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Updated: May 11, 2026
A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation
Published on: July 13, 2018
[Impact of Pax-8 gene interference on mitochondrial function and cardiomyocyte apoptosis]
Xiao-chun Dai1, Xi Zhou, Xiao-yan Huang
1Department of Cardiology, Institute for Cardiovascular Biology and Gene, Wenzhou, China.
Objective:
To observe the effects of paired box gene 8 (Pax-8) silencing by RNA interference on mitochondrial function and cardiomyocytes apoptosis.
Methods:
The cultured H9C2 (2-1) myocytes were divided into 3 groups: short interference RNA targeting Pax-8 (Pax-8 siRNA) group, non-specific siRNA group as the negative control (NC siRNA), and blank control group (BC siRNA). Fluorescence spectrophotometry was used to detect the activity of caspase-3. RT-PCR was performed to detect mRNA expression of Bcl2 and Bax. The protein expression of Bcl2, Bax and cytoplasm of Cytochrome was examined by Western blot. Changes of ΔΨm were detected by flow cytometry.ΔΨm with JC-1 monomer/polymer ratio was calculated for measuring mitochondrial depolarization proportion.
Results:
Compared to NC siRNA and BC siRNA group (0.075 ± 0.021, 0.072 ± 0.019), the activity of caspase-3 in Pax-8 siRNA group (0.167 ± 0.012) was significantly increased (P < 0.05); Bcl2 mRNA and protein expression in Pax-8 siRNA group (0.61 ± 0.06, 0.94 ± 0.11) were significantly downregulated compared with NC siRNA group (0.90 ± 0.070, 1.39 ± 0.15) and BC siRNA group (0.94 ± 0.087, 1.49 ± 0.20) (P < 0.05); Bax mRNA and protein expression in Pax-8 siRNA group (1.05 ± 0.10, 1.25 ± 0.12) were markedly upregulated compared with NC siRNA group (0.72 ± 0.03, 0.99 ± 0.12) and BC siRNA group (0.64 ± 0.03, 0.92 ± 0.06), P < 0.05; cytosolic cytochrome expression in Pax-8 siRNA group (0.75 ± 0.14) was significantly upregulated compared with NC siRNA group (0.51 ± 0.06) and BC siRNA group (0.48 ± 0.07) (P < 0.05); JC-1 monomer/polymer ratio in Pax-8 siRNA group (0.163 ± 0.011) was significantly increased compared with NC siRNA group (0.092 ± 0.015) and BC siRNA group (0.072 ± 0.025) (P < 0.05) indicating mitochondrial membrane potential was significantly reduced in Pax-8 siRNA group. Above parameters were similar between NC siRNA group and BC siRNA group (P > 0.05).
Conclusion:
Inhibiting Pax-8 results in enhanced cardiomyocytes apoptosis through the mitochondrial pathway.
Insights
Silencing paired box gene 8 (Pax-8) using RNA interference significantly increases caspase-3 activity and promotes cardiomyocyte apoptosis. This occurs via the mitochondrial pathway, indicated by altered Bcl2/Bax expression and reduced mitochondrial membrane potential.
Area of Science:
- Molecular Biology
- Cell Biology
- Cardiology
Background:
- Paired box gene 8 (Pax-8) plays a role in cellular development and function.
- Understanding Pax-8's role in cardiomyocytes is crucial for cardiac health.
- Mitochondrial dysfunction and apoptosis are key events in heart disease.
Purpose of the Study:
- To investigate the impact of Pax-8 gene silencing on mitochondrial function.
- To determine the effect of Pax-8 inhibition on cardiomyocyte apoptosis.
- To elucidate the underlying molecular mechanisms involving mitochondrial pathways.
Main Methods:
- Utilized RNA interference (siRNA) to silence Pax-8 in cultured H9C2 (2-1) myocytes.
- Assessed caspase-3 activity using fluorescence spectrophotometry.
- Quantified mRNA and protein expression of apoptosis markers (Bcl2, Bax) via RT-PCR and Western blot.
- Evaluated mitochondrial membrane potential (ΔΨm) using JC-1 staining and flow cytometry.
Main Results:
- Pax-8 silencing significantly increased caspase-3 activity compared to control groups.
- Downregulation of Bcl2 and upregulation of Bax mRNA and protein were observed post-Pax-8 silencing.
- Increased cytosolic cytochrome c release and reduced mitochondrial membrane potential (ΔΨm) were detected.
- These changes indicate enhanced mitochondrial depolarization and apoptosis.
Conclusions:
- Inhibition of Pax-8 expression promotes cardiomyocyte apoptosis.
- The observed apoptosis is mediated through the mitochondrial pathway.
- Pax-8 plays a protective role in cardiomyocytes by maintaining mitochondrial integrity.
