Leptin attenuates hypoxia/reoxygenation-induced activation of the intrinsic pathway of apoptosis in rat H9c2 cells

Cardiomyocyte apoptosis is a component of cardiac remodeling that can contribute to heart failure in obesity. A role for leptin in mediating this process has been suggested and the objective of this work was to investigate the effect of leptin on apoptosis and associated mechanisms in H9c2 cells which were subjected to hypoxia/reoxygenation (HR) to mimic myocardial ischemia/reperfusion. Qualitative immunofluorescent and quantitative laser scanning cytometry approaches demonstrated that exposure of cells to HR increased DNA fragmentation (TUNEL staining) which was attenuated by leptin (6 nM, 1 h) pretreatment. We also found increased annexin-V binding and caspase-3 activity in cells exposed to HR, both of which were attenuated by leptin pretreatment. Leptin reduced HR-induced translocation of the pro-apoptotic protein Bax to the mitochondrial membrane, which provides a mechanism to explain its protective effect. Consequently, leptin attenuated the HR-induced decrease in mitochondrial membrane potential and increase in cytochrome c release from mitochondria. Leptin treatment increased the phosphorylation of p38 MAPK and AMPK and respective inhibitors of these kinases, SB203580 and Compound C, prevented the ability of leptin to decrease HR-induced caspase-3 activity. In conclusion, we establish mechanisms via which leptin exerts anti-apoptotic effects that may be of significance in understanding the development of heart failure in obesity. J. Cell. Physiol. 221: 490–497, 2009. © 2009 Wiley-Liss, Inc.     

H2O2-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells

This study employed confocal laser scanning microscopy to monitor the effect of H2O2 on cytosolic as well as mitochondrial calcium (Ca2+) concentrations, mitochondrial inner membrane potential (psi m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H2O2, in the absence of extracellular Ca2+ ([Ca2+],) led to an increase either in cytosolic and in mitochondrial Ca2+ concentration. Additionally, H2O2 induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H2O2 on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H2O2. However, the H2O2-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca2+ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H2O2-evoked changes in mitochondrial Ca2+ concentration but not those in membrane potential and FAD state. The present results have indicated that H2O2 can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H2O2. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas.     

重组nkx2.5腺病毒抑制过氧化氢诱导的H9c2细胞凋亡

为研究心脏发育关键基因nkx2.5的功能及应用价值,构建Ad-Nkx2.5重组腺病毒,并检测nkx2.5过表达拮抗氧化应激损伤的效应及机制。采用AdEasy腺病毒表达系统构建Ad-Nkx2.5重组腺病毒,建立H2O2诱导H9c2心肌细胞凋亡模型,分别用Ad-Nkx2.5重组病毒或对照病毒感染细胞,采用Hoechst33342染色观察细胞形态变化、MTT法检测细胞存活率,免疫印迹检测caspase-3活化、细胞色素C的胞浆含量。并通过Real-timePCR检测凋亡相关基因bcl-2和bax表达。结果发现,nkx2.5过表达促进H9c2细胞存活,抑制H2O2诱导的caspase-3活化及线粒体细胞色素C的释放。Nkx2.5过表达上调bcl-2表达,显著下调H2O2诱导的bax表达。并发现H2O2对Nkx2.5核定位无明显影响。结果显示重组腺病毒介导的Nkx2.5过表达可通过调控凋亡相关基因表达,抑制线粒体凋亡途径,保护心肌细胞抗氧化损伤。     

Vitamin D sensitizes breast cancer cells to the action of H2O2: mitochondria as a convergence point in the death pathway

Calcitriol, the hormonal form of vitamin D3, sensitizes breast cancer cells to reactive oxygen species (ROS)-dependent cytotoxicity induced by various anticancer modalities. This effect could be due to increased generation of ROS and/ or to increased sensitivity of the target cells to ROS. This work examined the effect of calcitriol on the damage inflicted on breast cancer cells by the direct action of ROS represented by H2O2. Treatment of MCF-7 cells with H2O2 resulted in activation of caspase 7 as well as induction of caspase-independent cell death. Both were enhanced by 48-72 h of pretreatment with calcitriol. This effect was not due to modulation of H2O2 degradation or to a specific effect on *OH-mediated cytotoxicity. The H2O2-induced drop in mitochondrial membrane potential and release of cytochrome c were enhanced by calcitriol. These findings indicate that calcitriol sensitizes breast cancer cells to ROS-induced death by affecting event(s) common to both caspase-dependent and -independent modes of cell death upstream to mitochondrial damage.     

Protective effects of cynaroside against H₂O₂-induced apoptosis in H9c2 cardiomyoblasts

Flavonoids with potent anti-oxidative effects are the major effective components in traditional herbal medicine used in treating cardiovascular diseases. Cynaroside is a flavonoid compound that exhibits anti-oxidative capabilities. However, little is known about its effect on oxidative injury to cardiac myocytes and the underlying mechanisms. This study was designed to investigate the protective effects of cynaroside against H(2) O(2) -induced apoptosis in H9c2 cardiomyoblasts. H9c2 cells were pretreated with cynaroside for 4 h before exposure to 150 μM H(2) O(2) for 6 h. H(2) O(2) treatment caused severe injury to the H9c2 cells, which was accompanied by apoptosis, as revealed by analysis of cell nuclear morphology, through Annexin V FITC/PI staining and caspase proteases activation. Cynaroside pretreatment significantly reduced the apoptotic rate by enhancing the endogenous anti-oxidative activity of superoxide dismutase, glutathione peroxidase, and catalase, thereby inhibiting intracellular reactive oxygen species (ROS) generation. Moreover, cynaroside moderated H(2) O(2) -induced disruption of mitochondrial membrane potential, increased the expression of anti-apoptotic protein Bcl-2 while decreased the expression of pro-apoptotic protein Bax, and thereby inhibited the release of apoptogenic factors (cytochrome c and smac/Diablo) from mitochondria in H9c2 cells. Our data also demonstrated that cynaroside pretreatment showed an inhibitory effect on the H(2) O(2) -induced increase in c-Jun N-terminal kinase (JNK) and P53 protein expression. These results suggest that cynaroside prevents H(2) O(2) -induced apoptosis in H9c2 cell by reducing the endogenous production of ROS, maintaining mitochondrial function, and modulating the JNK and P53 pathways.     

Apollon/Bruce is upregulated by Humanin

Hydrogen sulfide (H₂S) protects cardiomyoblasts against high glucose (HG)-induced injury by inhibiting the activation of p38 mitogen-activated protein kinase (MAPK). This study aims to determine whether the leptin–p38 MAPK pathway is involved in HG-induced injury and whether exogenous H₂S prevents the HG-induced insult through inhibition of the leptin–p38 MAPK pathway in H9c2 cells. H9c2 cells were treated with 35 mM glucose (HG) for 24 h to establish a HG-induced cardiomyocyte injury model. Cell viability; mitochondrial membrane potential (ΔΨ _m); apoptosis; reactive oxygen species (ROS) level; and leptin, leptin receptor, and p38 MAPK expression level were measured by the methods indicated. The results showed pretreatment of H9c2 cells with NaHS before exposure to HG led to an increase in cell viability, decrease in apoptotic cells, ROS generation, and a loss of ΔΨ _m. Exposure of H9c2 cells to 35 mM glucose for 24 h significantly upregulated the expression levels of leptin and leptin receptors. The increased expression levels of leptin and leptin receptors were markedly attenuated by pretreatment with 400 μM NaHS. In addition, the HG-induced increase in phosphorylated (p) p38 MAPK expression was ameliorated by pretreatment with 50 ng/ml leptin antagonist. In conclusion, the present study has demonstrated for the first time that the leptin–p38 MAPK pathway contributes to the HG-induced injury in H9c2 cells and that exogenous H₂S protects H9c2 cells against HG-induced injury at least in part by inhibiting the activation of leptin–p38 MAPK pathway.     

Angiopoietin-1 protects H9c2 cells from H2O2-induced apoptosis through AKT signaling

Loss of cardiomyocytes by apoptosis is proposed to cause ventricular remodeling and heart failure. Reactive oxygen species-induced apoptosis of cardiomyocytes has been reported to play an important role in many types of pathological processes of the heart. We investigated whether angiopoietin-1 (Ang1) has direct cytoprotective effects on cardiomyocytes against oxidative stress. Cultured H9c2 cells (cardiomyocytes) were treated with hydrogen peroxide (H(2)O(2)). Apoptosis was evaluated by flow cytometry, TUNEL assay and DNA laddering. The H(2)O(2) treatment caused typical apoptosis of H9c2 cells in a time-dependent manner. Transfection of recombinant adenovirus expressing Ang1 resulted in a sustained phosphorylation of AKT and inhibition of H(2)O(2)-induced apoptosis in H9c2 cells. This effect could be reversed by AKT inhibition. These results suggest that Ang1 protects cardiomyocytes from oxidative stress-induced apoptosis by regulating the activity of AKT.     

Mitochondrial H2O2 regulates the angiogenic phenotype via PTEN oxidation

Recent studies have demonstrated that the tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10), the antagonist of the phosphosphoinositol-3-kinase (PI3K) signaling cascade, is susceptible to H2O2-dependent oxidative inactivation. This study describes the use of redox-engineered cell lines to identify PTEN as sensitive to oxidative inactivation by mitochondrial H2O2. Increases in the steady state production of mitochondrial derived H2O2, as a result of manganese superoxide dismutase (Sod2) overexpression, led to PTEN oxidation that was reversed by the coexpression of the H2O2-detoxifying enzyme catalase. The accumulation of an oxidized inactive fraction of PTEN favored the formation of phosphatidylinositol 3,4,5-triphosphate at the plasma membrane, resulting in increased activation of Akt and modulation of its downstream targets. PTEN oxidation in response to mitochondrial H2O2 enhanced PI3K signaling, leading to increased expression of the key regulator of angiogenesis, vascular endothelial growth factor. Overexpression of PTEN prevented the H2O2-dependent increase in vascular endothelial growth factor promoter activity and immunoreactive protein, whereas a mutant PTEN (G129R), lacking phosphatase activity, did not. Furthermore, mitochondrial generation of H2O2 by Sod2 promoted endothelial cell sprouting in a three-dimensional in vitro angiogenesis assay that was attenuated by catalase coexpression or the PI3K inhibitor LY2949002. Moreover, Sod2 overexpression resulted in increased in vivo blood vessel formation that was H2O2-dependent as assessed by the chicken chorioallantoic membrane assay. Our findings provide the first evidence for the involvement of mitochondrial H2O2 in regulating PTEN function and the angiogenic switch, indicating that Sod2 can serve as an alternative physiological source of the potent signaling molecule, H2O2.     

Progressive apoptotic cell death triggered by transient oxidative insult in H9c2 rat ventricular cells: a novel pattern of apoptosis and the mechanisms

Many pathophysiological processes are associated with oxidative stress and progressive cell death. Oxidative stress is an apoptotic inducer that is known to cause rapid cell death. Here we show that a brief oxidative insult (5-min exposure to 400 microM H(2)O(2)), although it did not kill H9c2 rat ventricular cells during the exposure, triggered an intracellular death cascade leading to delayed time-dependent cell death starting from 1 h after the insult had been withdrawn, and this post-H(2)O(2) cell death cumulated gradually, reaching a maximum level 8 h after H(2)O(2) withdrawal. By comparison, sustained exposure to H(2)O(2) caused complete cell death within a narrow time frame (2 h). The time-dependent post-H(2)O(2) cell death was typical of apoptosis, both morphologically (cell shrinkage and nuclear condensation) and biochemically (DNA fragmentation, extracellular exposure of phosphatidylserines, and caspase-3 activation). A dichlorofluorescein fluorescent signal showed a time-dependent endogenous increase of reactive oxygen species (ROS) production, which was almost abolished by inhibition of the mitochondrial electron transport chain. Application of antioxidants (vitamin E or DTT) before H(2)O(2) addition or after H(2)O(2) withdrawal prevented the H(2)O(2)-triggered progressive ROS production and apoptosis. Sequential appearance of events associated with activation of the mitochondrial death pathway was found, including progressive dissipation of mitochondrial membrane potential, cytochrome c release, and late activation of caspase-3. In conclusion, transient oxidative stress triggers an intrinsic program leading to self-sustained apoptosis in H9c2 cells via cumulative production of mitochondrial ROS and subsequent activation of the mitochondrial death pathway. This pattern of apoptosis may contribute to the progressive and long-lasting cell loss in some degenerative diseases.     

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.     

Mitochondria play an important role in 17beta-estradiol attenuation of H(2)O(2)-induced rat endothelial cell apoptosis

Studies have shown salutary effects of 17beta-estradiol following trauma-hemorrhage on different cell types. 17beta-Estradiol also induces improved circulation via relaxation of the aorta and has an anti-apoptotic effect on endothelial cells. Because mitochondria play a pivotal role in apoptosis, we hypothesized that 17beta-estradiol will maintain mitochondrial function and will have protective effects against H(2)O(2)-induced apoptosis in endothelial cells. Endothelial cells were isolated from rats' aorta and cultured in the presence or absence of H(2)O(2), a potent inducer of apoptosis. In additional studies, endothelial cells were pretreated with 17beta-estradiol. Flow cytometry analysis revealed H(2)O(2)-induced apoptosis in 80.9% of endothelial cells; however, prior treatment of endothelial cells with 17beta-estradiol resulted in an approximately 40% reduction in apoptosis. This protective effect of 17beta-estradiol was abrogated when endothelial cells were cultured in the presence ICI-182780, indicating the involvement of estrogen receptor (ER). Fluorescence microscopy revealed a 17beta-estradiol-mediated attenuation of H(2)O(2)-induced mitochondrial condensation. Western blot analysis demonstrated that H(2)O(2)-induced cytochrome c release from mitochondrion to cytosol and the activation of caspase-9 and -3 were decreased by 17beta-estradiol. These findings suggest that 17beta-estradiol attenuated H(2)O(2)-induced apoptosis via ER-dependent activation of caspase-9 and -3 in rat endothelial cells through mitochondria.     

Protective effects of combination of quercetin and α-tocopherol on mitochondrial dysfunction and myocardial infarct size in isoproterenol-treated myocardial infarcted rats: biochemical, transmission electron microscopic, and macroscopic enzyme mapping evidences

Mitochondrial dysfunction plays an important role in the pathology of myocardial infarction. We evaluated the combined protective effects of quercetin and α-tocopherol on mitochondrial damage and myocardial infarct size in isoproterenol-induced myocardia- infarcted rats. Rats were pretreated with quercetin (10 mg/kg) alone, α-tocopherol (10 mg/kg) alone, and combination of quercetin (10 mg/kg) and α-tocopherol (10 mg/kg) orally using an intragastric tube daily for 14 days. After pretreatment, rats were induced myocardial infarction by isoproterenol (100 mg/kg) at an interval of 24 h for 2 days. Isoproterenol treatment caused significant increase in mitochondrial lipid peroxides with significant decrease in mitochondrial antioxidants. Significant decrease in the activities of isocitrate, succinate, malate, and α-ketoglutarate and NADH dehydrogenases and cytochrome-c-oxidase, significant increase in calcium, and significant decrease in adenosine triphosphate were observed in mitochondria of myocardial infarcted rats. Combined pretreatment with quercetin and α-tocopherol normalized all the biochemical parameters and preserved the integrity of heart tissue and restored normal mitochondrial function in myocardial-infarcted rats. Transmission electron microscopic findings on heart mitochondria and macroscopic enzyme mapping assay on the size of myocardial infarct also correlated with these biochemical parameters. The present study showed that combined pretreatment was highly effective than single pretreatment.     

Mitochondrial monolysocardiolipin acyltransferase is elevated in the surviving population of H9c2 cardiac myoblast cells exposed to 2-deoxyglucose-induced apoptosis.

Cardiolipin (CL) is a major mitochondrial membrane phospholipid in the mammalian heart and the remodeling of CL is essential to maintain its unique unsaturated fatty acyl composition. We examined CL de novo biosynthesis and remodeling in the surviving population of H9c2 cardiac myoblast cells exposed to 2-deoxyglucose (2-DG). H9c2 cells were incubated in the absence or presence of 2-DG for 16 h with [1,3-3H]glycerol or [1-14C]linoleic acid (bound to albumin in a 1:1 molar ratio). Dead cells were removed and radioactivity was incorporated into CL. Its pool size, fatty acid composition, and the activities of the CL biosynthesis and remodeling enzymes were determined. The CL pool size, its fatty acid composition, and [1,3-3H]glycerol or [1-14C]linoleic acid incorporated into CL were unaltered in the surviving population of 2-DG-treated cells compared with controls. In addition, the activities of the CL de novo biosynthetic enzymes were unaltered. Cleaved caspase-3 and poly(ADP-ribose) polymerase were slightly elevated in the surviving population of 2-DG-treated cells compared with controls, indicating that apoptosis induction was occurring in these cells. Mitochondrial phospholipase A2 and monolysocardiolipin acyltransferase (MLCL AT) activities increased 33% (p < 0.05) and 63% (p < 0.05), respectively, in 2-deoxyglucose-treated cells compared with controls. In contrast, the activity of ALCAT1, an endoplasmic reticulum MLCL AT, decreased 77% (p < 0.05), but this was not due to a reduction in ALCAT1 mRNA expression. The mRNA expression of the Barth syndrome gene TAZ, encoding a mitochondrial CL transacylase, was unaltered in 2-DG treated cells. The increase in mitochondrial MLCL AT activity was due to an elevated expression in MLCL AT protein. Thus, an increase in MLCL AT activity and expression occurs to maintain the CL pool in the surviving population of H9c2 cells as a compensatory mechanism for the elevated phospholipase A2 activity seen in 2-DG-induced apoptosis. We hypothesize that increased mitochondrial MLCL AT activity and its expression, and hence, elevated CL resynthesis, may be a protective mechanism against monolysocardiolipin-mediated apoptosis.     

Ursolic acid stimulates UCP2 expression and protects H9c2 cells from hypoxia-reoxygenation injury via p38 signaling

Oxidative stress and apoptosis is involved in hypoxia-reoxygenation (H/R) induced myocardial injury. Increased expression of uncoupling protein 2 (UCP2), a cationic carrier protein, has protective effect against H/R injury. The present study aimed to find candidate drugs for H/R induced cardiac damage by identifying compounds regulating UCP2 expression. Here, among six natural compounds, ursolic acid (UA) had the most significant induction effect on UCP2 expression in H9c2 cells under H/R conditions. Subsequently, we found that UA significantly attenuated cell apoptosis and Caspase 3 activity, but increased nitric oxide (NO) release under H/R conditions. Additionally, UA pretreatment also decreased reactive oxygen species (ROS) production and malondialdehyde (MDA) content, but increased superoxide dismutase (SOD) activity. H/R caused a notable increase in the phosphorylation of p38, which was weakened by UA pretreatment. Moreover, p38 inhibitor (SB203580) showed the similar effects on H/R cells as UA pretreatment, while UCP2 knockdown had the reverse biological effects. More importantly, the effects of UA or p38 inhibitor exposure were partially rescued by UCP2 knockdown. Collectively, our data suggested the functions of UA on UCP2 expression and on the protection of H/R-stimulated H9c2 cells may be attributed to p38 signaling pathway.     

Neferine protected cardiomyocytes against hypoxia/oxygenation injury through SIRT1/Nrf2/HO-1 signaling

Acute myocardial infarction is regarded as myocardial necrosis resulting from myocardial ischemia/reperfusion (I/R) damage and retains a major cause of mortality. Neferine, which was extracted from the green embryos of mature seeds of Nelumbo nucifera Gaertn., has been reported to possess a broad range of biological activities. However, its underlying mechanism on the protective effect of I/R has not been fully clarified. A hypoxia/reoxygenation (H/R) model with H9c2 cells closely simulating myocardial I/R injury was used as a cellular model. This study intended to research the effects and mechanism underlying neferine on H9c2 cells in response to H/R stimulation. Cell Counting Kit-8 and lactate dehydrogenase (LDH) release assays were employed to measure cell viability and LDH, respectively. Apoptosis and reactive oxygen species (ROS) were determined by flow cytometry analysis. Oxidative stress was evaluated by detecting malondialdehyde, superoxide dismutase, and catalase. Mitochondrial function was assessed by mitochondrial membrane potential, ATP content, and mitochondrial ROS. Western blot analysis was performed to examine the expression of related proteins. The results showed that hypoxia/reoxygenation (H/R)-induced cell damage, all of which were distinctly reversed by neferine. Moreover, we observed that neferine inhibited oxidative stress and mitochondrial dysfunction induced by H/R in H9c2 that were concomitant with increased sirtuin-1 (SITR1), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 expression. On the contrary, silencing the SIRT1 gene with its small interferingRNA eliminated the beneficial effects of neferine. It is concluded that neferine preconditioning attenuated H/R-induced cardiac damage via suppressing apoptosis, oxidative stress, and mitochondrial dysfunction, which may be partially ascribed to the activation of SIRT1/Nrf2 signaling pathway.     

Tempol reduces infarct size in rodent models of regional myocardial ischemia and reperfusion.

Reactive oxygen species (ROS) contribute to ischemia-reperfusion injury of the heart. This study investigates the effects of tempol, a membrane-permeable radical scavenger on (i) the infarct size caused by regional myocardial ischemia and reperfusion of the heart in vivo (rat, rabbit) and in vitro (rat), and (ii) the cell injury caused by hydrogen peroxide (H2O2) in rat cardiac myoblasts (H9c2 cells). In the anesthetized rat, tempol reduced the infarct size caused by regional myocardial ischemia (25 min) and reperfusion (2 h) from 60 +/- 3% (control, n = 8) to 24 +/- 5% (n = 6, p < .05). In the anesthetized rabbit, tempol also attenuated the infarct size caused by myocardial ischemia (45 min) and reperfusion (2 h) from 59 +/- 3% (control, n = 6) to 39 +/- 5% (n = 5, p < .05). Regional ischemia (35 min) and reperfusion (2 h) of the isolated, buffer-perfused heart of the rat resulted in an infarct size of 54 +/- 4% (control n = 7). Reperfusion of hearts with buffer containing tempol (n = 6) caused a 37% reduction in infarct size (n = 6, p < .05). Pretreatment of rat cardiac myoblasts with tempol attenuated the impairment in mitochondrial respiration caused by H2O2 (1 mM for 4 h). Thus, the membrane-permeable radical scavenger tempol reduces myocardial infarct size in rodents.     

Effect of H2O2 on CCK-8-evoked changes in mitochondrial activity in isolated mouse pancreatic acinar cells

BACKGROUND INFORMATION: This paper studies the effect of H(2)O(2) on mitochondrial responses evoked by CCK-8 (cholecystokinin 8) in mouse pancreatic acinar cells. Cytosolic ([Ca(2+)](c)) and mitochondrial ([Ca(2+)](m)) free-calcium concentrations, mitochondrial inner membrane potential (psi(m)) and FAD autofluorescence were monitored using confocal laser scanning microscopy. RESULTS: CCK-8 induced an increase in [Ca(2+)](m) that slowly declined towards the prestimulation level. Depolarization of psi(m) that partially recovered, as well as increases in FAD autofluorescence, could also be observed in response to the hormone. Pretreatment of cells with 1 mM H(2)O(2) alone resulted in marked changes in mitochondrial parameters and, moreover, H(2)O(2) inhibited the CCK-8-evoked changes in [Ca(2+)](m), psi(m) and FAD autofluorescence. The results of the present study have demonstrated that CCK-8 can evoke marked changes in pancreatic acinar cell mitochondrial activity and that CCK-8-evoked responses are blocked by H(2)O(2). Additionally, H(2)O(2) releases Ca(2+) from intracellular stores and inhibits pancreatic acinar cell responses to CCK-8. CONCLUSION: The effects observed reflect an impairment of mitochondrial activity in the presence of H(2)O(2) that could represent some of its mechanisms of action to induce cellular damage leading to cell dysfunction and generation of pathologies.     

Ginsenoside Rg1 Protects against Cardiac Remodeling in Heart Failure via SIRT1/PINK1/Parkin-Mediated Mitophagy

Adverse cardiac remodeling may lead to the development and progression of heart failure, which is lack of effective clinical treatment. Ginsenoside Rg1 (GRg1), a primary ingredient of Panax ginseng, protects against diverse cardiovascular disease, but its effects on cardiac remodeling remain unclear. Thus, we investigated the protective effect and mechanism of GRg1 on cardiac remodeling after myocardial infarction. GRg1 significantly ameliorated cardiac remodeling in mice with left anterior descending coronary artery ligation, reflected by reduced left ventricular dilation and decreased cardiac fibrosis, accompanied by improved cardiac function. Mechanistically, GRg1 considerably increased mitophagosomes formation, ameliorated cardiac mitochondria damage, and enhanced SIRT1/PINK1/Parkin-mediated mitophagy during cardiac remodeling. Consistently, GRg1 increased cell viability and attenuated apoptosis and fibrotic responses in H₂O₂-treated H9c2 cells by promoting the SIRT1/PINK1/Parkin axis. Furthermore, SIRT1-specific inhibitor (EX527) or the use of small interfering RNA against Parkin abolished the protective effect of GRg1 in vitro. These findings reveal a novel mechanism of GRg1 alleviating cardiac remodeling via enhancing SIRT1/PINK1/Parkin-mediated mitophagy.     

Caffeic acid protects rat heart mitochondria against isoproterenol-induced oxidative damage

Cardiac mitochondrial dysfunction plays an important role in the pathology of myocardial infarction. The protective effects of caffeic acid on mitochondrial dysfunction in isoproterenol-induced myocardial infarction were studied in Wistar rats. Rats were pretreated with caffeic acid (15 mg/kg) for 10 days. After the pretreatment period, isoproterenol (100 mg/kg) was subcutaneously injected to rats at an interval of 24 h for 2 days to induce myocardial infarction. Isoproterenol-induced rats showed considerable increased levels of serum troponins and heart mitochondrial lipid peroxidation products and considerable decreased glutathione peroxidase and reduced glutathione. Also, considerably decreased activities of isocitrate, succinate, malate, α-ketoglutarate, and NADH dehydrogenases and cytochrome-C-oxidase were observed in the mitochondria of myocardial-infarcted rats. The mitochondrial calcium, cholesterol, free fatty acids, and triglycerides were considerably increased and adenosine triphosphate and phospholipids were considerably decreased in isoproterenol-induced rats. Caffeic acid pretreatment showed considerable protective effects on all the biochemical parameters studied. Myocardial infarct size was much reduced in caffeic acid pretreated isoproterenol-induced rats. Transmission electron microscopic findings also confirmed the protective effects of caffeic acid. The possible mechanisms of caffeic acid on cardiac mitochondria protection might be due to decreasing free radicals, increasing multienzyme activities, reduced glutathione, and adenosine triphosphate levels and maintaining lipids and calcium. In vitro studies also confirmed the free-radical-scavenging activity of caffeic acid. Thus, caffeic acid protected rat’s heart mitochondria against isoproterenol-induced damage. This study may have a significant impact on myocardial-infarcted patients.