cytochrome-c-t and Myocardial-Infarction

Refracterin therapy of patients with chronic heart failure caused by coronary heart disease and postinfarction cardiosclerosis markedly promoted improvement in the pulmonary and systemic circulation in comparison with patients receiving traditional therapy. The mean functional class of chronic cardiac failure decreased by 43% under the effect of refracterin vs. 27% decrease in the group receiving traditional therapy. After 1-month refracterin course the end-systolic and end-diastolic sizes of the left ventricle decreased by 12 and 7%, respectively, ejection fraction increased by 7.2% in comparison with the initial level, total oxidant activity and MDA content in the plasma decreased significantly, while total antioxidant activity, catalase and SOD activities, cytochrome C, NADH, and NADPH levels increased. The prooxidant-antioxidant system was shifted towards antioxidants, which attests to activation of the defense and adaptive mechanisms after administration of refracterin, which is especially important in elderly patients with initially decreased reserve potentialities of the antioxidant defense system.

Topics: Acetyldigoxins; Aged; Aged, 80 and over; Antioxidants; Cardiotonic Agents; Cytochromes c; Drug Combinations; Heart Failure; Humans; Inosine; Myocardial Infarction; NAD; Oxidative Stress; Oxyfedrine; Sclerosis

Topics: Alcohol Drinking; Alcoholism; Animals; Apoptosis; Calpain; Cardiolipins; Caspase 3; Cytochromes c; Dimethyl Sulfoxide; Ethanol; Isoproterenol; Male; Myocardial Infarction; Myocardium; Rats; Rats, Wistar

Thymoquinone (TQ) is the main active constituent of Nigella sativa. The present study aimed to investigate the effect of TQ on apoptotic parameters and MMP-9 expression in isoproterenol (ISP)-induced myocardial infarction (MI). TQ was given once daily for 7 days at doses of 10 and 20 mg/kg orally with ISP (86 mg/kg; s.c.) administered on the sixth and seventh days. TQ pre-treatment protected against ISP-induced MI as approved by normalisation of electrocardiogram (ECG) and b (CK)-MB, minimal histopathological changes, and reduction of the infarction size. Effects of TQ could be supported by its antioxidant activity, evidenced by the increase of cardiac reduced glutathione and total serum antioxidant capacity, and the inhibition of ISO-induced lipid peroxidation. TQ anti-inflammatory activity was associated with reduced expression of NF-κB and TNF-α. TQ ameliorated cardiomyocytes

Topics: Animals; Apoptosis; Benzoquinones; Biomarkers; Cytochromes c; Gene Expression Regulation, Enzymologic; Inflammation; Isoproterenol; Matrix Metalloproteinase 9; Myocardial Infarction; Random Allocation; Rats; Rats, Wistar

Mitochondrial dysfunction of cardiomyocytes (CMs) has been identified as a significant pathogenesis of early myocardial infarction (MI). However, only a few agents or strategies have been developed to improve mitochondrial dysfunction for the effective MI treatment. Herein, a reactive oxygen species (ROS)-responsive PAMB-G-TK/4-arm-PEG-SG hydrogel is developed for localized drug-loaded liposome delivery. Notably, the liposomes contain both elamipretide (SS-31) and sphingosine-1-phosphate (S1P), where SS-31 acts as an inhibitor of mitochondrial oxidative damage and S1P as a signaling molecule for activating angiogenesis. Liposome-encapsulated PAMB-G-TK/4-arm-PEG-SG hydrogels demonstrate myocardium-like mechanical strength and electrical conductivity, and ROS-sensitive release of SS-31 and S1P-loaded liposomes. Further liposomal release of SS-31, which can target cytochrome c in the mitochondrial inner membrane of damaged CMs, inhibits pathological ROS production, improving mitochondrial dysfunction. Meanwhile, S1P released from the liposome induces endothelial cell angiogenesis by activating the S1PR1/PI3K/Akt pathway. In a rat MI model, the resulting liposomal composite hydrogel improves cardiac function by scavenging excess ROS, improving mitochondrial dysfunction, and promoting angiogenesis. This study reports for the first time a liposomal composite hydrogel that can directly target mitochondria of damaged CMs for a feedback-regulated release of encapsulated liposomes to consume the overproduced pathological ROS for improved CM activity and enhanced MI treatment.

Topics: Animals; Biocompatible Materials; Cytochromes c; Hydrogels; Liposomes; Lysophospholipids; Mitochondria; Myocardial Infarction; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Reactive Oxygen Species; Sphingosine

Galectin 3 (GAL-3) is a beta galactoside binding lectin that has different roles in normal and pathophysiological conditions. GAL-3 has been associated with heart failure and was linked to increased risk of death in a number of studies. GAL-3 was found to be up regulated in animal models of heart failure as well as myocardial infarction (MI). The objective of his study is to test if high GAL-3 after myocardial infarction has a protective role on the heart through its anti-apoptotic and anti-necrotic functions.. Male C57B6/J mice and GAL-3 knockout (KO) mice were used for permanent ligation of the left anterior descending artery of the heart to create infarction in the anterior myocardium. Heart and plasma samples were collected 24 hours after the induction of MI and were used for immunohistochemistry, Tunnel procedure, electron microscopy and enzyme linked immunosorbent assay (ELISA).. Our results show that the significant increase in GAL-3 levels in the left ventricle at 24-hour following MI is associated with significant lower levels of pro-apoptotic proteins; cytochrome c, Bax, annexin V, cleaved caspase-3 and a higher levels of anti-apoptotic protein Bcl2 in GAL-3 wild MI group than GAL-3 KO group. We also have identified the anti-apoptotic activity of GAL-3 is mediated through a significant increase in Akt-1, NF kappa-B and beta- catenin proteins. In addition, we have identified the antiapoptotic activity is mediated through a significant lower levels of cathepsin-D protein.. We conclude that the increased levels of GAL-3 at 24-hour following MI regulate antiapoptotic mechanisms in the myocardium that will shape the future course of the disease. We also identified that the anti-apoptotic mechanisms are likely mediated through interaction of GAL-3 with Akt-1, NF kappa-B, beta- catenin and cathepsin D proteins.

Topics: Animals; Annexin A5; Apoptosis; bcl-2-Associated X Protein; beta Catenin; Caspase 3; Cathepsins; Cytochromes c; Disease Models, Animal; Galectin 3; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron, Transmission; Myocardial Infarction; Myocardium; Myocytes, Cardiac; NF-kappa B; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2

BACKGROUND The relationship between endoplasmic reticulum and mitochondria during acute myocardial ischemic injury is still unclear. Our study aimed to define the dynamics of endoplasmic reticulum stress and mitochondrial dysfunction during acute ischemic injury. MATERIAL AND METHODS A rat model of acute myocardial infarction and hypoxic cardiomyocytes were used in this study. Groups were set at 0 hours, 1 hour, 2 hours, 4 hours, and 6 hours after ischemic injury for both in vivo and in vitro studies. ATF6 and GRP-78 were examined to indicate endoplasmic reticulum stress. Cellular ATP and cytosolic levels of mitochondrial DNA and cytochrome c were detected to evaluate mitochondrial dysfunction. Caspase-3 was used for apoptosis analysis. RESULTS Our results showed that both mRNA and protein levels of ATF6 and GRP-78 were elevated from 1 hour after ischemic injury in vivo and in vitro (P<0.05). However, ATP levels were increased at 2 hours after ischemic injury and significantly decreased from 4 hours after ischemic injury in vivo, while ATP level of cultured cardiomyocytes decreased remarkably from 2 hours after ischemic injury (P<0.05). Cytosolic mitochondrial DNA levels began to increase from 2 hours after ischemic injury (P<0.05). Cytosolic levels of cytochrome c increased from 4 hours after ischemic injury. Additionally, both mRNA and protein expressions of caspase-3 started to significantly elevate at 6 hours after ischemic injury (P<0.05). CONCLUSIONS The present study suggested that mitochondrial dysfunction was secondary to endoplasmic reticulum stress, which provides a novel experimental foundation for further exploration of the detailed mechanism after ischemic injury, especially the interaction between endoplasmic reticulum and mitochondria.

Topics: Animals; Apoptosis; Cytochromes c; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Heart Injuries; Male; Mitochondria; Myocardial Infarction; Myocardial Ischemia; Myocardium; Myocytes, Cardiac; Oxidative Stress; Rats; Rats, Sprague-Dawley

Oxidative stress plays a critical role in the progression of myocardial injury. Increasing evidence suggests that hiruidin can treat patients with cardio-injury. However, the mechanism of hirudin against myocardial infraction remains unknown. In the present study, we evaluated the potential role and mechanism of hirudin on both isoproterenol (ISO)-induced myocardial infraction (MI) in rats and Hypoxia-Reoxygenation model in H9C2 cells. Compared with the model group, hirudin apparently decreased the levels of myocardial Creatine Kinase Isoenzyme-MB (CK-MB), lactate dehydrogenase (LDH), and alleviated myocardial histopathological changes induced by ISO injection. The underlying mechanisms were revealed by the following observations: Hirudin exerted its cardioprotective effect via restoring super oxide dismutase (SOD), attenuating reactive oxygen species (ROS) and malondialdehyde (MDA). It induced the activation of Nuclear factor erythroid 2-related factor 2 (Nrf2) signal pathway through disrupting Keap1-Nrf2 complex, thus Nrf2 translocated from cytoplasm to nucleus to regulate Nrf2-dependent gene (HO-1, SOD) expressions. Furthermore, it should be noted that hirudin restored mitochondrial membrane potential in addition to cytochrome C-related apoptosis.

Topics: Animals; Apoptosis; Cardiotonic Agents; Cell Hypoxia; Cell Line; Cell Survival; Creatine Kinase, MB Form; Cytochromes c; Heme Oxygenase (Decyclizing); Hirudins; Isoproterenol; Kelch-Like ECH-Associated Protein 1; L-Lactate Dehydrogenase; Male; Malondialdehyde; Membrane Potential, Mitochondrial; Myocardial Infarction; Myocardium; NF-E2-Related Factor 2; Oxidative Stress; Rats; Reactive Oxygen Species; Signal Transduction; Superoxide Dismutase

The voltage-dependent anion channel 1 (VDAC1) is a key player in mitochondrial function. VDAC1 serves as a gatekeeper mediating the fluxes of ions, nucleotides, and other metabolites across the outer mitochondrial membrane, as well as the release of apoptogenic proteins initiating apoptotic cell death. VBIT-4, a VDAC1 oligomerization inhibitor, was recently shown to prevent mitochondrial dysfunction and apoptosis, as validated in mouse models of lupus and type-2 diabetes. In the present study, we explored the expression of VDAC1 in the diseased myocardium of humans and rats. In addition, we evaluated the effect of VBIT-4 treatment on the atrial structural and electrical remodeling of rats exposed to excessive aldosterone levels. Immunohistochemical analysis of commercially available human cardiac tissues revealed marked overexpression of VDAC1 in post-myocardial infarction patients, as well as in patients with chronic ventricular dilatation\\dysfunction. In agreement, rats exposed to myocardial infarction or to excessive aldosterone had a marked increase of VDAC1 in both ventricular and atrial tissues. Immunofluorescence staining indicated a punctuated appearance typical for mitochondrial-localized VDAC1. Finally, VBIT-4 treatment attenuated the atrial fibrotic load of rats exposed to excessive aldosterone without a notable effect on the susceptibility to atrial fibrillation episodes induced by burst pacing. Our results indicate that VDAC1 overexpression is associated with myocardial abnormalities in common pathological settings. Our data also indicate that inhibition of the VDAC1 can reduce excessive fibrosis in the atrial myocardium, a finding which may have important therapeutic implications. The exact mechanism\\s of this beneficial effect need further studies.

Topics: Aldosterone; Animals; Apoptosis; Calcium Signaling; Cytochromes c; Disease Models, Animal; Fibrosis; Heart Atria; Humans; Hyperaldosteronism; Mitochondria; Myocardial Infarction; Myocardium; Protein Multimerization; Rats; Voltage-Dependent Anion Channel 1

To study the protective effects of late remote ischaemic preconditioning (RIPC) against myocardial ischaemia/reperfusion (I/R) injury and determine whether Stat5 is involved in this protection by using cardiomyocyte-specific Stat5 knockout mice (Stat5-cKO).. Mice were exposed to lower limb RIPC or sham ischaemia. After 24 h, the left anterior descending artery (LAD) was ligated for 30 min, then reperfused for 180 min. The myocardial infarct size (IS), apoptotic rate of cardiomyocytes, and serum myocardial enzymes were measured to evaluate for cardioprotective effects. Heart tissues were harvested to determine the cardiomyocytes' anti-apoptotic and survival signaling. When compared with the Stat5fl/fl mice without RIPC, Stat5fl/fl mice with RIPC (Stat5fl/fl+RIPC + I/R) displayed a decreased myocardial IS/LV (16 ± 1.5 vs. 30.1 ± 3.1%, P < 0.01; IS/ area at risk (AAR), 42.2 ± 3.5 vs. 69.2 ± 4.9%, P < 0.01), a reduced cardiomyocyte apoptotic rate (2.1 ± 0.37 vs. 5.5 ± 0.53%, P < 0.01), and lower creatine kinase (CK), lactate dehydrogenase (LDH), and creatine kinase-MB (CK-MB) levels. To the contrary, the Stat5-cKO mice (Stat5fl/fl; Tnnt2Cremice with Doxycycline treatment for 7 days) did not exhibit any effect of RIPC-induced cardioprotection. Activation of STAT5 protein was significantly higher in the Stat5fl/fl+RIPC + I/R group than in the Stat5fl/fl+I/R group, while there was no significant difference between the Stat5-cKO + RIPC + I/R and the Stat5-cKO + I/R group. Further analyses with heart tissues detected decreased protein expressions of cytochrome c (Cyt c) and cleaved Caspase-3 in the Stat5fl/fl+RIPC + I/R mice, along with increased anti-apoptotic molecules, including B-cell lymphoma-extra large (Bcl-xL) and B-cell lymphoma-2 (Bcl-2); such changes were not noted in the Stat5-cKO + RIPC + I/R mice. Additionally, RIPC increased cardiac hypoxia inducible factor-1 (HIF-1α) and interleukin-10 (IL10) protein levels and caused activation of AKT, phosphatidylinositol 3 kinase (PI3K), and vascular endothelial growth factor in the heart of the Stat5fl/fl mice. However, these changes were completely inhibited by the absence of Stat5.. These results suggest that RIPC-induced late cardioprotection against myocardial I/R injury is Stat5-dependent and is correlated with the activation of anti-apoptotic signaling and cardiomyocyte-survival signaling.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Creatine Kinase, MB Form; Cytochromes c; Disease Models, Animal; Femoral Artery; Hypoxia-Inducible Factor 1, alpha Subunit; Interleukin-10; Ischemic Preconditioning, Myocardial; L-Lactate Dehydrogenase; Ligation; Mice, Knockout; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Signal Transduction; STAT5 Transcription Factor; Vascular Endothelial Growth Factor A

To investigate the role and mechanism of micro ribonucleic acid (miR)-298 in myocardial apoptosis after myocardial infarction.. In vivo experiments, the rat model of myocardial infarction was established, and miR-298 was up-regulated via lentivirus with miR-298 overexpression. Cardiac function of rats was detected via echocardiography, Bcl-2 associated X protein (BAX) expressions in infarction border zone were detected via Real-time Quantitative PCR (qT-PCR) and Western blot, and TUNEL assay was used to detect the myocardial apoptosis. In vitro experiments, myocardial cells were isolated and cultured, an oxygen-glucose deprivation (OGD) model was established to mimicking the ischemic condition, the relationship between miR-298 and BAX was verified using luciferase reporter vector, lentivirus and small-interfering RNA (siRNA) in BAX.. In vivo experiments showed that the miR-298 expression was down-regulated at 2 and 4 weeks after myocardial infarction. The up-regulation of miR-298 significantly improved the cardiac function, decreased the expressions of BAX, reduced the myocardial apoptosis and inhibit the apoptosis proteins expression including cytochrome-c and cleaved caspase-3. In vitro experiments revealed that BAX was a target gene of miR-298 and further proof that miR-298 could inhibit the cytochrome-c and cleaved caspase-3 expression and myocardial apoptosis through BAX.. MiR-298 can improve the myocardial apoptosis through the target gene BAX.

Topics: 3' Untranslated Regions; Animals; Antagomirs; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Hypoxia; Cytochromes c; Down-Regulation; Glucose; Male; MicroRNAs; Myocardial Infarction; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Small Interfering; Up-Regulation

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal).\ \ This article has been retracted at the request of the Editor-in-Chief.\ \ Concerns were raised about the background pattern of the Western Blots from Figures 3D and 5A. Given the comments of Dr Elisabeth Bik regarding this article “This paper belongs to a set of over 400 papers (as per February 2020) that share very similar Western blots with tadpole-like shaped bands, the same background pattern, and striking similarities in title structures, paper layout, bar graph design, and - in a subset - flow cytometry panels”, the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Case-Control Studies; Cell Hypoxia; Cellular Microenvironment; Cytochromes c; Disease Models, Animal; Gene Expression Regulation; HEK293 Cells; Humans; Male; Mice, Inbred C57BL; MicroRNAs; Myocardial Infarction; Myocytes, Cardiac; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins c-akt; Rats; Signal Transduction

To investigate the role of Na+/Ca2+ exchanger (NCX) in myocardial ischemia-reperfusion injury and the underlying mechanisms.
 Methods: Forty Sprague-Dawley rats were divided into 4 groups randomly: a control group, a KB-R7943 group, an ischemia-reperfusion group (IR group), and an IR plus KB-R7943 group (KB-R7943+IR group). Isolated Sprague Dawley male rat hearts underwent Langendorff perfusion. The ratio of left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), the infarct size of myocardium, and the lactate dehydrogenase (LDH) activity in the coronary flow was determined. HE staining was used to assess the change of myocardial morphology. Western blot was used to determine the levels of cleaved caspase-3, cytochrome c and the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and the Thr17 site of phospholamban.
 Results: Compared with the control group, IR group significantly induced an enlarged infarct size, reduction of the ratio of LVDP, up-regulation of cytochrome c, cleaved caspase-3, p-CaMKII and p-phospholamban, and increased in the activity of LDH, the level of LVEDP (P<0.01) and the disordered myocardial morphology. These effects were significantly attenuated in the presence of KB-R7943 treatment (10 μmol/L).
 Conclusion: NCX mediates myocardial ischemia-reperfusion-induced cell apoptosis and necrosis through activation of CaMKII.. 目的：探讨钠钙交换体(Na+/Ca2+ exchanger，NCX)在离体心脏缺血再灌注损伤中的作用及其可能机制。方法：40只大鼠随机分为4组：正常对照组(Control组)、10 μmol/L KB-R7943药物对照组(KB-R7943组)、缺血再灌注(ischemia reperfusion，IR)组和10 μmol/L KB-R7943干预缺血再灌注组(KB-R7943+IR组)。采用Langendorff灌流装置建立离体心脏缺血再灌注模型；以再灌注末心肌纤维形态的变化、左室发展压(LVDP)比值、左室舒张末压(LVEDP)、冠状动脉流出液中乳酸脱氢酶(lactate dehydrogenase，LDH)活性、心肌梗死面积及细胞色素c和cleaved caspase-3蛋白的变化评价心肌损伤程度；Western印迹检测磷酸化钙/钙调蛋白依赖的蛋白激酶II(Ca2+/calmodulin-dependent protein kinase II，CaMKII)和受磷蛋白(phospholamban，PLN)苏氨酸17位点磷酸化(pThr17)水平的变化。结果：与Control组相比，IR组再灌注末心肌纤维排列紊乱，断裂明显，心肌梗死面积、LVEDP和冠状动脉流出液中LDH活性明显增加，LVDP比值降低，细胞色素c、cleaved caspase-3、磷酸化CaMKII及pThr17-PLN水平均上调(P<0.01)；KB-R7943+IR组心肌梗死面积明显减小，LVEDP降低，LVDP比值明显改善，LDH活性明显降低，细胞色素c和cleaved caspase-3蛋白水平明显降低，磷酸化CaMKII和pThr17-PLN水平也明显下调(P<0.01)。结论：NCX可通过激活CaMKII启动细胞凋亡和坏死，进而诱导心肌缺血再灌注损伤。.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Caspase 3; Cytochromes c; L-Lactate Dehydrogenase; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Necrosis; Random Allocation; Rats; Rats, Sprague-Dawley; Sodium-Calcium Exchanger

Researches have showed that cardiac shock wave therapy (CSWT) could improve left ventricular function and attenuate LV remodeling of the ischemic heart. Apoptosis plays an important role in myocardial infarction and determines heart function and prognosis. However, it is still not clear whether CSWT is sufficient to attenuate acute myocardial infarction (AMI) induced cardiomyocyte apoptosis in vivo. In this study, we used a rat model to examine whether CSWT could attenuate cardiomyocyte apoptosis after AMI and to explore potential mechanisms.. We generated an AMI rat model to investigate the function and possible regulatory mechanisms of CSWT. All rats were randomly divided into four groups: the sham-operated only group, sham-operated with SW treatment group, AMI only group, and AMI treated with SW treatment group.The rats were treated with a left anterior descending coronary artery ligation for 12h and then treated with or without CSWT (800 shots at 0.1 mJ/ mm2). Cytochrome c release was measured to analyze mitochondrial function and integrity. The apoptotic cell rate was determined by TUNEL assay. Western blot was used to analyze the cell apoptosis-, inflammation-, and survival-related signaling pathways.. First, the methodology of CSWT in the rat model of AMI was established. Second, CSWT attenuated the cardiomyocyte apoptosis rate in the infarct border zone. Third, CSWT suppressed the expression of apoptosis and inflammation molecules after AMI. Fourth, CSWT inhibited activation of the JNK pathway, which indicated inhibition of the cell inflammatory pathways and promotion of cardiomyocyte survival after AMI.. These results indicate that CSWT exerts a protective effect against AMI-induced cardiomyocyte apoptosis, potentially by attenuating cytochrome c release from the mitochondria and inhibiting of the mitochondrial-dependent intrinsic apoptotic pathway. We also demonstrate that CSWT suppresses the JNK pathway and cardiomyocyte inflammation, which may also decrease cardiomyocyte apoptosis in vivo.

Topics: Acute Disease; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cytochromes c; Extracorporeal Shockwave Therapy; JNK Mitogen-Activated Protein Kinases; Male; Mitochondria; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Signal Transduction; Ventricular Function, Left

Although preconditioning of sphingosine 1-phosphate (S1P) has been shown to protect myocytes from hypoxia reoxgenation injury in vitro, the role of S1P postconditioning on myocardial ischemia reperfusion injury (MIRI) in vivo and its related mechanism are unknown. The aim of this study was to investigate the protective role of sphingosine 1-phosphate (S1P) postconditioning in MIRI via its effects on mitochondrial signaling and Akt/Gsk3β phosphorylation.. Rats were subjected to MIRI, consisting of 30 min of ischemia followed by 120 min of reperfusion, with S1P administered at the beginning of the reperfusion. Myocardial infarct size and apoptotic index were measured by triphenyltetrazolium (TTC) and terminal deoxynucleotide transferase dUTP nick-end labeling (TUNEL) assays, respectively. Akt and Gsk3β phosphorylation, caspase-3 cleavage, and cytochrome c translocation were assessed by western blot. Mitochondrial permeability transition pore (MPTP) opening and mitochondrial membrane potential (MMP, ΔΨ) were also examined to determine overall mitochondrial function.. S1P postconditioning significantly decreased myocardial infarct size and apoptosis, as well as enhanced Akt and Gsk3β phosphorylation, attenuated caspase-3 cleavage and cytosolic cytochrome c translocation, and inhibited MPTP opening, which subsequently preserved Δψ. Electron microscopy also confirmed that S1P helped maintain myocardial mitochondria integrity. Moreover, the protective effects of S1P treatment were blocked by cotreatment with a PI3K inhibitor, LY294002.. These results suggest that S1P postconditioning protects against MIRI by regulating mitochondrial signaling and Akt/Gsk3β phosphorylation.

Topics: Animals; Chromones; Cytochromes c; Glycogen Synthase Kinase 3 beta; Ischemic Postconditioning; Lysophospholipids; Male; Membrane Potential, Mitochondrial; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Morpholines; Myocardial Infarction; Myocardial Reperfusion Injury; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Transport; Proto-Oncogene Proteins c-akt; Rats, Wistar; Signal Transduction; Sphingosine

To investigate the role of melatonin in calcium overload-induced heart injury.
 Methods: Thirty-two rats were divided into 4 groups: a control group (Control), a melatonin control group (Mel), a calcium overload group (CaP), and a calcium overload plus melatonin group (Mel+CaP). Isolated Sprague Dawley male rat hearts underwent Langendorff perfusion. Left ventricular developed pressure (LVDP) was calculated to evaluate the myocardial performance. Triphenyltetrazolium chloride staining was used to measure the infarct size of myocardium. Lactate dehydrogenase (LDH) activity in the coronary flow was determined. The expressions of caspase-3 and cytochrome c were determined by Western blot. The pathological morphological changes in myocardial fiber were analyzed by HE staining.
 Results: Compared with the control group, calcium overload significantly induced an enlarged infarct size (P<0.01), accompanied by the disordered arrangement of myocardial fiber, up-regulation of cytochrome c and caspase-3 (P<0.01), and the increased activity of LDH (P<0.01). These effects were significantly attenuated by 10 μmol/L melatonin (P<0.01).
 Conclusion: Melatonin can alleviate calcium overload-induced heart injury.. 目的：观察褪黑素(melatonin，Mel)对大鼠离体心脏急性钙超载损伤的保护作用。方法：将32只大鼠随机分为4组：对照组(Control)、褪黑素组(Mel)、钙反常组(calcium paradox，CaP)和Mel干预钙反常组(Mel+CaP)。大鼠离体心脏行Langendorff灌流，建立钙反常模型；观察左室发展压、心肌梗死面积、乳酸脱氢酶(lactate dehydrogenase，LDH)活性、细胞色素c及caspase-3蛋白的表达变化；HE染色观察心肌纤维形态学变化。结果：与Control组相比，CaP组心肌几乎全部梗死(P<0.01)，冠状动脉流出液中LDH活性显著增加(P<0.01)，心肌纤维排列紊乱，细胞色素c和caspase-3蛋白表达均上调(P<0.01)；Mel(10 μmol/L)干预钙反常组心脏损伤各项指标均明显降低(P<0.01)。结论：Mel可明显减轻大鼠离体心脏急性钙超载诱导的损伤。.

Topics: Animals; Calcium; Caspase 3; Coloring Agents; Cytochromes c; Heart; Heart Injuries; L-Lactate Dehydrogenase; Male; Melatonin; Myocardial Infarction; Myocardium; Perfusion; Rats; Rats, Sprague-Dawley; Tetrazolium Salts

Myocardin-related transcription factor-A (MRTF-A) can transduce both biomechanical and humoral signals, which can positively modulate cardiac damage induced by acute myocardial infarction. However, the molecular mechanism that underlies the contribution that MRTF-A provides to the myocardium is not completely understood. The objective of this study was to investigate the effects of MRTF-A on myocardium apoptosis and its mechanisms. Our experiment results showed that MRTF-A expression increased and Bcl-2 expression reduced during myocardial ischemia-reperfusion in rat. Meanwhile, primary cardiomyocytes were pretreated with wild-type MRTF-A or siRNA of MRTF-A before exposure to hypoxia. We found that overexpression of MRTF-A in myocardial cells inhibited apoptosis and the release of cytochrome c. MRTF-A enhanced Bcl-2, which contributes to MRTF-A interaction with Bcl-2 in the nuclei of cardiomyocytes. MRTF-A upregulation expression of Bcl-2 in cardiomyocytes induced by hypoxia was inhibited by PD98059, an ERK1/2 inhibitor. In conclusions, MRTF-A improved myocardial cell survival in a cardiomyocyte model of hypoxia-induced injury; this effect was correlated with the upregulation of anti-apoptotic gene Bcl-2 through the activation of ERK1/2.

Topics: Animals; Apoptosis; Cell Survival; Cytochromes c; Hypoxia; Male; MAP Kinase Signaling System; Myocardial Infarction; Myocardial Ischemia; Myocardium; Myocytes, Cardiac; Nuclear Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Trans-Activators; Transcription Factors; Transcriptional Activation; Up-Regulation

Carcinoembryonic antigen-related cell adhesion molecule1 (CEACAM1) is a tumor-associated factor that is known to be involved in apoptosis, but the role of CEACAM1 in cardiovascular disease is unclear. We aims to investigate whether CEACAM1 influences cardiac remodeling in mice with myocardial infarction (MI) and hypoxia-induced cardiomyocyte injury. Both serum in patients and myocardial CEACAM1 levels in mice were significantly increased in response to MI, while levels were elevated in neonatal rat cardiomyocytes (NRCs) exposed to hypoxia. Eight weeks after MI, a lower mortality rate, improved cardiac function, and less cardiac remodeling in CEACAM1 knock-out (KO) mice than in their wild-type (WT) littermates were observed. Moreover, myocardial expression of mitochondrial Bax, cytosolic cytochrome C, and cleaved caspase-3 was significantly lower in CEACAM1 KO mice than in WT mice. In cultured NRCs exposed to hypoxia, recombinant human CEACAM1 (rhCEACAM1) reduced mitochondrial membrane potential, upregulated mitochondrial Bax, increased cytosolic cytochrome C and cleaved caspase-3, and consequently increased apoptosis. RhCEACAM1 also increased the levels of GRP78 and CHOP in NRCs with hypoxia. All of these effects were abolished by silencing CEACAM1. Our study indicates that CEACAM1 exacerbates hypoxic cardiomyocyte injury and post-infarction cardiac remodeling by enhancing cardiomyocyte mitochondrial dysfunction and endoplasmic reticulum stress-induced apoptosis.

Topics: Animals; Antigens, CD; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Cell Adhesion Molecules; Cell Hypoxia; Cells, Cultured; Cytochromes c; Down-Regulation; Echocardiography; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Humans; Male; Membrane Potential, Mitochondrial; Mice; Mice, Knockout; Microscopy, Fluorescence; Mitochondria; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Recombinant Proteins; RNA Interference; RNA, Messenger; RNA, Small Interfering; Transcription Factor CHOP; Up-Regulation; Ventricular Remodeling

Ischemia impairs the adenine nucleotide translocase (ANT), which transports ADP and ATP across the inner mitochondrial membrane. We investigated whether ANT1 overexpression has protective effects on ischemic hearts. Myocardial infarction was induced in wild-type (WT) and heart-specific ANT1-transgenic (ANT1-TG) rats, and hypoxia was set in isolated cardiomyocytes. ANT1 overexpression reduced the myocardial infarct area and increased the survival rate of infarcted rats. Reduced ANT1 expression and increased 4-hydroxynonenal modification of ANT paralleled to impaired ANT function in infarcted WT hearts. ANT1 overexpression improved ANT expression and function. This was accompanied by reduced mitochondrial cytochrome C release and caspase-3 activation. ANT1-TG hearts suffered less from oxidative stress, as shown by lower protein carbonylation and 4-hydroxynonenal modification of ANT. ANT1 overexpression also increased cell survival of hypoxic cardiomyocytes and attenuated reactive oxygen species (ROS) production. This was linked to higher stability of mitochondrial membrane potential and lower activity of ROS detoxifying catalase. ANT1-TG cardiomyocytes also showed higher resistance against H2O2 treatment, which was independent of catalase activity. In conclusion, ANT1 overexpression compensates impaired ANT activity under oxygen-restricted conditions. It reduces ROS production and oxidative stress, stabilizes mitochondrial integrity, and increases survival, making ANT1 a component in ROS management and heart protection during ischemia.. ANT1 overexpression reduces infarct size and increases survival after infarction. ANT1 overexpression compensates restricted ANT expression and function in infarcted hearts. Increased ANT1 expression enhances mitochondrial integrity. ANT1-overexpressing hearts reduce oxidative stress by decreasing ROS generation. ANT1 is a component in ROS management and heart protection.

Topics: Adenine Nucleotide Translocator 1; Aldehydes; Animals; Caspase 3; Catalase; Cell Hypoxia; Cell Survival; Cytochromes c; Gene Expression Regulation; Hydrogen Peroxide; Male; Membrane Potential, Mitochondrial; Mitochondria, Heart; Myocardial Infarction; Myocytes, Cardiac; Oxidative Stress; Primary Cell Culture; Protein Carbonylation; Rats; Rats, Transgenic; Reactive Oxygen Species; Signal Transduction; Survival Analysis

Mitochondria-mediated cell death plays a critical role in myocardial ischemia-reperfusion (IR) injury. We hypothesized that nanoparticle-mediated drug delivery of mitochondrial division inhibitor 1 (Mdivi1) protects hearts from IR injury through inhibition of mitochondria outer membrane permeabilization (MOMP), which causes mitochondrial-mediated cell death.. We formulated poly (lactic-co-glycolic acid) nanoparticles containing Mdivi1 (Mdivi1-NP). We recently demonstrated that these nanoparticles could be successfully delivered to the cytosol and mitochondria of cardiomyocytes under H2O2-induced oxidative stress that mimicked IR injury. Pretreatment with Mdivi1-NP ameliorated H2O2-induced cell death in rat neonatal cardiomyocytes more potently than Mdivi1 alone, as indicated by a lower estimated half-maximal effective concentration and greater maximal effect on cell survival. Mdivi1-NP treatment of Langendorff-perfused mouse hearts through the coronary arteries at the time of reperfusion reduced infarct size after IR injury more effectively than Mdivi1 alone. Mdivi1-NP treatment also inhibited Drp1-mediated Bax translocation to the mitochondria and subsequent cytochrome c leakage into the cytosol, namely, MOMP, in mouse IR hearts. MOMP inhibition was also observed in cyclophilin D knockout (CypD-KO) mice, which lack the mitochondrial permeability transition pore (MPTP) opening. Intravenous Mdivi1-NP treatment in vivo at the time of reperfusion reduced IR injury in wild-type and CypD-KO mice, but not Bax-KO mice.. Mdivi1-NP treatment reduced IR injury through inhibition of MOMP, even in the absence of a CypD/MPTP opening. Thus, nanoparticle-mediated drug delivery of Mdivi1 may be a novel treatment strategy for IR injury.

Topics: Animals; bcl-2-Associated X Protein; Biocompatible Materials; Cell Survival; Cytochromes c; Drug Carriers; Drug Delivery Systems; Dynamins; Heart; Hydrogen Peroxide; Isolated Heart Preparation; Lactic Acid; Mice; Mitochondrial Membranes; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Nanoparticles; Oxidants; Permeability; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Protein Transport; Quinazolinones; Rats

In contrast to cardiomyocyte necrosis, which can be quantified by cardiac troponin, functional cardiomyocyte impairment, including mitochondrial dysfunction, has escaped clinical recognition in acute myocardial infarction (AMI) patients.. To investigate the diagnostic accuracy for AMI and prognostic prediction of in-hospital mortality of cytochrome c.. We prospectively assessed cytochrome c serum levels at hospital presentation in 2 cohorts: a diagnostic cohort of patients presenting with suspected AMI and a prognostic cohort of definite AMI patients. Diagnostic accuracy for AMI was the primary diagnostic end point, and prognostic prediction of in-hospital mortality was the primary prognostic end point. Serum cytochrome c had no diagnostic utility for AMI (area under the receiver-operating characteristics curve 0.51; 95% confidence intervals 0.44-0.58; P=0.76). Among 753 AMI patients in the prognostic cohort, cytochrome c was detectable in 280 (37%) patients. These patients had higher in-hospital mortality than patients with nondetectable cytochrome c (6% versus 1%; P<0.001). This result was mainly driven by the high mortality rate observed in ST-segment-elevation AMI patients with detectable cytochrome c, as compared with those with nondetectable cytochrome c (11% versus 1%; P<0.001). At multivariable analysis, cytochrome c remained a significant independent predictor of in-hospital mortality (odds ratio 3.0; 95% confidence interval 1.9-5.7; P<0.001), even after adjustment for major clinical confounders (odds ratio 4.01; 95% confidence interval 1.20-13.38; P=0.02).. Cytochrome c serum concentrations do not have diagnostic but substantial prognostic utility in AMI.

Topics: Aged; Aged, 80 and over; Biomarkers; Cohort Studies; Cytochromes c; Female; Hospital Mortality; Humans; Male; Middle Aged; Myocardial Infarction; Patient Admission; Prognosis; Prospective Studies

This study elucidates the effects of long-term nutritional preconditioning by resveratrol on ischemia/reperfusion (I/R) injury and its underlying mechanisms.. Mice were treated with resveratrol at 2.0 mg/kg/day by gastric gavages for 6 wk. Then hearts were isolated and subjected to I/R injury in a Langendorff apparatus. Resveratrol significantly improved left ventricular pressure, ±dp/dtmax, and coronary flow; decreased the lactate dehydrogenase and creatine phosphokinase activities; and reduced the infarction size. Additionally, long-term oral resveratrol intake prevented mitochondrial permeability transition pore opening and subsequently inhibited mitochondria-mediated apoptosis, as demonstrated by decrease of cytochrome c release, inactivation of caspase-3, and reduction of terminal deoxynucleotidyl transferase mediated nick end labeling positive cells. Furthermore, resveratrol inhibited the upregulation of voltage-dependent anion channel 1 (VDAC1) expression induced by I/R injury. Local left-ventricle overexpression of VDAC1 by adenovirus diminished the protective effect of resveratrol against I/R injury, indicating that VDAC1 plays an important role in resveratrol-mediated cardioprotection.. Our data revealed that long-term oral intake of resveratrol sets nutritional preconditioning to cope with myocardial I/R injury. Strikingly, we found that resveratrol downregulates VDAC1, leading to prevention of mitochondrial permeability transition pore opening and cardiomyocyte apoptosis.

Topics: Administration, Oral; Animals; Apoptosis; Cardiotonic Agents; Caspase 3; Cytochromes c; Down-Regulation; Gene Expression Regulation; Heart; Ischemic Preconditioning, Myocardial; Mice; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Infarction; Myocardial Reperfusion Injury; Organ Culture Techniques; Resveratrol; Stilbenes; Voltage-Dependent Anion Channel 1

Ischemic postcontioning (IPoC) is an effective method to prevent myocardial ischemia reperfusion injury (MIRI), but its cardioprotection is usually blocked in the presence of hypercholesterolemia (HC) and the potential mechanism is still unknown. In this study, we investigated the roles of reperfusion injury salvage kinase (RISK) and apoptosis-related pathways in the attenuation of cardioprotection of IPoC in the presence of HC. The results showed that IPoC significantly decreased the infarct size and apoptosis, improved the recovery of ischemic myocardium, but these beneficial effects were reversed by high cholesterol diet-induced HC. Moreover, we also found that HC inhibited the phosphorylation of Akt and ERK1/2 which usually activated by IPoC in normal heart, induced excessive apoptosis by down-regulating Bcl-2 and up-regulating Bax, cytochrome c, caspase 9 and caspase 3 when compared with that in normal heart. Taken together, our results demonstrated that the cardioprotection of IPoC was abolished by HC, which was associated with inactivation of RISK signal pathway and dysregulation of downstream apoptosis-related pathway.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Coronary Vessels; Cytochromes c; Heart; Hypercholesterolemia; Ischemic Postconditioning; Lipids; Male; Mitogen-Activated Protein Kinase 3; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Proto-Oncogene Proteins c-akt; Rats, Wistar; Signal Transduction

Febuxostat is a selective inhibitor of xanthine oxidase (XO). XO is a critical source of reactive oxygen species (ROS) during myocardial ischemia/reperfusion (I/R) injury. Inhibition of XO is therapeutically effective in I/R injury. Evidence suggests that febuxostat exerts antioxidant effects by directly scavenging ROS. The present study was performed to investigate the effects of febuxostat on myocardial I/R injury and its underlying mechanisms.. We utilized an in vivo mouse model of myocardial I/R injury and an in vitro neonatal rat cardiomyocyte (NRC) model of hypoxia/reoxygenation (H/R) injury. Mice were randomized into five groups: Sham, I/R (I/R + Vehicle), I/R + FEB (I/R + febuxostat), AL + I/R (I/R + allopurinol) and FEB (febuxostat), respectively. The I/R + FEB mice were pretreated with febuxostat (5 mg/kg; i.p.) 24 and 1 h prior to I/R. NRCs received febuxostat (1 and 10 µM) at 24 and 1 h before exposure to hypoxia for 3 h followed by reoxygenation for 3 h. Cardiac function, myocardial infarct size, serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH), and myocardial apoptotic index (AI) were measured in order to ascertain the effects of febuxostat on myocardial I/R injury. Hypoxia/reperfusion (H/R) injury in NRCs was examined using MTT, LDH leakage assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The underlying mechanisms were determined by measuring ROS production, mitochondrial membrane potential (ΔΨm), and expression of cytochrome c, cleaved caspases as well as Bcl-2 protein levels.. Myocardial I/R led to an elevation in the myocardial infarct size, serum levels of CK and LDH, cell death and AI. Furthermore, I/R reduced cardiac function. These changes were significantly attenuated by pretreatment with febuxostat and allopurinol, especially by febuxostat. Febuxostat also protected the mitochondrial structure following myocardial I/R, inhibited H/R-induced ROS generation, stabilized the ΔΨm, alleviated cytosolic translocation of mitochondrial cytochrome C, inhibited activation of caspase-3 and -9, upregulated antiapoptotic proteins and downregulated proapoptotic proteins.. This study revealed that febuxostat pretreatment mediates the cardioprotective effects against I/R and H/R injury by inhibiting mitochondrial-dependent apoptosis.

Topics: Animals; Animals, Newborn; Apoptosis; Caspase 3; Caspase 9; Cytochromes c; Febuxostat; Heart Function Tests; Heart Ventricles; Hypoxia; Male; Membrane Potential, Mitochondrial; Mice, Inbred C57BL; Mitochondria; Models, Biological; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Necrosis; Rats, Sprague-Dawley; Reactive Oxygen Species; Signal Transduction

It was previously shown that total saponins extracted from Aralia taibaiensis (sAT) have potent antioxidant activities for treating diabetes mellitus and attenuate d-galactose-induced aging. Since diabetes mellitus and aging are closely associated with cardiac dysfunction, particularly ischemic heart disease, sAT may have potential protective activity against myocardial ischemia/reperfusion injury (MI/RI). However, the anti-MI/RI effects of sAT have yet to be examined, and the possible molecular mechanisms remain to be determined. The present study was undertaken to investigate the anti-MI/RI activities of sAT and to elucidate the mechanisms underlying these effects in rats using TUNEL and Hoechst 33258 staining. The results confirmed the cardioprotective effects in vivo and elucidated the potential molecular mechanisms of sAT in vitro. Pretreatment with sAT significantly reduced infarct size, decreased the levels of lactate dehydrogenase and creatine kinase in the serum and blocked apoptosis. In addition, sAT inhibited A/R-induced apoptosis by decreasing DNA strand breaks, caspase-3 activity and cytochrome c release in H9c2 cells. Furthermore, sAT markedly increased the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl CoA carboxylase and elevated the Bcl2/Bcl-2-associated X protein ratio. These effects were blocked by compound C. The results suggested that sAT pretreatment exerts protective effects on myocardial cells in vitro and in vivo against MI/RI-induced apoptosis by activating AMPK pathway.

Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Aralia; bcl-2-Associated X Protein; Blotting, Western; Cardiotonic Agents; Caspase 3; Cell Line; Creatine Kinase; Cytochromes c; L-Lactate Dehydrogenase; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Phosphorylation; Phytotherapy; Proto-Oncogene Proteins c-bcl-2; Rats, Sprague-Dawley; Saponins; Signal Transduction

Volatile anesthetic postconditioning reduces myocardial infarct size against ischemia/reperfusion (I/R) injury. We tested the hypothesis that emulsified isoflurane (EIso) administrated after ischemia exerts cardioprotection in a rat model of myocardial I/R. Male SD rats underwent 30-min coronary occlusion followed by 3-h reperfusion except for sham rats. All vehicles were administrated intravenously at reperfusion onset for 30 min. In the first study, 56 rats were given saline (CON), 30% intralipid (IL) and 1, 2, 4, 8 or 16 mL/kg EIso for infarct size measurement. In a second study, 32 rats were randomized to four groups and administrated saline in sham (sham) and control (CON) groups, 30% intralipid in IL group and 2 mL/kg emulsified isoflurane in EIso group. Cardiomyocytic enzyme activity was determined. Myocardial mitochondria and cytosol were isolated to determine mitochondrial energy metabolism, cytochrome c release, mitochondrial membrane potential (ΔΨm) and opening of the mitochondrial permeability transition pore (mPTP). Morphologic changes in mitochondria were observed by transmission electron microscopy. Compared with CON and IL, 2, 4 and 8 mL/kg EIso limited infarct size (P < 0.01). Serum levels of cardiac enzyme leakage were reduced in EIso-treated hearts compared with CON (P < 0.01 or P < 0.05). EIso preserved the ultrastructure of mitochondria, protected against mPTP opening, decreased cytochrome c release and preserved ATP production and ΔΨm . In conclusion, EIso is effective in reducing infarct size and in preserving mitochondrial function after ischemia and reperfusion injury.

Topics: Anesthetics, Inhalation; Animals; Cytochromes c; Disease Models, Animal; Emulsions; Energy Metabolism; Ischemic Postconditioning; Isoflurane; Male; Membrane Potential, Mitochondrial; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Infarction; Myocardial Reperfusion Injury; Rats; Rats, Sprague-Dawley

Myocardial ischemia and reperfusion injury in ST-segment elevation myocardial infarction (STEMI) can trigger no-flow, resulting in myocardial necrosis and apoptosis, even a poor prognosis. Cytochrome c can induce an apoptotic process. The aim of our study was to assess the relationship between systemic cytochrome c levels and the occurrence of no-reflow in STEMI.. One hundred and sixty patients with STEMI undergoing a primary percutaneous coronary intervention (PPCI) were randomly chosen. Patients were divided into two groups defined by the mean cytochrome c peak level after PPCI. No-reflow was assessed using three different methods after PPCI: myocardial blush grade, electrocardiographic ST-resolution, and microvascular obstruction (MO) assessed by cardiovascular magnetic resonance imaging. The primary clinical end points were major adverse cardiovascular events (defined as cardiac death, reinfarction, or new congestive heart failure). Clinical follow-up was carried out for 1 year.. Patients with a cytochrome c level of at least the mean peak level had a greater creatine kinase-MB isoenzyme peak level (P=0.044), a lower left ventricular ejection fraction (P=0.029), a significantly higher occurrence of early MO (P=0.008), and a significantly larger extent of early MO (P=0.020). The cytochrome c peak level was elevated in patients with early MO (P=0.025), myocardial blush grade 0-1 (P=0.002), and ST-resolution less than 30% (P=0.003) after PPCI. A higher incidence of cardiac death at the 1-year follow-up was found in the patients with cytochrome c levels of at least the mean peak level (log rank, P=0.029).. Cytochrome c levels above the mean peak level were related to no-reflow and mortality in patients with STEMI.

Topics: Aged; Biomarkers; Contrast Media; Coronary Angiography; Coronary Circulation; Creatine Kinase, MB Form; Cytochromes c; Electrocardiography; Female; Humans; Magnetic Resonance Imaging, Cine; Male; Middle Aged; Myocardial Infarction; Myocardial Perfusion Imaging; No-Reflow Phenomenon; Organometallic Compounds; Percutaneous Coronary Intervention; Predictive Value of Tests; Risk Factors; Stroke Volume; Time Factors; Treatment Outcome; Ventricular Function, Left

The aim of this study was to investigate whether the mitochondrial permeability transition pore (MPTP) opening was involved in the protective effects of CB2 receptor against ischemia-reperfusion (I-R) injury. For this, isolated perfused rat hearts were subjected to 30 min global ischemia followed by 120 min reperfusion, and left ventricle function was recorded. At the end of reperfusion, the infarct size in the hearts was measured by staining with triphenyltetrazolium chloride. MPTP opening and the mitochondrial membrane potential (ΔΨ(m)) were measured by flow cytometry. Western blot analysis of cytochrome c in the mitochondrion and cytosol, as well as ERK1/2 and p-ERK1/2 were performed. Administration of CB2 receptor agonist JWH133 before ischemia significantly improved the recovery of cardiac ventricular function during reperfusion, increased coronary flow, reduced infarct size, prevented the loss of ΔΨ(m) and MPTP opening, reduced the release of cytochrome c from mitochondria, and increased levels of p-ERK1/2. These effects of JWH133 were abolished by pretreatment with CB2 receptor antagonist AM630, or ERK1/2 inhibitor PD98059. Furthermore, JWH133 reversed the MPTP opening induced by atractyloside. The protective effect of JWH133 on the heart against I-R injury may be through increased ERK1/2 phosphorylation, inhibiting MPTP opening.

Topics: Animals; Cannabinoids; Cytochromes c; Male; MAP Kinase Signaling System; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Infarction; Myocardial Reperfusion Injury; Rats; Rats, Sprague-Dawley; Receptor, Cannabinoid, CB2

Inflammatory responses play an important role in the pathogenesis of adverse ventricular remodeling after myocardial infarction (MI). We previously demonstrated that interleukin (IL)-17A plays a pathogenic role in myocardial ischemia/reperfusion injury and viral myocarditis. However, the role of IL-17A in post-MI remodeling and the related mechanisms have not been fully elucidated. Acute MI was induced by permanent ligation of the left anterior descending coronary artery in C57BL/6 mice. Repletion of IL-17A significantly aggravated both early- and late-phase ventricular remodeling, as demonstrated by increased infarct size, deteriorated cardiac function, increased myocardial fibrosis, and cardiomyocyte apoptosis. By contrast, genetic IL-17A deficiency had the opposite effect. Additional studies in vitro indicated that IL-17A induces neonatal cardiomyocyte (from C57BL/6 mice) apoptosis through the activation of p38, p53 phosphorylation, and Bax redistribution. These data demonstrate that IL-17A induces cardiomyocyte apoptosis through the p38 mitogen-activated protein kinase (MAPK)-p53-Bax signaling pathway and promotes both early- and late-phase post-MI ventricular remodeling. IL-17A might be an important target in preventing heart failure after MI. Key message: We demonstrated that IL-17A plays a pathogenic role both in the early and late stages of post-MI remodeling. IL-17A induces murine cardiomyocyte apoptosis. IL-17A induces murine cardiomyocyte apoptosis through the p38 MAPK-p53-Bax signaling pathway.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cytochromes c; Fibrosis; Interleukin-17; Male; Mice, Inbred C57BL; Myocardial Infarction; Myocardium; Myocytes, Cardiac; p38 Mitogen-Activated Protein Kinases; Receptors, Interleukin-17; RNA, Messenger; Tumor Suppressor Protein p53; Ventricular Remodeling

AB We evaluated the post-myocardial infarction (MI) therapeutic effects of Bendavia. Two hours after coronary artery ligation, rats were randomized to receive chronic Bendavia treatment (n = 28) or water (n = 26). Six weeks later, Bendavia significantly reduced scar circumference (39.7% +/- 2.2%) compared with water treatment (47.4% +/- 0.03%, P = 0.024) and reduced left ventricular (LV) volume by 8.9% (P = 0.019). LV fractional shortening was significantly improved by Bendavia (28.8% +/- 1.7%) compared with water treatment (23.8% +/- 1.8%, P = 0.047). LV ejection fraction was higher with Bendavia (55.3% +/- 1.4%) than water treatment (49.3% +/- 1.4%, P = 0.005). Apoptosis, within the MI border zone, was significantly less in the Bendavia group (32% +/- 3%, n = 12) compared with the water group (41% +/- 2%, n = 12; P = 0.029). Bendavia reversed mitochondrial function-related gene expression in the MI border, which was largely reduced in water-treated rats. Bendavia improved complex-I and -IV activity, and reduced production of reactive oxygen species and cytosolic cytochrome c level in the peri-infarcted region. Bendavia improved post-MI cardiac function, prevented infarct expansion and adverse LV remodeling, and restored mitochondria-related gene expression, complex-I and -IV activity, and reduced reactive oxygen species and cardiomyocyte apoptosis in the noninfarcted MI border.

Topics: Animals; Apoptosis; Cytochromes c; Disease Models, Animal; Electron Transport Complex I; Electron Transport Complex IV; Female; Gene Expression Regulation; Mitochondria; Myocardial Infarction; Myocytes, Cardiac; Oligopeptides; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Ventricular Function, Left; Ventricular Remodeling

Studies from our lab have shown that decreasing myocardial G protein-coupled receptor kinase 2 (GRK2) activity and expression can prevent heart failure progression after myocardial infarction. Since GRK2 appears to also act as a pro-death kinase in myocytes, we investigated the effect of cardiomyocyte-specific GRK2 ablation on the acute response to cardiac ischemia/reperfusion (I/R) injury. To do this we utilized two independent lines of GRK2 knockout (KO) mice where the GRK2 gene was deleted in only cardiomyocytes either constitutively at birth or in an inducible manner that occurred in adult mice prior to I/R. These GRK2 KO mice and appropriate control mice were subjected to a sham procedure or 30 min of myocardial ischemia via coronary artery ligation followed by 24 hrs reperfusion. Echocardiography and hemodynamic measurements showed significantly improved post-I/R cardiac function in both GRK2 KO lines, which correlated with smaller infarct sizes in GRK2 KO mice compared to controls. Moreover, there was significantly less TUNEL positive myocytes, less caspase-3, and -9 but not caspase-8 activities in GRK2 KO mice compared to control mice after I/R injury. Of note, we found that lowering cardiac GRK2 expression was associated with significantly lower cytosolic cytochrome C levels in both lines of GRK2 KO mice after I/R compared to corresponding control animals. Mechanistically, the anti-apoptotic effects of lowering GRK2 expression were accompanied by increased levels of Bcl-2, Bcl-xl, and increased activation of Akt after I/R injury. These findings were reproduced in vitro in cultured cardiomyocytes and GRK2 mRNA silencing. Therefore, lowering GRK2 expression in cardiomyocytes limits I/R-induced injury and improves post-ischemia recovery by decreasing myocyte apoptosis at least partially via Akt/Bcl-2 mediated mitochondrial protection and implicates mitochondrial-dependent actions, solidifying GRK2 as a pro-death kinase in the heart.

Topics: Animals; Apoptosis; bcl-X Protein; Cytochromes c; G-Protein-Coupled Receptor Kinase 2; Hemodynamics; Isoproterenol; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; Oxidative Stress; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Up-Regulation

The mitogen-activated protein kinases (MAPKs) play an important role in ischemia/reperfusion (I/R) injury. Previous evidence suggests that p38 MAPK inhibition before ischemia is cardioprotective. However, whether p38 MAPK inhibition during ischemia or reperfusion provides cardioprotection is not well known. We tested the hypothesis that p38 MAPK inhibition at different times during I/R protects the heart from arrhythmias, reduces the infarct size, and attenuates ventricular dysfunction. Adult Wistar rats were subject to a 30-minute left anterior descending coronary artery occlusion, followed by a 120-minute reperfusion. A p38 MAPK inhibitor, SB203580, was given intravenously before left anterior descending coronary artery occlusion, during ischemia, or at the onset of reperfusion. The results showed that SB203580 given either before or during ischemia, but not at the onset of reperfusion, decreased the ventricular tachycardia/ventricular fibrillation (VT/VF) incidence and heat shock protein 27 phosphorylation, and increased connexin 43 phosphorylation. The infarct size and cytochrome c level was decreased in all SB203580-treated rats, without the alteration of the total Bax/Bcl-2 expression. The ventricular function was improved only in SB203580-pretreated rats. These findings suggest that timing of p38 MAPK inhibition with respect to onset of ischemia is an important determinant of therapeutic efficacy.

Topics: Administration, Intravenous; Animals; Arrhythmias, Cardiac; Cytochromes c; Disease Models, Animal; Enzyme Inhibitors; Imidazoles; Male; Myocardial Infarction; Myocardial Reperfusion Injury; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Rats; Rats, Wistar; Time Factors; Ventricular Function

The mechanisms by which ischemic preconditioning (IP) inhibits mitochondrial permeability transition pore opening and, hence, ischemia-reperfusion injury remain unclear. Here we investigate whether and how mitochondria-bound hexokinase 2 (mtHK2) may exert part of the cardioprotective effects of IP.. Control and IP Langendorff-perfused rat hearts were subject to ischemia and reperfusion with measurement of hemodynamic function and infarct size. Outer mitochondrial membrane (OMM) permeabilization after ischemia was determined by measuring rates of respiration and H2O2 production in the presence and absence of added cytochrome c in isolated mitochondria and permeabilized fibers. IP prevented OMM permeabilization during ischemia and reduced the loss of mtHK2, but not Bcl-xL, observed in control ischemic hearts. By contrast, treatment of permeabilized fibers with glucose-6-phosphate at pH 6.3 induced mtHK2 loss without OMM permeabilization. However, metabolic pretreatments of the perfused heart chosen to modulate glucose-6-phosphate and intracellular pHi revealed a strong inverse correlation between end-ischemic mtHK2 content and infarct size after reperfusion. Loss of mtHK2 was also associated with reduced rates of creatine phosphate generation during the early phase of reperfusion. This could be mimicked in permeabilized fibers after mtHK2 dissociation.. We propose that loss of mtHK2 during ischemia destabilizes mitochondrial contact sites, which, when accompanied by degradation of Bcl-xL, induces OMM permeabilization and cytochrome c loss. This stimulates reactive oxygen species production and mitochondrial permeability transition pore opening on reperfusion, leading to infarction. Consequently, inhibition of mtHK2 loss during ischemia could be an important mechanism responsible for the cardioprotection mediated by IP and other pretreatments.

Topics: Animals; bcl-2-Associated X Protein; bcl-X Protein; Cytochromes c; Disease Models, Animal; Glucose-6-Phosphate; Hemodynamics; Hexokinase; Hydrogen Peroxide; Ischemic Preconditioning, Myocardial; Male; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Permeability Transition Pore; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Permeability; Phosphocreatine; Rats; Rats, Wistar; Reactive Oxygen Species

This study was designed to evaluate the anti-inflammatory and anti-apoptotic effects of the alcoholic extract of the berries of Crataegus oxyacantha (AEC), a medicinal herb, on isoproterenol-induced myocardial infarction (MI) in a rat model. Three groups of Wistar albino rats, each comprising six animals, were selected for this study. Group I rats served as control. Group II rats were given isoproterenol (85 mg/kg body weight) subcutaneously on 59th and 60th days. Group III rats were given AEC (0.5 ml/100 g body weight/day), orally on a daily basis for 60 days, and isoproterenol (85 mg/kg body weight, subcutaneously) was given on 59th and 60th days. On the 61st day, the animals were sacrificed, and marker enzymes like lactate dehydrogenase (LDH) and creatine kinase (CK) were estimated in serum. In the heart tissue sample, antioxidant status, lipid peroxidation and anti-inflammatory properties of AEC were determined. Isoproterenol significantly increased the release of LDH, CK in serum, decreased the antioxidant status in the heart along with an increase in lipid peroxidation. Nitritive stress and apoptosis were seen in isoproterenol-induced rat heart. Pre-treatment with the AEC for 60 days had a significant effect on all the above factors and maintained near normal status. The study confirms the protective effect of AEC against isoproterenol-induced inflammation and apoptosis-associated MI in rats.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; Catalase; Crataegus; Creatine Kinase; Cyclooxygenase 2; Cytochromes c; DNA Fragmentation; Drug Evaluation, Preclinical; Fruit; Isoproterenol; L-Lactate Dehydrogenase; Lipid Peroxidation; Male; Mitochondria, Heart; Myocardial Infarction; Myocardium; Nitric Oxide Synthase Type II; Nitrites; Phytotherapy; Plant Extracts; Rats; Rats, Wistar; Superoxide Dismutase

HF (heart failure) after MI (myocardial infarction) is a major cause of morbidity and mortality worldwide. Recent studies have shown that hydrogen sulfide (H2S) has cardioprotective effects. Hence, we aimed to elucidate the potential effects of H2S on HF after MI in rats. The HF model after MI was made by ligating the left anterior descending coronary artery. HF groups and sham-operated groups of rats were treated with vehicle, sodium hydrosulfide (NaHS) or PAG (propagylglycine). Equal volumes of saline, 3.136 mg · kg-1 · day-1 NaHS or 37.5 mg · kg-1 · day-1 PAG, were intraperitoneally injected into rats for 6 weeks after operation. Survival, lung-to-body weight ratio and left ventricular haemodynamic parameters were measured. The protein and gene expression of Bcl-2, Bax, caspase 3 and cytochrome c were analysed by Western blotting and RT-PCR (reverse transcription-PCR). TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) and EM (electron microscopy) were used to examine apoptosis of heart tissues. NaHS was found to improve the survival and lower the lung-to-body weight ratio. It increased the LVSP (left ventricular systolic pressure) and the maximum rate of pressure and decreased LVEDP (left ventricular end-diastolic pressure). Furthermore, NaHS promoted Bcl-2 protein and mRNA expression and demoted Bax, caspase 3 protein and mRNA expression in HF rats. We also showed that NaHS decreased the leakage of cytochrome c protein from the mitochondria to the cytoplasm. Histological observation by TUNEL and EM proved that NaHS inhibited cardiac apoptosis in HF hearts and improved mitochondrial derangements, but that PAG aggravated those indices. Hence, H2S has protective effects in HF rats.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blood Pressure; Cardiotonic Agents; Caspase 3; Cytochromes c; Disease Models, Animal; Gene Expression; Genes, bcl-2; Heart Failure; Heart Ventricles; Hydrogen Sulfide; Male; Mitochondria, Heart; Mitochondrial Proteins; Myocardial Infarction; Rats; Sulfides; Ventricular Function, Left

In vivo hyperoxic preconditioning (PC) has been shown to protect against ischemia/reperfusion (I/R) myocardial damage. Mitochondrial permeability transition pore (MPTP) opening is an important event in cardiomyocyte cell death occurring during I/R and therefore a possible target for cardioprotection. We tested the hypothesis that in vivo hyperoxic PC, obtained by mechanical ventilation of animals, could protect heart against I/R injury by inhibiting MPTP opening and cytochrome c release from mitochondria. Mechanically ventilated rats were first exposed to a short period of hyperoxia and isolated hearts were subsequently subjected to I/R in a Langendorff apparatus. Hyperoxic PC significantly improved the functional recovery of hearts on reperfusion, reduced the infarct size, and decreased necrotic damage as shown by the reduced release of lactate dehydrogenase. Mitochondria from hyperoxic PC hearts were less sensitive than mitochondria from reperfused heart to MPTP opening. In addition, hyperoxic PC prevented mitochondrial NAD(+) depletion, an indicator of MPTP opening, and cytochrome c release as well as cardiolipin oxidation/depletion associated with I/R. Together, these results demonstrate that hyperoxic PC protects against heart I/R injury by inhibiting MPTP opening and cytochrome c release. Thus, in vivo hyperoxic PC may represent a useful strategy for the treatment of cardiac I/R injury and could have potential applications in clinical practice.

Topics: Animals; Calcium; Cardiolipins; Cytochromes c; Hyperoxia; Male; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Permeability Transition Pore; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; NAD; Necrosis; Oxygen; Rats; Rats, Wistar

Because myocardial infarction is a major cause of morbidity and mortality worldwide, protecting the heart from the ischaemia and reperfusion (I/R) damage is the focus of intense research. Based on our in vitro findings showing that TVP1022 (the S-enantiomer of rasagiline, an anti-Parkinsonian drug) possesses cardioprotective effects, in the present study we investigated the hypothesis that TVP1022 can attenuate myocardial damage in an I/R model in rats.. The model consisted of 30-min occlusion of the left anterior descending artery followed by 4 or 24 h reperfusion. In addition, we investigated the possible mechanisms of cardioprotection in H9c2 cells and neonatal rat ventricular myocytes (NRVM) exposed to oxidative stress induced by H(2) O(2) .. TVP1022 (20 and 40 mg·kg(-1) ) administered 5 min before reperfusion followed by an additional dose 4 h after reperfusion reduced the infarct size and attenuated the decline in ventricular function. TVP1022 also attenuated I/R-induced deterioration in cardiac mitochondrial integrity evaluated by mitochondrial swelling capacity. In vitro, using H9c2 cells and NRVM, TVP1022 attenuated both serum free- and H(2) O(2) -induced damage, preserved mitochondrial membrane potential and Bcl-2 levels, inhibited mitochondrial cytochrome c release and the increase in cleaved caspase 9 and 3 levels, and enhanced the phosphorylation of protein kinase C and glycogen synthase kinase-3β.. TVP1022 provided cardioprotection in a model of myocardial infarction, and therefore should be considered as a novel adjunctive therapy for attenuating myocardial damage resulting from I/R injuries.

Topics: Animals; Cardiotonic Agents; Caspase 3; Caspase 9; Cells, Cultured; Cytochromes c; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Indans; Male; Membrane Potentials; Mitochondria; Myocardial Infarction; Myocardium; Protein Kinase C; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reperfusion Injury

Apoptosis causes myocardiocyte loss during and after myocardial infarction. Therapeutic approaches designed to arrest apoptosis would be a significant new development in the recovery of acute myocardial infarction (AMI). In order to examine apoptotic markers in the circulation, serum levels of p53 and cytochrome c were assessed in patients with AMI.. Blood samples were taken on admission (before initiation of therapy) and on the 3rd and 7th days of hospitalization. Serum levels of p53 and cytochrome c were measured by enzyme-linked immunassay.. The serum level of p53 was higher in AMI patients on admission compared to the control group. A time-dependent decrease was observed in the serum level of p53, but there was no significant change in the serum level of cytochrome c during therapy.. p53, but not cytochrome c, appears to have potential as a biomarker for reporting on apoptosis following myocardial infarction.

Topics: Aged; Apoptosis; Biomarkers; Cytochromes c; Female; Humans; Male; Middle Aged; Myocardial Infarction; Tumor Suppressor Protein p53

The chronically infarcted rat heart has multiple defects in metabolism, yet the location of the primary metabolic abnormality arising after myocardial infarction is unknown. Therefore, we investigated cardiac mitochondrial metabolism shortly after infarction.. Myocardial infarctions (n = 11) and sham operations (n = 9) were performed on Wistar rats, at 2 weeks cardiac function was assessed using echocardiography, and rats were grouped into failing (ejection fraction < or =45%), moderately impaired (46-60%), and sham-operated (>60%). Respiration rates were decreased by 28% in both subsarcolemmal and interfibrillar mitochondria isolated from failing hearts, compared with sham-operated controls. However, respiration rates were not impaired in mitochondria from hearts with moderately impaired function. The mitochondrial defect in the failing hearts was located within the electron transport chain (ETC), as respiration rates were suppressed to the same extent when fatty acids, ketone bodies, or glutamate were used as substrates. Complex III protein levels were decreased by 46% and complex III activity was decreased by 26%, in mitochondria from failing hearts, but all other ETC complexes were unchanged. Decreased complex III activity was accompanied by a three-fold increase in complex III-derived H(2)O(2) production, decreased cardiolipin content, and a 60% decrease in mitochondrial cytochrome c levels from failing hearts. Respiration rates, complex III activity, cardiolipin content, and reactive oxygen species generation rates correlated with ejection fraction.. In conclusion, a specific defect in complex III occurred acutely after myocardial infarction, which increased oxidative damage and impaired mitochondrial respiration. The extent of mitochondrial dysfunction in the failing heart was proportional to the degree of cardiac dysfunction induced by myocardial infarction.

Topics: Animals; Cardiolipins; Citric Acid Cycle; Cytochromes c; Electron Transport Complex III; Fatty Acids; Heart; Hydrogen Peroxide; Male; Mitochondria, Heart; Mitochondrial Proteins; Myocardial Infarction; Oxygen Consumption; Rats; Rats, Wistar; Reactive Oxygen Species

Remifentanil is a commonly used opioid in anesthesia with cardioprotective effect in ischemia-reperfused (I/R) heart. We evaluated the influence of remifentanil on myocardial infarct size and expressions of proteins involved in apoptosis in I/R rat heart following various time protocols of remifentanil administration. Artificially ventilated anesthetized Sprague-Dawley rats were subjected to a 30 min of left anterior descending coronary artery occlusion followed by 2 h of reperfusion. Rats were randomly assigned to one of five groups; Sham, I/R only, remifentanil preconditioning, postconditioning and continuous infusion group. Myocardial infarct size, the phosphorylation of ERK1/2, Bcl2, Bax and cytochrome c and the expression of genes influencing Ca2+ homeostasis were assessed. In remifentanil-administered rat hearts, regardless of the timing and duration of administration, infarct size was consistently reduced compared to I/R only rats. Remifentanil improved expression of ERK1/2 and anti-apoptotic protein Bcl2, and expression of sarcoplasmic reticulum genes which were significantly reduced in the I/R rats only. Remifentanil reduced expression of pro-apoptotic protein, Bax and cytochrome c. These suggested that remifentanil produced cardioprotective effect by preserving the expression of proteins involved in anti-apoptotic pathways, and the expression of sarcoplasmic reticulum genes in I/R rat heart, regardless of the timing of administration.

Topics: Adjuvants, Anesthesia; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Calcium; Cell Survival; Cytochromes c; Disease Models, Animal; Gene Expression Regulation; Hemodynamics; Homeostasis; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Phosphorylation; Piperidines; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Remifentanil; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sarcoplasmic Reticulum; Time Factors

Glutamate-cysteine ligase (GCL), a rate-limiting enzyme for glutathione (GSH) synthesis, is composed of catalytic and modifier subunits. This study examined the pathogenic role of GCL modifier subunits (GCLM) in myocardial ischaemia-reperfusion (I/R) injury using mice lacking the GCLM (GCLM(-/-)).. The GCLM(-/-)mice had an increase in myocardial I/R injury and apoptosis in ischaemic myocardium compared with GCLM(+/+) mice. There was a decrease in mitochondrial glutathione (GSH) levels in ischaemic myocardium that was more pronounced in GCLM(-/-) mice than in GCLM(+/+) mice (12 vs. 55% of baseline GCLM(+/+), respectively). The ESR signal intensity of the dimethyl-1-pyrroline-N-oxide-hydroxyl radical adducts in ischaemic myocardium was higher in GCLM(-/-) mice than in GCLM(+/+) mice. Hypoxia-reoxygenation induced greater mitochondrial damage in cultured cardiomyocytes from GCLM(-/-) mice than from GCLM(+/+) mice, as evidenced by a reduced membrane potential and increased protein carbonyl content in isolated mitochondria, together with enhanced cytochrome c translocation into the cytosol. Administration of GSH ethyl-ester attenuated myocardial I/R injury and reversed the mitochondrial damage in parallel with the mitochondrial GSH restoration in the myocardium or the cardiomyocytes of GCLM(-/-) mice.. GCLM(-/-) mice were susceptible to myocardial I/R injury partly through an increased vulnerability of mitochondria to oxidative damage owing to mitochondrial GSH reduction.

Topics: Animals; Caspase 3; Caspase 9; Cells, Cultured; Cyclic N-Oxides; Cytochromes c; Echocardiography; Glutamate-Cysteine Ligase; Glutathione; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mitochondria; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; Oxidative Stress; Reactive Oxygen Species; RNA, Messenger; Ventricular Function, Left

Post-myocardial infarction ventricular remodeling is associated with the expression of a variety of factors including S100B that can potentially modulate myocyte apoptosis.. This study was undertaken to investigate the expression and function of S100B and its receptor, the receptor for advanced glycation end products (RAGE) in both postinfarction myocardium and in a rat neonatal myocyte culture model.. In a rat model of myocardial infarction following coronary artery ligation, we demonstrate in periinfarct myocytes, upregulation of RAGE, induction of S100B, and release into plasma with consequent myocyte apoptosis. Using a coimmunoprecipitation strategy, we demonstrate a direct interaction between S100B and RAGE. In rat neonatal cardiac myocyte cultures, S100B at concentrations > or = 50 nmol/L induced myocyte apoptosis, as evidenced by increased terminal DNA fragmentation, TUNEL, cytochrome c release from mitochondria to cytoplasm, phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p53, increased expression and activity of proapoptotic caspase-3, and decreased expression of antiapoptotic Bcl-2. Transfection of a full-length cDNA of RAGE or a dominant-negative mutant of RAGE resulted in increased or attenuated S100B-induced myocyte apoptosis, respectively. Inhibition of ERK1/2 by U0126/PD-98059 or overexpression of a dominant negative p53 comparably inhibited S100B-induced myocyte apoptosis.. These results suggest that interaction of RAGE and its ligand S100B after myocardial infarction may play a role in myocyte apoptosis by activating ERK1/2 and p53 signaling. This receptor-mediated mechanism is uniquely amenable to therapeutic intervention.

Topics: Animals; Apoptosis; Butadienes; Caspase 3; Cell Line; Cytochromes c; Cytosol; Disease Models, Animal; DNA Fragmentation; Enzyme Inhibitors; Flavonoids; Gene Expression Regulation; Humans; Mitochondria, Heart; Mitogen-Activated Protein Kinase 3; Muscle Proteins; Myocardial Infarction; Myocytes, Cardiac; Nerve Growth Factors; Nitriles; Phosphorylation; Rats; Rats, Sprague-Dawley; Receptor for Advanced Glycation End Products; Receptors, Immunologic; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Signal Transduction; Tumor Suppressor Protein p53; Ventricular Remodeling

The release of cytochrome c from the mitochondria to the cytosol is a critical step for downstream caspase-mediated apoptotic signal transduction in ischemia-reperfusion (I/R)-induced myocardial tissue injury. 10-N-nonyl acridine orange (NAO), a cardiolipin-specific dye, has been shown to inhibit Bid-mediated cytochrome c release from isolated mitochondria in vitro; however, the possible protective effects of NAO and the mechanisms underlying the protection from myocardial I/R-induced tissue injury in a rat model are unknown. Male Sprague-Dawley rats were subjected to a 30-min coronary arterial occlusion followed by reperfusion. All rats received either vehicle or NAO (100 microg/kg iv) 10 min before the occlusion. The infarct size in the heart at 24 h after reperfusion was significantly reduced in NAO-treated rats compared with vehicle-treated rats. NAO treatment significantly reduced the cytosolic cytochrome c contents and caspase-9 activity in the ischemic region but did not affect caspase-8 activity. Furthermore, NAO treatment markedly suppressed the translocation of truncated Bid, a proapoptotic Bcl-2 family member, to the mitochondrial fraction. NAO also suppressed the mitochondrial swelling and oxygen uptake stimulated by calcium overload. The results suggest that NAO possesses protective effects against myocardial I/R injury, which may be due to the suppression of cytochrome c release through blockade of truncated Bid translocation to mitochondria and inhibition of the opening of mitochondrial permeability transition pores.

Topics: Aminoacridines; Animals; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Cardiolipins; Caspase 8; Caspase 9; Cytochromes c; Cytosol; Disease Models, Animal; Fluorescent Dyes; Male; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Infarction; Myocardial Reperfusion Injury; Rats; Rats, Sprague-Dawley; Time Factors

Myocardial infarction (MI) causes myocardium injury and scar formation, and the transmural infarction is associated with ventricular hypofunction. Stem cell transplantation therapy has improved cardiac function in animal models of MI. However, the poor survival of the donor cells in the host myocardium hampers the therapeutic efficacy of stem cell transplantation. Diazoxide, a mitochondrial ATP-sensitive potassium channel opener, has been applied to suppress cell apoptosis and promote cell survival. We therefore assessed the effects of diazoxide on the selected mesenchymal stem cells (SMSCs). Pretreatment of SMSCs with diazoxide (200 micromol/L) for 30 min protected cells from oxidative stress injury by upregulating the expression of basic fibroblast growth factor and hepatocyte growth factor mRNAs and phospho-Akt and by preventing mitochondral cytochrome c translocation into the cytoplasm. Expression of mRNAs and proteins was detected by RT-PCR and western blot analyses. Thirty min after establishment of MI (the ligation of the left anterior descending of coronary artery) in female rats, the male rat SMSCs preconditioned with diazoxide were injected at four sites on the edge of the infarcted area. At 4 weeks after cell tranplantation, the donor cells in the recipient myocardium were tracked with Y chromosome. Preconditioning with diazoxide improved the survival rate of the transplanted SMSCs, compared to the untreated SMSCs. Moreover, transplantation of the diazoxide-pretreated SMSCs reduced the infarct size and increased left ventricular function, as judged by transthoracic echocardiography. In conclusion, diazoxide preconditioning is effective to promote SMSCs survival under oxidative stress and attenuates cardiac injury in MI.

Topics: Animals; Apoptosis; Cell Count; Cell Survival; Collagen; Cytochromes c; Cytoprotection; Decanoic Acids; Diazoxide; Female; Fibroblast Growth Factor 2; Gene Expression; Heart Ventricles; Hepatocyte Growth Factor; Hydrogen Peroxide; Hydroxy Acids; Ion Channel Gating; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mitochondrial Membranes; Muscle Development; Myocardial Infarction; Myocardium; Neovascularization, Physiologic; Phosphorylation; Potassium Channels; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Troponin T; Vascular Endothelial Growth Factor A; Ventricular Function, Left

We tested whether shock wave (SW) offers additional benefits in improving left ventricular (LV) function after acute myocardial infarction (AMI) in rabbits receiving SW-treated autologous bone marrow-derived mononuclear cells (BMDMNCs) transplantation.. Saline (750 microL; group 2), BMDMNCs (1.0 x 10(7); group 3), or preimplant SW-treated BMDMNCs (group 4) were implanted into the infarct area of male rabbits 15 minutes after left coronary artery ligation, whereas eight rabbits without AMI served as controls (group 1; n = 8 per group). The results showed that in infarct area of LV, protein expressions of Cx43 and cytochrome C in mitochondria and endothelial nitric oxide synthase mRNA expression were lower in group 2 than in other groups, and decreased in group 3 as compared with groups 1 and 4 (all p values < 0.01). Conversely, mRNA expressions of endothelin-1 and matrix metalloproteinase-9, mitochondrial oxidative stress, and total fibrotic area were higher in group 2 than in other groups (all p values < 0.05). Furthermore, 6-month LV function by 2-D echo/angiogram showed significant impairment in group 2 than in other groups and in group 3 than in groups 1 and 4 (all p values < 0.005).. Application of SW-treated autologous BMDMNCs is superior to BMDMNCs alone for preserving LV function after AMI.

Topics: Animals; Bone Marrow Cells; Bone Marrow Transplantation; Cells, Cultured; Connexin 43; Cytochromes c; Disease Models, Animal; Endothelin-1; Fibrosis; Gene Expression Regulation; High-Energy Shock Waves; Male; Matrix Metalloproteinase 9; Mitochondria, Heart; Myocardial Infarction; Myocardium; Nitric Oxide Synthase Type III; Oxidative Stress; Rabbits; Recovery of Function; RNA, Messenger; Time Factors; Transplantation, Autologous; Ventricular Function, Left

In patients with ST-segment elevation acute myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (pPCI), abrupt reperfusion can induce myocardial injury and apoptotic cell death. Reperfusion-induced myocardial damage, however, cannot be easily evaluated in clinical practice because of the lack of specific biomarkers. Cytochrome c, a mitochondrial protein, is released on reperfusion into the cytosol, where it triggers the apoptotic process. It can reach the external fluid and circulating blood when cell rupture occurs. We measured the cytochrome c circulating levels in patients with STEMI undergoing pPCI, and correlated them with the clinical signs of myocardial necrosis and reperfusion. The plasma creatine kinase-MB mass and serum cytochrome c (enzyme-linked immunosorbent assay method) were serially measured in 55 patients with STEMI undergoing pPCI. The angiographic and electrocardiographic signs of myocardial reperfusion were also assessed. Cytochrome c transiently increased in all patients with STEMI, with a curve that paralleled that of creatine kinase-MB. A significant relation was found between the peak values of the 2 biomarkers (R = 0.35, p = 0.01) and between the areas under the 2 curves (R = 0.33, p = 0.02). The creatine kinase-MB peak value correlated significantly with the clinical features of infarct extension. In contrast, the cytochrome c peak value correlated inversely with the myocardial blush grade. Patients with clinical signs of myocardial reperfusion injury had a significantly greater cytochrome c peak value than patients without reperfusion injury (median 1.65 ng/ml, interquartile range 1.20 to 2.20, vs 1.1 ng/ml, interquartile range 0.65 to 1.55; p = 0.04). In conclusion, serum cytochrome c is detectable in the early phase of STEMI treated with pPCI and is associated with clinical signs of impaired myocardial reperfusion.

Topics: Aged; Angioplasty, Balloon, Coronary; Biomarkers; Cytochromes c; Female; Humans; Male; Middle Aged; Myocardial Infarction; Myocardial Reperfusion Injury; Prospective Studies

Myocyte apoptosis is considered to be the major causative factor of left ventricular (LV) remodeling following myocardial infarction (MI). We previously reported that 3', 4'-dihydroxyflavonol (DiOHF), was able to suppress oxidative stress and preserve the expression of endothelial nitric oxide synthase during myocardial reperfusion injury, which may benefit the reduction of myocyte apoptosis. We therefore aimed to evaluate the potential actions of DiOHF against myocyte apoptosis and post-infarction LV remodeling in this study.. Following experimental MI, surgical instrumented goats were randomly assigned into vehicle and DiOHF (2 mg/kg; i.v., daily) groups to receive 4 weeks of reperfusion with corresponding treatments. LV pressure recordings and echocardiogram were performed at baseline, 2 and 4 weeks of reperfusion. Myocardial tissues were collected in the end to determine infarct size and apoptosis related assays.. LV end-diastolic volume and diameter were significantly increased 4 weeks after MI in the vehicle group, accompanied by reduced posterior wall thickness, septal thickness and LV mass, whereas those changes were markedly prevented by DiOHF treatment. Similarly, significantly reduced infarct size was found in DiOHF group as compared to vehicle group, and DiOHF dramatically inhibited the increase in LV end-diastolic pressure and the reductions in ejection fraction, fraction shortening and dP/dt(max). Moreover, DiOHF treatment significantly reduced the extent of myocyte apoptosis detected by TUNEL assay, enhanced the protein expression of caspase-3, Fas, Bax and cytochrome c in the non-infarcted myocardium in comparison to vehicle.. Daily DiOHF treatment during the reperfusion period after MI in the ovine hearts markedly reduced the magnitude of post-infarction LV remodeling through the inhibition of myocyte apoptosis in the remote non-infarcted myocardium.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cytochromes c; Echocardiography; fas Receptor; Female; Flavonols; Goats; In Situ Nick-End Labeling; Male; Myocardial Infarction; Myocytes, Cardiac; Random Allocation; Ventricular Remodeling

alphaB-crystallin (alphaBC) is a small heat shock protein expressed at high levels in the myocardium where it protects from ischemia-reperfusion damage. Ischemia-reperfusion activates p38 MAP kinase, leading to the phosphorylation of alphaBC on serine 59 (P-alphaBC-S59), enhancing its ability to protect myocardial cells from damage. In the heart, ischemia-reperfusion also causes the translocation of alphaBC from the cytosol to other cellular locations, one of which was recently shown to be mitochondria. However, it is not known whether alphaBC translocates to mitochondria during ischemia-reperfusion, nor is it known whether alphaBC phosphorylation takes place before or after translocation. In the present study, analyses of mitochondrial fractions isolated from mouse hearts subjected to various times of ex vivo ischemia-reperfusion showed that alphaBC translocation to mitochondria was maximal after 20 min of ischemia and then declined steadily during reperfusion. Phosphorylation of mitochondrial alphaBC was maximal after 30 min of ischemia, suggesting that at least in part it occurred after alphaBC association with mitochondria. Consistent with this was the finding that translocation of activated p38 to mitochondria was maximal after only 10 min of ischemia. The overexpression of alphaBC-AAE, which mimics alphaBC phosphorylated on serine 59, has been shown to stabilize mitochondrial membrane potential and to inhibit apoptosis. In the present study, infection of neonatal rat cardiac myocytes with adenovirus-encoded alphaBC-AAE decreased peroxide-induced mitochondrial cytochrome c release. These results suggest that during ischemia alphaBC translocates to mitochondria, where it is phosphorylated and contributes to modulating mitochondrial damage upon reperfusion.

Topics: alpha-Crystallin B Chain; Animals; Animals, Newborn; Cells, Cultured; Cytochromes c; Hydrogen Peroxide; Kinetics; Mice; Mice, Inbred C57BL; Mitochondria, Heart; Myocardial Infarction; Myocardial Reperfusion; Myocardial Reperfusion Injury; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Transport; Rats; Transduction, Genetic

Label-free imaging is desirable for elucidating morphological and biochemical changes of heart tissue in vivo. Spontaneous Raman microscopy (SRM) provides high chemical contrast without labeling, but presents disadvantage in acquiring images due to low sensitivity and consequent long imaging time. Here, we report a novel technique for label-free imaging of rat heart tissues with high-speed SRM combined with resonance Raman effect of heme proteins. We found that individual cardiomyocytes were identified with resonance Raman signal arising mainly from reduced b- and c-type cytochromes, and that cardiomyocytes and blood vessels were imaged by distinguishing cytochromes from oxy- and deoxy-hemoglobin in intact hearts, while cardiomyocytes and fibrotic tissue were imaged by distinguishing cytochromes from collagen type-I in infarct hearts with principal component analysis. These results suggest the potential of SRM as a label-free high-contrast imaging technique, providing a new approach for studying biochemical changes, based on the molecular composition, in the heart.

Topics: Animals; Coronary Vessels; Cytochromes b; Cytochromes c; Cytoplasm; Female; Fibrosis; Image Interpretation, Computer-Assisted; Microscopy, Confocal; Myocardial Infarction; Myocytes, Cardiac; Rats; Rats, Wistar; Spectrum Analysis, Raman

Melatonin, a well-known antioxidant, has been shown to protect against ischemia-reperfusion myocardial damage. Mitochondrial permeability transition pore (MPTP) opening is an important event in cardiomyocyte cell death occurring during ischemia-reperfusion and therefore a possible target for cardioprotection. In the present study, we tested the hypothesis that melatonin could protect heart against ischemia-reperfusion injury by inhibiting MPTP opening. Isolated perfused rat hearts were subjected to global ischemia and reperfusion in the presence or absence of melatonin in a Langerdoff apparatus. Melatonin treatment significantly improves the functional recovery of Langerdoff hearts on reperfusion, reduces the infarct size, and decreases necrotic damage as shown by the reduced release of lactate dehydrogenase. Mitochondria isolated from melatonin-treated hearts are less sensitive than mitochondria from reperfused hearts to MPTP opening as demonstrated by their higher resistance to Ca(2+). Similar results were obtained following treatment of ischemic-reperfused rat heart with cyclosporine A, a known inhibitor of MPTP opening. In addition, melatonin prevents mitochondrial NAD(+) release and mitochondrial cytochrome c release and, as previously shown, cardiolipin oxidation associated with ischemia-reperfusion. Together, these results demonstrate that melatonin protects heart from reperfusion injury by inhibiting MPTP opening, probably via prevention of cardiolipin peroxidation.

Topics: Animals; Antioxidants; Calcium; Cardiolipins; Cardiovascular Agents; Cyclosporine; Cytochromes c; Heart Rate; In Vitro Techniques; L-Lactate Dehydrogenase; Lipid Peroxidation; Male; Melatonin; Membrane Potential, Mitochondrial; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; NAD; Necrosis; Perfusion; Rats; Rats, Wistar; Recovery of Function; Time Factors; Ventricular Function, Left; Ventricular Pressure

Ischemic preconditioning (IPC) and anesthetic-induced preconditioning against myocardial infarction are mediated via protein kinase B. Pim-1 kinase acts downstream of protein kinase B and was recently shown to regulate cardiomyocyte survival. The authors tested the hypothesis that IPC and anesthetic-induced preconditioning are mediated by Pim-1 kinase.. Pentobarbital-anesthetized male C57Black/6 mice were subjected to 45 min of coronary artery occlusion and 3 h of reperfusion. Animals received no intervention, Pim-1 kinase inhibitor II (10 microg/g intraperitoneally), its vehicle dimethyl sulfoxide (10 microl/g intraperitoneally), or 1.0 minimum alveolar concentration desflurane alone or in combination with Pim-1 kinase inhibitor II (10 microg/g intraperitoneally). IPC was induced by three cycles of 5 min ischemia-reperfusion each, and animals received IPC either alone or in combination with Pim-1 kinase inhibitor II (10 microg/g intraperitoneally). Infarct size was determined with triphenyltetrazolium chloride, and area at risk was determined with Evans blue (Sigma-Aldrich, Taufkirchen, Germany). Protein expression of Pim-1 kinase, Bad, phospho-Bad, and cytosolic content of cytochrome c were measured using Western immunoblotting.. Infarct size in the control group was 47 + or - 2%. Pim-1 kinase inhibitor II (44 + or - 2%) had no effect on infarct size. Desflurane (17 + or - 3%) and IPC (19 + or - 2%) significantly reduced infarct size compared with control (both P < 0.05 vs. control). Blockade of Pim-1 kinase completely abrogated desflurane-induced preconditioning (43 + or - 3%), whereas IPC (35 + or - 3%) was blocked partially. Desflurane tended to reduce cytosolic content of cytochrome c, which was abrogated by Pim-1 kinase inhibitor II.. These data suggest that Pim-1 kinase mediates at least in part desflurane-induced preconditioning and IPC against myocardial infarction in mice.

Topics: Anesthetics, Inhalation; Animals; bcl-Associated Death Protein; Blood Pressure; Blotting, Western; Cytochromes c; Data Interpretation, Statistical; Desflurane; Enzyme Activation; Heart Rate; Ischemic Preconditioning, Myocardial; Isoflurane; Male; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocardium; Proto-Oncogene Proteins c-pim-1; Reperfusion Injury; Signal Transduction

The activation of extracellular signal-regulated kinase (ERK)1/2 protects against ischemic-reperfusion injury. Whether ERK1/2 mediates the cardioprotection of sevoflurane postconditioning is unknown. We tested whether sevoflurane postconditioning produces cardioprotection via an ERK1/2-dependent mechanism.. In protocol 1, Langendorff-perfused Sprague-Dawley rat hearts (n=84, 12 per group), with the exception of the Sham group, were subjected to 30 min ischemia followed by 90 min reperfusion and were assigned to the untreated (control) group, followed by 4 cycles of ischemic postconditioning (25 s of each), 3% (v/v) sevoflurane postconditioning (for 5 min and 10 min of washout), and the PD98059 solvent DMSO (<0.2%), ERK1/2 inhibitor PD98059 (20 micromol/L), and Sevo+PD administration. Left ventricular hemodynamics and coronary flow at 30 min of equilibrium were recorded at 30, 60, and 90 min of reperfusion, respectively. Acute infarct size was measured by triphenyltetrazolium chloride staining. The configuration of mitochondria was observed by an electron microscope. Western blot analysis was used to determine the contents of cytosolic and mitochondrial cytochrome c at the end of reperfusion. In protocol 2, after 15 min of reperfusion, the expression of total and phosphorylated forms of ERK1/2 and its downstream target p70S6K was determined by Western blotting.. No differences in baseline hemodynamics were observed among the experimental groups (P>0.05). After reperfusion, compared with the control group, sevoflurane postconditioning and ischemic postconditioning significantly(P<0.05) improved functional recovery and largely (P<0.05) decreased myocardial infarct size (22.9%+/-4.6% and 21.2%+/-3.8%, vs 39.4%+/- 5.7%, both P<0.05). Sevoflurane-mediated protection was abolished by PD98059.. Anesthetic postconditioning by sevoflurane effectively protects against reperfusion damage by activating ERK1/2 in vitro.

Topics: Anesthetics, Inhalation; Animals; Cardiotonic Agents; Cytochromes c; Cytosol; Enzyme Activation; Heart Function Tests; Hemodynamics; In Vitro Techniques; Male; Methyl Ethers; Mitochondria, Heart; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocardial Infarction; Myocardium; Rats; Rats, Sprague-Dawley; Sevoflurane

Considerable evidence indicates that apoptosis plays a critical role in acute myocardial infarction. We have previously shown that Guan-Xin-Er-Hao (GXEH), a Chinese medicine formula, attenuates postischemia myocardial apoptosis. The present study was designed to determine the mechanisms by which GXEH exerts its antiapoptotic effect. Adult male Sprague-Dawley rats were randomized to receive vehicle or GXEH (5 or 15 g/kg) orally 30 min before ischemia and subjected to myocardial ischemia of 3 h (apoptosis peak) or 24 h (necrosis peak) for determination of infarct size. Compared with rats receiving vehicle, those rats treated with GXEH (15 g/kg) showed significantly reduced infarct size, the reduced myocardial apoptosis, as judged by the decreases in TUNEL-positive staining (22.40 +/- 5.68% vs. 40.31 +/- 10.58%, p < 0.01), and the decrease in the degree of caspase-3 activation (82.97 +/- 10.54 vs. 159.95 +/- 9.16 mumol cleaved acetyl-Asp-Glu-Val-Asp-p-nitroanilide/mg protein, p < 0.01). Treatment with GXEH (15 g/kg) significantly reduced the release of mitochondrial cytochrome c, a primary mediator of apoptosis, the degree of caspase-9 activation, and the Bax/Bcl-2 ratio. Caspase-9 cleaves and activates caspase-3. Bax promotes apoptosis, while Bcl-2 inhibits apoptosis. Thus, the antiapoptotic mechanisms of GXEH may involve the mitochondrial cytochrome c-mediated caspase-3 activation in cardiomyocytes after acute myocardial infarction. Taken together, GXEH tilted the balance between Bax and Bcl-2 toward an antiapoptotic state, decreased mitochondrial cytochrome c release, reduced caspase-9 activation, and attenuated subsequent caspase-3 activation and postischemic myocardial apoptosis in rats. GXEH may be used as a promising agent for future treatment of cardiovascular diseases.

Topics: Animals; Apoptosis; Caspase 3; Caspase 9; Cell Death; Cytochromes c; Drug Evaluation, Preclinical; Drugs, Chinese Herbal; Enzyme Activation; Male; Medicine, Chinese Traditional; Mitochondria, Heart; Myocardial Infarction; Myocardial Ischemia; Random Allocation; Rats; Rats, Sprague-Dawley

Ischemic preconditioning (IPC) strongly protects against ischemia-reperfusion injury; however, its effect on subsequent myocardial oxygenation is unknown. Therefore, we determine in an in vivo mouse model of regional ischemia and reperfusion (I/R) if IPC attenuates postischemic myocardial hyperoxygenation and decreases formation of reactive oxygen/nitrogen species (ROS/RNS), with preservation of mitochondrial function. The following five groups of mice were studied: sham, control (I/R), ischemic preconditioning (IPC + I/R, 3 cycles of 5 min coronary occlusion/5 min reperfusion) and IPC + I/R N(G)-nitro-L-arginine methyl ester treated, and IPC + I/R eNOS knockout mice. I/R and IPC + I/R mice were subjected to 30 min regional ischemia followed by 60 min reperfusion. Myocardial Po(2) and redox state were monitored by electron paramagnetic resonance spectroscopy. In the IPC + I/R, but not the I/R group, regional blood flow was increased after reperfusion. Po(2) upon reperfusion increased significantly above preischemic values in I/R but not in IPC + I/R mice. Tissue redox state was measured from the reduction rate of a spin probe, and this rate was 60% higher in IPC than in non-IPC hearts. Activities of NADH dehydrogenase (NADH-DH) and cytochrome c oxidase (CcO) were reduced in I/R mice after 60 min reperfusion but conserved in IPC + I/R mice compared with sham. There were no differences in NADH-DH and CcO expression in I/R and IPC + I/R groups compared with sham. After 60 min reperfusion, strong nitrotyrosine formation was observed in I/R mice, but only weak staining was observed in IPC + I/R mice. Thus IPC markedly attenuates postischemic myocardial hyperoxygenation with less ROS/RNS generation and preservation of mitochondrial O(2) metabolism because of conserved NADH-DH and CcO activities.

Topics: Animals; Coronary Vessels; Cytochromes c; Ischemic Preconditioning, Myocardial; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria, Heart; Myocardial Infarction; Myocardium; NADH Dehydrogenase; Nitric Oxide Synthase Type III; Oxidation-Reduction; Oxidative Stress; Oxygen Consumption; Random Allocation; Reactive Oxygen Species; Regional Blood Flow; Tyrosine

Despite major advances in treating patients with coronary heart disease, reperfusion injury is still considered to be a major problem, especially in surgical settings. Here, we demonstrate the protective effects of a novel bisindolylmaleimide derivative, MS1 (2-[1-(3-aminopropyl)indol-3-yl]-3-(indol-3-yl)-N-methylmaleimide), against reperfusion injury of the heart. After anesthesia and artificial ventilation, Wistar rats were subjected to 30 min of left coronary artery occlusion followed by 120 min of reperfusion with or without treating the rats with MS1 (2.25 mumol.L-1.kg-1) before left coronary artery occlusion. Compared with the untreated hearts, MS1 treatment significantly reduced myocardial infarct size (35.1% +/- 3% vs. 75.5% +/- 5%, p < 0.001), reduced prevalence of apoptotic cells (2.6% +/- 0.5% vs. 12.2% +/- 2.1%, p < 0.001), prevented mitochondrial swelling and cytochrome c release, inhibited downregulation of antiapoptotic protein Bcl-2 expression, and suppressed caspase-3 activation. In contrast, pretreatment with atractyloside, a mitochondrial permeability transition pore opener, abolished the protective effects of MS1. In conclusion, MS1 inhibits pathologic opening of permeability transition pores and protects the heart against reperfusion injury and pathologic apoptosis.

Topics: Acute Disease; Animals; Apoptosis; Cardiotonic Agents; Caspase 3; Cytochromes c; Disease Models, Animal; Indoles; Infusions, Intravenous; Male; Maleimides; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Infarction; Myocardial Reperfusion Injury; Rats; Rats, Wistar

The role of mitochondrial permeability transition pore (MPTP) in the mechanism of intermittent high altitude (IHA) hypoxic adaptation is not understood. Therefore, we study whether the protective effect of IHA hypoxia against ischemia-reperfusion injury is accompanied by inhibition of MPTP opening. IHA hypoxia significantly improved the functional recovery of Langendorff hearts on reperfusion and limited infarct size. In isolated myocytes, IHA hypoxia significantly improved the recovery of cell length, lowered ischemia-reperfusion-induced [Ca2+]c and [Ca2+]m overloading. Furthermore, IHA hypoxia accelerated [Ca2+]c decline during reperfusion. Opening the MPTP with atractyloside immediately at reperfusion abolished these cardioprotective effects of IHA hypoxia, but had no appreciable influence on those of normoxic hearts. IHA hypoxia prolongs the time taken to induce MPTP opening and the time taken to induce rigor contracture when myocytes subjected to oxidative stress. The data from isolated mitochondria demonstrated that IHA hypoxia prevented the decrease of ADP/O ratio, the opening of MPTP and the release of cytochrome c from mitochondria at high Ca2+ concentrations (100 and 200 microM). Inhibition of MPTP opening in the first few minutes of reperfusion accelerated [Ca2+]c decline and attenuated [Ca2+]c and [Ca2+]m overloading, which contributed to the cardioprotection of IHA hypoxic adaptation. Enhancement of the tolerance of mitochondria against Ca2+ might underlie the protective mechanism of IHA hypoxia.

Topics: Adaptation, Physiological; Altitude; Altitude Sickness; Animals; Calcium; Cell Size; Cytochromes c; Intracellular Membranes; Male; Mitochondria, Heart; Myocardial Contraction; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Permeability; Rats; Rats, Sprague-Dawley; Ventricular Function

Pharmacological modulation of heme oxygenase (HO) gene expression may have significant therapeutic potential in oxidant-induced disorders, such as ischemia reperfusion (I/R) injury. Higenamine is known to reduce ischemic damages by unknown mechanism(s). The protective effect of higenamine on myocardial I/R-induced injury was investigated. Ligation of rat left anterior descending coronary artery for 30 min under anesthesia was done and followed by 24 h reperfusion before sacrifice. I/R-induced myocardial damages were associated with mitochondria-dependent apoptosis as evidenced by the increase of cytochrome c release and caspase-3 activity. Administration of higenamine (bolus, i.p) 1 h prior to I/R-injury significantly decreased the release of cytochrome c, caspase-3 activity, and Bax expression but up-regulated the expression of Bcl-2, HO-1, and HO enzyme activity in the left ventricles, which were inhibited by ZnPP IX, an enzyme inhibitor of HO-1. In addition, DNA-strand break-, immunohistochemical-analysis, and TUNEL staining also supported the anti-apoptotic effect of higenamine in I/R-injury. Most importantly, administration of ZnPP IX inhibited the beneficial effect of higenamine. Taken together, it is concluded that HO-1 plays a core role for the protective action of higenamine in I/R-induced myocardial injury.

Topics: Alkaloids; Animals; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cardiotonic Agents; Caspase 3; Caspases; Cytochromes c; Cytochromes c2; Enzyme Inhibitors; Glutathione; Heme Oxygenase (Decyclizing); In Situ Nick-End Labeling; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Oxidation-Reduction; Peroxynitrous Acid; Proto-Oncogene Proteins c-bcl-2; Protoporphyrins; Rats; Rats, Sprague-Dawley; Tetrahydroisoquinolines

In ischemia-reperfusion injuries, elevated calcium and reactive oxygen species (ROS) induce mitochondrial permeability transition (mPT), which plays a pivotal role in mediating damages and cell death. Inhibition of mPT decreases necrotic cell death; however, during reperfusion, the continuous production of ROS may contribute to the temporary opening of the pore and thus the onset of the delayed apoptotic cell death. Based on amiodarone structure, we developed the first SOD-mimetic mPT inhibitor (HO-3538) that can eliminate ROS in the microenvironment of the permeability pore. In isolated mitochondria, HO-3538 inhibited mPT and the release of proapoptotic mitochondrial proteins. It had a ROS scavenging effect and antiapoptotic effect in a cardiomyocyte line and it diminished release of mitochondrial proapoptotic proteins. Furthermore, HO-3538 significantly enhanced the recovery of mitochondrial energy metabolism and functional cardiac parameters; decreased infarct size, lipid peroxidation, and protein oxidation; and suppressed necrotic as well as apoptotic cell death pathways in Langendorff-perfused hearts. In these respects it was somewhat superior to its two constituents, amiodarone and a pyrrol-derivative free radical scavenger. These data suggest that the SOD-mimetic mPT inhibitors are ideal candidates for drug development for the alleviation of postinfarct myocardial injuries.

Topics: Amiodarone; Animals; Apoptosis; Cytochromes c; Humans; Ischemia; Jurkat Cells; Magnetic Resonance Spectroscopy; Mice; Mitochondria; Myocardial Infarction; Necrosis; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase

Cardiac mitochondria sustain damage during ischemia and reperfusion, contributing to cell death. The reversible blockade of electron transport during ischemia with amobarbital, an inhibitor at the rotenone site of complex I, protects mitochondria against ischemic damage. Amobarbital treatment immediately before ischemia was used to test the hypothesis that damage to mitochondrial respiration occurs mainly during ischemia and that protection of mitochondria during ischemia leads to decreased cardiac injury with reperfusion. Langendorff-perfused Fischer-344 rat hearts were treated with amobarbital (2.5 mM) or vehicle for 1 min immediately before 25 min of global ischemia. Both groups were reperfused for 30 min without additional treatment. Subsarcolemmal (SSM) and interfibrillar (IFM) populations of mitochondria were isolated after reperfusion. Ischemia and reperfusion decreased state 3 and increased state 4 respiration rate in both SSM and IFM. Amobarbital treatment protected oxidative phosphorylation measured following reperfusion and improved the coupling of respiration. Cytochrome c content measured in SSM and IFM following reperfusion decreased in untreated, but not in amobarbital-treated, hearts. H(2)O(2) release from SSM and IFM isolated from amobarbital-treated hearts during reperfusion was markedly decreased. Amobarbital treatment before ischemia improved recovery of contractile function (percentage of preischemic developed pressure: untreated 51 +/- 4%, n = 12; amobarbital 70 +/- 4%, n = 11, p < 0.01) and substantially reduced infarct size (untreated 32 +/- 2%, n = 7; amobarbital 13 +/- 2%, n = 7, p < 0.01). Thus, mitochondrial damage occurs mainly during ischemia rather than during reperfusion. Reperfusion in the setting of preserved mitochondrial respiratory function attenuates the mitochondrial release of reactive oxygen species, enhances contractile recovery, and decreases myocardial infarct size.

Topics: Amobarbital; Animals; Citrate (si)-Synthase; Cytochromes; Cytochromes c; Electron Transport; Hydrogen Peroxide; In Vitro Techniques; Male; Mitochondria, Heart; Myocardial Contraction; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Oxidative Phosphorylation; Oxygen Consumption; Rats; Rats, Inbred F344; Sarcolemma

The Fas/Fas ligand and mitochondria pathways have been involved in cell death in several cell types. We combined the genetic inactivation of the Fas receptor (lpr mice), on the one hand, to the pharmacological inhibition of the mitochondrial permeability transition pore (mPTP), on the other hand, to investigate which of these pathways is predominantly activated during prolonged ischemia-reperfusion. Anesthetized C57BL/6JICO (control) and C57BL/6-lpr mice were pretreated with either saline or cyclosporin A (CsA; 40 mg/kg, 3 times a day), an inhibitor of the mPTP, and underwent 25 min of ischemia and 24 h of reperfusion. After 24 h of reperfusion, hearts were harvested: infarct size was assessed by 2,3,5-triphenyltetrazolium chloride staining, myocardial apoptosis by caspase 3 activity, and mitochondrial permeability transition by Ca2+-induced mPTP opening using a potentiometric approach. Infarct size was comparable in untreated control and lpr mice, ranging from 77 +/- 5% to 83 +/- 3% of the area at risk. CsA significantly reduced infarct size in control and lpr hearts. Control and lpr hearts exhibited comparable increase in caspase 3 activity that averaged 57 +/- 18 and 49 +/- 5 pmol x min(-1) x mg(-1), respectively. CsA treatment significantly reduced caspase 3 activity in control and lpr hearts. The Ca2+ overload required to open the mPTP was decreased to a similar extent in lpr and controls. CsA significantly attenuated Ca2+-induced mPTP opening in both groups. Our results suggest that the Fas pathway likely plays a minor role, whereas mitochondria are preferentially involved in mice cardiomyocyte death after a lethal ischemia-reperfusion injury.

Topics: Animals; Apoptosis; Blotting, Western; Calcium; Caspase 3; Caspases; Cell Death; Cyclosporine; Cytochromes c; Enzyme Activation; fas Receptor; In Situ Nick-End Labeling; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria, Heart; Myocardial Infarction; Myocardial Reperfusion Injury; Myocytes, Cardiac; Permeability

The opening of mitochondrial permeability transition pore (PTP) during reperfusion injury of heart has been well demonstrated and thus controlling PTP would attenuate the myocardial damage and cell death. Ursodeoxycholic acid (UDCA) is a hydrophilic bile salt and has been shown to prevent apoptosis in hepatocytes by inhibiting the opening of PTP. Here we demonstrate the role of UDCA in preventing the reperfusion injury of heart through its ability to inhibit PTP. Wistar rats underwent 30 min left coronary artery occlusion (LCA) followed by 180 min reperfusion after treatment with 40 mg/kg per iv infusion of UDCA over 30 min before LCA occlusion. Other groups of rats were treated with PTP agonist atractyloside(5 mg/kg) or PI3 kinase inhibitor wortmannin (16 ug/kg) before UDCA treatment. UDCA treatment prior to LCA occlusion, activated phosphorylation of Akt and Bad. Phosphorylating Bad prevented its translocation in to mitochondria, there by preventing the down regulation of Bcl-2 expression and PTP opening. This was confirmed by reduced cytochrome C release from intramitochondrial space in to the cytosol and hence reduced cell death either by apoptosis (4.8 vs 11.8%, P<0.001, UDCA treated against control group) or necrosis (reduced MI area in UDCA treated group (22.1%) compared to control group(46.4%), P<0.001). In contrast, inhibition of Akt activation with PI3K inhibitor wortmannin or opening the PTP with atractyloside abolished, UDCA mediated cytoprotective effects. Studies on primary culture cardiomyocytes also confirmed our in vivo results of UDCA on cell survival. These results altogether demonstrate that UDCA protect the heart against reperfusion injury by inhibiting the PTP in a PI3K/Akt dependent pathway.

Topics: Adenosine Triphosphate; Androstadienes; Animals; Apoptosis; Atractyloside; bcl-Associated Death Protein; Cell Hypoxia; Cell Survival; Cells, Cultured; Cytochromes c; Male; Mitochondrial Swelling; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; Oxygen; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Ursodeoxycholic Acid; Wortmannin

This study is aimed at investigating the novel use of minocycline for cardiac protection during ischemia/reperfusion (I/R) injury, as well as its mechanism of action.. Minocycline is a tetracycline with anti-inflammatory properties, which is used clinically for the treatment of diseases such as urethritis and rheumatoid arthritis. Experimentally, minocycline has also been shown to be neuroprotective in animal models of cerebral ischemia and to delay progression and improve survival in mouse models of neurodegenerative diseases.. We studied 62 rat intact hearts exposed to I/R and cell cultures of neonatal and adult rat ventricular myocytes.. Minocycline significantly reduced necrotic and apoptotic cell death, both in neonatal and adult myocytes, not only when given prior to hypoxia (p < 0.001), but also at reoxygenation (p < 0.05). Moreover, in the intact heart exposed to I/R, in vivo treatment with minocycline promoted hemodynamic recovery (p < 0.001) and cell survival, with reduction of infarct size (p < 0.001), cardiac release of creatine phosphokinase (p < 0.001), and apoptotic cell death (p < 0.001). In regard to its antiapoptotic mechanism of action, minocycline significantly reduced the expression level of initiator caspases, increased the ratio of XIAP to Smac/DIABLO at both the messenger RNA and protein level, and prevented mitochondrial release of cytochrome c and Smac/DIABLO (all, p < 0.05). These synergistic actions dramatically prevent the post-ischemic induction of caspase activity associated with cardiac I/R injury.. Because of its safety record and multiple novel mechanisms of action, minocycline may be a valuable cardioprotective agent to ameliorate cardiac dysfunction and cell loss associated with I/R injury.

Topics: Animals; Animals, Newborn; Anti-Bacterial Agents; Apoptosis; Apoptosis Regulatory Proteins; Carrier Proteins; Caspase Inhibitors; Cells, Cultured; Cytochromes c; Down-Regulation; Enzyme Inhibitors; Minocycline; Mitochondria, Heart; Mitochondrial Proteins; Myocardial Infarction; Myocardial Ischemia; Myocytes, Cardiac; Oxygen; Proteins; Rats; Rats, Sprague-Dawley; Reperfusion Injury; X-Linked Inhibitor of Apoptosis Protein

Myocardial ischemia/reperfusion (I/R) is associated with an extensive loss of myocardial cells. The apoptosis repressor with caspase recruitment domain (ARC) is a protein that is highly expressed in heart and skeletal muscle and has been demonstrated to protect the heart against I/R injury (Gustafsson, A. B., Sayen, M. R., Williams, S. D., Crow, M. T., and Gottlieb, R. A. (2002) Circulation 106, 735-739). In this study, we have shown that transduction of TAT-ARCL31F, a mutant of ARC in the caspase recruitment domain, did not reduce creatine kinase release and infarct size after I/R. TAT-ARCL31F also failed to protect against hydrogen peroxide-mediated cell death in H9c2 cells, suggesting that the caspase recruitment domain is important in mediating ARC's protective effects. In addition, we report that ARC co-immunoprecipitated with the pro-apoptotic protein Bax, which causes cytochrome c release when activated. TAT-ARC, but not TAT-ARCL31F, prevented Bax activation and cytochrome c release in hydrogen peroxide-treated H9c2 cells. TAT-ARC was also effective in blocking cytochrome c release after ischemia and reperfusion, whereas TAT-ARCL31F had no effect on cytochrome c release. In addition, recombinant ARC protein abrogated Bax-induced cytochrome c release from isolated mitochondria. This suggests that ARC can protect against cell death by interfering with activation of the mitochondrial death pathway through the interaction with Bax, preventing mitochondrial dysfunction and release of pro-apoptotic factors.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Caspase Inhibitors; Cell Death; Cell Line; Creatine Kinase; Cytochromes c; Muscle Proteins; Myocardial Infarction; Myocardium; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Recombinant Fusion Proteins

Reperfusion after a period of ischemia is associated with the formation of reactive oxygen species (ROS) and Ca2+ overload resulting in the opening of a nonspecific pore in the inner membrane of the mitochondria, called the mitochondrial permeability transition pore (PTP), leading to cell damage. Although endogenous antioxidants are activated because of oxidative stress following ischemia, their levels are not high enough to prevent reperfusion injury. Hence there is always a need for exogenous supplement of antioxidants, especially after acute ischemia. Here we demonstrated the effects of the antioxidant 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186) in preventing reperfusion injury of the heart by inhibition of PTP opening. Ischemia (30 min) by left coronary artery (LCA) occlusion and reperfusion (120 min) in Wistar rats after pretreatment with MCI-186 (10 mg/kg iv) infusion starting from 30 min before LCA occlusion resulted in 1) less area of myocardial infarction (19.2% vs. 61.6%), 2) well-maintained myocardial ATP content (P < 0.03 vs. control), 3) decreased mitochondrial swelling and reduced cytochrome c release, 4) increased expression of BCl-2, 5) lower prevalence of apoptotic cells (14.3% vs. 2.9%), and 6) reduced DNA fragmentation in the MCI-186-treated group. These cytoprotective effects of MCI-186 were inhibited on opening PTP before MCI-186 treatment with the PTP activators lonidamine (10 mg/kg iv) or atractyloside (5 mg/kg iv) but failed to inhibit the protective effects exerted by another antioxidant, allopurinol, suggesting that the PTP inhibiting property is specific for MCI-186. These results demonstrate that the radical scavenger MCI-186, by inhibiting the opening of the PTP, prevents necrosis and cytochrome c release and hence pathological apoptosis.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Antioxidants; Antipyrine; Atractyloside; Blood Pressure; Cytochromes c; DNA Fragmentation; Edaravone; Enzyme Inhibitors; Heart Rate; Indazoles; Male; Mitochondrial Swelling; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Up-Regulation

Topics: Animals; Aspartate Aminotransferases; Cardiovascular Diseases; Cytochromes; Cytochromes c; Dogs; Electron Transport Complex IV; Myocardial Infarction; Myocardium; Oxidoreductases; Transaminases