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Updated: Jun 6, 2025
A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation
Published on: July 13, 2018
Targeted Delivery and ROS-Responsive Release of Lutein Nanoassemblies Inhibit Myocardial Ischemia-Reperfusion Injury
Pilong Shi1, Yuetong Sha1, Xinran Wang1
1Department of Pharmacology, Harbin Medical University, Heilongjiang, 163319, People's Republic of China.
Purpose:
Myocardial ischemia-reperfusion injury (MI/RI) is associated with increased oxidative damage and mitochondrial dysfunction, resulting in an elevated risk of mortality. MI/RI may be alleviated by protecting cardiomyocytes from oxidative stress. Lutein, which belongs to a class of carotenoids, has proven to be effective in cardiovascular disease treatment due to its remarkable antioxidant properties, but its application is limited due to its poor stability and low bioavailability in vivo.
Methods:
In this study, a delivery system was developed based on distearoyl phosphatidyl ethanolamine (DSPE)-thiol-ketone (TK)-PEG2K (polyethylene glycol 2000) (abbreviated as DTP) and PCM-SH (CWLSEAGPVVTVRALRGTGSW) to deliver lutein (abbreviated as lutein@DTPP) to damaged myocardium. First, lutein, lutein@DTP, or lutein@DTPP were injected through the tail vein once a day for 3 days and then MI/RI model rats were established by exposing rats to ischemia for 45 min and reperfusion for 6 h. We employed a range of experimental techniques including qRT-PCR, Western blotting, transmission electron microscopy, immunohistochemistry, immunofluorescence, flow cytometry, immunoprecipitation, molecular docking, and molecular dynamics simulations.
Results:
Lutein@DTPP exhibited good myocardial targeting and ROS-responsive release. Our data suggested that lutein@DTPP effectively suppresses ferroptosis in cardiomyocytes. Mechanistically, we observed an upregulation of mouse double minute-2 (MDM2) in the hearts of MI/RI models and cardiomyocytes exposed to hypoxia/reoxygenation (H/R) conditions. In addition, NADH-ubiquinone oxidoreductase 75 kDa Fe-S protein 1 (NDUFS1) translocation from the cytosol to the mitochondria was inhibited by MDM2 upregulation. Notably, no significant variation in the total NDUFS1 expression was observed in H/R-exposed cardiomyocytes following treatment with siMDM2. Further study indicated that lutein facilitates the translocation of NDUFS1 from the cytosol to mitochondria by directly binding and sequestering MDM2, thereby improving mitochondrial function and inhibiting ferroptosis.
Conclusion:
Lutein@DTPP promoted the mitochondrial translocation of NDUFS1 to restore mitochondrial function and inhibited the ferroptosis of cardiomyocytes by directly binding and sequestering MDM2.
Insights
This study developed lutein@DTPP to treat myocardial ischemia-reperfusion injury by improving mitochondrial function and inhibiting ferroptosis. The delivery system targets damaged heart tissue, enhancing lutein
Area of Science:
- Cardiovascular Research
- Mitochondrial Biology
- Nanomedicine
Background:
- Myocardial ischemia-reperfusion injury (MI/RI) increases mortality due to oxidative damage and mitochondrial dysfunction.
- Lutein, a carotenoid, has antioxidant properties beneficial for cardiovascular diseases but suffers from poor stability and bioavailability.
- Effective delivery systems are needed to enhance lutein's therapeutic potential in MI/RI.
Purpose of the Study:
- To develop a novel delivery system, lutein@DTPP, for targeted delivery of lutein to damaged myocardium.
- To investigate the protective effects of lutein@DTPP against MI/RI by suppressing ferroptosis in cardiomyocytes.
- To elucidate the underlying mechanism involving MDM2 and NDUFS1 in lutein-mediated cardioprotection.
Main Methods:
- A distearoyl phosphatidyl ethanolamine (DSPE)-thiol-ketone (TK)-PEG2K delivery system (DTP) was synthesized to encapsulate lutein (lutein@DTPP).
- MI/RI rat models were established, and rats were treated with lutein, lutein@DTP, or lutein@DTPP.
- Various techniques including qRT-PCR, Western blotting, electron microscopy, and molecular simulations were used to assess myocardial protection and mechanism.
Main Results:
- Lutein@DTPP demonstrated effective myocardial targeting and reactive oxygen species (ROS)-responsive release.
- Lutein@DTPP significantly suppressed ferroptosis in cardiomyocytes and protected against MI/RI.
- The mechanism involves lutein binding to MDM2, promoting NDUFS1 translocation to mitochondria, restoring mitochondrial function, and inhibiting ferroptosis.
Conclusions:
- Lutein@DTPP effectively delivers lutein to the myocardium, offering protection against MI/RI.
- Lutein@DTPP mitigates ferroptosis by restoring mitochondrial function through the MDM2-NDUFS1 pathway.
- This novel nanomedicine approach holds promise for treating cardiovascular diseases associated with oxidative stress and mitochondrial dysfunction.
