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Oxidative stress developed during the reperfusion of ischemic myocardium induces apoptosis
1Cardiovascular Division, Department of Surgery, University of Connecticut School of Medicine, Farmington 06030-1110, USA.
Abstract:
Apoptosis or programmed cell death is a genetically controlled response for cells to commit suicide and, is associated with DNA fragmentation or laddering. The common inducers of apoptosis include oxygen free radicals/oxidative stress and Ca2+ which are also implicated in the pathogenesis of myocardial ischemic reperfusion injury. To examine whether ischemic reperfusion injury is mediated by apoptotic cell death, isolated perfused rat hearts were subjected to 15, 30 or 60 min of ischemia as well as 15 min of ischemia followed by 30, 60 or 120 min of reperfusion. At the end of each experiment, hearts were processed for the evaluation of apoptosis, DNA laddering. Apoptosis was studied by visualizing the apoptotic cardiomyocytes by direct fluorescence detection of digoxigenin-labeled genomic DNA using APOPTAG in situ apoptosis detection kit. DNA laddering was evaluated by subjecting the DNA obtained from the hearts to 1.8% agarose gel electrophoresis and photographed under UV illumination. The results of our study revealed apoptotic cells only in the 60 and 120 min reperfused hearts as demonstrated by the intense fluorescence of the immunostained digoxigenin-labeled genomic DNA when observed under fluorescence microscopy. None of the ischemic hearts showed any evidence of apoptosis. These results corroborated with the findings of DNA fragmentation which showed increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the intenucleosomal DNA length (about 180 bp). The presence of apoptotic cells and DNA fragmentation in the myocardium were abolished by preperfusing the hearts in the presence of ebselen, which also removed the oxidative stress developed in the heart. Taken together, these results clearly demonstrate that oxidative stress developed in the ischemic reperfused myocardium induces apoptosis.
Insights
Myocardial ischemic reperfusion injury induces apoptosis, or programmed cell death, mediated by oxidative stress. This process, characterized by DNA fragmentation, was prevented by ebselen treatment.
Area of Science:
- Cardiovascular Research
- Cellular Biology
- Pathophysiology
Background:
- Apoptosis, a genetically controlled cell death, involves DNA fragmentation and is linked to oxidative stress.
- Oxidative stress and Ca2+ are implicated in myocardial ischemic reperfusion injury.
- Investigating the role of apoptosis in this injury is crucial for understanding cardiac damage.
Purpose of the Study:
- To determine if apoptotic cell death mediates myocardial ischemic reperfusion injury.
- To analyze the temporal relationship between ischemia, reperfusion, and apoptosis in the heart.
- To evaluate the effect of oxidative stress on apoptosis during reperfusion.
Main Methods:
- Isolated perfused rat hearts subjected to varying durations of ischemia and reperfusion.
- Apoptosis detection using the APOPTAG in situ apoptosis detection kit for visualizing digoxigenin-labeled DNA.
- DNA fragmentation assessed via agarose gel electrophoresis and UV illumination.
Main Results:
- Apoptotic cells and DNA fragmentation were observed in hearts subjected to 60 and 120 minutes of reperfusion following ischemia.
- No evidence of apoptosis was found in hearts that underwent ischemia alone.
- Preperfusion with ebselen abolished apoptosis and DNA fragmentation, concurrently reducing oxidative stress.
Conclusions:
- Oxidative stress in ischemic reperfused myocardium is a key inducer of apoptosis.
- Apoptotic cell death plays a significant role in the pathogenesis of myocardial ischemic reperfusion injury.
- Ebselen demonstrates a protective effect by mitigating oxidative stress and subsequent apoptosis.