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Distinct Role of Autophagy in Myocardial Ischemia/Reperfusion Injury

Author: XuQiuLin
Tutor: LiaoYuLin; BinJianPing
School: Southern Medical University,
Course: Department of Cardiology
Keywords: autophagy apoptosis myocardial ischemia-reperfusion injury anoxia-reoxygenation mPTP
CLC: R363
Type: PhD thesis
Year: 2012
Downloads: 593
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BackgroundCoronary artery disease is the leading cause of death in cardiovascular diseases. Coronary stenosis and/or thrombosis may result in myocardial ischemia or even infarction. The persistence and the extent of myocardial ischemia or infarction are important factors closely related to the prognosis. Prompt restore of coronary perfusion is the principle therapeutic strategy to limit infarct size and rescue myocardium at risk. However, for a given duration of ischemia, infarct size can be limited by reducing reperfusion injury. Ischemia/reperfusion (I/R) injury involves a variety of molecular and cellular mechanisms, such as increased production of reactive oxygen species (ROS), calcium overload, inflammation, mitochondrial damage, apoptosis and autophagy. Autophagy is believed to play a critical role in myocardial I/R injury.Autophagy, a lysosome-mediated recycling of long-lived proteins and damaged cells, plays a key role in the degradation and turnover of mitochondria and other organelles. During I/R injury, both intracellular ATP reduction and increased AMPK activity can activate autophagy signaling pathway. Upregulated BH3domain protein, calcium concentration increase, reactive oxygen species, excessive nitric oxide, the openning of the mitochondrial permeability transition pore, endoplasmic reticulum stress and unfolded protein response are also reported to promote myocardial autophagy. The mammalian target of rapamycin (mTOR) signaling pathway, Ras/cAMP-dependent protein kinase A (PKA) pathway, insulin/growth factor signaling pathway and LKB1-AMPK signal pathway can regulate activity of autophagy. In eukaryotic cells, rapamycin enhances autophagy by inhibiting mTOR, while3-methyladenine or wortmannin attenuates autophagy by inhibiting the phosphatidylinositol3-kinase (PI3K).Mitochondrion is the most abundant organelle in cardiomyocytes and plays critical role in maintaining the cellular function. Myocardial I/R injury may induce mitochondrial dysfunction characterized by mitochondrial membrane potential depolarization and opening of mitochondrial permeability transition pore (mPTP). Mitochondrial dysfunction may lead to ATP reduction, generation of ROS and promotion of the apoptotic protein release, and consequently result in cell death. ROS in turn accelerates mitochondrial injury to form a vicious circle. Autophagy activated by mitochondrial membrane potential depolarization, opening of mPTP or ROS may serve to clear out the damaged mitochondria so as to save the cardiomyocytes at risk. However, excessive autophagy may be harmful if autophagic death occurs by excessive elimination of organelles.It is well known that anoxia/reoxygenation (A/R) and myocardial I/R can increase autophagy activity, but there are controversies on the relation between time course of A/R or I/R and activity of autophagy. There are reports showed that short duration of ischemia or anoxia exerted no significant influence on the activity of autophagy, while others reported that autophagy was decreased by ischemia and enhanced by reperfusion, moreover, there is evidence that enhanced autophagy is protective during ischemia while detrimental during reperfusion phase. It is generally believed that ischemia for more than30min or reperfusion for more than2h in rodents usually promoted myocardial autophagy. Many studies support that enhanced autophagy is protective while there are also substantial evidence that increased autophagy is detrimental to ischemic myocardium.The controversies on the role of autophagy in myocardial I/R injury may be partially attributable to the limitations of the experimental design and the different research approaches. Most of the reported studies usually focus on the role of autophagy under single condition of pathological stress rather than comparative investigations under different conditions. For example, the role of autophagy may be different between moderate and severe I/R injuries. We hypothesized that insufficient or excessive autophagy would be harmful for I/R injured myocardium. The conclusions from autophagy genes related transgenic and knockout mice should be carefully interpreted. Determinating the safety range of autophagy would provide clues to develop autophagy into a therapeutic target.ObjectiveTo explore the role and mechanism of autophagy in A/R or I/R injury in cultured cardiomyocytes or whole heart subjected to distinct severities of A/R and I/R.MethodsIn vitro studies:Neonatal rat cardiomyocytes were isolated and cultured. The following items were examined.1. Cell viability:cardiomyocytes were seeded in96well plates. Three groups were divided:control group, the rapamycin group and wortmannin group. Drugs were given30min before anoxia. Culture plates were placed in a hypoxic incubator for0h,6h,12h and24h, respectively, and then reoxygenation for4h. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay was used to measure cell viability.2. Cardiomyocyte apoptosis detection:Hoechst33258dye was used to stain DNA in the nucleolus and then observed under fluorescence microscopy.3. Autophagy detection:Activity of autophagy was measured by detection of fluorescence signal of monodansylcadaverine (MDC) staining with a laser confocal microscopy, or by detection of autophagosome formation with electronic microscopy, or by western blot analysis of autophagy related protein LC3-Ⅱ and Beclin1.4. Detection of mPTP opening of cardiomyocytes:Myocytes were co-incubated with calcein and cobalt chloride, and the fluorescence intensity was detected with flow cytometry or laser confocal microscopy.5. Detection of mitochondrial membrane potential depolarization:JC-1probe and cardiomyocytes were incubated for30min, then washed with PBS, mitochondrial JC-1aggregations were determined by a laser scanning confocal microscopy.In vivo studyC57BL/6J mice were used to generate myocardial I/R and infarct models. After15min or40min ischemia, reperfusion was carried out to persist for24h. A permanent ligation of left coronary artery for24h was also set as a group. Rapamycin and wortmannin was used to check the role of autophagy on myocardial injury. The following indexes were examined:1. Serum concentrations of lactate dehydrogenase (LDH) were measured with ELISA kits. 2. Myocardial infarct size was determined with TTC (2,3,5-triphenyltetrazolium chloride) staining.3. Autophagy was detected by western blot of protein LC3-Ⅱ and electronic microscopy.Statistical analysisFor all statistical tests, multiple comparisons were performed by one-way ANOVA with LSD multiple comparison exact probability test. Comparisons of enumeration data were performed with chi-square test (SPSS16.0). The results thus obtained were reported in terms of the mean±SE, and P-values of less than0.05were considered to be statistically significant.Results1. Autophagy activity was enhanced dependent of the anoxia durationThe autofluorescent drug MDC can combine with the acid material of autophagosome to display bright blue fluorescent and form massive convergence. With the prolongation of anoxia time, the incorporation of MDC (number and size of blue particles) in cardiomyocytes was significantly increased (P<0.05). Similarly, the number of autophagosome in cardiomyocytes observed by electronic microscope markedly increased dependent of anoxia duration (P<0.05). LC3-II and Bclinl protein expression levels detected by western blot were enhanced with the prolongation of anoxia.2. Influence of autophagy manipulation on cell viability in response to A/R In6h anoxia A/R group, rapamycin. an autophagy enhancer, significantly improved the cell viability, while autophagy inhibitor wortmannin reduced the cell survival rate. In contrast, in24h anoxia A/R group, rapamycin reduced and wortmannin increased cell viability. These results suggest that there is a safety range of autophagy for cardiomyocytes during A/R injury.3. Effects of autophagy manipulationon apoptosis andautophagic cell deathHoechst33258stained apoptotic nuclear condensation appeared bright blue. The results showed that the number of apoptotic cell increased along with the prolongation of anoxia. Rapamycin and wortmannin did not increase the apoptosis rate in non-anoxia group. In6h,24h anoxia A/R groups, rapamycin slightly but significantly reduced the apoptosis, while wortmannin increased the apoptosis. These findings suggest that the reduced cell viability by rapamycin under the conditions of severe A/R injury is due to the net effect of increased autophagic death.4. Effects of autophagy manipulation on mitochondrial functionCalcein and cobalt chloride were co-incubated with cardiomyocytes to detect the opening of mPTP. With the prolongation of anoxia time, the fluorescence intensity of mitochondrial detected by flow cytometry decreased, indicating the opening of mPTP. The intensity of mitochondrial green fluorescence (it would be quenched in the cytoplasm) detected by laser confocal microscopy also reflects the openness of mPTP, similar results to flow cytometry were noted. The fluorescent probe JC-1can be used as an indicator of mitochondrial membrane potential to represent mitochondrial permeability. We noted that the red/green fluorescence ratio decreased significantly along with duration of anoxia, indicating increased potential depolarization in mitochondrial membrane.Neither rapamycin nor inhibitor wortmannin changes the openness of mPTP in normoxia. In6h anoxia/4h reoxygenation group, rapamycin reduced the mPTP opening, whereas wortmannin increased it. In24h anoxia/4h reoxygenation group, both of rapamycin and wortmannin did decrease fluorescent intensity significantly.5. The influence of autophagy intervention on myocardial I/R injuryMyocardial infarct size (IS) and serum concentrations of LDH were ischemic severity-dependent increased from ischemia15min/reperfusion24h to ischemia40min/reperfusion24h to permanent ischemia for24h. In ischemia40min I/R group, rapamycin treatment decreased LDH release and reduced IS significantly, while wortmannin exerted opposite effects. In ischemia24h group, rapamycin treatment increased LDH release and IS significantly, while wortmannin reduced serum LDH concentrations and reduced IS. Autophagic activity evaluated with electronic microscopy and western blot was increased dependent of ischemia duration. Rapamycin increased myocardial autophagy, while wortmannin decreased it.Conclusions1. Activity of autophagy in cardiomyocytes increases dependent on the duration of anoxia or ischemia, indicating an association between autophagic activity and injury severity.2. When the autophagic level is low, autophagy enhancer treatment is cardioprotective, potentially mediated by eliminating cellular garbage and improving mitochondrial dysfunction and consequently inhibiting apoptosis.3. Excessive autophagic activity induced by severe A/R or I/R injury destroys a large fraction of mitochondria. In such situation, autophagy enhance is detrimental to cardiomyocytes, while inhibition of autophagy is beneficial for increasing cardiomyocyte survival and limiting myocardial infarct size.

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