Dataset Information

Astragaloside IV inhibits oxidative stress-induced mitochondrial permeability transition pore opening by inactivating GSK-3? via nitric oxide in H9c2 cardiac cells.

ABSTRACT:

Objective

This study aimed to investigate whether astragaloside IV modulates the mitochondrial permeability transition pore (mPTP) opening through glycogen synthase kinase 3? (GSK-3?) in H9c2 cells.

Methods

H9c2 cells were exposed to astragaloside IV for 20 min. GSK-3? (Ser(9)), Akt (Ser(473)), and VASP (Ser(239)) activities were determined with western blot. The mPTP opening was evaluated by measuring mitochondrial membrane potential (??(m)). Nitric oxide (NO) generation was measured by 4-amino-5-methylamino-2', 7'-difluorofluorescein (DAF-FM) diacetate. Fluorescence images were obtained with confocal microscopy.

Results

Astragaloside IV significantly enhanced GSK-3? phosphorylation and prevented H(2)O(2)-induced loss of ??(m). These effects of astragaloside IV were reversed by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, the NO sensitive guanylyl cyclase selective inhibitor ODQ, and the PKG inhibitor KT5823. Astragaloside IV activated Akt and PKG. Astragaloside IV was also shown to increase NO production, an effect that was reversed by L-NAME and LY294002. Astragaloside IV applied at reperfusion reduced cell death caused by simulated ischemia/reperfusion, indicating that astragaloside IV can prevent reperfusion injury.

Conclusions

These data suggest that astragaloside IV prevents the mPTP opening and reperfusion injury by inactivating GSK-3? through the NO/cGMP/PKG signaling pathway. NOS is responsible for NO generation and is activated by the PI3K/Akt pathway.

SUBMITTER: He Y

PROVIDER: S-EPMC3463196 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Publications

Astragaloside IV inhibits oxidative stress-induced mitochondrial permeability transition pore opening by inactivating GSK-3β via nitric oxide in H9c2 cardiac cells.

He Yonggui Y Xi Jinkun J Zheng Huan H Zhang Yidong Y Jin Yuanzhe Y Xu Zhelong Z

Oxidative medicine and cellular longevity 20120925

<h4>Objective</h4>This study aimed to investigate whether astragaloside IV modulates the mitochondrial permeability transition pore (mPTP) opening through glycogen synthase kinase 3β (GSK-3β) in H9c2 cells.<h4>Methods</h4>H9c2 cells were exposed to astragaloside IV for 20 min. GSK-3β (Ser(9)), Akt (Ser(473)), and VASP (Ser(239)) activities were determined with western blot. The mPTP opening was evaluated by measuring mitochondrial membrane potential (ΔΨ(m)). Nitric oxide (NO) generation was meas ...[more]

PMID: 23050041

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Project description:A gentle optical examination of the mitochondrial permeability transition pore (mPTP) opening events was carried out in isolated quiescent ventricular myocytes by tracking the inner membrane potential (ΔΨM) using TMRM (tetramethylrhodamine methyl ester). Zeiss Airyscan 880 ″super-resolution" or "high-resolution" imaging was done with very low levels of illumination (0.009% laser power). In cellular areas imaged every 9 s (ROI or regions of interest), transient depolarizations of variable amplitudes occurred at increasing rates for the first 30 min. The time to first depolarization events was 8.4 min (±1.1 SEM n = 21 cells). At longer times, essentially permanent and irreversible depolarizations occurred at an increasing fraction of all events. In other cellular areas surrounding the ROI, mitochondria were rarely illuminated (once per 5 min) and virtually no permanent depolarization events occurred for over 1 h of imaging. These findings suggest that photon stress due to the imaging itself plays an important role in the generation of both the transient mPTP opening events as well as the permanent mPTP opening events. Consistent with the evidence that photon "stress" in mitochondria loaded with virtually any photon absorbing substance, generates reactive oxygen species (ROS) [1-5], we show that cyclosporine-A (CsA, 10 μM) and the antioxidant n-acetyl cysteine (NAC, 10 mM), reduced the number of events by 80% and 93% respectively. Furthermore, CsA and NAC treatment led to the virtual disappearance of permanent depolarization events. Nevertheless, all transient depolarization events in any condition (control, CsA and NAC) appeared to repolarize with a similar half-time of 30 ± 6 s (n = 478) at 37 °C. Further experiments showed quantitatively similar results in cerebral vascular smooth muscle cells, using a different confocal system, and different photon absorbing reagent (TMRE; tetramethylrhodamine ethyl ester). In these experiments, using modest power (1% laser power) transient depolarization events were seen in only 8 out of 23 cells while with higher power (8%), all cells showed transient events, which align with the level of photon stress being the driver of the effect. Together, our findings suggest that photon-induced ROS is sufficient to cause depolarization events of individual mitochondria in quiescent cells; without electrical or mechanical activity to stimulates mitochondrial metabolism, and without raising the mitochondrial matrix Ca2+. In a broad context, these findings neither support nor deny the relevance or occurrence of ΔΨM depolarization events in specific putatively physiologic mitochondrial behaviors such as MitoFlashes [6,7] or MitoWinks [8]. Instead, our findings raise a caution with regards to the physiological and pathophysiological functions attributed to singular ΔΨM depolarization events when those functions are investigated using photon absorbing substances. Nevertheless, using photon stress as a tool ("Optical Stress-Probe"), we can extract information on the activation, reversibility, permanency and kinetics of mitochondrial depolarization. These data may provide new information on mPTP, help identify the mPTP protein complex, and establish the physiological function of the mPTP protein complex and their links to MitoFlashes and MitoWinks.