Sirtuin 6 protects the heart from hypoxic damage

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http://dx.doi.org/10.10014-4827/& 2014 E

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Research Article

Sirtuin 6 protects the heart from hypoxic damage

Anna Maksin-Matveeva, Yariv Kanfia, Edith Hochhauserb, Ahuva Isaka, Haim Y. Cohena,Asher Shainberga,n

aThe Mina and Everard Goodman, Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, IsraelbThe Laboratory of the Department of Cardiothoracic Surgery, Felsenstein Medical Research Center, Rabin Medical Center, Petach Tikva, Israel

a r t i c l e i n f o r m a t i o n

Article Chronology:

Received 19 May 2014Received in revised form9 July 2014Accepted 11 July 2014

Keywords:

Cardiac cell culturesCardioprotectionHypoxiaOver-expression of Sirtuin 6

016/j.yexcr.2014.07.013lsevier Inc. All rights reser

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a b s t r a c t

Sirtuin 6 (SIRT6) is a protein associated with prolonged life expectancy. We investigated whetherlife extension is associated with cardioprotection against hypoxia. The proposed study is to developapproaches to reduce hypoxic damage through the use of the sirtuin pathway and to elucidate themechanism involved. For that purpose we subjected cardiomyocytes from transgenic mice (TG)with over-expression of SIRT6, to hypoxic stress in cell cultures. We hypothesized that cardiomyo-cytes from transgenic mice subjected to prolonged hypoxia may release survival factors or fewerdamage markers to protect them from hypoxic stress compared with wild type (WT) mice. Lactatedehydrogenase (LDH) and creatine kinase (CK) released to the medium and propidium iodide (PI)

binding, were markedly decreased following hypoxia in TG cardiomyocytes. The protective mechan-ism of SIRT6 over-expression includes the activation of pAMPKα pathway, the increased protein levelof B-cell lymphoma 2 (Bcl2), the inhibition of nuclear factor kappa-light-chain-enhancer of activatedB cells (NFκB), the decrease of reactive oxygen species (ROS) and the reduction in the protein level ofphospho-protein kinase B (pAkt) during hypoxia. Together, all these processes impede the necrosis/apoptosis pathways leading to the improved survival of cardiomyocytes following hypoxia, whichmight explain life extension.

& 2014 Elsevier Inc. All rights reserved.

Introduction

Ischemic injury to the myocardium in response to coronary occlusionis the leading cause of morbidity and mortality in the developedworld [1,2]. Enormous interest has therefore arisen concerning themechanisms capable of limiting myocardial damage. However, noagent has yet gained widespread clinical use [3]. Recent studies havedemonstrated that heart failure can be prevented or averted, at leastin experimental models, by caloric restriction, a dietary regimen thatlimits calorie intake [4,5]. Additionally, for many years, it has beenknown that a calorie restricted diet slows the rate of aging andextends the life span of many organisms [6]. One family of proteins

ved.

m, [email protected]

ev, et al., Sirtuin 6 protect

linking the regulation of caloric restriction pathway and the heartfunction is the sirtuins [7].Sirtuins (SIRTs) are a family of class III histone deacetylases

(HDACs), distinguished from other HDAC classes by their requirementfor nicotinamide adenine dinucleotide (NAD) in the deacetylationreaction [4,8]. Seven SIRT homologs exist in the mammalian genome,termed SIRT1 to SIRT7. These proteins regulate a wide range ofbiological processes including; metabolism [9], gene silencing [10],aging and lifespan extension [11,12], and cell survival in response tostress [13,14].SIRTs were first discovered in yeast, in which an extra copy of SIR2

was shown to extend the lifespan by 50%, whereas its deletion

.il (A. Shainberg).

s the heart from hypoxic damage, Exp Cell Res (2014), http://dx.

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shortened the lifespan. In addition, SIRT6-deficient mice show themost severe phenotype of all SIRT gene knockouts, with prematureaging that includes features of osteoporosis, absence of subcutaneousfat, severe metabolic imbalance, lymphopenia and acute onsethypoglycemia that result in the death of mice within 1 month ofage [15]. In contrast, transgenic over-expression of SIRT6 protectsmice against the adverse consequences of a high-fat diet, which iscommonly considered a forerunner of cardiovascular morbidity[16–19]. Thus, SIRT6 protects against pathological damage causedby diet-induced obesity and extends lifespan like caloric restriction[16,18].In this study, we show that over-expression of SIRT6 protects

cardiomyocytes against hypoxia. The protective mechanism stimu-lated by the over-expression of SIRT6 include the activation ofpAMPKα pathway, an increase in protein level of B-cell lymphoma2 (Bcl2), inhibition of nuclear factor kappa-light-chain-enhancerof activated B cells (NFκβ), a decrease in reactive oxygen species(ROS) and reduction in the protein level of protein kinase B (pAkt)during hypoxia. All these pathways preclude necrosis/apoptosisand protect cardiac cells from hypoxic stress and might explainlifespan extension.

Methods

Chemicals

All chemicals were acquired from Sigma (St Louis, Mo) unlessotherwise stated. Propidium iodide (PI) was acquired fromMolecular Probes (Eugene, Or) and LDH/CK kits bought fromThermo Thermo Electron (Melbourne, Australia).

Solutions

Standard phosphate-buffered saline (PBS) solution, pH 7.4, contained135 mM NaCl, 8.09 mM Na2HPO4, 1.47 mM KH2PO4, 2.68 mM KCl,0.9 mM CaCl2, 0.48 mM MgCl2, and 5.55 mM glucose.

Preparation of heart cultures

Mice were purchased from Harlan Labs (Jerusalem, Israel). Theexperiments were carried out in accordance with the guidelinesof the Animal Care and Use Committee of Bar-Ilan University, andthe Guide for the Care and Use of Laboratory Animals publishedby the US National Institute of Health. Hearts of SIRT6-transgenicmice from CB6F1 (background was generated as described pre-viously [18]; animals were 2–3 days old) were removed understerile conditions and washed three times in PBS to remove excessblood cells. The hearts were minced into small fragments andthen gently agitated in RDB, a solution of proteolytic enzymesprepared from fig tree extract (Biological Institute, Ness-Ziona,Israel). RDB was diluted 1:100 in Ca2þ- and Mg2þ-free PBS at25 1C, and incubated with the heart fragments for several cycles of10 min each, as mentioned before [20]. Dulbecco's modifiedEagle's medium, supplemented with 10% inactivated horse serum(Biological Industries, Kibbutz Beit Haemek, Israel) and 0.5% chickembryo extract, was added to the supernatant containing asuspension of dissociated cells. The mixture was centrifuged at300� g for 5 min. The supernatant was discarded and the cellswere re-suspended. The cell suspension was diluted to 1.0�106

Please cite this article as: A. Maksin-Matveev, et al., Sirtuin 6 protectdoi.org/10.1016/j.yexcr.2014.07.013

cells/ml, and 1.5 ml of the suspension was placed in 35-mmplastic culture dishes or glass cover slips coated with collagen/gelatin. The cultures were incubated in a humidified atmosphereof 5% CO2–95% air at 37 1C. A confluent monolayer exhibitingspontaneous contractions developed within 1–2 days. The experi-ments were performed on 4–6 day-old cardiomyocyte cultures.

Hypoxic experiments

Heart-culture dishes were washed in glucose-free PBS. Thenglucose-free PBS containing 1 mg/ml BSA was added to the disheswhich were transferred for 120 min to hypoxic chambers at 37 1Cwith a constant flow of argon. Lactate dehydrogenase (LDH) orcreatine kinase (CK) was determined in the PBS immediately afterthe hypoxia, as previously described [20]. An amount of 25 μl ofthe supernatant of growing cells was transferred into a 96-welldish, and LDH or CK activities were determined using LDH-L or CKkits (Thermo Electron, Melbourne, Australia). The product of theenzyme, NADH, was measured spectrometrically at 30 1C at awavelength of 340 nm. Experiments were performed with 4–8replicas each and were repeated at least 3 times.

Propidium iodide assay

The assay is based on binding of propidium iodide (PI) to thenuclei of cells the plasma membranes of which have becomepermeable due to cell damage. Cell viability was determined by PIfluorometry using a multi-well plate reader (TECAN SpectraFluorPlus, Austria) [21]. Cardiomyocytes following hypoxia receivedPBS containing glucose and 5 μM PI were incubated for 30 min at37 1C. Fluorescence (A) from each plate/well was measured atexcitation and emission wavelengths of 540 and 630 nm, respec-tively. The background fluorescence (B) was assessed from unstainedplate. Experiments were terminated by permeabilizing plasmamembranes with 300 μM digitonin to label all nuclei with PI.A final fluorescence (C) was measured 30 min after digitonin treat-ment. The percentage of damaged cells (V) was calculated as V¼100(A�B)/(C�B).

Western blot analysis

Heart cultures were homogenized on ice in a tissue homogeniza-tion lysis buffer containing 50 mM tris–HCl, 150 mM NaCl, 1% NP-40, and 0.1% SDS, pH¼8. Heart homogenate was incubated at 4 1Cwith lysis buffer, with the addition of protease and phosphataseinhibitor cocktail (Sigma), and with constant mixing for 30 min.The amount of total protein was then determined by the Bradfordmethod using a commercial assay kit (Sigma). Sample buffer wasadded to the protein samples (35–50 μg per lane). Samples werethen separated on 10% polyacrylamide-SDS gels (1.5 h, 100 V) inrunning buffer, and transferred (in transfer buffer) to nitrocellu-lose membranes (Bio-Rad, Israel) for Western blotting (1.5 h,250 mA). The nitrocellulose membranes were incubated for 1 hin blocking solution containing 5% milk powder in Tris-bufferedsaline (TBS) at 25 1C, and then incubated with the desired anti-body (rabbit polyclonal anti-SIRT6 or rabbit anti-pAMPKα, or anti-pAkt, or anti-Bcl2 (1:1000, Santa Cruz Biotechnology)) in ablocking solution overnight. The membrane was then washed 4times in Tris-buffered saline tween (TBST) for 10 min each time,and then incubated for 1 h in TBS with the secondary antibody

s the heart from hypoxic damage, Exp Cell Res (2014), http://dx.

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(goat polyclonal anti-rabbit, Santa Cruz Biotechnology, 1:10,000)at 25 1C and exposed to Kodak X-ray film.

Immunoprecipitation

Cells were washed twice with 5 ml of PBS followed by incubation onice with lysis buffer containing 0.5% NP-40, 50 mM Tris, 150 mMNaCl, 1 mM phenylmethylsulfonyl fluoride, 5 mg/ml leupeptin,2 mg/ml aprotinin, 1 mM sodium orthovanadate, and 1 mM EDTAfor 5 min. Cells were harvested from the plates and transferred to a1.5 ml tube. The lysate was centrifuged at 16,000g for 5 min at 4 1Cand the supernatant was transferred to a new tube. Lysates werequantified by the Bradford assay and equal amounts of total proteinwere used for co-immunoprecipitation with either the anti-SIRT6 oranti-igG rabbit Ab. The immunocomplexes were then subjected toSDS-PAGE.

ROS assay

Cardiac cells were seeded in 96-well black tissue culture micro-plates to which 2 mM of DCFH-DA was added for 30 min at 371Cand then the dye was removed from the cells. After removal of theDCFH-DA, the cells were subjected to hypoxia. After that, cellfluorescence was determined with a Tecan fluorometer withexcitation and emission wavelengths of 485 and 530 nm aspreviously described [21].

siRNA experiments

The SIRT6 siRNA (SASI_Mm01_00089564) and negative controlGFP siRNA from Sigma Aldrich were used. Heart cells werecultured on collagen for 2 days; 20 nM SIRT6 siRNA, or GFP siRNAoligonucleotides were introduced into each well with siIMPORTERtransfection reagent (Millipore, Billerica, MA, USA). All siRNAexperiments were carried out in triplicate. The cells were harvested72 h after transfection and the hypoxic assay and Western blotanalysis were performed.

Mice

Mice over-expressing SIRT6 were generated as described pre-viously [18] and were kept under specific pathogen free (SPF) and12 h day/night conditions. For genotyping, DNA was isolated fromtails of mice according to the manufacturer's protocol (5ʹ) andamplified by PCR using the following primers for transgenic micegenotyping:

Pd

Forward: 50-GAGCTGCACGGAAACATG-30

Reverse: 50-CCCATAATTTTTGGCAGAGG-30 (unique to the30UTR)

Statistical analysis

Statistical analysis was performed by analysis of variance with theapplication of a posthoc Tukey–Kramer test. Data shown repre-sent mean of at least six replicates in three separate experi-ments7SEM. po0.05 was accepted as indicating statisticalsignificance.

lease cite this article as: A. Maksin-Matveev, et al., Sirtuin 6 protectoi.org/10.1016/j.yexcr.2014.07.013

Results

Effect of SIRT6 over-expression on hypoxic damage

Since changes in the SIRT6 routes have been proposed to beassociated with an anti-aging process [22–24], we investigatedwhether activation of this pathway in the cardiomyocytes in amouse model protects cells from hypoxic damage. Cardiomyo-cytes from WT and from transgenic (TG) mice with the over-expression of SIRT6 were subjected to prolonged hypoxia (2 h) incell cultures. To confirm that SIRT6 pathway enhanced thesurvival of cardiomyocytes, we determined the level of lactatedehydrogenase (LDH) and creatine kinase (CK) released to themedium, and propidium iodide (PI) binding to the cells, immedi-ately after hypoxia. We found that cardiomyocytes from TG miceshowed higher levels of survival, as revealed by a lower level ofLDH and CK release (4574%, po0.01), and a reduced level of PIbinding (8274.2%, po0.01, Fig. 1A–D). In addition, we tested twosubtypes of transgenic mice lines (line 85 and line 91), to makesure that the results obtained were due to the over-expression ofSIRT6 and not due to the intake of the plasmid by the cardiac cells(in additional experiments we used line 85 of the transgenicmice).Western blotting of the homogenates from these groups was

performed to confirm the over-expression of SIRT6. It was foundthat the expression of SIRT6 was 4–6 times higher in the cardiaccells of transgenic mice than in the control cells (Fig. 1E and F).

Effect of siRNA against SIRT6 on cardioprotection

In order to confirm that cardioprotection against hypoxia is due toover-expression of SIRT6, siRNA [25] against SIRT6 was applied onTG mice cardiomyocyte cultures. First we investigated whethersiRNA reduced SIRT6 protein level in the cells. Then we subjectedthe cells to hypoxia. It was found that siRNA reduced SIRT6protein level (by 8577.4%, po0.01) (Fig. 2A and B). When theprotein level of SIRT6 was reduced, the cardio protection fromhypoxia was abrogated. The LDH and CK levels in the mediumwere higher (by 4273.5%, po0.01) in heart cultures from TG micetreated with siRNA than in cultures from TG mice withouttreatment with siRNA against SIRT6 protein (Fig. 2C and D). Fora positive control we used siRNA against GFP (a protein that doesnot exist in myocardial cells), to ensure that the results obtainedwere due to the reduction of the SIRT6 protein level and not dueto siRNA intake.

Determining the pathway for cardioprotection via SIRT6

To study the effects of SIRT6 on the myocardial cells we challengedsome key survival proteins such as reduction of reactive oxygenspecies, anti-apoptotic proteins family, anti-inflammation proteins,exchange pathway for reproducing energy during hypoxia etc.

Anti-oxidant function

During hypoxia, reactive oxygen species (ROS) increase dramatically[26]. These reactive molecules may result in significant damage tocell structures. Cultured cardiomyocytes from WT and TG mice wereloaded with DCFH-DA (dichloro-dihydro-fluorescein diacetate) in

s the heart from hypoxic damage, Exp Cell Res (2014), http://dx.

Fig. 1 – Over-expression of SIRT6 enhances the survival of cardiomyocytes in-vitro. Heart cultures from wild type (WT) and fromtransgenic (TG) mice were subjected to 120 min hypoxia. (A) LDH released to the medium. (B) CK released to the medium. (C, D)Propidium iodide (PI) binding quantification and representative slides. PI are arbitrary units normalized to the total number ofcells. (E, F) Protein expression using Western blot analysis of SIRT6 in cardiomyocytes subjected to hypoxia compared to normoxia,normalized to β-actin. Over-expression of SIRT6 decreased LDH and CK release and PI binding level. Values are average7SEM ofenzyme released from the cells. npo0.01 vs WT under the same conditions, n¼6.

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order to measure ROS concentration. Then the cells were subjectedto hypoxia. The results demonstrated that SIRT6 functions as ananti-oxidant and reduces ROS formation by 3972.8% (po0.05,Fig. 3).

Anti-apoptosis function

Cell damage caused by hypoxia can lead to planned cell death(apoptosis) [7]. We investigated whether SIRT6 prevents theapoptotic process. For this purpose we analyzed Bcl2 (B celllymphoma 2), a regulator protein that controls apoptotic celldeath and has anti-apoptotic activity [27–29]. It was found thatSIRT6 increased Bcl2 protein level by 9776.5% (po0.05, Fig. 4).

pAkt

Akt, also known as protein kinase B (PKB), is a serine/threoninespecific protein kinase, playing a key role in multiple cellularprocesses such as glucose metabolism, cell motility and cell survival[30,31]. We wondered whether SIRT6 affects the pAkt level. For thispurpose we conducted Western blot analysis to investigate pAktlevel. It can be seen that in heart homogenate cultures from TG micesubjected to hypoxia the pAkt protein level is reduced by 8278%(po0.05) than in cardiac cells from WT mice. Namely, SIRT6decreases the pAkt protein level in hypoxia (Fig. 5).

Please cite this article as: A. Maksin-Matveev, et al., Sirtuin 6 protectdoi.org/10.1016/j.yexcr.2014.07.013

NO synthase (NOS)

Nitric oxide synthases (NOS) are a family of enzymes catalyzingthe production of nitric oxide (NO) from L-arginine. NOS regulatecardiac function and protect heart during hypoxia [26]. Weinvestigated whether NOS participates in SIRT6 cardioprotectingpathway. For this purpose we treated the cardiocytes from TG andWT mice for 24 h with 100 μM L-NAME (NOS activity inhibitor),and after that subjected the cultures to hypoxia. It was found thatwhen the cells were treated with L-NAME, cardioprotection wasreduced by 6874.5% (po0.05), as revealed by LDH release.Namely, SIRT6 affects cardioprotection also via NOS activity(Fig. 6).

NFκB

NFκB is a major factor that regulates inflammatory genes expres-sion, as does TNF-α. During hypoxia, inflammation occurs. SIRT6physically binds to one of the subunits of NFκB – RelA (p-65) anddeacetylates H3K9 on RelA dependent promoters and repressestheir expression [32]. To determine whether SIRT6 binds to NFκBwe conducted an immunoprecipitation assay. Results of theWestern blot analysis of the co-precipitate showed that SIRT6indeed binds physically to p-65 subunit of NFκB (Fig. 7).

s the heart from hypoxic damage, Exp Cell Res (2014), http://dx.

Fig. 2 – siRNA against SIRT6 reduced the survival of cardiomyocytes from TG mice. Cardiomyocytes from wild type (WT) and fromtransgenic (TG) mice were subjected to 120 min hypoxia. (A, B) Protein expression in cardiomyocyte lysates using Western blot.(C) LDH released to the medium. (D) CK released to the medium. siRNA against SIRT6 reduced SIRT6 protein level on TG mice, thathave over-expression of SIRT6 and reduced the influence of over-expression on cardioprotection. siRNA against GFP was used as anegative control. Values are average7SEM of enzyme released from the cells. Western blot calculations are normalized to β-actin.npo0.01, TG vs WT. #po0.05 TG siRNA vs TG, n¼3.

Fig. 3 – SIRT6 functions as an anti-oxidant. Heart cultures fromwild type (WT) and from transgenic (TG) mice were subjected to120 min hypoxia. ROS level was then measured usingDCFH-DA with excitation at 485 nm and emission at 530 nm.SIRT6 decreased ROS levels in hypoxic myocardial cells. npo0.05,n¼4.

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pAMPKα

AMP-activated protein kinase (AMPK) is a heterotrimeric Ser/Thrprotein kinase that acts as a cellular energy sensor controllingseveral metabolic pathways [5,33]. Recently, we demonstrated that

Please cite this article as: A. Maksin-Matveev, et al., Sirtuin 6 protectdoi.org/10.1016/j.yexcr.2014.07.013

SIRT6 activates AMPK in the liver [34]. Thus, we further investigatedwhether, in our system too, SIRT6 activates pAMPKα. For thispurpose we analyzed pAMPKα protein level and found that in TGmice heart homogenate pAMPKα was elevated 8.571.5%-fold innormoxia and 771.4% in hypoxia (po0.05, Fig. 8A and B). Inaddition, we questioned whether pAMPKα participates in cardio-protection. For this purpose we treated WT and TG mice cardiac cellcultures with 100 μM Compound C (pAMPKα inhibitor) for 24 h andthen subjected the cells to hypoxia. The damage to the cells wasdetermined with LDH released to the medium. The results revealedthat elevated pAMPKα by SIRT6 over-expression confer protectionagainst hypoxia. When the activity of pAMPKα was inhibited,cardioprotection from hypoxia was reduced by 6673.9% (po0.01,Fig. 8C–E).

Discussion

In this study, we elucidated the SIRT6 pathway. Our data indicatethat over-expression of SIRT6 confers cardioprotection by posi-tively and negatively regulating key survival proteins which mightlead to an extended lifespan [16]. The results show that cardio-myocyte cultures with over-expression of SIRT6 (TG mice) wereassociated with a significant reduction in the LDH and CK release,and PI binding following hypoxic stress as compared to WT mice.The protective mechanism of SIRT6 over-expression includes

s the heart from hypoxic damage, Exp Cell Res (2014), http://dx.

Fig. 4 – SIRT6 functions as an anti-apoptotic protein.Cardiomyocyte cultures fromwild type (WT) and from transgenic(TG) mice were subjected to 120 min hypoxia. Bcl-2 expressionwas measured by Western blot analysis. SIRT6 increased Bcl2protein level in myocardial cells of TG mice. Western blotcalculations are normalized to β-actin. npo0.05, n¼6.

Fig. 5 – pAkt protein level. Cardiomyocyte cultures from wildtype (WT) and from transgenic (TG) mice were subjected to120 min hypoxia. The pAkt level was measured by Western blotanalysis. SIRT6 decreased pAkt protein level of myocardial cellsin hypoxia, while during hypoxia the pAkt protein level in WTcells increased. Western blot calculations are normalized toβ-actin. npo0.05 vs WT, n¼4.

Fig. 6 – NOS activity in SIRT6 protection pathway.Cardiomyocyte cultures from wild type (WT) and fromtransgenic (TG) mice treated for 24 h with L-NAME (100 lM)and then were subjected to 120 min hypoxia. LDH released tothe medium was determined. Cells from TG mice that weretreated with L-NAME showed increased LDH release. Thus,L-NAME prevented SIRT6 protection. Values are average7SEM ofenzyme released from the cells. npo0.01, #po0.05 vs WT. n¼4.

Fig. 7 – Immunoprecipitation of SIRT6 together with p-65.Cardiomyocytes from wild type (WT) and from transgenic(TG) mice were subjected to 120 min hypoxia. Then the cellswere homogenized and immunoprecipitated with antiSIRT6.The co-precipitate was then subjected to Western blot analysiswith anti p-65.

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activation of pAMPKα pathway, increased Bcl2 protein, inhibitionof NFκB, a decrease in ROS formation and pAkt activity. Theseprocesses prevent necrosis/apoptosis pathways, protect cardiac

Please cite this article as: A. Maksin-Matveev, et al., Sirtuin 6 protectdoi.org/10.1016/j.yexcr.2014.07.013

cells from hypoxic stress and lead to the improved survival ofcardiomyocytes following hypoxia.

The roles of sirtuins, a family of NAD-dependent histonedeacetylase, in age-related diseases have been intensivelyexplored in the last decade. The best-characterized sirtuin, SIRT1,has been indicated to protect cardiomyocytes from oxidativestress-mediated cell death and retard certain cardiac degenerativechanges associated with aging. There are indications that there isa cross talk between SIRT1 and SIRT6. It was demonstrated thatSIRT1 regulates SIRT6 by forming a complex with FOXO3a andnuclear respiratory factor 1 (NRF1) on the promoter of SIRT6.

s the heart from hypoxic damage, Exp Cell Res (2014), http://dx.

Fig. 8 – pAMPKα a major pathway in SIRT6 cardioprotection. Cardiomyocyte cultures fromwild type (WT) and from transgenic (TG)mice were subjected to 120 min hypoxia and their pAMPKα expression was analyzed using Western blotting (A, B). Heart culturesfrom WT and from TG mice were treated for 24 h with 100 lM Compound C (ComC, pAMPKα inhibitor), and then subjected to120 min hypoxia. LDH released to the medium was assayed (C). ComC reduced p-AMPKα protein level (D, E) and the protection bySIRT6 was reduced (C). Values are average7SEM of enzyme released from the cells. Western blot calculations are normalized toβ-actin. npo0.01, #po0.05, nnpo0.005, n¼4.

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In turn, SIRT6 deacetylates lysine 9 of histone H3 (H3K9) on thepromoters of many genes, which have an essential role inglycolysis and lipid metabolism. Over-expression of SIRT1 raisesa different protection pathway than SIRT6. SIRT1 causes Aktactivation leading to cardioprotection from hypertrophy, whereasSIRT6 reduces Akt and also leads to cardioprotection [30,35,36].We tested SIRT6 protein in cardioprotection against hypoxicdamage.

During hypoxia, the heart cells are damaged by lack of oxygen andthe cell membrane is impaired; therefore LDH or CK is released tothe medium. We found that cardiomyocyte cultures from the over-expression of SIRT6 mice released reduced LDH or CK to the mediumcompared to wild type mice. We also measured the uptake of PIwhich is an intercalating agent and a fluorescent molecule thatbinds to nucleic acids. PI is commonly used to identify damaged cellsin a population. TG mice cardiomyocytes were more resistant tohypoxic stress than the heart cells from WT mice. After thisnoteworthy finding we investigated the molecular mechanismthrough which SIRT6 offers cardioprotection. We tested some keyproteins that are known to change during hypoxia. We examinedthe anti-oxidative function of SIRT6. During hypoxia, ROS increase

Please cite this article as: A. Maksin-Matveev, et al., Sirtuin 6 protectdoi.org/10.1016/j.yexcr.2014.07.013

dramatically [21,26]. Those reactive molecules may cause significantdamage to the cell structure and inflict apoptosis or necrosis to thecells. We found that ROS levels in TG cardiomyocytes compared toWT cells were reduced following hypoxia. This finding may suggestthe existence of an anti-oxidant function of SIRT6 that could explaina mechanism of cardioprotection.The next step was to examine the anti-apoptotic function of SIRT6.

We therefore studied Bcl2 protein levels, a regulator protein thatdemonstrates anti-apoptotic activity [27–29]. We found that Bcl2 inTG mice cells was elevated; therefore SIRT6 increases Bcl2 proteinlevels, and thus SIRT6 acts like an anti-apoptotic factor also possiblyexplaining the route to cardioprotection.Another protein that was investigated was pAkt. It was found

that resveratrol (RSV), an activator of SIRT1, induced SIRT1 over-expression, protected cardiomyocytes from oxidative injury andcell death induced by ischemia-reperfusion, through activation ofmitogen-activated protein kinase (MAPK) pathway via Akt signal-ing [36]. The latter is known as an activator of mTOR (mammaliantarget of rapamycine). Akt is also known to regulate cell growth,cell proliferation and cell survival [30,31]. We wondered whetherSIRT6 decreases pAkt activation and thus confers protection on

s the heart from hypoxic damage, Exp Cell Res (2014), http://dx.

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cardiomyocytes. The results show that pAkt is reduced in hypoxia inthe over-expression of SIRT6; therefore m-TOR activity might also bereduced which might clarify the mode of cardioprotection. However,pAkt is increased in hypoxia in WT cells. The explanation could bethat this is a defense protein and cells try to activate defensepathways to survive. Yet not all protection pathways are vital forsurvival. Similarly for the pAkt pathway, which raises the activity ofthe m-TOR protein, can cause damage to cardiomyocytes.During hypoxia, inflammation factors, such as NFκB, increase

and cause cell damage. It is known that SIRT6 binds physically top-65 (RELA), a subunit of NFκB and inhibits it [24,32]. Therefore,several inflammatory genes or age-related diseases that areincited by p-65 are silenced. Using a co-immunoprecipitationassay, we examined SIRT6–p-65 physical binding complex. Theresults confirmed that SIRT6 binds physically to p-65, and thusinhibits p-65 function as a transcription factor. In addition, wetried to inhibit NFκB with Pyrrolidinedithiocarbamic acid ammo-nium salt (PLDC, 100 μM for 24 h) and to investigate whether asynergistic effect with SIRT6 is obtained (data not shown). Thiseffect was not observed. A possible explanation for these results isthat the PLDC inhibitor inhibits phosphorylation of the inhibitorysubunits of NFκB, iκb-α, and so NFκB remains in the cytoplasmand is not free to move to the nucleus for its activity as atranscription factor. Therefore, NFκB was hardly observed in thenucleus (data not shown). It is worth mentioning that SIRT6 alsoprotected cardiomyocytes from hypertrophy via the inhibition ofNFκB-dependent transcriptional activity [35]. In addition, SIRT1overexpression prevented the age-dependent increase in cardiachypertrophy and cardiac dysfunction [37].NOS are a family of enzymes catalyzing the production of nitric

oxide (NO) from L-arginine. NO is an important cellular signalingmolecule. It modulates vascular tone, insulin secretion, airwaytone, peristaltic movements, angiogenesis and neural develop-ment. In addition, NOS activates cyclic guanosine monophosphate(cGMP), which induces mitochondrial KATP channel opening. Thispathway confers a “pre-conditioning effect” resulting in cardio-protection from hypoxia [38]. It was demonstrated that theexpression of eNOS and SIRT1 is up-regulated by resveratrol.These data help in the identification of molecular mechanismsthat may contribute to the beneficial effects on the cardiovascularsystem of resveratrol [39].To test the hypothesis that elevation of SIRT6 increases NOS

activity thereby producing cGMP, which opens the KATP channeland affords cardioprotection, we treated cardiac cells from WTand TG mice with L-NAME (NOS inhibitor, 100 μM) for 24 h beforehypoxia. Our results demonstrated that inhibition of NO produc-tion reduced the cardioprotection in TG mice, suggesting thatSIRT6 increases NOS activity, thereby leading to cardioprotection.AMPK is a heterotrimeric Ser/Thr protein kinase that acts as a

cellular energy sensor and controls several metabolic pathways[5,33,40]. There is accumulating evidence that once activated,such is the case during myocardial ischemia, AMPK not onlyswitches on ATP-generating pathways, but also switches off ATP-consuming biosynthetic pathways, including protein synthesis. Inaddition recently we published that SIRT6 activates AMPK in theliver [34]. Therefore, we investigated whether SIRT6 increasespAMPKα (active form of AMPKα), and thus confers cell home-ostasis during hypoxic stress, and preserves energy and survival ofthe cardiomyocytes under hypoxia. The results reveal that SIRT6elevates pAMPKα protein level. When phosphorylation of AMPKα

Please cite this article as: A. Maksin-Matveev, et al., Sirtuin 6 protectdoi.org/10.1016/j.yexcr.2014.07.013

was inhibited by Compound C (100 μM for 24 h before hypoxia),the protection against hypoxia of cardiac cells from TG mice wasreduced. Therefore, pAMPKα contributes significantly to thecardioprotection pathway through SIRT6.

Recently, it has been shown [35] that in angiotensin II-inducedhypertrophic cardiomyocytes the expression of SIRT6 and SIRT1proteins was upregulated. Over-expression of wild-type SIRT6attenuated angiotensin II-induced cardiomyocyte hypertrophy.It has also been demonstrated that a physical interaction betweenSIRT6 and NFκB catalytic subunit p65, could be repressed by SIRT6over-expression [35]. The authors concluded that the protectionfrom hypertrophy is via the inhibition of NFκB transcriptionalactivity [35]. In our work, we also demonstrated that the protec-tion from hypoxia depends on NFκB but not exclusively. Evenwhen SIRT6 is over-expressed additional proteins are alsoinvolved in the cardioprotection from hypoxia.

In summary, we have shown that age-related deterioration ofthe heart can be inhibited by the over-expression of SIRT6.Furthermore, we demonstrated for the first time the cardiopro-tection pathways used by SIRT6. As SIRT6 plays a direct, causativerole in cardioprotection, our findings support a rationale for usingSIRT6 inducers as a therapeutic means for the hypoxic heart.

Disclosures

I hereby confirm that the authors have no conflict of interest.

Acknowledgments

This research was conducted through the generous support of TheAdar Program for the Advancement of Research in Heart Functionat Bar Ilan University (Grant No. 20-6366).

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