Title: Redox regulation of resveratrolmediated switching of death signal into survival signal
1Redox regulation of resveratrol-mediated
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
Survival
Resveratrol Preconditioning
Intracellular Oxidation
Ischaemia/ Reperfusion
ROS
Apoptosis
2The 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
3Resveratrol 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 reduction- oxidation agent
and can be pro-oxidant under certain conditions.
Antioxidant Potential as a Free Radical Scavenger
.
(3,4,5-trihydroxystilbene)
R
RH
4Resveratrol the all-protective polyphenol?
Das, D. and Maulik N. Resveratrol in
Cardioprotection. 2006. Mol Interventions.
636-47.
5Resveratrol in cardioprotection
Das, D. and Maulik N. Resveratrol in
Cardioprotection. 2006. Mol Interventions.
636-47.
6 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.
7Thioredoxin system plays a crucial role in
defense against oxidative stress
Main function in cytoprotection against ROS is to
reduce intracellular disfulides to free thiols
8Thioredoxin system plays a crucial role in redox
signal transduction
9Preconditioning 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/reperfusio
n 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
10Aim 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.
11Animal 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
12Cisplatin 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.
13Cisplatin, but not shRNA-Trx-1, abolishes the
effects of resveratrol on percentage of damged
heart tissue and myocyte apoptosis.
Resveratrol
Resveratrol
Treated with I/R
Treated with I/R
14Measurement of malonaldehyde for assessment of
oxidative stress
- Malonaldehyde is an end product of lipid
oxidation, and was measured as MDA-DNPH
derivative by HPLC -
15GSH/GSSG ratio is an indicator of intracellular
oxidative stress
- Concentration of GSH and GSSG were determined in
vitro by a glutathione reductase cycling
procedure.
16Cisplatin, 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 2 345
1 2 3 4 5 1 2 345
Increased survival is accompanied by
reduced oxidative stress
17Survival Signaling of Resveratrol
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
Am J Physiol Heart Circ Physiol. 2005
Jan288(1)H328-35. Epub 2004
18Sample 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)
19Cisplatin 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
20Cisplatin Decreases Protein Levels of Trx-2
Cisplatin
-
I/R Resveratrol
- -
- 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
21Cisplatin abolishes resveratrol-mediated increase
in Trx-1/2 RNA levels after I/R
Cisplatin
-
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
22Role 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.
23Construction 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
24Dn-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.
25Trx-1 has no role in resveratrol-mediated
upregulation of Akt-P and Bcl-2
Akt phosphorylation levels
Bcl-2 levels
26How 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.
27Electron 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.
28Resveratrol-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
29Summary 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
- ResveratrolshRNA-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.
30Critique 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
31Critique of Paper Cont. (Western RT-PCR Data)
Western Blot RT-PCR
32Critique of Paper Cont. (Western Data)
Western Blot
33Critique of Paper Cont. (Western Data)
Western Blot