Transcript Redox regulation of resveratrol-mediated switching of death signal

Redox regulation of resveratrolmediated switching of death signal
into survival signal
Das 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
Resveratrol Preconditioning
Ischaemia/
Reperfusion
ROS
Intracellular
Oxidation
Survival
Apoptosis
The French Paradox and Polyphenols
-Ethanol ingestion enhances ROS production
and lipid peroxidation, which are regarded as
underlying factors in cardiovascular disease
-The French, despite having high-fat diets
enriched with red wine, have exceptionally
low incidences of coronary heart disease and
reduced 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 wine
which is fermented with the skin.
-red wine can contain 2-12 mg/L resveratrol.
Polyphenols are generally thought to be protective due to
antioxidant activity. However, every antioxidant is a reductionoxidation agent and can be pro-oxidant under certain conditions.
Antioxidant Potential as a Free Radical Scavenger
(3,4’,5-trihydroxystilbene)
.
R
RH
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.
Ischaemia-Reperfusion
Ischaemia: shortage in blood supply to an organ,
resulting in hypoxia, or lack of oxygen.
Reperfusion: resumption of blood flow resulting in
reoxygenation
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 reperfusion
phase can be more harmful than ischaemic phase.
Thioredoxin system plays a crucial role
in defense against oxidative stress
Main function in cytoprotection
against ROS is to reduce
intracellular disfulides to free
thiols
Thioredoxin system plays a crucial role
in redox signal transduction
Preconditioning potentiates the survival of
cardiac tissue after ischaemia/reperfusion
Classic Preconditioning: short cycles of reversible 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 results in an immediate resistance effect lasting
several hours and a delayed resistance effect appearing after 12-24 hours
and 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
Results:
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
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.
Resveratrol
Treated with I/R
Resveratrol
Treated with I/R
Measurement of malonaldehyde for
assessment of oxidative stress
• Malonaldehyde is an end product of lipid
oxidation, and was measured as MDADNPH 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 heart
Resveratrol
Resveratrol
1 2 3 4 5 1’ 2’ 3’4’5’
1 2345
1’ 2’ 3’4’5’
Increased survival is accompanied by reduced
oxidative stress
Survival Signaling of Resveratrol
Bcl-2 is outer mitochondrial membrane protein
Bcl-2 /Bad heterodimer induces apoptosis
Akt dependent Bad phosphorylation disrupts dime
Destabilized heterodimer is anti-apoptotic
Cell Survival
Apoptosis
Am J Physiol Heart Circ Physiol. 2005 Jan;288(1):H328-35. Epub 2004
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
shRNA-Trx1 Cisplatin
I/R: Resveratrol: -
•
•
•
•
+
-
+
+
+
+
+
+
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-mediated
increase in Trx-1/2 RNA levels after I/R
I/R:
Resveratrol:
-
+
-
+
+
+
+
Cisplatin
• 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
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.
Trx-1 has no role in resveratrol-mediated
upregulation of Akt-P and Bcl-2
Akt phosphorylation levels
Bcl-2 levels
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 Resonance
EPR 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.
Resveratrol-pretreatment potentiates reduction
of free radicals in myocardial tissue during ischemia
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).
Reduction Rates:
Control: 4.2 ± 0.9 s-1
RSV: 7.58 ± 1.5 s-1
Summary and Conclusion
Resveratrol 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 resveratrolmediated 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.
Critique of Paper (Western and RT-PCR Data)
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 Cont.
(Western & RT-PCR Data)
Western Blot
RT-PCR
Critique of Paper Cont. (Western Data)
Western Blot
Critique of Paper Cont. (Western Data)
Western Blot