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. 2014;2014:345105.
doi: 10.1155/2014/345105. Epub 2014 Jan 12.

Antiaging Properties of a Grape-Derived Antioxidant Are Regulated by Mitochondrial Balance of Fusion and Fission Leading to Mitophagy Triggered by a Signaling Network of Sirt1-Sirt3-Foxo3-PINK1-PARKIN

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Free PMC article

Antiaging Properties of a Grape-Derived Antioxidant Are Regulated by Mitochondrial Balance of Fusion and Fission Leading to Mitophagy Triggered by a Signaling Network of Sirt1-Sirt3-Foxo3-PINK1-PARKIN

Somak Das et al. Oxid Med Cell Longev. .
Free PMC article

Abstract

It was proposed that resveratrol, a polyphenolic antioxidant and a calorie restriction mimetic could promote longevity but subsequent studies could not prove this. The original proposal was based on the fact that a grape-derived antioxidant could activate the antiaging gene Sirt1. Most studies agree that indeed grape activates Sirt1, but a question remains whether Sirt1 is the cause or consequence of resveratrol treatment. Subsequently, mitochondrial Sirt3 was found to be activated. The present study on ischemic reperfusion (I/R) in rat hearts demonstrates that Foxo3a is activated subsequent to Sirt3 activation, which then activates PINK1. PINK1 potentiates activation of PARKIN leading to the activation of mitochondrial fission and mitophagy. Confocal microscopy conclusively shows the coexistence of Sirt3 with Foxo3a and Foxo3a with PINK1 and PARKIN. Mitophagy was demonstrated both by confocal microscopy and transmission electron microscopy. Western blot analyses data are consistent with the results of confocal microscopy. It appears that the grape-derived antioxidant modifies the intracellular environment by changing the oxidizing milieu into a reducing milieu and upregulating intracellular glutathione, potentiates a signal transduction cascade consisting of Sirt1/Sirt3-Foxo3a-PINK1-PARKIN-mitochondrial fusion fission-mitophagy that leads to cardioprotection, and paves the way to an anti-aging environment.

Figures

Figure 1
Figure 1
(a) Intracellular quantification of ROS by DCFDA in the samples. (b) Intracellular concentration of glutathione (GSH).
Figure 2
Figure 2
Immunofluorescence multichromatic projections of mitochondrial Sirt3 and the nuclear factor Foxo3a in the ischaemic (IR) cardiac specimens after treatments with resveratrol or longevinex. Panels (a1–a4) are administration of vehicle only; panels (b1–b4) are administration of resveratrol; panels (c1–c4) are administration of longevinex, where (1) is counterstaining of nuclei with Hoechst 33342 (blue), (2) is the immunofluorescence images of Sirt3 (green), (3) is the immunofluorescence images of Foxo3a (red), and (4) is overlay of (1–3) images. Nuclear localization of Foxo3a is indicated with white arrows. The confocal images were taken with optical Z-step of 0.5 µm.
Figure 3
Figure 3
Immunofluorescence multichromatic projections of PARKIN1, the nuclear factor Foxo3a, and the mitochondrial marker TOM20 in the ischaemic (IR) cardiac specimens after treatments with resveratrol or longevinex. Panels (a1) and (a2) are administration of vehicle only; panels (b1) and (b2) are administration of resveratrol; panels (c1) and (c2) are administration of longevinex, where: (1) are projections of PARKIN1 (green), Foxo3a (red), and nuclei (blue); nuclear localization of Foxo3a is indicated with white arrows in panels (b1) and (c1); (2) are projections of PARKIN1 (green), TOM20 (red), and nuclei (blue); and mitochondrial fission is indicated with white arrows in panels (b2) and (c2). The confocal images were taken with optical Z-step of 0.5 µm.
Figure 4
Figure 4
Immunofluorescence multichromatic projections of mitochondrial Sirt3 and TOM20 along with LC3 protein, a marker of autophagosome formation, in the ischaemic (IR) cardiac specimens after treatments with resveratrol or longevinex. Panels (a) and (d) are administration of vehicle only; panels (b) and (e) are administration of resveratrol; panels (c) and (f) are administration of longevinex. Panel (g) is 5x magnification of the selected area indicated in panel (f) where mitochondrial fission and mitophagy are indicated with white arrows. Overlay of the immunofluorescence images of Sirt3 (green), TOM20 (red), and nuclei (blue) is shown in panels (a), (b), and (c); mitochondrial fission is indicated in panels (b) and (c) with white arrows. Overlay of the immunofluorescence images of LC3 (green), TOM20 (red), and nuclei (blue) is shown in panels (d–g); mitochondrial fission is shown in panel (f) with white arrows. The confocal images were taken with optical Z-step of 0.5 µm.
Figure 5
Figure 5
Immunofluorescence multichromatic projections of PINK1, LC3 protein, a marker of autophagosome formation, and the mitochondrial TOM20 in the ischaemic (IR) cardiac specimens after treatments with resveratrol or longevinex. Panel (a) is administration of vehicle only; panel (b) is administration of resveratrol; panels (c–e) are administration of longevinex. Panel (f) is 5x magnification of the selected area indicated in panel (e) where mitochondrial fission and mitophagy are indicated with white arrows. Overlay of the immunofluorescence images of LC3 (green), PINK1 (red), and nuclei (blue) is shown in panels (a), (b), and (c); autophagy activation is indicated in panels (b) and (c) with white arrows. Overlay of the immunofluorescence images of PINK1 (green), TOM20 (red), and nuclei (blue) is shown in panel (d). Overlay of the immunofluorescence images of LC3 (green), TOM20 (red), and nuclei (blue) is shown in panel (e); mitochondrial fission is shown in panel (f) with white arrows. The confocal images were taken with optical Z-step of 0.5 µm.
Figure 6
Figure 6
Transmission electron microscopy images showing the ultrastructure findings in control and resveratrol treated hearts. Cardiomyocytes from control show normal ultrastructure and resveratrol treated hearts show mitophagy. Autophagosomes in resveratrol treated samples TEM images show early autophagic vacuoles containing still identifiable organelles (a–c) and late autophagosomes containing lamellar and vesicular structures (d–f). Autophagosomes preferentially contain mitochondria (m) enclosed by distinctive double membrane (arrows).
Figure 7
Figure 7
Western blot analysis of Sirt3, Foxo3a, and PINK-19 (a) and Sirt1 and PARKIN (b). Mitochondrial marker and cytosolic marker GAPDH are shown below.

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