, and phosphorylated Bad in normocholesterolemic rats was not affected by delayed sevoflurane preconditioning but was decreased in the hypercholesterolemic control group, and this was not reversed by sevoflurane, compared with the normocholesterolemic control group. Taken together, these results indicate that sevoflurane preconditioning exerts delayed cardioprotection against IR injury in normocholesterolemic rats, which is blocked by hypercholesterolemia potentially via interference with the iNOS/mitochondrial ATP-dependent K? channel pathway.     

Peroxisome proliferator-activated receptor-gamma antagonists GW9662 and T0070907 reduce the protective effects of lipopolysaccharide preconditioning against organ failure caused by endotoxemia

OBJECTIVE: There is evidence that a) ligands of the nuclear receptor peroxisome proliferator-activated receptor (PPAR)-gamma and b) lipopolysaccharide preconditioning protect the organs against the multiple organ injury and dysfunction caused by endotoxemia. Here we investigate the hypothesis that the protective effects of lipopolysaccharide preconditioning are due to an enhanced formation of endogenous ligands of PPAR-gamma. DESIGN: Prospective, randomized study. SETTING: University-based research laboratory. SUBJECTS: Ninety-nine anesthetized male Wistar rats. INTERVENTIONS: Rats were pretreated with low-dose lipopolysaccharide (1 mg/kg intraperitoneally, 24 hrs before induction of endotoxemia) in the absence or presence of the selective PPAR-gamma antagonists GW9662 (1 mg/kg intraperitoneally) or T0070907 (1 mg/kg intraperitoneally) or the selective cyclooxygenase-2 inhibitor parecoxib (20 mg/kg intraperitoneally). At 24 hrs after preconditioning with low-dose lipopolysaccharide, the rats were subjected to acute severe endotoxemia (lipopolysaccharide 6 mg/kg intravenously). MEASUREMENTS AND MAIN RESULTS: Lipopolysaccharide preconditioning significantly attenuated the development of renal dysfunction (serum creatinine), hepatocellular injury (serum alanine aminotransferase and aspartate aminotransferase), and circulatory failure (hypotension) as well as the increase in the plasma levels of interleukin-1beta caused by severe endotoxemia. All of these beneficial effects afforded by preconditioning with lipopolysaccharide were attenuated by the specific PPAR-gamma antagonists used. In contrast, the cyclooxygenase-2 inhibitor parecoxib did not affect the beneficial effects afforded by preconditioning with lipopolysaccharide. CONCLUSIONS: We propose that endogenous ligands of PPAR-gamma contribute to the protection afforded by lipopolysaccharide preconditioning against the organ injury and dysfunction associated with severe endotoxemia in the rat.     

Mechanisms of 5-hydroxytryptamine(2A) receptor activation of the mitogen-activated protein kinase pathway in vascular smooth muscle

5-Hydroxytryptamine (5-HT) activates the extracellular signal-regulated kinase (Erk) mitogen-activated protein kinases (MAPKs) in the vasculature, resulting in contraction. The mechanisms by which this occurs are unclear. G protein-coupled receptors can activate Erk MAPK pathways through a variety of mechanisms, including stimulation of Src, phosphoinositide-3 kinase (PI-3-K), protein kinase C (PKC), or the epidermal growth factor (EGF) receptor tyrosine kinase. We hypothesize that 5-HT uses one or more of these pathways. In isolated strips of rat aorta, the MAPK/Erk kinase inhibitor U0126 (50 microM), Src inhibitor PP1 (0.5 microM), PKC inhibitors calphostin C (1 microM) and chelerythrine (10 microM), and the PI-3-K inhibitor LY294002 (1-20 microM) reduced 5-HT-induced contraction. The EGF receptor tyrosine kinase inhibitor AG1478 (0.25-1 microM) was without effect. Thus, 5-HT activates PKC, Src, and possibly PI-3-K to result in contraction. In rat aortic myocytes, 5-HT (1 microM) activated Erk MAPK proteins 2- to 3-fold over basal values; activation was reduced by U0126, PP1, and LY294002 and unaffected by calphostin C or chelerythrine, wortmannin, or AG1478. The lack of effect of EGF receptor tyrosine kinase and PI-3-K inhibitors was confirmed in that the EGF receptor immunoprecipitated from 5-HT-exposed cells did not display an increase in autophosphorylation, nor did 5-HT significantly increase activation of Akt/protein kinase B, a downstream substrate for PI-3-K. These data suggest that the rat aortic 5-HT(2A) receptor uses Src but not PKC, PI-3-K, or the EGF receptor tyrosine kinase in stimulating Erk MAPK activation.     

Protein kinase C betaII peptide inhibitor exerts cardioprotective effects in rat cardiac ischemia/reperfusion injury

Ischemia followed by reperfusion (I/R) in the presence of polymorphonuclear leukocytes (PMNs) results in a marked cardiac contractile dysfunction. A cell-permeable protein kinase C (PKC) betaII peptide inhibitor was used to test the hypothesis that PKC betaII inhibition could attenuate PMN-induced cardiac dysfunction by suppression of superoxide production from PMNs and increase NO release from vascular endothelium. The effects of the PKC betaII peptide inhibitor were examined in isolated ischemic (20 min) and reperfused (45 min) rat hearts with PMNs. The PKC betaII inhibitor (10 microM; n = 7) significantly attenuated PMN-induced cardiac dysfunction compared with I/R hearts (n = 9) receiving PMNs alone in left ventricular developed pressure (LVDP) and the maximal rate of LVDP (+dP/dt(max)) cardiac function indices (p < 0.01). The PKC betaII inhibitor at 10 microM significantly increased endothelial NO release from a basal value of 1.85 +/- 0.18 pmol NO/mg tissue to 3.49 +/- 0.62 pmol NO/mg tissue from rat aorta. It also significantly inhibited superoxide release (i.e., absorbance) from N-formyl-L-methionyl-L-leucyl-L-phenylalanine-stimulated rat PMNs from 0.13 +/- 0.01 to 0.02 +/- 0.004 (p < 0.01) at 10 microM. Histological analysis of the left ventricle of representative rat hearts from each group showed that the PKC betaII peptide inhibitor-treated hearts experienced a marked reduction in PMN vascular adherence and infiltration into the postreperfused cardiac tissue compared with I/R + PMN hearts (p < 0.01). These results suggest that the PKC betaII peptide inhibitor attenuates PMN-induced post-I/R cardiac contractile dysfunction by increasing endothelial NO release and by inhibiting superoxide release from PMNs.     

Erythropoietin protects against doxorubicin-induced cardiomyopathy via a phosphatidylinositol 3-kinase-dependent pathway

Doxorubicin (DOX) is an effective antineoplastic agent whose use has been limited by its cardiotoxic side effects. Recent studies have established that erythropoietin (EPO), a cytokine essential for red blood cell production, protects against ischemic injury in the heart and other organs. The purpose of this study was to assess whether EPO protects the heart against cardiotoxicity induced by DOX. We found that DOX-induced apoptosis and impaired heart function in mice were largely prevented by EPO administration. To investigate the mechanism of protection by EPO, cultured neonatal mouse ventricular myocytes were treated with EPO at therapeutic levels (i.e., 1 U/ml), before application of DOX (0.1-1.0 microM). EPO protected against DOX-induced cardiomyocyte death (by approximately 50%) and apoptosis assessed by annexin-V labeling, DNA fragmentation, and caspase-3 activity. DOX-mediated increases in reactive oxygen species, which trigger cardiotoxicity, were also reversed by preconditioning with EPO. These functional effects of EPO correlated with increased Akt/protein kinase B ( approximately 2-fold) and glycogen synthase kinase 3 (GSK-3; approximately 1.3-fold) phosphorylations, suggesting protection by EPO was mediated by phosphatidylinositol 3-kinase activation. Indeed, preventing Akt and GSK-3beta phosphorylations by phosphatidylinositol 3-kinase (PI3K) inhibition abolished protection by EPO against cardiomyocyte loss, apoptosis, and oxidative stress. Thus, pretreatment with therapeutic levels of EPO can protect the myocardium against DOX-induced impaired heart function and cardiomyocyte apoptosis by activating PI3K-Akt cell survival pathways.     

Hypoxic preconditioning induces delayed cardioprotection through p38 MAPK-mediated calreticulin upregulation

The protective mechanisms of hypoxic preconditioning (HPC) involve the mitigation of cellular calcium overload in cardiomyocytes. The sarcoplasmic reticulum (SR) chaperone calreticulin (CRT) plays an important role in regulating calcium homeostasis and is upregulated by HPC. The goal of this study was to show whether the late cardioprotection of HPC is mediated by calreticulin upregulation and to demonstrate whether the calreticulin induction is mediated by p38 MAPK phosphorylation. Hypoxic preconditioning was induced by hypoxemic hypoxic exposure by a 24-h period of normoxic reoxygenation before undergoing LAD occlusion in rats or hypoxia/reoxygenation (H/R) in cardiomyocytes. Ca uptake and release of the SR vesicles was determined by use of Ca and the Millipore filtration technique. Western blotting analysis was used to detect calreticulin expression and activity of p38 MAPK. Hypoxic preconditioning induced calreticulin upregulation and attenuated H/R injury in neonatal cardiomyocytes and myocardial ischemia injury by increasing calcium uptake and reducing calcium release in SR. Hearts from the HPC group were more resistant to sustained ischemia and had much stronger phosphorylation of p38 MAPK than sham operation. Inhibition of p38 MAPK with SB202190 (a selective p38 MAPK inhibitor) abolished the calreticulin upregulation and cardioprotection by HPC. Hypoxic preconditioning upregulates calreticulin expression through a p38 MAPK signaling pathway and protects cardiomyocytes from H/R (and ischemia) injury.     

Tedisamil blocks the transient and delayed rectifier K+ currents in mammalian cardiac and glial cells

The potassium currents in rat and guinea pig ventricular myocytes and mouse astrocytes were studied using tedisamil, a novel antiarrhythmic agent. A 1 to 20 microM dosage of tedisamil caused marked prolongation of the action potential in isolated rat ventricular myocytes, mimicking its reported effects on multicellular rat heart preparations. Under voltage clamp conditions, tedisamil caused a dose-dependent increase in the speed of inactivation of the transient outward K+ current (Ito), the predominant outward current in rat ventricular myocytes. In cardiac myocytes, the tedisamil block was neither use- nor voltage-dependent. The slow reversibility of drug action when applied from the outside, and its effectiveness when applied intracellularly, suggested an internal site of drug action. In guinea pig ventricular myocytes, tedisamil blocked the slowly developing time-dependent delayed rectifier K+ current (IK) over the same concentration range as that found for Ito in the rat myocytes. Tedisamil reduced this current without changing the characteristics of its slow (tau approximately 1 sec) activation. The effects of tedisamil on Ito and IK were independent of the phosphorylation state of the channel, as assessed by the equal effectiveness of the drug in the presence or absence of isoproterenol. Tedisamil also blocked the transient K+ current and the delayed rectifier current (IK) in mouse astrocytes over the same concentration range as that found in the cardiac myocytes and by a process that accelerated (transient K+ current) or mimicked (IK) inactivation. At concentrations of up to 50 microM, tedisamil had little effect on the time-dependent inward rectifier K+ current, or inward calcium current in rat or guinea pig ventricular myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

长期运动预适应模型大鼠心脏蛋白激酶C和热休克蛋白70的表达

背景:研究表明蛋白激酶C和热休克蛋白70可能参与运动预适应的心脏保护作用。目的:观察长期运动预适应对大鼠心脏蛋白激酶C和热休克蛋白70的影响及其对心脏保护的作用机制。方法:将SD大鼠随机分为对照组,力竭运动组和运动预适应组。对照组和力竭运动组大鼠常规饲养3周,运动预适应组大鼠进行3周的间歇性游泳运动建立长期运动预适应动物模型。3周后,力竭运动组和运动预适应组大鼠进行一次性力竭游泳运动。结果与结论:大鼠力竭运动后,力竭运动组心脏热休克蛋白70表达高于对照组(P<0.05);先经3周运动预适应再进行力竭运动后,运动预适应组心脏蛋白激酶C和热休克蛋白70的表达高于力竭运动组(P<0.05)。结果证实,长期运动预适应能激活心脏蛋白激酶C,诱导热休克蛋白70的合成增多,从而发挥其心脏保护作用。