Opening of potassium channels modulates mitochondrial function in rat skeletal muscle

We have investigated the presence of diazoxide- and nicorandil-activated K+ channels in rat skeletal muscle. Activation of potassium transport in the rat skeletal muscle myoblast cell line L6 caused a stimulation of cellular oxygen consumption, implying a mitochondrial effect. Working with isolated rat skeletal muscle mitochondria, both potassium channel openers (KCOs) stimulate respiration, depolarize the mitochondrial inner membrane and lead to oxidation of the mitochondrial NAD-system in a strict potassium-dependent manner. This is a strong indication for KCO-mediated stimulation of potassium transport at the mitochondrial inner membrane. Moreover, the potassium-specific effects of both diazoxide and nicorandil on oxidative phosphorylation in skeletal muscle mitochondria were completely abolished by the antidiabetic sulfonylurea derivative glibenclamide, a well-known inhibitor of ATP-regulated potassium channels (K(ATP) channels). Since both diazoxide and nicorandil facilitated swelling of de-energised mitochondria in KSCN buffer at the same concentrations, our results implicate the presence of a mitochondrial ATP-regulated potassium channel (mitoK(ATP) channel) in rat skeletal muscle which can modulate mitochondrial oxidative phosphorylation.     

Diazoxide triggers cardioprotection against apoptosis induced by oxidative stress

Although mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels have been reported to reduce the extent of apoptosis, the critical timing of mitoK(ATP) channel opening required to protect myocytes against apoptosis remains unclear. In the present study, we examined whether the mitoK(ATP) channel serves as a trigger of cardioprotection against apoptosis induced by oxidative stress. Apoptosis of cultured neonatal rat cardiomyocytes was determined by flow cytometry (light scatter and propidium iodide/annexin V-FITC fluorescence) and by nuclear staining with Hoechst 33342. Mitochondrial membrane potential (DeltaPsi) was measured by flow cytometry of cells stained with rhodamine-123 (Rh-123). Exposure to H(2)O(2) (500 microM) induced apoptosis, and the percentage of apoptotic cells increased progressively and peaked at 2 h. This H(2)O(2)-induced apoptosis was associated with the loss of DeltaPsi, and the time course of decrease in Rh-123 fluorescence paralleled that of apoptosis. Pretreatment of cardiomyocytes with diazoxide (100 microM), a putative mitoK(ATP) channel opener, for 30 min before exposure to H(2)O(2) elicited transient and mild depolarization of DeltaPsi and consequently suppressed both apoptosis and DeltaPsi loss after 2-h exposure to H(2)O(2). These protective effects of diazoxide were abrogated by the mitoK(ATP) channel blocker 5-hydroxydecanoate (500 microM) but not by the sarcolemmal K(ATP) channel blocker HMR-1098 (30 microM). Our results suggest for the first time that diazoxide-induced opening of mitoK(ATP) channels triggers cardioprotection against apoptosis induced by oxidative stress in rat cardiomyocytes.     

Diazoxide-induced cardioprotection via DeltaPsim loss depending on timing of application

Although the role of mitochondrial ATP-sensitive potassium (mitoKATP) channels in cardioprotection is widely accepted, it remains unclear when their opening is critical for protection. We tested the hypothesis that the mitoKATP channel acts as a trigger or mediator of protection against apoptosis through loss of mitochondrial inner membrane potential (DeltaPsim). Exposure of neonatal rat cardiomyocytes to H2O2 (0.5 mmol/L) resulted in apoptosis associated with severe DeltaPsim loss. Pretreatment with diazoxide (20 to 100 micromol/L) prevented H2O2-induced apoptosis and DeltaPsim loss at 2 but not 18 h after exposure, while the latter was prevented by cotreatment with diazoxide. Lack of protection by pretreatment with diazoxide was observed in cardiomyocytes cultured in a medium containing H2O2 for 2 h and then not containing for 16 h. The slopes of the regression lines of the relationship between the proportion of apoptotic cells and DeltaPsim loss (y = -0.89 vs. -0.42) and the proportion of cells with high side scatter signal differed between cardiomyocytes exposed H2O2 for 2 and 18 h. Diazoxide per se caused a transient DeltaPsim loss (within 30 min) with a recovery followed by persistent DeltaPsim loss (after 6 h). Inhibition of the former by 5-hydroxydecanoate (5-HD, 0.5 mmol/L) abolished protection of pretreatment with diazoxide (trigger phase), while that of the latter prevented the protection of cotreatment with diazoxide (mediator phase). Our results suggest that mitoKATP channels act as a trigger and mediator of cardioprotection through a transient or persistent DeltaPsim loss depending on phenotypic consequence in response to oxidants.     

Single channel studies of the ATP-regulated potassium channel in brain mitochondria

Mitochondrial potassium channels in the brain have been suggested to have an important role in neuroprotection. The single channel activity of mitochondrial potassium channels was measured after reconstitution of the purified inner membrane from rat brain mitochondria into a planar lipid bilayer. In addition to a large conductance potassium channel that was described previously, we identified a potassium channel that has a mean conductance of 219 ± 15 pS. The activity of this channel was inhibited by ATP/Mg²⁺ and activated by the potassium channel opener BMS191095. Channel activity was not influenced either by 5-hydroxydecanoic acid, an inhibitor of mitochondrial ATP-regulated potassium channels, or by the plasma membrane ATP-regulated potassium channel blocker HMR1098. Likewise, this mitochondrial potassium channel was unaffected by the large conductance potassium channel inhibitor iberiotoxin or by the voltage-dependent potassium channel inhibitor margatoxin. The amplitude of the conductance was lowered by magnesium ions, but the opening ability was unaffected. Immunological studies identified the Kir6.1 channel subunit in the inner membrane from rat brain mitochondria. Taken together, our results demonstrate for the first time the single channel activity and properties of an ATP-regulated potassium channel from rat brain mitochondria.     

Quinine Inhibits Mitochondrial ATP-regulated Potassium Channel from Bovine Heart

The mitochondrial ATP-regulated potassium (mitoK_ATP) channel has been suggested as trigger and effector in myocardial ischemic preconditioning. However, molecular and pharmacological properties of the mitoK_ATP channel remain unclear. In the present study, single-channel activity was measured after reconstitution of the inner mitochondrial membrane from bovine ventricular myocardium into bilayer lipid membrane. After incorporation, a potassium-selective current was recorded with mean conductance of 103 ± 9 pS in symmetrical 150 mM KCl. Single-channel activity of this reconstituted protein showed properties of the mitoK_ATP channel: it was blocked by 500 μM ATP/Mg, activated by the potassium-channel opener diazoxide at 30 μM, inhibited by 50 μM glibenclamide or 150 μM 5-hydroxydecanoic acid, and was not affected by the plasma membrane ATP-regulated potassium-channel blocker HMR1098 at 100 μM. We observed that the mitoK_ATP channel was blocked by quinine in the micromolar concentration range. The inhibition by quinine was additionally verified with the use of ⁸⁶Rb⁺ flux experiments and submitochondrial particles. Quinine inhibited binding of the sulfonylurea derivative [³H]glibenclamide to the inner mitochondrial membrane. We conclude that quinine inhibits the cardiac mitoK_ATP channel by acting on the mitochondrial sulfonylurea receptor.(P. Bednarczyk and A. Kicińska) These authors contributed equally to this work.This revised version was published online in August 2005 with a corrected cover date.     

Clusterin: a protective mediator for ischemic cardiomyocytes?

We examined the relationship between clusterin and activated complement in human heart infarction and evaluated the effect of this protein on ischemic rat neonatal cardiomyoblasts (H9c2) and isolated adult ventricular rat cardiomyocytes as in vitro models of acute myocardial infarction. Clusterin protects cells by inhibiting complement and colocalizes with complement on jeopardized human cardiomyocytes after infarction. The distribution of clusterin and complement factor C3d was evaluated in the infarcted human heart. We also analyzed the protein expression of clusterin in ischemic H9c2 cells. The binding of endogenous and purified human clusterin on H9c2 cells was analyzed by flow cytometry. Furthermore, the effect of clusterin on the viability of ischemically challenged H9c2 cells and isolated adult ventricular rat cardiomyocytes was analyzed. In human myocardial infarcts, clusterin was found on scattered, morphologically viable cardiomyocytes within the infarcted area that were negative for complement. In H9c2 cells, clusterin was rapidly expressed after ischemia. Its expression was reduced after reperfusion. Clusterin bound to single annexin V-positive or annexin V and propidium iodide-positive H9c2 cells. Clusterin inhibited ischemia-induced death in H9c2 cells as well as in isolated adult ventricular rat cardiomyocytes in the absence of complement. We conclude that ischemia induces the upregulation of clusterin in ischemically challenged, but viable, cardiomyocytes. Our data suggest that clusterin protects cardiomyocytes against ischemic cell death via a complement-independent pathway.     

Berberine Inhibits Doxorubicin-Triggered Cardiomyocyte Apoptosis via Attenuating Mitochondrial Dysfunction and Increasing Bcl-2 Expression

Cardiomyocyte apoptosis is an important event in doxorubicin (DOX)-induced cardiac injury. The aim of the present study was to investigate the protection of berberine (Ber) against DOX- triggered cardiomyocyte apoptosis in neonatal rat cardiomyocytes and rats. In neonatal rat cardiomyocytes, Ber attenuated DOX-induced cellular injury and apoptosis in a dose-dependent manner. However, Ber has no significant effect on viability of MCF-7 breast cancer cells treated with DOX. Ber reduced caspase-3 and caspase-9, but not caspase-8 activity in DOX-treated cardiomyocytes. Furthermore, Ber decreased adenosine monophosphate-activated protein kinase α (AMPKα) and p53 phosphorylation at 2 h, cytosolic cytochrome c and mitochondrial Bax levels and increased Bcl-2 level at 6 h in DOX-stimulated cardiomyocytes. Pretreatment with compound C, an AMPK inhibitor, also suppressed p53 phosphorylation and apoptosis in DOX-treated cardiomyocytes. DOX stimulation for 30 min led to a loss of mitochondrial membrane potential and a rise in the AMP/ATP ratio. Ber markedly reduced DOX-induced mitochondrial membrane potential loss and an increase in the AMP/ATP ratio at 1 h and 2 h post DOX exposure. In in vivo experiments, Ber significantly improved survival, increased stroke volume and attenuated myocardial injury in DOX-challenged rats. TUNEL and Western blot assays showed that Ber not only decreased myocardial apoptosis, caspase-3 activation, AMPKα and p53 phosphorylation, but also increased Bcl-2 expression in myocardium of rats exposed to DOX for 84 h. These findings indicate that Ber attenuates DOX-induced cardiomyocyte apoptosis via protecting mitochondria, inhibiting an increase in the AMP/ATP ratio and AMPKα phosphorylation as well as elevating Bcl-2 expression, which offer a novel mechanism responsible for protection of Ber against DOX-induced cardiomyopathy.     

RXR agonists inhibit oxidative stress-induced apoptosis in H9c2 rat ventricular cells

Retinoid X receptor (RXR) plays a central role in the regulation of intracellular receptor signaling pathways. We examined its role in regulating oxidative stress-induced apoptosis in H9c2 rat ventricular cells. We showed for the first time that functional RXR protein was downregulated by hydrogen peroxide (H₂O₂) in H9c2 cardiomyocytes. Natural and synthetic agonists of RXR, 9-cis-RA, and LGD1069 respectively, prevented H₂O₂-triggered apoptosis, and this anti-apoptotic effect was inhibited by the RXR antagonist HX531. Further investigation into the protective mechanisms of RXR demonstrated that H₂O₂-induced loss of mitochondrial membrane potential, mitochondrial release of cytochrome c and caspase-3 activation were all significantly attenuated by pretreatment with RXR agonists. Furthermore, this protection was associated with a reduction in intracellular reactive oxygen species and an upregulation in catalase activity. Thus, these data indicate that pharmacological activation of RXR exerts protective effects against H₂O₂-induced apoptosis in H9c2 rat ventricular cells through antioxidant and mitochondria-protective mechanisms.     

Recombinant PTD-Cu/Zn SOD attenuates hypoxia–reoxygenation injury in cardiomyocytes

Abstract

Background. Oxidative stress plays a pivotal role in myocardial ischemia–reperfusion injury. Increasing the protein expression of intracellular Cu/Zn SOD, which is the major endogenous antioxidant enzyme, may attenuate or prevent hypoxia–reoxygenation injury (HRI) in cultured cardiomyocytes. However, ectogenic Cu/Zn-SOD can hardly be transferred into cells to exert biological effects. In this study, we constructed PTD-Cu/Zn SOD plasmid with a kind of translocation structure-Protein transduction domain (PTD) and detected its transmembrane ability and antioxidant effects in H9c2 rat cardiomyocytes subjected to hypoxia/reoxygenation injury (HRI). Methods. We constructed the pET-PTD-Cu/Zn SOD (CDs) prokaryotic expression vectors in plasmid that were inserted into E. coli BL21 to induce the protein expression of PTD-Cu/Zn SOD. H9c2 cardiomyocyte HRI was achieved by exposing cardiomyocytes to 12 h hypoxia followed by 2 h reoxygenation. Protein expression of PTD-Cu/Zn SOD in cardiomyocytes was assayed by Western blot and their enzyme activities were investigated by immunohistochemistry and flow cytometry. Results. In cultured cardiomyocytes hypoxia–reoxygenation injury model, exogenous PTD-Cu/Zn SOD could penetrate cell membrane to clear superoxide anion and decrease hydrogen peroxide level in H9c2 cardiomyocytes subjected to HRI. The level of mitochondrial membrane potential was restored to normal, and the cell apoptosis was reduced in cardiomyocytes with PTD-Cu/Zn SOD treatment during HRI. Conclusion. Recombinant PTD-Cu/Zn SOD could scavenge intracellular-free superoxide anion, protect mitochondria from damages, and attenuate the hypoxia–reoxygenation injury in cultured cardiomyocytes.     

Exogenous hydrogen sulfide attenuates diabetic myocardial injury through cardiac mitochondrial protection

In the study, we investigated how exogenous H₂S (hydrogen sulfide) influenced streptozotocin (STZ)-induced diabetic myocardial injury through cardiac mitochondrial protection and nitric oxide (NO) synthesis in intact rat hearts and primary neonatal rat cardiomyocytes. Diabetes was induced by STZ (50?mg/kg) and the daily administration of 100?μM NaHS (sodium hydrosulfide, an H₂S donor) in the diabetes?+?NaHS treatment group. At the end of 4, 8, and 12?weeks, the morphological alterations and functions of the hearts were observed using transmission electron microscopy and echocardiography system. The percentage of apoptotic cardiomyocytes, the mitochondrial membrane potential, the production of reactive oxygen species (ROS) and the level of NO were measured. The expressions of cystathionine-γ-lyase (CSE), caspase-3 and -9, the mitochondrial NOX4 and cytochrome c were analyzed by western blotting. The results showed the cardiac function injured, morphological changes and the apoptotic rate increased in the diabetic rat hearts. In the primary neonatal rat cardiomyocytes of high glucose group, ROS production was increased markedly, whereas the expression of CSE and the level of NO was decreased. However, treatment with NaHS significantly reversed the diabetic rat hearts function, the morphological changes and decreased the levels of ROS and NO in the primary neonatal rat cardiomyocytes administrated with high glucose group. Furthermore, NaHS down-regulated the expression of mitochondrial NOX4 and caspase-3 and -9 and inhibited the release of cytochrome c from mitochondria in the primary neonatal rat cardiomyocytes. In conclusion, H₂S is involved in the attenuation of diabetic myocardial injury through the protection of cardiac mitochondria.     

KPC1 alleviates hypoxia/reoxygenation-induced apoptosis in rat cardiomyocyte cells though BAX degradation

Bax triggers cell apoptosis by permeabilizing the outer mitochondrial membrane, leading to membrane potential loss and cytochrome c release. However, it is unclear if proteasomal degradation of Bax is involved in the apoptotic process, especially in heart ischemia-reperfusion (I/R)-induced injury. In the present study, KPC1 expression was heightened in left ventricular cardiomyocytes of patients with coronary heart disease (CHD), in I/R-myocardium in vivo and in hypoxia and reoxygenation (H/R)-induced cardiomyocytes in vitro. Overexpression of KPC1 reduced infarction size and cell apoptosis in I/R rat hearts. Similarly, the forced expression of KPC1 restored mitochondrial membrane potential (MMP) and cytochrome c release driven by H/R in H9c2 cells, whereas reducing cell apoptosis, and knockdown of KPC1 by short-hairpin RNA (shRNA) deteriorated cell apoptosis induced by H/R. Mechanistically, forced expression of KPC1 promoted Bax protein degradation, which was abolished by proteasome inhibitor MG132, suggesting that KPC1 promoted proteasomal degradation of Bax. Furthermore, KPC1 prevented basal and apoptotic stress-induced Bax translocation to mitochondria. Bax can be a novel target for the antiapoptotic effects of KPC1 on I/R-induced cardiomyocyte apoptosis and render mechanistic penetration into at least a subset of the mitochondrial effects of KPC1.     

线粒体钾通道参与葛根素抗心肌细胞缺氧/复氧损伤的作用

目的:探讨线粒体ATP敏感性钾通道和线粒体钙激活钾通道在葛根素预处理抗心肌细胞缺氧/复氧损伤中的作用。方法:采用酶解分离大鼠心肌细胞复制心肌细胞缺氧/复氧模型,台盼蓝拒染法测定心肌细胞存活率;四甲基罗丹明乙酯(TMRE)孵育测定线粒体膜电位值;分离线粒体测定线粒体渗透性转换孔开放程度。结果:与缺氧/复氧组相比,葛根素(0.24mmol/L)预处理5min可明显增加心肌细胞的存活率,线粒体ATP敏感性钾通道抑制剂5-羟基癸酸(100μmol/L,预处理20min)或线粒体钙激活钾通道阻断剂paxilline(1μmol/L,预处理5min)均可拮抗葛根素的作用。葛根素预处理可明显减弱缺氧引起的线粒体膜电位的耗损,5-羟基癸酸和paxilline都能明显拮抗其作用。在分离心肌线粒体模型上,葛根素显著减弱CaCl2诱导的线粒体在A520处吸光度降低,其作用与单独应用线粒体渗透性转换孔抑制剂环孢菌素A相似;5-羟基癸酸和paxilline可拮抗葛根素的保护作用。结论:在大鼠分离心肌细胞模型或分离线粒体模型上,葛根素预处理具有抗缺氧/复氧损伤的作用,这种保护作用可能与其促进线粒体ATP敏感性钾通道和线粒体钙激活钾通道的开放,进而稳定线粒体膜电位,抑制线粒体渗透性转换孔开放有关。     

Mechanisms by which opening the mitochondrial ATP- sensitive K(+) channel protects the ischemic heart

Diazoxide opening of the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel protects the heart against ischemia-reperfusion injury by unknown mechanisms. We investigated the mechanisms by which mitoK(ATP) channel opening may act as an end effector of cardioprotection in the perfused rat heart model, in permeabilized fibers, and in rat heart mitochondria. We show that diazoxide pretreatment preserves the normal low outer membrane permeability to nucleotides and cytochrome c and that these beneficial effects are abolished by the mitoK(ATP) channel inhibitor 5-hydroxydecanoate. We hypothesize that an open mitoK(ATP) channel during ischemia maintains the tight structure of the intermembrane space that is required to preserve the normal low outer membrane permeability to ADP and ATP. This hypothesis is supported by findings in mitochondria showing that small decreases in intermembrane space volume, induced by either osmotic swelling or diazoxide, increased the half-saturation constant for ADP stimulation of respiration and sharply reduced ATP hydrolysis. These effects are proposed to lead to preservation of adenine nucleotides during ischemia and efficient energy transfer upon reperfusion.     

ATP-sensitive potassium channel in mitochondria of the eukaryotic microorganism Acanthamoeba castellanii

We describe the existence of a potassium ion transport mechanism in the mitochondrial inner membrane of a lower eukaryotic organism, Acanthamoeba castellanii. We found that substances known to modulate potassium channel activity influenced the bioenergetics of A. castellanii mitochondria. In isolated mitochondria, the rate of resting respiration is increased by about 10% in response to potassium channel openers, i.e. diazoxide and BMS-191095, during succinate-, malate-, or NADH-sustained respiration. This effect is strictly dependent on the presence of potassium ions in an incubation medium and is reversed by glibenclamide (a potassium channel blocker). Diazoxide and BMS-191095 also caused a slight but statistically significant depolarization of mitochondrial membrane potential (measured with a TPP(+)-specific electrode), regardless of the respiratory substrate used. The resulting steady state value of membrane potential was restored after treatment with glibenclamide or 1 mM ATP. Additionally, the electrophysiological properties of potassium channels present in the A. castellanii inner mitochondrial membrane are described in the reconstituted system, using black lipid membranes. Conductance from 90 +/- 7 to 166 +/- 10 picosiemens, inhibition by 1 mM ATP/Mg(2+) or glibenclamide, and activation by diazoxide were observed. These results suggest that an ATP-sensitive potassium channel similar to that of mammalian mitochondria is present in A. castellanii mitochondria.     

Ischemic Postconditioning Reduces NMDA Receptor Currents Through the Opening of the Mitochondrial Permeability Transition Pore and KATP Channel in Mouse Neurons

Ischemic postconditioning (PostC) is known to reduce cerebral ischemia/reperfusion (I/R) injury; however, whether the opening of mitochondrial ATP-dependent potassium (mito-K_ATP) channels and mitochondrial permeability transition pore (mPTP) cause the depolarization of the mitochondrial membrane that remains unknown. We examined the involvement of the mito-K_ATP channel and the mPTP in the PostC mechanism. Ischemic PostC consisted of three cycles of 15 s reperfusion and 15 s re-ischemia, and was started 30 s after the 7.5 min ischemic load. We recorded N-methyl-d-aspartate receptors (NMDAR)-mediated currents and measured cytosolic Ca²⁺ concentrations, and mitochondrial membrane potentials in mouse hippocampal pyramidal neurons. Both ischemic PostC and the application of a mito-K_ATP channel opener, diazoxide, reduced NMDAR-mediated currents, and suppressed cytosolic Ca²⁺ elevations during the early reperfusion period. An mPTP blocker, cyclosporine A, abolished the reducing effect of PostC on NMDAR currents. Furthermore, both ischemic PostC and the application of diazoxide potentiated the depolarization of the mitochondrial membrane potential. These results indicate that ischemic PostC suppresses Ca²⁺ influx into the cytoplasm by reducing NMDAR-mediated currents through mPTP opening. The present study suggests that depolarization of the mitochondrial membrane potential by opening of the mito-K_ATP channel is essential to the mechanism of PostC in neuroprotection against anoxic injury.

     

Adenosine A1 and A3 receptor agonists reduce hypoxic injury through the involvement of P38 MAPK

Activation of either the A₁ adenosine receptor (A₁R) or the A₃ adenosine receptor (A₃R), by their specific agonists CCPA and Cl-IB-MECA, respectively, protects cardiac cells in culture against ischemic injury. Yet the full protective mechanism remains unclear. In this study, we therefore examined the involvement of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK) phosphorylation in this protective intracellular signaling mechanism. Furthermore, we investigated whether p38 MAPK phosphorylation occurs upstream or downstream from the opening of mitochondrial ATP-sensitive potassium (K_ATP) channels. The role of p38 MAPK activation in the intracellular signaling process was studied in cultured cardiomyocytes subjected to hypoxia, that were pretreated with CCPA or Cl-IB-MECA or diazoxide (a mitochondrial K_ATP channel opener) with and without SB203580 (a specific inhibitor of phosphorylated p38 MAPK). Cardiomyocytes were also pretreated with anisomycin (p38 MAPK activator) with and without 5-hydroxy decanoic acid (5HD) (a mitochondrial K_ATP channel blocker). SB203580 together with the CCPA, Cl-IB-MECA or diazoxide abrogated the protection against hypoxia as shown by the level of ATP, lactate dehydrogenase (LDH) release, and propidium iodide (PI) staining. Anisomycin protected the cardiomyocytes against ischemic injury and this protection was abrogated by SB203580 but not by 5HD. Conclusions Activation of A₁R or A₃R by CCPA or Cl-IB-MECA, respectively, protects cardiomyocytes from hypoxia via phosphorylation of p38 MAPK, which is located downstream from the mitochondrial K_ATP channel opening. Elucidating the signaling pathway by which adenosine receptor agonists protect cardiomyocytes from hypoxic damage, will facilitate the development of anti ischemic drugs.     

活性氧自由基对心肌细胞损伤效应研究

为探讨活性氧自由基对心肌细胞的影响 ,采用胰蛋白酶酶消化法分离SD乳鼠心肌细胞 ,培养于适当的条件并观察其形态学和生理学方面的特征 ;加入H_{2O2 活性氧刺激心肌细胞 ,模拟氧自由基损伤心肌细胞方式 ,构建心肌细胞缺血再灌注损伤的模型并了解H2O2 对心肌细胞的损伤作用。结果表明胰蛋白酶消化分离的心肌细胞能够在体外完好生长 ,并能够在一段时间内维持其原有的生理特性 ;MTT检测结果和形态学观察结果表明H2O2 对心肌细胞的损伤与其浓度和作用时间呈正比关系 ,TUNEL和DNA凝胶电泳分析结果显示 ,H2O2 在心肌细胞中的积累是造成细胞凋亡的主要因素之一。}     

Do modulators of the mitochondrial K(ATP) channel change the function of mitochondria in situ?

Pharmacological opening of mitochondrial cardiac ATP-sensitive potassium (K(ATP)) channels has the chance to be a promising but still controversial cardioprotective mechanism. Physiological roles of mitochondrial K(ATP) channels in the myocardium remain unclear. We studied the effects of diazoxide, a specific opener of these channels, on the function of rat mitochondria in situ in saponin-permeabilized fibers using an ionic medium that mimics the cytosol. In the presence of NADH-producing substrates (malate + glutamate), neither 100 microm diazoxide nor 100 microm glibenclamide (a K(ATP) channel blocker) changed the mitochondrial respiration in the absence or presence of ADP. Because the K(ATP) channel function could be modified by changes in adenine nucleotide concentrations near the mitochondria, we studied the effects of diazoxide and glibenclamide on the functional activity of mitochondrial kinases. Both diazoxide and glibenclamide did not change the in situ ADP sensitivity in the presence or absence of creatine (apparent K(m) values for ADP were, respectively, 59 +/- 9 and 379 +/- 45 microm). Similarly, stimulation of the mitochondrial respiration with AMP in the presence of ATP due to adenylate kinase activity was not affected by the modulators of K(ATP) channels. However, when succinate was used as substrate, diazoxide significantly inhibited basal respiration by 22% and maximal respiration by 24%. Thus, at a cardioprotective dose, the main functional effect of diazoxide depends on respiratory substrates and seems not to be related to K(ATP) channel activity.     

Activation of mitochondrial ATP-sensitive potassium channels increases cell viability against rotenone-induced cell death

We recently showed that activation of ATP-sensitive potassium (KATP) channels in PC12 cells induces protection against the neurotoxic effect of rotenone, a mitochondrial complex I inhibitor. In this study, we sought to determine the locus of the KATP channels that mediate this protection in PC12 cells. We found that pretreatment of PC12 cells with diazoxide, a mitochondrial KATP channel selective opener, dose-dependently increases cell viability against rotenone-induced cell death as indicated in trypan blue exclusion assays. The protective effect of this preconditioning is attenuated by 5-hydroxydecanoic acid (5-HD), a selective mitochondrial KATP channel antagonist but not in the presence of HMR-1098, a selective plasma membrane KATP potassium channel antagonist. In contrast, P-1075, a selective plasma membrane KATP channel opener, does not induce protection. Using specific antibodies against SUR1 and Kir6.1, we detected immunoreactive proteins of apparent molecular masses 155 and 50 kDa, corresponding to those previously reported for SUR1 and Kir6.1, respectively, in the mitochondria-enriched fraction of PC12 cells. In addition, whole cell patch-clamp studies revealed that inward currents in PC12 cells are insensitive to P-1075, HMR-1098, glibenclamide and diazoxide, indicating that functional plasma membrane KATP channels are negligible. Taken together, our results demonstrate for the first time that activation of mitochondrial KATP channels elicits protection against rotenone-induced cell death.