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A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation
Published on: July 13, 2018
Antioxidants ameliorates glucose/glucose oxidase-induced myocardial damage through mitochondrial and MAPK pathway
Santosh Kumar1, Prachi Agrawal1, Prachi Mendhey1
1Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh 495009 India.
Abstract:
Diabetes is characterized by high blood glucose concentration that leads to the generation of elevated levels of free radicals (oxidative stress) via auto-oxidation. Oxidative stress plays a key role in diabetes-associated progressive pathologies including myocardial complications. The aim of the present study is to investigate the protective effects of antioxidants in glucose/glucose oxidase (G/GO)-dependent oxidative stress-induced cardiac cell damage. We found that exposure of G (33mM)/GO (1.6 milliunits) to cardiac muscle H9c2 cells resulted in a significant increase in apoptosis as indicated by accumulation of membrane phospholipid phosphatidylserine, DNA damage, and intracellular esterase activity. Confocal microscopy and FACS analysis further showed that G/GO induced the production of reactive oxygen and reactive nitrogen species which led to the loss of mitochondrial membrane potential and release of cytochrome c in H9c2 cells. Treatment of H9c2 cells with antioxidants like N-Acetyl Cysteine, catalase or glutathione abolished the G/GO-induced free radicals, perturbed the mitochondrial membrane potential, and induced cytochrome c release. These antioxidants also inhibited G/GO-induced cell death, caspases, and cleavage of PARP. In addition, antioxidants restored G/GO-induced suppression of antiapoptotic proteins, Bcl-2, Bcl-xL, cFLIP, XIAP, and survivin. Furthermore, G/GO impacted the MAPK pathway via activation of Raf1, MEK1 and ERK1/2 in oxidative stress-dependent manner. Pharmacologic inhibition of Raf1 also abolished G/GO-induced apoptosis. Thus, our data suggest that antioxidants have a strong protective efficacy against G/GO-induced oxidative stress through inhibition of mitochondrial and MAPK-mediated pathways in cardiac cells.
Insights
Antioxidants protect cardiac cells from diabetes-induced oxidative stress by inhibiting mitochondrial damage and MAPK pathways. This study highlights their potential in preventing heart complications associated with high blood glucose.
Area of Science:
- Biochemistry
- Cardiovascular Biology
- Cell Biology
Background:
- Diabetes mellitus is characterized by hyperglycemia, leading to oxidative stress.
- Oxidative stress is a significant contributor to diabetes-associated cardiac complications.
- Glucose/glucose oxidase (G/GO) systems can mimic diabetes-induced oxidative stress in vitro.
Purpose of the Study:
- To investigate the protective effects of antioxidants against G/GO-induced oxidative stress in cardiac H9c2 cells.
- To elucidate the mechanisms underlying G/GO-induced cardiac cell damage.
- To evaluate the role of mitochondrial and MAPK pathways in this process.
Main Methods:
- Cardiac H9c2 cells were exposed to G/GO to induce oxidative stress.
- Apoptosis was assessed via phosphatidylserine accumulation, DNA damage, and esterase activity.
- Reactive oxygen/nitrogen species, mitochondrial membrane potential, and cytochrome c release were analyzed.
- The impact on MAPK pathway (Raf1, MEK1, ERK1/2) was investigated.
- Antioxidant treatments (N-Acetyl Cysteine, catalase, glutathione) were applied.
Main Results:
- G/GO exposure significantly increased apoptosis, reactive oxygen/nitrogen species, and mitochondrial dysfunction.
- Antioxidants effectively scavenged free radicals, preserved mitochondrial integrity, and inhibited apoptosis.
- Antioxidants restored levels of antiapoptotic proteins (Bcl-2, Bcl-xL, cFLIP, XIAP, survivin).
- G/GO activated the MAPK pathway (Raf1, MEK1, ERK1/2) in an oxidative stress-dependent manner.
- Inhibition of Raf1 abolished G/GO-induced apoptosis.
Conclusions:
- Antioxidants demonstrate significant protective effects against G/GO-induced oxidative stress in cardiac cells.
- These protective effects are mediated through the inhibition of mitochondrial dysfunction and MAPK signaling pathways.
- The findings suggest a therapeutic potential for antioxidants in managing diabetes-related cardiac pathologies.
