Redox regulation of resveratrol-mediated switching of death

signal into survival signalDas S, Khan N, Mukherjee S, Bagchi D, Gurusamy N, Swartz H, Das DK.Cardiovascular Research Center, University of Connecticut School of Medicine, Farmington, CT 06030, USA.

Tad Kawashima, Kevin Roy, Letian Xie, Yuchen Shi, and Carolina Zapata

Ischaemia/Reperfusion ROS Apoptosis

SurvivalResveratrol Preconditioning

IntracellularOxidation

The French Paradox and Polyphenols

-Ethanol ingestion enhances ROS production and lipid peroxidation, which are regarded asunderlying factors in cardiovascular disease

-The French, despite having high-fat diets enriched with red wine, have exceptionallylow incidences of coronary heart disease andreduced injury due to ischemia-reperfusion

Because French wine is rich in polyphenols, these compounds have been presumed to be responsible for the protective benefits

Resveratrol: an abundant polyphenol-found in a variety of dietary sources, including grapes, plums, and peanuts

-resveratrol is mainly found in the skins of grapes, thus it is enriched in red winewhich is fermented with the skin.

-red wine can contain 2-12 mg/L resveratrol.

R.

RH

Antioxidant Potential as a Free Radical Scavenger

(3,4’,5-trihydroxystilbene)

Polyphenols are generally thought to be protective due toantioxidant activity. However, every antioxidant is a reduction-oxidation agent and can be pro-oxidant under certain conditions.

Resveratrol: the all-protective polyphenol?

Das, D. and Maulik N. Resveratrol in Cardioprotection. 2006. Mol Interventions. 6:36-47.

Resveratrol in cardioprotection

Das, D. and Maulik N. Resveratrol in Cardioprotection. 2006. Mol Interventions. 6:36-47.

IschaemiaIschaemia--ReperfusionReperfusion Ischaemia: shortage in blood supply to an organ, resulting in hypoxia, or lack of oxygen.

Reperfusion: resumption of blood flow resulting inreoxygenation

Cells made hypoxic can survive variable lengths time depending on the tissue.However, reperfusion often results in reoxygenation injury.Due to excessive ROS production, the reperfusionphase can be more harmful than ischaemic phase.

Thioredoxin system plays a crucial role in defense against oxidative stress

Main function in cytoprotectionagainst ROS is to reduceintracellular disfulides to freethiols

Thioredoxin system plays a crucial role in redox signal transduction

Preconditioning potentiates the survival of cardiac tissue after ischaemia/reperfusion

Previous studies in rodents have shown that classic preconditioning renders the heart more resistant to subsequent lethal ischemic/reperfusion injury.This resistance has been shown to be the result of an increase in endogenous defense mechanisms.

Classic Preconditioning: short cycles of reversible ischaemia/reperfusion

Classic preconditioning results in an immediate resistance effect lastingseveral hours and a delayed resistance effect appearing after 12-24 hoursand lasting up to 72 hours.

The mechanism of cardiac preconditioning is COMPLEX and debated,But appears to involve… - upregulation of antioxidant defenses- activation of adenosine receptors (A1), kinases (PKC, MAPK, and Y-kinase),the mitochondrial KATP channel- upregulation of HO and iNOS/eNOS during resveratrol preconditioning

Aim: Determine the role of thioredoxin 1 and 2

in resveratrol-mediated cardioprotection

Relative to untreated controls, resveratrol treatment… - lowered % damaged heart tissue- lowered % apoptotic cells- lowered MDA level- increased GSH/GSSG- upregulated anti-apoptosis through Akt-P and Bcl-2- upregulated thioredoxin system, including trx2- reduced free radicals introduced during ischaemia faster

Results:

All of these resveratrol-mediated effects were abolished by inhibition of both Trx-1 and Trx-2 but not by inhibition of Trx-1 alone. These results implicate Trx-2 as having a crucial role in preconditioning-mediated cardioprotection.

Animal Treatment• - Rats were randomly assigned to one of the

following groups:

I.  Control Group

  II. Ischemia/Reperfusion (I/R)

  III. Resveratrol + I/R

  IV. Resveratrol + I/R + shRNA-Trx-1

  V. Cisplatin + Resveratrol + I/R

Cisplatin and shRNA-Trx-1 Treatments

- For group 2-5, resveratrol (2.5 mg/kg body wt/day) was fed by gavaging for 10 days before the experiment.

- For group 5, cisplatin, which inhibits both Trx-1 and Trx 2, was injected on days 1, 3, 5, 7 and 9 intravenously during the gavaging of resveratrol.

- For group 4, a single dose of 100ul shRNA – Trx-1 was injected into the anterior wall of the left ventricle.

- After 10 days, isolated rat and mouse hearts were subject to ischaemia for 30 mins followed by 2 hour of reperfusion.

Cisplatin, but not shRNA-Trx-1, abolishes the effects of resveratrol on percentage of

damged heart tissue and myocyte apoptosis.

Treated with I/R Treated with I/R

Resveratrol Resveratrol

Measurement of malonaldehyde for assessment of oxidative stress

• Malonaldehyde is an end product of lipid oxidation, and was measured as MDA-DNPH derivative by HPLC

GSH/GSSG ratio is an indicator of intracellular oxidative stress

•Concentration of GSH and GSSG were determined in vitro by a glutathione reductase cycling procedure.

Cisplatin, but not shRNA-Trx-1, abolishes the effects of resveratrol on MDA formation and GSH/GSSG levels of

the heartResveratrol

Resveratrol

Increased survival is accompanied by reducedoxidative stress

1 2 3 4 5 1’ 2’ 3’4’5’ 1 2 3 4 5 1’ 2’ 3’4’5’

Survival Signaling of Resveratrol

Am J Physiol Heart Circ Physiol. 2005 Jan;288(1):H328-35. Epub 2004

Cell Survival

Bcl-2 is outer mitochondrial membrane protein

Bcl-2 /Bad heterodimer induces apoptosis

Akt dependent Bad phosphorylation disrupts dimer

Destabilized heterodimer is anti-apoptotic

Apoptosis

Sample Preparation for Western Blot Analysis

Interested in looking at anti-apoptotic pathway markers Akt-P and Bcl-2:

Left ventricles from hearts homogenized in buffer containing:

• Sodium Orthovanadate (Na3VO4): Inhibitor of phosphatases

• Okadaic Acid: Inhibits Serine/Threonine Phosphatases• PMSF: Serine Protease Inhibitor

Purpose: to protect phosphoylation states and protein degradation

Blotted with antibody against:

• NF-kB, Akt, Akt-P, Bcl-2, GAPDH (loading control)

Cisplatin and not Trx-1 Decreases Survival Markers

I/R:Resveratrol:

- + + + +- - + + +

shRNA-Trx1 Cisplatin

• I/R treatment decreases Survival Marker levels• Resveratrol enhances levels of Akt-P & Bcl-2• Addition of shRNA-Trx1 had no effect on markers• Addition of Cisplatin negates enhancement of RSV

Cisplatin Decreases Protein Levels of Trx-2

I/R:Resveratrol :

- + + + - - + +

Cisplatin

• Trx-2 protein expression levels dropped during I/R• Resveratrol was able to restore Trx-2 levels• Cisplatin abolished the effects of resveratrol• Trx-1 protein “could not be detected”

Cisplatin abolishes resveratrol-mediatedincrease in Trx-1/2 RNA levels after I/R

I/R:Resveratrol:

• Trx-1 and Trx-2 transcripts were reduced after I/R• Resveratrol treatment increased expression levels• Cisplatin diminished Trx-1/2 even in presence of RSV

- + + + - - + +

Cisplatin

Role of thioredoxin system in resveratrol-mediated

preconditioning• So far the data indicated that resveratrol provided

cardioprotection by triggering a survival signal through phosphorylation of Akt and activation of Bcl-2.

• Thioredoxin system seems to have a role because cisplatin abolished the survival signal and cardioprotection generated with resveratrol.

• However, the inability of shRNA directed against Trx-1 to block the effects of resveratrol indicated that Trx-1 had no role in resveratrol-mediated cardioprotection.

Construction of Dominate-negative (Dn) Trx-1 transgenic mice to confirm shRNA-

Trx1 results• Dn-Trx-1 transgenic mice

were generated by mutation of Cys32 and Cys35 of hTrx-1 to Ser by PCR

• These transgenic mice are shown to function as a dominant negative for endogenous Trx-1

Dn-Trx-1 mice confirm that Trx-1 has no role in resveratrol-mediated cardioprotection

LVDP: left ventricular develop pressure (recovery of function)With resveratrol treatment to both wild type and Dn-Trx-1, recovery function in both increased significantly, and the infarct size for both decreased significantly.

Akt phosphorylation levels Bcl-2 levels

Trx-1 has no role in resveratrol-mediated upregulation of Akt-P and Bcl-2

How does resveratrol-preconditioning affect the ability to reduce free

radicals? Strategy: determine redox status of the isolated perfused heart during ischaemia.

•Perfuse rat heart with 0.2 mM TEMPO nitroxide, a free radical used as biological probe, for 15 minutes•Perform ischemia for 30 minutes.•The reduction of the nitroxide during ischemia results in a decrease in signal intensity over time.

Electron Paramagnetic ResonanceEPR is used to detect and identify free radicals

(unpaired electrons)

•Similar to NMR, except that electron spins are excited and not atomic nuclei.•Generated by exposing paramagnetic molecules to microwaves at a constant frequency and increasing the magnetic field until the gap between spin states matches the energy of the microwaves. There is a net absorption of energy and this absorption is measured and converted to a spectrum.

The difference in energy states matches the frequency of the microwaves.

Result: Both groups of myocardial tissue had a decrease in signal intensity with time, but resveratrol-treated hearts exhibited a more rapid decrease in signal intensity as compared to the untreated heart (control).

Resveratrol-pretreatment potentiates reduction of free radicals in myocardial tissue during ischemia

Reduction Rates:Control: 4.2 ± 0.9 s-1

RSV: 7.58 ± 1.5 s-1

Summary and ConclusionResveratrol increased the rate of decay of free radicals

Relative to resveratrol only, treatment with cisplatin and resveratrol prior to I/R caused:

- Increasing infarct size and myocyte apoptosis - Increased oxidative state(measured via MDA level and GSH/GSSG

ratio) - Decrease survival signaling markers- Trx-2 protein level decreased- Inhibition of Trx-1 and Trx-2 RNA levels

Resveratrol+shRNA-Trx1 treated rats showed Trx-1 plays no role in resveratrol-mediated cardioprotection. This is further confirmed by Dn-Trx-1 transgenic mice.

Trx-2 is likely to play a role in switching I/R-induced death signal into survival signals. Mitochondria are the primary ROS source. The mitochondrial localization of Trx-2 suggests that mitochondria redox regulation and signaling is the primary target of resveratrol-mediated cardio-protection.

Western:NF-kB Ab was not used in any Westerns shown or stated, but indicated in protocol

Trx-1 Western “not detected” suggests that Ab was working and no protein detected:• However no positive control was shown for Ab function

RT-PCR:Need control for RT PCR for normalization (issues with input variability, etc.)

shRNA for Trx-2 data needed to get a complete story

Critique of Paper (Western and RT-PCR Data)

Critique of Paper Cont. (Western & RT-PCR Data)

Western Blot RT-PCR

Western Blot

Critique of Paper Cont. (Western Data)

Western Blot

Critique of Paper Cont. (Western Data)