1017

Effects of Longevinex (modified resveratrol) on

cardioprotection and its mechanisms of action
Subhendu Mukherjee, Diptarka Ray, Istvan Lekli, Istvan Bak, Arpad Tosaki, and
Dipak K. Das

Abstract: Although resveratrol has been proven to possess diverse health benefits, several recent reports have demonstrated conflicting results on some aspects of its effects, including its anti-aging properties. Considerable debate appears to
exist on the dose and bioavailability of resveratrol, leading to the controversies on its effectiveness. To resolve the problem, we designed a study with a resveratrol formulation that contained resveratrol supplemented with 5% quercetin and
5% rice bran phytate (commercially known as Longevinex). These ingredients were micronized to increase the bioavailability. SpragueDawley rats were gavaged with either Longevinex or vehicle (5% quercetin plus 5% rice bran phytate),
and rats were sacrificed after 1 or 3 months, when isolated working hearts were subjected to 30 min ischemia followed by
2 h of reperfusion. Longevinex-treated hearts, irrespective of the duration of treatments, revealed superior cardiac performance, reduced infarct size, and induction of survival signals as evidenced by increased Bcl2/Bax ratio and enhanced Akt
phosphorylation. In contrast, LC3-II and Beclin were enhanced significantly after 3 months of Longevinex treatment, suggesting that autophagy occurred only after feeding Longevinex to rats for a prolonged period of time. Corroborating with
the results of autophagy, Sirt1 and Sirt3 increased significantly only after 3 months of Longevinex treatment, suggesting
that enhanced expression of Sirts correlated with induction of autophagy. In concert, Longevinex caused phosphorylation
and nuclear translocation of FoxO1, FoxO3a, and FoxO4, indicating involvement of FoxOs with autophagy. Since Sirts
and FoxOs are reliable markers of longevity, the results appear to suggest that Longevinex induces longevity after prolonged feeding via induction of autophagy, while it converts death signals into survival signals and provides cardioprotection within a relatively shorter period of time.
Key words: resveratrol, Longevinex, cardioprotection, survival signal, autophagy, longevity.
Resume : Des etudes ont prouve que le resveratrol presente divers avantages pour la sante, mais plusieurs rapports recents
ont demontre des resultats contradictoires sur certains aspects de ses effets dont les proprietes antivieillissement. Un debat
important semble exister sur la dose et la biodisponibilite du resveratrol a` lorigine de la controverse sur son efficacite.
Pour mettre un terme a` ce debat, nous avons concu une etude avec une preparation contenant du resveratrol additionne de
5 % de quercetine et de 5 % de phylate issu du riz (connu commercialement sous le nom de Longevinex). Nous avons micronise ces ingredients pour augmenter la biodisponibilite. Nous avons gave des rats SpragueDawley avec ce Longevinex
ou avec un vehicule (5 % de quercetine plus 5 % de phylate issu du riz). Nous avons sacrifie les rats apre`s 1 ou 3 mois, et
nous avons soumis les curs battants isoles a` une ischemie de 30 min suivie dune reperfusion de 2 heures. Le rats traites
au Longevinex, sans egard pour la duree du traitement, ont montre une performance cardiaque superieure, une taille dinfarctus reduite, et une induction de signaux de survie demontree par une augmentation du rapport Bcl2/Bax et une stimulation de la phosphorylation de lAkt. En revanche, les proteines LC3-II et Beclin ont augmente de manie`re significative
apre`s 3 mois de traitement au Longevinex, ce qui laisse supposer que lautophagie sest produite seulement apre`s une ingestion prolongee de Longevinex. Corroborant les resultats relatifs a` lautophagie, Sirt1 et Sirt3 ont augmente significativement apre`s seulement 3 mois de traitement au Longevinex, ce qui donne a` penser que laugmentation de lexpression des
Sirts a ete correlee avec linduction de lautophagie. Longevinex a entrane la phosphorylation et la translocation nucleaire
des FoxO1, FoxO3a et FoxO4, ce qui montre la participation de FoxOs dans lautophagie. Etant donne que les Sirts et les
FoxOs sont des marqueurs plausibles de longevite, les resultats semblent indiquer que le Longevinex induit un allongement
de la duree de vie apre`s une ingestion de longue duree via linduction de lautophagie, alors quil convertit le signal
dapoptose en signal de survie et fournit une cardioprotection a` linterieur dune periode de temps relativement plus courte.
Mots-cles : resveratrol, Longevinex, cardioprotection, signal de survie, autophagie, longevite.
[Traduit par la Redaction]

Received 13 April 2010. Accepted 24 May 2010. Published on the NRC Research Press Web site at cjpp.nrc.ca on 8 October 2010.
S. Mukherjee and D. Ray. Cardiovascular Research Center, University of Connecticut School of Medicine, Farmington, CT 060301912, USA.
I. Lekli, I. Bak, and A. Tosaki. School of Pharmacy, University of Debrecen, Debrecen H-4102, Hungary.
D.K. Das.1 Cardiovascular Research Center, University of Connecticut School of Medicine, Farmington, CT 06030-1912, USA; School
of Pharmacy, University of Debrecen, Debrecen H-4102, Hungary.
1Corresponding

author (e-mail: ddas@neuron.uchc.edu).

Can. J. Physiol. Pharmacol. 88: 10171025 (2010)

doi:10.1139/Y10-082

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Introduction
A growing body of evidence indicates that mild to moderate wine drinking attenuates cardiovascular, cerebrovascular,
and peripheral vascular risk due to reduced platelet and
monocyte adhesion and attenuates the risk of developing
prostate as well as other types of cancers, including pancreatic, gastric, and thyroid tumors (Bhat et al. 2001). Studies
on the cardioprotective effects of wine, especially red wine,
strongly suggest that resveratrol, a polyphenolic antioxidant
present in red wine, is responsible for most of the heart
health benefits of wine. While a large number of experimental data exist supporting the cardioprotective effects of resveratrol, epidemiological evidence also exists in support of
its health benefits (Das and Maulik 2006).
Resveratrol exerts its cardioprotective actions through its
ability to modulate cardiovascular disorders due to ischemic
heart disease (Das and Das 2007), atherosclerosis (Bertelli
and Das 2009), hypertension (Wu 2010), diabetes (Ramadori
et al. 2009), obesity (van der Spuy and Pretorius 2009), and
aging (Azzi 2009). Several lines of evidence suggest that resveratrol protects the heart through preconditioning; a stateof-the-art technique for cardioprotection, by converting disease-mediated death signals into survival signals (Das et al.
2004). More recently, resveratrol has been found to induce
autophagy in the ischemic myocardium (Gurusamy et al.
2010; Lekli et al. 2009, 2010).
Despite knowledge of the large number of health benefits
of resveratrol, several issues remain unresolved. The most
important of these is the required dose of resveratrol. While
most recent reports of the health benefits of resveratrol are
based on its use at a low dose, there are conflicting reports
regarding its use at relatively higher doses (Dudley et al.
2009; Mukherjee et al. 2010). Poor bioavailability of resveratrol remains another important issue (Cottart et al. 2010).
The most controversial issue in recent years is probably the
anti-aging effects of resveratrol. There are many conflicting
reports on the anti-aging effects of resveratrol. While earlier
studies showed the anti-aging role of resveratrol, more recent studies failed to support it. Even significant controversies exist regarding the induction of the anti-aging gene
Sirt1 by resveratrol. Careful studies, however, support the
notion that several longevity proteins can be induced with
wines and polyphenols, including resveratrol, when used at
lower doses. To resolve this controversy, the present study
was designed to test the cardioprotective effects of a resveratrol-based formulation, Longevinex, which has been found
to possess heart health effects superior to those of resveratrol.

Materials and methods

Animals
All animals used in this study received humane care in
compliance with regulations relating to animals and experiments involving animals and adheres to principles stated in
the Guide for the care and use of laboratory animals (Institute of Laboratory Animal Research 1996). All the protocols
were approved by the Institutional Animal Care Committee
of the University of Connecticut Health Center, Farmington,
Connecticut, USA. Male SpragueDawley rats weighing between 250 and 300 g were fed regular rat chow ad libitum

Can. J. Physiol. Pharmacol. Vol. 88, 2010

with free access to water until the start of the experimental

procedure. Animals were randomly subdivided into 3 groups
(n = 6 for ventricular function and infarct size; n = 4 for
biochemical parameters), of which the control group was
gavaged with 1 mL of water containing 5% quercetin and
5% phytate (ingredients of Longevinex minus resveratrol).
The other 2 groups were gavaged with Longevinex
(10 mg
kg1
day1) for 1 and 3 months, respectively.
Isolated working heart preparation
After completing the feeding protocol, the animals were
anesthetized with sodium pentobarbital (80 mg/kg, i.p.) (Abbott Laboratories, North Chicago, Ill., USA), and heparin sodium (500 IU/kg., i.p.) (Elkins-Sinn Inc., Cherry Hill, N.J.,
USA) was used as an anticoagulant. Under anesthesia, the
hearts were excised, the aorta was canulated, and the hearts
were perfused through the aorta in Langendorff mode at a
constant (100 cm of water) perfusion pressure at 37 8C with
KrebsHenseleit solution for a 5 min washout period, as described previously (Hattori et al. 2002). The perfusion medium consisted of a modified KrebsHenseleit bicarbonate
buffer (118 mmol/L sodium chloride, 4.7 mmol/L potassium
chloride, 1.7 mmol/L calcium chloride, 25 mmol/L sodium
bicarbonate, 0.36 mmol/L potassium dihydrogenphosphate,
1.2 mmol/L magnesium sulfate, and 10 mmol/L glucose),
and after its oxygenation the pH was 7.4 at 37 8C. During
the washout period, the left atrium was canulated, and the
Langendorff preparation was switched to the working mode
for 10 min with a left atrial filling pressure of 17 cm H2O
and aortic afterload pressure set to 100 cm of water. After
10 min, baseline cardiac functions such as heart rate (HR,
beats/min), aortic flow (AF, mL/min), coronary flow (CF,
mL/min), left ventricular developed pressure (LVDP, mm
Hg), and the first derivative of LVDP (LV dP/dt, mm Hg/s)
were recorded. Next, 30 min of global ischemia was initiated by clamping the left atrial inflow and aortic outflow
lines at a point close to their origins. At the end of the
30 min period of ischemia, reperfusion was initiated for
120 min by unclamping the atrial inflow and aortic outflow
lines. During the first 10 min, reperfusion was in Langendorff mode to prevent ventricular fibrillations, and afterwards the hearts were switched to anterograde working
mode (Tosaki et al. 1996; Hattori et al. 2001).
Cardiac function assessment
After 10 min of working mode perfusion, baseline parameters were recorded. To monitor the recovery of the heart,
the left ventricular cardiac function was recorded after 60
and 120 min of reperfusion. A calibrated flow meter (Gilmont Instrument Inc., Barrington, Ill., USA) was used to
measure AF. CF was measured by timed collection of the
coronary effluent dripping from the heart. During the entire
experiment, aortic pressure was monitored using a Gould
P23XL pressure transducer (Gould Instrument Systems Inc.,
Valley View, Ohio, USA) connected to a side arm of the
aortic cannula, and the signal was amplified using a Gould
6600 series signal conditioner CORDAT II real-time data
acquisition and analysis system (Triton Technologies, San
Diego, Calif., USA) (Hattori et al. 2001; Das et al. 2004).
HR, LVDP, and LV dP/dt) were calculated from a continuously generated pressure signal.
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1019

Fig. 1. Effects of Longevinex on ventricular function. Rats were fed Longevinex by gavaging for a period of 1 or 3 months, while control
experiments were performed by gavaging rats with vehicle (1 mL water with quercetin and phytate). At the end of 1 and 3 months, isolated
hearts perfused by working mode were subjected to 30 min ischemia followed by 2 h of reperfusion. Cardiac function was monitored at
baseline and during reperfusion. Results are expressed as means SE of 6 animals per group. *, p < 0.05 vs. control. BL, baseline. LVDP,
left ventricular developed pressure; LVmaxdP/dt, maximum first derivative of LVDP.

Infarct size estimation

Infarct size was measured according to the triphenyl tetrazolium chloride (TTC) method (Lekli et al. 2010). After 2 h
of reperfusion, 40 mL of 1% (w/v) solution of TTC in phosphate buffer was infused through the aortic cannula, and the
heart samples were stored at 70 8C for subsequent analysis.
Sections (0.8 mm) of frozen heart were fixed in 2% paraformaldehyde, placed between 2 cover slips, and digitally imaged using a Microtek ScanMaker 600z (Microtek, Cerritos,
Calif.). To quantitate the areas of infarct in pixels, a standard NIH image program was used. The infarct size was expressed in pixels (Hattori et al. 2001; Das et al. 2004).
Western blot analysis
Left ventricles from the hearts were homogenized in
1 mL of buffer (25 mmol/L TrisHCl, 25 mmol/L NaCl,
1 mmol/L orthovanadate, 10 mmol/L NaF, 10 mmol/L pyrophosphate, 10 mmol/L okadaic acid, 0.5 mmol/L EDTA,
1 mmol/L PMSF, and 1 protease inhibitor cocktail). The
homogenates were centrifuged at 2000 r/min at 4 8C for
10 min. The supernatant was centrifuged at 10 000 r/min at

4 8C for 20 min. The resultant supernatant was the cytosolic

fraction. The cytosolic extracts were aliquoted, snap frozen,
and stored at 80 8C until use. Total protein concentrations
in cytosolic extracts were determined using a BCA Protein
Assay Kit (Pierce, Rockford, Ill., USA) (Das et al. 2008a).
Proteins were separated by SDSPAGE and transferred to
nitrocellulose membranes. The membranes were blocked in
5% nonfat dry milk and probed with a 1:1000 dilution of
primary antibody overnight. The following primary antibodies were obtained from Cell Signaling Technology (Boston,
Mass., USA): Bcl-2, Bax, phospho (p)-Akt, Akt, PBEF,
Beclin-1, LC3, Sirt 1, Sirt 3, FoxO1 (Forkhead box O), pFoxO1, FoxO3a, p-FoxO 3a, Foxo4, p-FoxO4, and Sirt 4
from Abcam, whereas glyceraldehyde-6-phosphate dehydrogenase (GAPDH) was obtained from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). The protein bands were
detected using horseradish peroxidase conjugated secondary
antibody (1:2000 dilution) and Western blot luminol reagent
(Santa Cruz Biotechnology). GAPDH, histone, and Cox 4
were used as loading controls for the cytosolic, nuclear, and
mitochondrial fractions, respectively. The bands were digiPublished by NRC Research Press

1020

tized, subjected to densitometric scanning using a standard

image program, and normalized against the loading control.
Statistical analysis
The values for myocardial function parameters, infarct
size, and apoptosis were expressed as means SE. A oneway analysis of variance was first carried out to test for any
differences in mean values between groups. If differences
were established, the values of the drug-treated groups were
compared with those of the drug-free group by a modified
Students t test. The results were considered significant if
p < 0.05.

Can. J. Physiol. Pharmacol. Vol. 88, 2010

Fig. 2. Myocardial infarct size determined by TTC staining method.
Rats were fed Longevinex or vehicle as described in the legend of
Fig. 1. At the end of the 30 min ischemia and 120 min reperfusion,
the hearts were perfused with TTC. Infarct size was calculated as
the ratio of the infarcted area (white) to the risk area (red). Values
are expressed as means SE (n = 3). *, p < 0.05 vs. control. IR,
ischemia and reperfusion.

Results
Effects of Longevinex on left ventricular function
The baseline values of left ventricular function did not
vary appreciably between the groups (Fig. 1). During postischemic reperfusion, all the parameters were depressed appreciably as expected except for CF and HR, which did not
change significantly. Longevinex-treated hearts, both after
1 and 3 months, revealed improved aortic flow and LVmax
dP/dt during the reperfusion period. As shown in Fig. 1, at
30, 60, and 120 min after reperfusion, these functional parameters were higher for Longevinex-treated hearts than for
the vehicle control. In contrast, Longevinex, irrespective of
the number of days of treatment, did not significantly alter
HR and CF.
Effects of Longevinex on myocardial infarct size
As shown in Fig. 2, infarct sizes of hearts treated with
Longevinex for either 1 or 3 months, expressed as the percent area of risk area, were lower compared with those of
the vehicle control. There was no significant difference in
infarct size between hearts treated with Longevinex for 1 or
3 months.
Effects of Longevinex on the generation of survival signal
We monitored the expression of the induction of the antiapoptotic protein Bcl-2 and pro-apoptotic protein Bax in
hearts treated with Longevinex. Figure 3 shows the results.
As expected, Longevinex, both after 1 and 3 months of
treatment, enhanced the expression of Bcl-2 and reduced
the expression of Bax. Thus, the Bcl-2/Bax ratio was enhanced significantly with Longevinex, with no difference
found as a result of treatment duration. Since most of the
survival signals follow the PI-3-kinase/Akt signaling pathway, we also monitored the activation of Akt by determining Akt phosphorylation. As depicted in Fig. 3, the
phosphorylation of Akt was reduced significantly after ischemia and reperfusion (IR). The phosphorylation of Akt
was significantly enhanced after Longevinex treatment. Akt
phosphorylation was significantly higher in hearts treated
with Longevinex for 3 months compared with those treated
for 1 month.
Effects of Longevinex on the generation of autophagy
Since resveratrol has been found to induce the production
of survival signals through autophagy, we examined the effects of Longevinex on 2 reliable markers of autophagy,
LC3-I/II and Beclin-1. As shown in Fig. 4, the formation of

LC3-II (from LC3-I) as well as Beclin-1 was significantly

enhanced in the hearts treated with Longevinex for 3 months
compared with those treated with both vehicle and Longevinex for 1 month, suggesting that the degree of autophagy is
enhanced depending on the duration of treatment. IR reduced the expression of both LC3-II and Beclin-1.
Effects of Longevinex on longevity proteins
We examined the induction of the expression of several
longevity proteins by Western blot analysis: The results are
shown in the hearts, treated or untreated with Longevinex,
that were subjected to 30 min of ischemia and 2 h of reperfusion. There was no difference in Sirt1 and Sirt3 expression
between the baseline and the IR hearts. The expression of
both Sirt1 (nuclear) and Sirt3 (mitochondria) was enhanced
slightly, but not significantly, after 1 month of Longevinex
treatment (Fig. 5). The induction of the expression of Sirt1
and Sirt3 was significantly enhanced after 3 months of Longevinex treatment (Fig. 5).
Unlike Sirts, the induction of the expression of PBEF was
increased significantly after IR, indicating that ischemic
stress up-regulates PBEF expression. Longevinex treatment
for 1 month increased PBEF expression slightly but appreciably after 3 months of treatment. However, after IR, PBEF
expression levels were similar in all groups.
We then determined FoxOs in the mitochondrial as well
as in the nuclear fractions of the hearts subjected to IR. Similar to Sirts, the expression of FoxOs was also not affected
by IR. In the nucleus, phosphorylation of FoxO1, FoxO3a,
and FoxO4 was reduced progressively as a function of the
duration of Longevinex treatment (Fig. 6). For example,
phosphorylation of FoxOs was reduced after 1 month and
further reduced after 2 months of Longevinex treatment. In
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1021

Fig. 3. Western blot analysis of Akt, phospho-Akt, Bcl-2, and Bax proteins in tissues obtained from hearts treated with vehicle or Longevinex for 1 or 3 month, with or without subjecting them to ischemia and reperfusion (IR). GAPDH was used as the loading control. BL,
baseline.

Fig. 4. Western blot analysis of LC3 (L) and Beclin-1 (B) in tissues
obtained from hearts treated with vehicle or Longevinex for 1 or 3
months, with or without subjecting them to ischemia and reperfusion (IR). GAPDH was used as the loading control.

contrast, phosphorylation of FoxOs in the mitochondrial

fractions was progressively and steadily increased as a function of the duration of Longevinex treatment (Fig. 7). Thus,
the phosphorylation of all FoxOs was increased after
1 month and further increased after 3 months in the experimental groups compared with phosphorylation in the untreated groups.

Discussion
The results of the present study demonstrated that the resveratrol formulation Longevinex, similar to resveratrol
alone, triggered a short-term physiological effect resulting
in cardioprotection by converting an ischemia-induced death
signal into a survival signal by activating Akt and enhancing
anti-apoptotic factor Bcl-2. In contrast, Longevinex potentiated autophagy programming, as indicated by the activation
of Beclin-1 and formation of LC3-II only after a prolonged
period, leading to the activation of longevity proteins Sirt1
and Sirt3 as well resulting in the phosphorylation and nuclear translocation of FoxO1, FoxO3a, and FoxO4. In concert, PBEF also increased significantly after 3 months (from
the baseline level), while IR enhanced PBEF at all levels.
Resveratrol, a polyphenol phytoalexin, exerts a variety of
physiological actions resulting in cardioprotection, neuropro-

tection, and cancer preventive activities. Resveratrol potentiates cardioprotection by attenuating a variety of heart
diseases, including IR injury, atherosclerosis, and hypertension (Das and Das 2007; Azzi 2009; Bertelli and Das 2009;
Ramadori et al. 2009; van der Spuy and Pretorius 2009; Wu
2010). It appears that the heart health effect of resveratrol is
mediated by preconditioning effects rather than direct protection (Hattori et al. 2002; Saleh et al. 2010). The idea of
preconditioning was developed from the fact that resveratrol
could fulfill many criteria for preconditioning, including activation of adesosine A1 and A3 receptors, PKC, MAPKs,
KATP channels, and nitric oxide (Das et al. 2006a, 2006b;
Tan et al. 2008). Although most studies support heart health
properties of resveratrol that include, in addition to the
aforementioned, cardiac disorders, diabetes, obesity, and
aging (Azzi 2009; Ramadori et al. 2009; van der Spuy and
Pretorius 2009), many controversies are being generated
about its anti-aging properties. The anti-aging action of resveratrol was initially supported by a study that showed activation of the anti-aging gene Sirt1 (Baur et al. 2006).
While there is little controversy regarding the ability of resveratrol to activate Sirt1 (Sulaiman et al. 2010), considerable debate now exists on whether activation of Sirt1 is the
cause or the consequence of resveratrol action. A previous
study indicated that both red and white wines, tyrosol, and
hydroxytyrosol could also activate Sirt1 in addition to resveratrol, when used at low doses (Mukherjee et al. 2009).
The same study also revealed that resveratrol activated Sirt3
and Sirt4 as well as FoxO1, FoxO3a, FoxO4, and PBEF.
Sirt1 is located in the nuclear compartment, while Sirt3 and
Sirt4 are present in mitochondria. The role of Sirt1 as a longevity gene has been extensively studied (Wilusz and Wilusz 2007), but a recent report puts emphasis on Sirt3 and
Sirt4 as the gatekeepers for cellular longevity (Yang et al.
2010). Thus, it appears that mitochondrial Sirts play a vital
role in longevity by keeping the cells alive when they are
destined to die. Sirt3 deacetylates and activates the mitochondrial AceCS2, which converts acetate into acetyl CoA,
whereas Sirt4 ADP-ribosylates mitochondrial glutamate
GDH, which converts glutamate into a-ketoglutarate. When
a cell becomes stressed, a signaling pathway promotes an increase in PBEF (NAMPT), thereby activating NAD+, leading to the activation of Sirt3 and Sirt4. Consistent this, we
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Can. J. Physiol. Pharmacol. Vol. 88, 2010

Fig. 5. Western blot analysis of Sirt1, Sirt3, and PBEF in tissues obtained from hearts treated with vehicle or Longevinex for 1 or 3 months,
with or without subjecting them to ischemia and reperfusion (IR). GAPDH, COX4, and histone were used as loading controls. Representative images of 3 different groups are shown. Each experiment was repeated at least 3 times.

Fig. 6. Western blot analysis of cytosolic phosphorylated and nonphosphorylated FoxO1 and FoxO3a in tissues obtained from hearts treated
with vehicle or Longevinex for 1 or 3 months, with or without subjecting them to ischemia and reperfusion (IR). GAPDH was used as the
loading control. Representative images of 3 different groups are shown. Each experiment was repeated at least 3 times.

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1023

Fig. 7. Western blot analysis of nuclear phosphorylated and nonphosphorylated FoxO1 and FoxO3a in tissues obtained from hearts treated
with vehicle or Longevinex for 1 or 3 months, with or without subjecting them to ischemia and reperfusion (IR). Representative images of 3
different groups are shown. Each experiment was repeated at least 3 times.

found activation of PBEF in concert with the activation of

Sirt3 and Sirt4 (Mukherjee et al. 2009).
Similar to Sirts, FoxO transcription factors are important
regulators of stress signaling, thereby rendering resistance
to stress and promoting longevity. FoxOs contain 4 members
in mammals, FoxO1, FoxO3a, FoxO4, and FoxO6 (Greer
and Brunet 2005). They are abundant in all mammalian tissues, FoxO4 being particularly abundant in the heart. An increasing number of reports now imply that FoxOs increase
life span (Morris 2005). FoxOs are the major substrates of
Akt, which phosphorylates the FoxOs (van der Spuy and
Pretorius 2009). FoxOs are relocalized from the nucleus to
the cytoplasm in the presence of growth factors or insulin
followed by degradation via the ubiquitinproteasome pathway. Stress signals cause the nuclear translocation of FoxOs,
thereby allowing an adaptive response to stress.
Interestingly enough, our results indicate induction of autophagy with Longevinex only after prolonged treatment. In
contrast, pure resveratrol can rapidly induce autophagy
(Lekli et al. 2009; Morselli et al. 2009; Gurusamy et al.
2010). Evidence is rapidly accumulating indicating that autophagy is an essential element for life-span extension, and
many autophagy genes or proteins are regulated by longevity pathway (Madeo et al. 2010). Recent studies implicate a
direct role of Sirt1 in the regulation of autophagic degradation and autophagocytosis (Vellai et al. 2008). Not only
Sirt1 deacetylase regulates autophagosome formation;
FoxOs are equally involved in this signalling pathways and
exert a significant role in life-span extension. Our results indicate that autophagy parallels with the induction of Sirts,

FoxOs, and PBEF, suggesting that Longevinex induces lifespan extension through autophagy, which is in turn triggered
by several longevity genes. Thus, it is tempting to speculate
that Longevinex-mediated induction of life-span extension is
directly related to autophagy.
Finally, like pure resveratrol, Longevinex potentiates cardioprotection within a short period of time by generating a
survival signal through the PI-3-kinaseAkt pathway (Das
et al. 2008b). Phosphorylation of Akt occurred after only 1
month of resveratrol treatment. Also, similar to pure resveratrol, Longevinex triggered the stimulation of anti-apoptotic
Bcl2 protein.
In summary, our results clearly demonstrated that the resveratrol formulation Longevinex rapidly potentiated cardioprotective effects by converting the IR-mediated death
signal into a survival signal. However, unlike resveratrol,
which can rapidly activate autophagy and longevity genes,
Longevinex activated a coordinated programming by stimulating autophagy through several longevity proteins. In the
present study, one important issue is that resveratrol was
present at a low dose in Longevinex, consistent with our
previous observations. Although doseresponse studies were
not undertaken with Longevinex, it is tempting to speculate
that a higher dose might result in adverse effects. If the effectiveness of Longevinex is in fact mediated via survival
proteins through autophagy, then it is likely to exert prosurvival and pro-aging effects at higher doses. Indeed,
longevity genes like Sirt1 exert pro-aging effects, both in
yeast and in mammals (Wu 2010) as well as in the myocardium of transgenic mice (Yang et al. 2010).
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1024

Acknowledgements
This study was supported by grants from OTKA72315,
GVOP 3.2.1.2004-04-0269/3.0, TAMOP-4.2.2-08/1-20080007, and TAMOP-4.2.1B-09/1.

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