research article rapamycin attenuated cardiac hypertrophy...

Research ArticleRapamycin Attenuated Cardiac HypertrophyInduced by Isoproterenol and Maintained EnergyHomeostasis via Inhibiting NF-120581B Activation

Xi Chen1 Siyu Zeng1 Jian Zou2 Yanfang Chen3 Zhongbao Yue1

Ying Gao1 Luankun Zhang1 Weiwei Cao1 and Peiqing Liu1

1 Department of Pharmacology and Toxicology School of Pharmaceutical Sciences Sun Yat-sen University Guangzhou 510006 China2Department of Pharmacy Chengdu Fifth Peoplersquos Hospital Chengdu 611130 China3The Second Affiliated Hospital of Guangzhou Medical University Guangzhou 510260 China

Correspondence should be addressed to Peiqing Liu liupqmailsysueducn

Received 10 March 2014 Revised 11 May 2014 Accepted 14 May 2014 Published 19 June 2014

Academic Editor Elaine Hatanaka

Copyright copy 2014 Xi Chen et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Rapamycin also known as sirolimus is an immunosuppressant drug used to prevent rejection organ (especially kidney)transplantation However little is known about the role of Rapa in cardiac hypertrophy induced by isoproterenol and its underlyingmechanism In this study Rapa was administrated intraperitoneally for one week after the rat model of cardiac hypertrophyinduced by isoproterenol established Rapa was demonstrated to attenuate isoproterenol-induced cardiac hypertrophy maintainthe structure integrity and functional performance of mitochondria and upregulate genes related to fatty acid metabolism inhypertrophied hearts To further study the implication of NF-120581B in the protective role of Rapa cardiomyocytes were pretreatedwith TNF-120572 or transfected with siRNA against NF-120581Bp65 subunit It was revealed that the upregulation of extracellular circulatingproinflammatory cytokines induced by isoproterenol was able to be reversed by Rapa which was dependent on NF-120581B pathwayFurthermore the regression of cardiac hypertrophy and maintaining energy homeostasis by Rapa in cardiomyocytes may beattributed to the inactivation of NF-120581B Our results shed new light on mechanisms underlying the protective role of Rapa againstcardiac hypertrophy induced by isoproterenol suggesting that blocking proinflammatory response by Rapamight contribute to themaintenance of energy homeostasis during the progression of cardiac hypertrophy

1 Introduction

Cardiac hypertrophy was induced by many kinds of physi-ological and pathophysiological stimuli including exercisepressure or volume overload endocrine disorders and valvu-lar heart diseases [1] Although it has been viewed as atemporary compensatory mechanism for hearts to diminishwall stress cardiac hypertrophy is associated with increasedrisks of developing decompensatory heart failure [2]

Hearts consume more energy than any other organs tomaintain excitation-contraction coupling As a result com-mon heart diseases either myocardial ischemia or myocar-dial infarction were characterized by energymetabolismdys-regulation It has been demonstrated that fatty acid utilizationwas decreased significantly resulting in energy insufficiency

that exacerbated the progression of heart diseases [3] Thereduction in fatty oxidation led to the abnormal accumula-tion of intracellular lipid which could be lipotoxic to hearts[4] Mitochondria provide ATP to hearts for preservingcontractile and relaxation functions through conversion ofmetabolism substrates to usable energy [5] Thus it is ofgreat importance for hearts to maintain structure integrityand functional performance of mitochondria as well as theefficiency in utilization of fatty acid [6]

Inducements of cardiac hypertrophy are of great com-plexities The retention of salt and water as well as excessiveperipheral vasoconstriction was to blame for the disease pro-gression And prior to overtly symptomatic decompensatorycardiac hypertrophy sympathetic nervous system (SNS) acti-vation was induced to increase the cardiac output at the onset

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 868753 15 pageshttpdxdoiorg1011552014868753

2 Mediators of Inflammation

of heart disease [7] however the persistent stimulation ofneurohormonal factors contributes to the mechanical dys-function of hearts ultimately [8] It had been demonstratedthat overexpression of biologically active neurohormonalmolecules contributed to the progression of decompensatedcardiac hypertrophy independently of the hemodynamicstatus by virtue of the direct toxic effects of these neuro-hormonal molecules exert on hearts [9] Isoproterenol isone of these biologically active neurohormonal molecules Inaddition to its direct cardiac effect isoproterenol was provedto have a cardiac trophic effect via stimulation of the reninangiotensin system (RAS) [10]

Rapamycin is a clinically used immunosuppressor in pre-venting transplant rejection [11] and restenosis in coronaryarteries following balloon angioplasty Recently Rapa wasdemonstrated to ameliorate cardiac remodeling and improvethe left ventricular function after myocardial infarction [12]Besides it has been reported that Rapa regressed left ventric-ular hypertrophy in renal transplant recipients regardless ofblood pressure changes suggesting nonhemodynamic-effectmechanisms of rapamycin on left ventricular mass [13]Thusit is worthy of investigation that whether the treatment ofRapa was capable of regressing cardiac hypertrophy inducedby persistent stimulation of sympathetic nervous systemAndthe underlying mechanisms of Rapa in protecting againstcardiac hypertrophy induced by isoproterenol remain to beelucidated

Based on these considerations mentioned above weestablished the rat model of cardiac hypertrophy inducedby isoproterenol and then Rapa was administrated to eval-uate the cardiac function and mitochondrial functionalperformance In this study Rapa was observed to attenu-ate isoproterenol-induced cardiac hypertrophy and main-tain energy homeostasis in adult rats Increased levels ofproinflammatory cytokines were detected in cardiomyocytesin response to isoproterenol which could be substantiallyattenuated byRapaMoreover these protective effects of Rapamight be attributed to the inactivation of NF-120581BThe presentdata revealed that the role of Rapa in protecting hearts fromisoproterenol-induced cardiac hypertrophy and maintainingenergy homeostasis was dependent on NF-120581B pathway

2 Materials and Methods

21 Animal Models of Cardiac Hypertrophy and ExperimentalProtocols Male Sprague-Dawley rats weighing 225 g to 250 gwere purchased from Animal Breeding Center of Sun Yat-sen University Isoproterenol (55mgKg Merck) dissolvedin saline was injected subcutaneously for consecutive tendays Control animals underwent the same procedure exceptthat saline vehicle was injected subcutaneously instead ofisoproterenol After the infusion of isoproterenol rats sur-viving were assigned to the following groups randomly avehicle-treated group (vehicle 119899 = 6) a rapamycin-treatedgroup (12mgkg Selleck 119899 = 6) Treatments of Rapa wereinjected intraperitoneally daily for seven days The doses weadministrated here were comparable with those used in micestudies when normalized by body surface area [12 14] The

experiments were approved by the ethics Committees of SunYat-sen University of medical Science

22 Echocardiographic Studies andHistological Analysis Ratswere anesthetized with 2 isoflurane allowing spontaneousbreathing The Vevo 2100 scanner equipped with a 16MHzProbe (Visual Sonics Toronto Canada) was used to per-form echocardiography analysis After a good-quality two-dimensional image was obtained M-mode images of leftventricles were recorded Left ventricular anterior and poste-rior wall thickness (LVAWand LVPW) fractional shortening(LVFS) and ejection fraction (LVEF) were measured andanalyzed

Once all rats were sacrificed the hearts were washedwith PBS and dried to weigh and fixed within 4 paraformovernight Paraffin-embedded sections (7 120583m) were stainedwith hematoxylin and eosin Slides were examined withbrightfield microscopy and times400 images were capturedThe mean myocyte diameter was calculated by randomlymeasuring 100 cardiomyocytes in 10 randomly chosen fieldsaccording to method and criteria reported [15 16]

23 Transmission Electron Microscopy Analysis Cardiac tis-sues were cut into 1mm cubes immediately after sacri-fice and fixed in 25 glutaraldehyde and then postfixedin 1 osmium tetroxide After acetone dehydration andembedding the ultrathin sections were stained as methodsdescribed [17] Images were taken at 120KV with JEM1400electron microscopy equipped with a high-performanceCCD camera

24Mitochondrial Oxygen Consumption Hearts were freshlyexcised and the apex of left ventricle (200mg) was obtainedfrom hearts in three experimental groups Then freshmitochondria were isolated using mitochondria extrac-tion and isolation kit (GenMed Scientifics) according tothe manufacturerrsquos instruction Clark-type oxygen electrode(Strathkelvin Scotland) was used to determine the rate ofmitochondrial respiration (25∘C) The respiration mediumwas prepared asmethod reported [18] Following the additionof 1mM adenosine diphosphate (ADP) the state III respira-tion was recorded After the complete phosphorylation of theadded ADP the state IV respiration was recorded as wellTherespiration control rate (RCR) which reflects the function ofmitochondria was represented by the ratio of rate of state IIIrespiration to the rate of state IV respiration Mitochondrialprotein concentration was determined by BCA protein assaykit (Thermo Scientifics) The rates of oxygen consumptionwere expressed as nmol O

2minmg

25 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) According to the manufacturerrsquos instruction myocar-dial RNA was extracted from snap-frozen tissues with Trizolreagent (Invitrogen) The SYBR-Green Quantitative PCR kit(Takara) was used to determine the mRNA expression levelsof each target gene by iCycler iQ system (Bio-Rad) AllPCR reactions were performed in triplicate Specific primer

Mediators of Inflammation 3

Table 1 Primer sequences for qRT-PCR

Target gene Sequence

ANF Forward 51015840-GGAAGTCAACCCGTCTCA-31015840

Reverse 51015840-AGCCCTCAGTTTGCTTTT-31015840

BNP Forward 51015840-TTTGGGCAGAAGATAGACCG-31015840

Reverse 51015840-AGAAGAGCCGCAGGCAGAG-31015840

120573-MHC Forward 51015840-GACAACGCCTATCAGTACATG-31015840

Reverse 51015840-TGGCAGCAATAACAGCAAAA-31015840

GAPDH Forward 51015840-AGGAGTAAGAAACCCTGGAC-31015840

Reverse 51015840-CTGGGATGGAATTGTGAG-31015840

CPT-1120573 Forward 51015840-TCAAGGTTTGGCTCTATGAGGGCT-31015840

Reverse 51015840-TCCAGGGACATCTTGTTCTTGCCA-31015840

CPT-2 Forward 51015840-TCCTGCATACCAGCAGATGAACCA-31015840

Reverse 51015840-TATGCAATGCCAAAGCCATCAGGG-31015840

MCAD Forward 51015840-CTGCTCGCAGAAATGGCGATGAAA-31015840

Reverse 51015840-CAAAGGCCTTCGCAATAGAGGCAA-31015840

LCAD Forward 51015840-AATGGGAGAAAGCCGGAGAAGTGA-31015840

Reverse 51015840-GATGCCGCCATGTTTCTCTGCAAT-31015840

IL-1120573 Forward 51015840-TCCTCTGTGACTCGTGGGAT-31015840

Reverse 51015840-TCAGACAGCACGAGGCATTT-31015840

IL-2 Forward 51015840-CCAAGCAGGCCACAGAATTG-31015840

Reverse 51015840-TCCAGCGTCTTCCAAGTGAA-31015840

TNF-120572 Forward 51015840-TGGCGTGTTCATCCGTTCTC-31015840

Reverse 51015840-CCCAGAGCCACAATTCCCTT-31015840

PGC-1120572 Forward 51015840-ACGAAAGGCTCAAGAGGGACGAAT-31015840

Reverse 51015840-CACGGCGCTCTTCAATTGCTTTCT-31015840

PPAR120572 Forward 51015840-AGCTCAGGACACAAGACGTTGTCA-31015840

Reverse 51015840-AGGGACTTTCCAGGTCATCTGCTT-31015840

120573-actin Forward 51015840-TCGTGCGTGACATTAAAGAG-31015840

Reverse 51015840-ATTGCCGATAGTGATGACCT-31015840

sequences synthesized by Invitrogen were listed in Table 1GAPDH and 120573-actin served as an endogenous control

26 ATP Concentration Measurements Using an ATP biolu-minescent assay kit (Sigma) myocardial ATP concentrationwasmeasured according to themanufacturerrsquos recommenda-tions Experiments were done in triplicate for each group

27 Western Blot Analysis Using a commercial availableNuclear and Cytoplasm Extraction Kit (Active Motif)nuclear proteins were isolated from left ventricles of ratsor primary neonatal rat ventricular cardiomyocytes Thehomogenate proteins were separated on SDS-PAGE and thentransferred to PVDFmembrane (Millipore)Themembraneswere incubated with primary polyclonal antibodies againstp65 (Cell Signal Technology) I120581B-120572 (Cell Signal Technol-ogy) Histone-H3 (Sigma) p-mTOR (Cell Signal Technol-ogy)mTOR (Cell Signal Technology) PPAR120572 (Sigma) PGC-1120572 (Calbiochem) GAPDH (Sigma) and 120572-tubulin (Sigma)overnight at 4∘C After washing in TBS-T buffer the secondantibodies (Promega) were conjugated at room temperature

and then protein bands were visualized using enhancedSuper-Signal West Pico substrates (Pierce)

28 Primary Culture of Neonatal Rat Ventricular Cardiomy-ocytes and Treatments Primary culture of neonatal rat ven-tricular cardiomyocytes was prepared using 1- to 3-day-oldSprague-Dawley rats as methods we reported [19] Cells werecultured in 10 serum for 24 hours after isolation 48 hourslater the serum-containing culture medium was replacedwith DMEM containing 01 FBS After incubation for 24hours the cells were further treated with isoproterenol (1120583molL) or in combinationwith rapamycin (100 nmolL)wasadministrated to cells and maintained for 24 hours

29 Small Interference RNA The small interference RNA(siRNA) sequences against rat NF-120581B p65 subunit weresynthesized by GenePharma NF-120581B p65 antisense sequenceswere 51015840-UCUAUGGGAACUUGAAAGGTT-31015840 the scram-bled sequence was used as a negative control (NC) Smallinterference RNA (100 nmolL) was transiently transfectedinto cardiomyocytes by adding it to lipofectamine 2000


(Invitrogen) according to the manufacturerrsquos instructionsThe knockdown of p65 protein expression was confirmed bywestern blot analysis

210 TNF-120572 IL-1120573 and IL-2 Measurement An ELISA kit(Dakewe) was used to determine the concentrations of IL-1120573 IL-2 and TNF-120572 in the culture medium according to themanufacturerrsquos instructions

211 Cell Surface Area Quantification To determine the cellsurface area cells were fixed with 4 paraform and thenpermeabilized within 05 TritonX-100 after which cellswere incubated with TRITC-labeled phalloidin (Sigma) for30min at room temperature as methods reported [20]

212 Mitochondrial Membrane Potential Analysis TMRE(10 nmolL) a mitochondrial potential-indicating fluoro-phore (Invitrogen Molecular Probes) was added to cells for30min after which cells were visualized under confocal laserscanning microscopy (LSM710 Zeiss) Images were analyzedas reported previously [21]

213 Statistical Analysis Data were expressed as mean plusmn SDAll results were obtained from three independent experi-ments SPSS 170 for statistic software was used to performall data analyses Statistical significance of difference amongexperimental groups was assessed with one-way analysis ofvariance (ANOVA) followed by the Bonferroni post hoc testThe level of 119875 lt 005 was considered to be statisticallysignificant in all cases

3 Results

31 Rapa Attenuated Cardiac Hypertrophy Induced by Isopro-terenol in Adult Rats We first determined whether the treat-ment of Rapa after consecutive isoproterenol-infusion for tendays reverses indices ofmyocardial hypertrophyThe ratios ofheart weight to body weight (HWBW) and left ventricularweight to body weight (LVWBW) were significantly upreg-ulated in isoproterenol-infusionmodel group compared withthose in control group (119875 lt 001 versus control) As shown inFigure 1(a) rats treated with Rapa reduced the isoproterenol-induced increase in the ratios of bothHWBWand LVWBWsignificantly (HWBW 246 plusmn 035 versus 315 plusmn 039 119875 lt001 versus model LVWBW 166 plusmn 028 versus 240 plusmn 055119875 lt 001 versus model) Histological analysis revealedthat hearts under isoproterenol stimulation demonstrateddirect evidence of increased mean myocyte diameters whichwas reversed in the Rapa-treated hearts (Figures 1(b) and1(c)) Consistently echocardiographic analysis showed thatRapa reduced the isoproterenol-induced increases in thethicknesses of left ventricular anterior and posterior wallsduring systole (LVAWs LVPWs) (LVAWs 252 plusmn 021 versus322 plusmn 020 119875 lt 0001 versus model LVPWs 280 plusmn 027versus 322plusmn016119875 lt 005 versusmodel) On the other handthe cardiac function which was represented by LVEF andLVFS was not changed (119875 = ns versus model) (Figure 1(d))

Additionally real-time PCR analysis was performed toquantify the expression of hypertrophicmarker genes includ-ing ANF BNP and 120573-MHC (Figure 1(e)) As expected levelsof ANF BNP and 120573-MHC mRNA were increased about 2-fold in model group (119875 lt 005 versus control) In contrastRapa treatment significantly decreased the expression ofthese marker genes comparing with the model group (119875 lt005 versus model)

32 Effects of Rapa on the Energy Homeostasis in Hyper-trophied Hearts TEM analyses were used to examine theultrastructures of mitochondria As seen from Figure 2(a)disorganized cristae and vacuoles of mitochondria wereobserved in the hypertrophied hearts in contrast the struc-ture of mitochondria was well organized and the cristaestayed intact and evident in Rapa-treated hearts

In the hypertrophied hearts treated with Rapa stateIII respiration rates were significantly increased comparedwith those in hypertrophied hearts (119875 lt 001 versusmodel) This meant the dysfunction of mitochondria in thehypertrophied hearts could be attenuated by Rapa treat-ment (Figure 2(b)) Consistently the production of ATP wassignificantly decreased in the hypertrophied hearts infusedwith isoproterenol which was substantially reversed by Rapatreatment (6774plusmn1004 versus 4978plusmn1109 119875 lt 005 versusmodel) (Figure 2(d)) suggesting that the efficiency of energysupply was maintained by Rapa

In order to investigate the role of Rapa in regulatingfatty acid metabolism genes in hypertrophied hearts wesubsequently performed quantitative real time-PCR analysisamong experimental groups As shown in Figure 2(e) thetreatment of Rapa prevented the declines in representativemetabolic target genes associated with fatty acid metabolismincluding carnitine palmitoyl transferase-1120573 and -2 (CPT-1120573 and CPT-2) and medium- and long-chain acyl-CoAdehydrogenase (MCADandLCAD) (119875 lt 005 versusmodel)

33 The Activation of NF-120581B Pathway in HypertrophiedHearts Was Inhibited by Rapa Treatment Upon activationby hypertrophic stimuli p65 the subunit of NF-120581B rapidlyenters the nucleus which resulted in elevated expression ofhypertrophic genes [22] We next examined the effects ofRapa treatment on the nuclear translocation of p65 and thedegradation of I120581B-120572 in adult rats The persistent stimulationof isoproterenol significantly promoted the translocation ofp65 from cytoplasm to the nucleus which were preventedby Rapa treatment (119875 lt 005 versus model) (Figures 2(f)and 2(g)) Consistently as shown in Figures 2(h) and 2(i) thedegradation of I120581B-120572 was detected to increase in response toisoproterenol stimulation (119875 lt 005 versus control) and thiseffect can be reversed by Rapa (119875 lt 005 versus model)

34 Effects of Rapa on Proinflammatory Cytokines Releaseand Gene Expression in Cardiomyocytes via NF-120581B PathwayP65 was knocked down in neonatal rat cardiomyocytes usingsiRNA and TNF-120572 a cytokine known to activate NF-120581Bsignal pathway was used to further clarify the role of NF-120581B in the protective effects of Rapa in cardiomyocytes It has


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Figure 1 Rapa attenuated cardiac hypertrophy induced by consecutive infusion of isoproterenol (a) Changes in the ratio of heart weightto body weight (HWBW) and the ratio of left ventricular weight to body weight (LVWBW) Values represented mean plusmn SD of 6 rats ineach group (b) Representative high magnification (times400) of left ventricular stained with hematoxylin and eosin from each group (c) Meanmyocyte diameter was calculated by randomly measuring 100 cells from 5 sections in each group (d)The thickness of left ventricular anteriorand posterior wall (left) and left ventricular fraction shortening and left ventricular ejection fraction Echocardiographic measurements wereperformed and values represented as mean plusmn SD of 6 rats in each group (e) Gene expression patterns of ANF BNP and 120573-MHC amongdifferent groupsThemRNAwas prepared and normalized to GAPDH geneModel the cardiac hypertrophymodel induced by isoproterenolRapa isoproterenol-infused rats treated with Rapa Values represented as mean plusmn SD of 4 to 5 rats in each group lowast119875 lt 005 versus controlgroup lowastlowast119875 lt 001 versus control group lowastlowastlowast119875 lt 0001 versus control group 119875 lt 005 versus model group 119875 lt 001 versus model groupand 119875 lt 0001 versus model group

been shown that the protein expression of p65 in culturedcardiomyocytes was increased by TNF-120572 in a concentration-dependent manner by western blot analysis (see Supplemen-tary Figure 1(a) in Supplementary Material available onlineat httpdxdoiorg1011552014868753) and p65 depletionwas proved to decrease the protein expression of p65 (Sup-plementary Figure 1(c)) in cardiomyocytes

To investigate whether the activation of the NF-120581Bpathway is implicated in proinflammatory gene expressionsand cytokines releases induced by isoproterenol the extra-cellular production of cytokines was measured by ELISA Asshown in Figures 3(a) 3(b) and 3(c) releases of IL-1120573 IL-2 and TNF-120572 were significantly induced by isoproterenol(119875 lt 001 versus control) In contrast the administration ofRapa to cardiomyocytes attenuated the increases in releasesof proinflammatory cytokines (119875 lt 005 versus model)and this effect could be abrogated by TNF-120572 (20 ngmL)Then real-time PCR analysis was performed to quantify theexpression of representative cytokine genes The stimulationof isoproterenol led to increases in mRNA levels of IL-1120573 IL-2 and TNF-120572 (119875 lt 005 versus control) whichcould be reversed by Rapa (Figure 3(d)) Furthermore itwas revealed that this downregulation of proinflammatorycytokines by Rapa was reversed by TNF-120572 (20 ngmL)

whereas the anti-inflammatory effect of Rapa was preservedwhen NF-120581Bp65 was depleted in cardiomyocytes

35 NF-120581B Participates in the Antihypertrophic Effect ofRapa In order to explore whether the activation of theNF-120581B pathway is implicated in the underlying mechanismof the inhibitory effects of Rapa on cardiac hypertrophyimmunofluorescence analyses and real-time PCR analyseswere performed in primary cultured neonatal cardiomy-ocytes As shown in Figures 4(a) and 4(b) the surface areaof cardiomyocytes as well as mRNA levels of hypertrophiedmaker genes was increased significantly in response to iso-proterenol stimulation for 24 hours (119875 lt 005 versus control)In contrast the administration of Rapa to cardiomyocytesattenuated the increases in hypertrophy biomarkers (119875 lt 005versus model) and this protective effect could be abrogatedby TNF-120572 (20 ngmL) In contrast this antihypertrophiceffect of Rapa was maintained after p65 depletion in car-diomyocytesThese results suggested the involvement of NF-120581B inactivation in the protective effect of Rapa against cardiachypertrophy induced by isoproterenol

36 The Role of Rapa in Maintaining Energy HomeostasisWas Dependent on NF-120581B To investigate the role of NF-120581B



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Figure 2 Rapa maintained energy homeostasis and inhibited NF-120581B activation in hypertrophied hearts induced by isoproterenol (a) Theultrastructures of the mitochondria were analyzed under TEM Transmission electron micrographs of histological sections from the apexof left ventricle with different treatments were shown Black arrows indicate disorganized cristae and vacuoles of mitochondria in thehypertrophied hearts White arrows indicate autophagosomes Scale bars 1 120583m (b) State III and state IV respiration rates (c) Respiratorycontrol rate (RCR) was calculated as the ratio of state III respiration rates to state IV respiration rates Values represented as mean plusmn SD of 6rats in each group (d) ATP production Values represented as mean plusmn SD of 6 rats in each group (e) Expression of metabolic genes associatedwith fatty acid metabolism at mRNA levels The mRNA was prepared and normalized to 120573-actin gene Values represented as mean plusmn SD of 4to 5 rats in each group ((f) and (h)) Cytoplasmic and nuclear extracts from left ventricles of three groups of rats were immunoblotted withanti-p65 and anti-I120581B-120572 antibodies ((g) and (i)) Densitometric analysis of the relative protein expression of p65 and I120581B-120572 Histone H3 and120572-tubulin were used to normalize the nuclear and cytosolic proteins loading respectively Values represented as mean plusmn SD of 4 to 5 rats ineach group Model the cardiac hypertrophy model induced by isoproterenol Rapa isoproterenol-infused rats treated with Rapa lowast119875 lt 005versus control group lowastlowast119875 lt 001 versus control group 119875 lt 005 versus model group and

119875 lt 001 versus model group

pathway in preserving mitochondrial function in cardiomy-ocytes TMRE a sensitive fluorescent probe that reflects thelevel of membrane potential were used to assess the capacityof mitochondria to produce ATP As seen from Figure 4(c)a significant loss of membrane potential was shown in

cardiomyocytes treated with isoproterenol (119875 lt 0001 versuscontrol) which could be abolished partially by Rapa (119875 lt001 versus model) Consistent with TMRE staining theATP production in cardiomyocytes was decreased notably inresponse to isoproterenol stimulation compared with vehicle


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Figure 3 NF-120581B participates in the role of Rapa in reducing pro-inflammatory cytokines release and gene expressions in cardiomyocytes((a) (b) and (c)) ELISA was used to determine the release of IL-1120573 IL-2 and TNF-120572 into the cultured medium (d) Real-time PCR analysiswas performed to quantify the mRNA levels of proinflammatory cytokines The mRNA was prepared and normalized to 120573-actin gene 119899 = 3experiments in duplicate Error bars SD lowastlowast119875 lt 001 versus control group lowastlowastlowast119875 lt 0001 versus control group 119875 lt 005 versus model groupand 119875 lt 001 versus model group

control (119875 lt 001 versus control) And this reductionin conversion of metabolic substrates into usable energycan be substantially attenuated by Rapa (119875 lt 005 versusmodel) (Figure 4(d)) Moreover it was observed that theeffect of Rapa on preservingmitochondrial function andATPproduction in hypertrophied cardiomyocytes was abrogatedby TNF-120572 (20 ngmL) In contrast the protective role of Rapa

in maintaining energy homeostasis was preserved after p65depletion in cardiomyocytes

37 The Role of Rapa in Upregulating Genes Associated withFatty Acid Metabolism Was Dependent on NF-120581B In orderto explore the mechanisms underlying the protective role ofRapa in maintaining energy homeostasis the activation of


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Rela

tive T

MRE

fluor

esce

nce

P lt 001P lt 005

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

lowastlowastlowast lowastlowastlowast

(c)

0

10

20

30

40

50

ATP

conc

entr

atio

n(120583

Mg

cell

prot

ein)

Con

trol

Mod

elM

odel+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

P lt 0001

P lt 0001


(d)

Figure 4 NF-120581B participates in the protective role of Rapa in regressing cardiac hypertrophy and energy homeostasis (a) Cardiac myocyteswere stained with rhodamine-phalloidin followed by cell surface area quantitation (b) Gene expression pattern of atrial natriuretic factor(ANF) brain natriuretic peptide (BNP) and 120573-myosin heavy chain (120573-MHC) among different groups The mRNA was prepared andnormalized to GAPDH gene (c) Fluorescence of mitochondrial in TMRE loaded active cardiac myocytes under different treatments Therelative values of TMRE fluorescence intensity was normalized to 10 Scale bar 20120583m The results were obtained from three independentexperiments (d) ATP production 119899 = 3 experiments in duplicate Error bars SD lowast119875 lt 005 versus control group lowastlowast119875 lt 001 versus controlgroup lowastlowastlowast119875 lt 0001 versus control group 119875 lt 005 versus model group 119875 lt 001 versus model group and

119875 lt 0001 versus modelgroup


mTOR which serves as a sensor of the energy state [23]was determined by western blot analysis It was observedthat the administration of isoproterenol to cardiomyocytesfor 24 hours decreased the phosphorylation of mTOR atthe site of serine 2448 (119875 lt 001 versus control) whichcould be substantially attenuated by Rapa (Figures 5(a) and5(b)) Furthermore the mRNA and protein expression of itsdownstream transcriptional factor PPAR120572 and coactivatorPGC-1120572 were determined As shown in Figures 5(c) 5(d)and 5(e) the downregulation of PPAR120572 and PGC-1120572 in bothmRNA and protein levels could be inhibited by Rapa (119875 lt005 versus model) and these effects of Rapa were abrogatedby TNF-120572 (20 ngmL)

Besides the mRNA levels of medium- and long-chainacyl-CoA dehydrogenase (MCAD and LCAD) and carnitinepalmitoyl transferase-1120573 and -2 (CPT-1120573 and CPT-2) werenotably decreased by isoproterenol which could be partiallyreversed by Rapa (119875 lt 001 versusmodel) however the effectof Rapa on upregulating these genes associated with fattyacidmetabolismwas abrogated by the pretreatment of TNF-120572(20 ngmL) (Figures 5(f) and 5(g)) Collectively these resultssuggested the involvement of NF-120581B inactivation in the roleof Rapa in upregulating genes associated with fatty acidmetabolism in cardiomyocytes treated with isoproterenol

4 Discussion

In this study it was demonstrated that the regression ofcardiac hypertrophy by Rapa in adult rat was associated withattenuation of the increases inmyocyte cell size andHWBWwithout loss of left ventricular function Activated NF-120581Bpathway was detected in the hypertrophied hearts whichwas substantially reversed byRapaMoreover Rapa protectedcardiomyocytes from the mitochondrial dysfunction andabnormal energy utilization which could be attributed tothe inactivation of NF-120581B Conversely activation of NF-120581Bpathway resulted in the catastrophic loss of ATP productionin cultured cardiomyocytes and abrogated the protectiverole of Rapa in regressing cardiac hypertrophy Our resultsrevealed the mechanisms underlying the protective role ofRapa against cardiac hypertrophy induced by isoproterenolsuggesting that blocking proinflammatory response by Rapamight contribute to the maintenance of energy homeostasisduring the progression of cardiac hypertrophy

Long-lived postmitotic cells including neurons and car-diomyocytes are most vulnerable to mitochondrial dysfunc-tion [24] In order to evaluate the effect of Rapa on thefunctional performance of mitochondria in hypertrophiedhearts a well-established method was performed [25] RCRwhich reflects the mitochondrial function decreased signif-icantly in the hypertrophied hearts induced by isoproterenol(Figure 2(c)) and these functional changes were associatedwith evidences of structural abnormalities in mitochondria(Figure 2(a)) And consistently these injuries on structureand functional performance of mitochondria caused bypersistent stimulation of isoproterenol could be substantiallyattenuated by Rapa

NF-120581B had been demonstrated to be involved in patho-geneses of inflammatory disorders [26] and viewed as a linkbetween cardiomyopathy and the dysregulation of energymetabolism [27] In this study we observed that the adminis-tration of Rapa to the hypertrophied hearts could reverse theactivation of NF-120581B induced by isoproterenol (Figure 2(f))Rapa was demonstrated to inhibit mTOR activity througha mechanism involving interaction with FKBP12 [28] Andprior to our study this inhibition ofmTORwas proved to sup-press the activity of I-120581B kinase (an essential activator of NF-120581B) through a mechanism that might involve dissociation ofRaptor frommTOR [29] In keeping with these observationswe found that the degradation of I120581B-120572 induced by isopro-terenol was substantially attenuated by Rapa administration(Figure 2(h)) therebymaintainingNF-120581B in an inactive stateHerein we found that the administration of isoproterenolto cardiomyocytes led to an aberrant production of varietiesof proinflammatory cytokines (Figures 3(a) 3(b) and 3(c))which was in line with the activation of NF-120581B pathwayinduced by isoproterenol The nuclear translocated NF-120581Bwas demonstrated to regulate the expression of various geneswhich encode proinflammatory cytokines including TNF-120572[30] Meanwhile the downregulation of cytokine releases byRapa was impaired under TNF-120572 treatment indicating thatthe role of Rapa in restraining the inflammatory responses incardiomyocytes under persistent stimulation of isoproterenolwas NF-120581B-dependent

In the present study the stimulation of isoproterenolpromoted inflammatory responses in hypertrophied car-diomyocytes and Rapa could partially restrain the downregu-lation of PPAR120572 induced by isoproterenol (Figure 5(a)) Onepotential explanation to this effect of Rapa in upregulatingPPAR120572 may be its activity in inactivation of mTOR justas we found in the decrease in the ratio of phosphor-mTOR to total mTOR (Figure 5(b)) Recently it has beenreported that pharmacological or genetic inhibition ofmTORC1 was able to promote the translocation of tran-scription factor EB (TFEB) to the nucleus and increaseits transcription activity [31] More importantly it wasdemonstrated that TFEB controls cellular lipid metabolismtrough a mechanism of upregulation of PGC-1120572 and PPAR120572[32] which were proved to play prominent roles in con-trolling genes mediating process from fatty acid uptake tooxidation including medium-chain acyl-CoA dehydroge-nase (MCAD) and carnitine palmitoyl transferase (CPT)[33 34]

Herein the present results revealed that the adminis-tration of isoproterenol to hearts weakened the expressionof CPT-1120573 and CPT-2 which were proved to be crucial inmediating the rate-limiting reaction of fatty acid transport[35] and this effect could be partially abrogated by Rapa(Figure 2(e)) Moreover clusters of genes associated withfatty acid metabolism downregulated by isoproterenol inhypertrophied cardiomyocytes were activated byRapa whichcould be abrogated by TNF-120572 In contrast the role of Rapain upregulating these genes was maintained by inactivationof NF-120581B in cardiomyocytes (Figures 5(f) and 5(g)) Ourobservations reconciled with those findings concerning thatTNF-120572 impaired fatty acid oxidation in the heart through


-Tubulin

Con

trol

p-mTOR

Total m-TOR

Isop

rote

reno

l

Iso+

Rapa

Iso+

Rapa

+TN

F-120572

Iso+

Rapa

+si

p65

PPAR120572

PGC1120572

120572

(a)

0

1

2

3

p-m

TOR

tota

l mTO

R(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowast

Mod

el+

Rapa

(b)

00

05

10

15

Relat

ive p

rote

in le

vels

of P

PAR120572

(fo

ld ch

ange

)

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(c)

00

05

10

15Re

lativ

e pro

tein

leve

ls of

PG

C-1120572

(fo

ld ch

ange

)

P lt 001

P lt 001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(d)

00

05

10

15

Rela

tive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5



PPAR120572

PGC-1120572

P lt 0001

P lt 001

P lt 001

P lt 001

Mod

el+

Rapa

(e)

00

05

10

15

MCADLCAD

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 001

P lt 001

P lt 001



(f)

00

05

10

15

CPT2

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

CPT1-120573

lowast lowast

lowast

lowastlowastlowast

Mod

el+

Rapa

P lt 001

P lt 001

P lt 005

P lt 005

(g)

Figure 5 NF-120581B participates in the role of Rapa in upregulating fatty acid metabolism in cardiomyocytes induced by isoproterenol (a)Cytoplasmic proteins extracted from cardiomyocytes were immunoblotted with anti-p-mTOR mTOR PPAR120572 and PGC-1120572 antibodies ((b)(c) and (d)) Densitometric analysis of the relative protein expression of the ratio of p-mTORmTOR PPAR120572 and PGC-1120572 And 120572-tubulinwas used to normalize proteins loading ((d) (e) and (f)) Expression of metabolic genes associated with fatty acid metabolism (PPAR120572PGC1120572 MCAD LCAD CPT-1120573 and CPT-2) at mRNA levels The mRNA was prepared and normalized to 120573-actin gene 119899 = 3 experimentsin duplicate Error bars SD lowast119875 lt 005 versus control group lowastlowast119875 lt 001 versus control group lowast119875 lt 0001 versus control group 119875 lt 005versus model group and



activation ofNF-120581Bwhich underlies cardiac dysfunction andheart failure in metabolic diseases [36]

It has been reported that the activation of S6 Kinasewas critical for the development of cardiac hypertrophyin response to pressure overload And Rapa was able toattenuate this induction of cardiac hypertrophy which couldbe attributed to the inhibition of S6 Kinase [37 38] Simi-larly previous in vitro studies had reported that Rapa wasable to attenuate angiotensin-II-induced cardiac hypertro-phy through inactivation of S6 Kinase [39] These resultsindicated that the role of Rapa in regressing of cardiachypertrophy induced by pressure-overload or angiotensin-IIwas associated with inactivation of S6 Kinase which playsan important role in increases in overall protein synthesisin cardiomyocytes In the present study we establishedthe model of cardiac hypertrophy induced by consecutiveinfusion of isoproterenol to mimic the sympathetic stimula-tion [40] Herein proinflammatory response was associatedwith energy metabolism alterations in the model of cardiachypertrophy induced by isoproterenol Rapa was demon-strated to ameliorate energy homeostasis in cardiomyocytesvia inactivation of NF-120581B thereby attenuating establishedcardiac hypertrophy after isoproterenol stimulation Thesefindings suggested that blocking proinflammatory responseby Rapa might contribute to the maintenance of energyhomeostasis and provided rational basis for developmentof novel strategies to prevent cardiac hypertrophy aftersympathetic stimulation

In this study although the thickness of LVAW increasedmarkedly in the rat model of cardiac hypertrophy inducedby isoproterenol the left ventricular function (represented byLVEF and LVFS) was maintained One potential explanationto this paradox might be the compensatory ability of theheart and it would develop heart failure after the infusionof isoproterenol for longer time [41] And our findingswere consistent with the reported data that there was nosignificant decrease in cardiac function of the hypertrophicheart [42] Herein we observed that during the compensatedstate of cardiac hypertrophy induced by isoproterenol Rapawas able to attenuate cardiac remodeling and maintainedenergy homeostasis without loss of cardiac function in adultrat This present study reconciled with the earlier find-ings that autophagy activated by Rapa reduced endotoxin-induced inflammatory responses in epithelial cells maintain-ing intestinal homeostasis ultimately [43] Interestingly ithas been reported that the administration of Rapa was lesseffective in regressing decompensated cardiac hypertrophyin mice than compensated cardiac hypertrophy (40 and70 regression resp) [38] Therefore further studies arerequired to investigatemechanisms underlying the differenceof Rapa in treating cardiac hypertrophy under differentstates

In summary these present in vivo and in vitro studiesdemonstrated the protective role of Rapa in isoproterenol-induced cardiac hypertrophy which was dependent on NF-120581B pathway These findings may provide clues to the patho-physiology of proinflammatory response associated withenergy metabolism alterations during the progression ofcardiac hypertrophy and shed new light on the underlying

mechanism of Rapa in protecting hearts from cardiac hyper-trophy under the persistent stimulation of neurohormonalfactors

Abbreviations

Rapa RapamycinmTOR Mammalian target of rapamycinHW Heart weightLVW Left ventricle weightBW Body weightLVEF Left ventricular ejection fractionLVFS Left ventricular fractional shorteningLVAW Left ventricular anterior wallLVPW Left ventricular posterior wallRCR Respiratory control rateANF Atrial natriuretic factorBNP Brain natriuretic peptideMHC Myosin heavy chainNF-120581B Nuclear factor-kappa BTEM Transmission electron microscopyFBS Fetal calf serumDMEM Dulbecco Modified Eagle MediumTRITC Tetraethyl rhodamine isothiocyanateTNF-120572 Tumor necrosis factor-120572TMRE Tetramethylrhodamine ethyl esterPPAR Peroxisome proliferator-activated receptorPGC-1120572 PPAR120574 coactivator-1120572

Conflict of Interests

The authors have no conflict of interests to disclose

Authorsrsquo Contribution

Xi Chen and Siyu Zeng contributed equally to this paper

Acknowledgments

This work was supported by grants from the National NaturalScience Foundation of China (nos 81072641 81273499)The authors gratefully acknowledge Kunpeng Li (ElectronMicroscopy Research Laboratory Sun Yat-senUniversity) forexcellent assistance with the TEM analysis

References

[1] B H Lorell and B A Carabello ldquoLeft ventricular hypertrophypathogenesis detection and prognosisrdquo Circulation vol 102no 4 pp 470ndash479 2000

[2] P M Okin D A Hille S E Kjeldsen B Dahlof and R BDevereux ldquoPersistence of left ventricular hypertrophy is asso-ciated with increased cardiovascular morbidity and mortalityin hypertensive patients with lower achieved systolic pressureduring antihypertensive treatmentrdquo Blood Pressure vol 23 no2 pp 71ndash80 2013

[3] S Neubauer ldquoThe failing heartmdashan engine out of fuelrdquoTheNewEngland Journal of Medicine vol 356 no 11 pp 1140ndash1151 2007


[4] G D Lopaschuk J R Ussher C D L Folmes J S Jaswal andW C Stanley ldquoMyocardial fatty acid metabolism in health anddiseaserdquo Physiological Reviews vol 90 no 1 pp 207ndash258 2010

[5] A Terman and U T Brunk ldquoMyocyte aging and mitochondrialturnoverrdquo Experimental Gerontology vol 39 no 5 pp 701ndash7052004

[6] M G Rosca B Tandler and C L Hoppel ldquoMitochondria incardiac hypertrophy and heart failurerdquo Journal of Molecular andCellular Cardiology vol 55 no 1 pp 31ndash41 2013

[7] I T Meredith G Eisenhofer G W Lambert E M DewarG L Jennings and M D Esler ldquoCardiac sympathetic nervousactivity in congestive heart failure evidence for increased neu-ronal norepinephrine release and preserved neuronal uptakerdquoCirculation vol 88 no 1 pp 136ndash145 1993

[8] J D Bisognano H D Weinberger T J Bohlmeyer etal ldquoMyocardial-directed overexpression of the human 1205731-adrenergic receptor in transgenic micerdquo Journal of Molecularand Cellular Cardiology vol 32 no 5 pp 817ndash830 2000

[9] K Rivard P Paradis M Nemer and C Fiset ldquoCardiac-specificoverexpression of the human type 1 angiotensin II receptorcauses delayed repolarizationrdquoCardiovascular Research vol 78no 1 pp 53ndash62 2008

[10] M Shimizu H Sasaki J Sanjo et al ldquoEffect of captoprilon isoproterenol-induced cardiac hypertrophy and polyaminecontentsrdquo Japanese Circulation Journal vol 56 no 11 pp 1130ndash1137 1992

[11] R N Saunders M S Metcalfe and M L NicholsonldquoRapamycin in transplantation a review of the evidencerdquoKidney International vol 59 no 1 pp 3ndash16 2001

[12] H Kanamori G Takemura K Goto et al ldquoThe role ofautophagy emerging in postinfarction cardiac remodellingrdquoCardiovascular Research vol 91 no 2 pp 330ndash339 2011

[13] E Paoletti M Amidone P Cassottana M Gherzi L Marsanoand G Cannella ldquoEffect of sirolimus on left ventricular hyper-trophy in kidney transplant recipients a 1-year nonrandomizedcontrolled trialrdquo American Journal of Kidney Diseases vol 52no 2 pp 324ndash330 2008

[14] G Y OuditM A Crackower U Eriksson et al ldquoPhosphoinosi-tide 3-kinase 120574-deficientmice are protected from isoproterenol-induced heart failurerdquoCirculation vol 108 no 17 pp 2147ndash21522003

[15] J Zou K Le S Xu et al ldquoFenofibrate ameliorates cardiachypertrophy by activation of peroxisome proliferator-activatedreceptor-120572 partly via preventing p65-NF120581Bbinding toNFATc4rdquoMolecular and Cellular Endocrinology vol 370 no 1-2 pp 103ndash112 2013

[16] W Oriyanhan H Tsuneyoshi T Nishina S Matsuoka TIkeda and M Komeda ldquoDetermination of optimal durationof mechanical unloading for failing hearts to achieve bridgeto recovery in a rat heterotopic heart transplantation modelrdquoJournal of Heart and Lung Transplantation vol 26 no 1 pp 16ndash23 2007

[17] R Singh S Kaushik Y Wang et al ldquoAutophagy regulates lipidmetabolismrdquo Nature vol 458 no 7242 pp 1131ndash1135 2009

[18] H Bugger M Schwarzer D Chen et al ldquoProteomicremodelling of mitochondrial oxidative pathways in pressureoverload-induced heart failurerdquo Cardiovascular Research vol85 no 2 pp 376ndash384 2010

[19] J J Fu H Gao R B Pi and P Q Liu ldquoAn optimized protocolfor culture of cardiomyocyte fromneonatal ratrdquoCytotechnologyvol 49 no 2-3 pp 109ndash116 2005

[20] Y Li H Zhang W Liao et al ldquoTransactivated EGFR mediates120572 1-AR-induced STAT3 activation and cardiac hypertrophyrdquoAmerican Journal of Physiology Heart and Circulatory Physiol-ogy vol 301 no 5 pp H1941ndashH1951 2011

[21] Y Nakagawa K Kuwahara G Takemura et al ldquoP300 plays acritical role in maintaining cardiac mitochondrial function andcell survival in postnatal heartsrdquo Circulation Research vol 105no 8 pp 746ndash754 2009

[22] J D Molkentin J-R Lu C L Antos et al ldquoA calcineurin-dependent transcriptional pathway for cardiac hypertrophyrdquoCell vol 93 no 2 pp 215ndash228 1998

[23] T P Neufeld ldquoContribution of Atg1-dependent autophagy toTOR-mediated cell growth and survivalrdquo Autophagy vol 3 no5 pp 477ndash479 2007

[24] A Terman T Kurz M Navratil E A Arriaga and U T BrunkldquoMitochondrial Turnover and aging of long-lived postmitoticcells themitochondrial-lysosomal axis theory of agingrdquoAntiox-idants and Redox Signaling vol 12 no 4 pp 503ndash535 2010

[25] A Fritah J H Steel D Nichol et al ldquoElevated expressionof the metabolic regulator receptor-interacting protein 140results in cardiac hypertrophy and impaired cardiac functionrdquoCardiovascular Research vol 86 no 3 pp 443ndash451 2010

[26] S Vallabhapurapu and M Karin ldquoRegulation and function ofNF-120581B transcription factors in the immune systemrdquo AnnualReview of Immunology vol 27 pp 693ndash733 2009

[27] A Planavila J C Laguna and M Vazquez-Carrera ldquoNuclearfactor-120581B activation leads to down-regulation of fatty acidoxidation during cardiac hypertrophyrdquoThe Journal of BiologicalChemistry vol 280 no 17 pp 17464ndash17471 2005

[28] C L Sawyers ldquoWill mTOR inhibitors make it as cancer drugsrdquoCancer Cell vol 4 no 5 pp 343ndash348 2003

[29] H C Dan M J Cooper P C Cogswell J A Duncan J P-YTing and A S Baldwin ldquoAkt-dependent regulation of NF-120581B iscontrolled bymTORandRaptor in associationwith IKKrdquoGenesand Development vol 22 no 11 pp 1490ndash1500 2008

[30] K J Tracey S F Lowry and A Cerami ldquoThe pathophysiologicrole of cachectinTNF in septic shock and cachexiardquoAnnales delInstitut Pasteur Immunology vol 139 no 3 pp 311ndash317 1988

[31] J AMartina Y ChenMGucek andR Puertollano ldquoMTORC1functions as a transcriptional regulator of autophagy by pre-venting nuclear transport of TFEBrdquoAutophagy vol 8 no 6 pp903ndash914 2012

[32] C Settembre R de Cegli G Mansueto et al ldquoTFEB controlscellular lipidmetabolism through a starvation-induced autoreg-ulatory looprdquo Nature Cell Biology vol 15 no 8 pp 647ndash6582013

[33] B N Finck and D P Kelly ldquoPeroxisome proliferator-activatedreceptor 120574 coactivator-1 (PGC-1) regulatory cascade in cardiacphysiology and diseaserdquo Circulation vol 115 no 19 pp 2540ndash2548 2007

[34] K Watanabe H Fujii T Takahashi et al ldquoConstitutive reg-ulation of cardiac fatty acid metabolism through peroxisomeproliferator-activated receptor 120572 associated with age-dependentcardiac toxicityrdquo The Journal of Biological Chemistry vol 275no 29 pp 22293ndash22299 2000

[35] M A Martın M A Gomez F Guillen et al ldquoMyocardialcarnitine and carnitine palmitoyltransferase deficiencies inpatientswith severe heart failurerdquoBiochimica et BiophysicaActavol 1502 no 3 pp 330ndash336 2000

[36] X Palomer D Alvarez-Guardia R Rodrıguez-Calvo et alldquoTNF-120572 reduces PGC-1120572 expression through NF-120581B and p38


MAPK leading to increased glucose oxidation in a humancardiac cell modelrdquo Cardiovascular Research vol 81 no 4 pp703ndash712 2009

[37] T Shioi J R McMullen O Tarnavski et al ldquoRapamycin atten-uates load-induced cardiac hypertrophy in micerdquo Circulationvol 107 no 12 pp 1664ndash1670 2003

[38] J R McMullen M C Sherwood O Tarnavski et al ldquoInhibi-tion of mTOR signaling with rapamycin regresses establishedcardiac hypertrophy induced by pressure overloadrdquoCirculationvol 109 no 24 pp 3050ndash3055 2004

[39] J Sadoshima and S Izumo ldquoRapamycin selectively inhibitsangiotensin II-induced increase in protein synthesis in car-diac myocytes in vitro potential role of 70-kD S6 kinasein angiotensin II-induced cardiac hypertrophyrdquo CirculationResearch vol 77 no 6 pp 1040ndash1052 1995

[40] F Jaffre J Callebert A Sarre et al ldquoInvolvement of theserotonin 5-HT2B receptor in cardiac hypertrophy linked tosympathetic stimulation control of interleukin-6 interleukin-1120573 and tumor necrosis factor-120572 cytokine production by ventric-ular fibroblastsrdquo Circulation vol 110 no 8 pp 969ndash974 2004

[41] S J Matkovich A Diwan J L Klanke et al ldquoCardiac-specificablation of G-protein receptor kinase 2 redefines its roles inheart development and 120573-adrenergic signalingrdquo CirculationResearch vol 99 no 9 pp 996ndash1003 2006

[42] O Drews O Tsukamoto D Liem J Streicher Y Wangand P Ping ldquoDifferential regulation of proteasome func-tion in isoproterenol-induced cardiac hypertrophyrdquo CirculationResearch vol 107 no 9 pp 1094ndash1101 2010

[43] Y Fujishima S Nishiumi A Masuda et al ldquoAutophagy in theintestinal epithelium reduces endotoxin-induced inflammatoryresponses by inhibiting NF-120581B activationrdquoArchives of Biochem-istry and Biophysics vol 506 no 2 pp 223ndash235 2011

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

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OphthalmologyJournal of


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Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


of heart disease [7] however the persistent stimulation ofneurohormonal factors contributes to the mechanical dys-function of hearts ultimately [8] It had been demonstratedthat overexpression of biologically active neurohormonalmolecules contributed to the progression of decompensatedcardiac hypertrophy independently of the hemodynamicstatus by virtue of the direct toxic effects of these neuro-hormonal molecules exert on hearts [9] Isoproterenol isone of these biologically active neurohormonal molecules Inaddition to its direct cardiac effect isoproterenol was provedto have a cardiac trophic effect via stimulation of the reninangiotensin system (RAS) [10]

Rapamycin is a clinically used immunosuppressor in pre-venting transplant rejection [11] and restenosis in coronaryarteries following balloon angioplasty Recently Rapa wasdemonstrated to ameliorate cardiac remodeling and improvethe left ventricular function after myocardial infarction [12]Besides it has been reported that Rapa regressed left ventric-ular hypertrophy in renal transplant recipients regardless ofblood pressure changes suggesting nonhemodynamic-effectmechanisms of rapamycin on left ventricular mass [13]Thusit is worthy of investigation that whether the treatment ofRapa was capable of regressing cardiac hypertrophy inducedby persistent stimulation of sympathetic nervous systemAndthe underlying mechanisms of Rapa in protecting againstcardiac hypertrophy induced by isoproterenol remain to beelucidated

Based on these considerations mentioned above weestablished the rat model of cardiac hypertrophy inducedby isoproterenol and then Rapa was administrated to eval-uate the cardiac function and mitochondrial functionalperformance In this study Rapa was observed to attenu-ate isoproterenol-induced cardiac hypertrophy and main-tain energy homeostasis in adult rats Increased levels ofproinflammatory cytokines were detected in cardiomyocytesin response to isoproterenol which could be substantiallyattenuated byRapaMoreover these protective effects of Rapamight be attributed to the inactivation of NF-120581BThe presentdata revealed that the role of Rapa in protecting hearts fromisoproterenol-induced cardiac hypertrophy and maintainingenergy homeostasis was dependent on NF-120581B pathway

2 Materials and Methods

21 Animal Models of Cardiac Hypertrophy and ExperimentalProtocols Male Sprague-Dawley rats weighing 225 g to 250 gwere purchased from Animal Breeding Center of Sun Yat-sen University Isoproterenol (55mgKg Merck) dissolvedin saline was injected subcutaneously for consecutive tendays Control animals underwent the same procedure exceptthat saline vehicle was injected subcutaneously instead ofisoproterenol After the infusion of isoproterenol rats sur-viving were assigned to the following groups randomly avehicle-treated group (vehicle 119899 = 6) a rapamycin-treatedgroup (12mgkg Selleck 119899 = 6) Treatments of Rapa wereinjected intraperitoneally daily for seven days The doses weadministrated here were comparable with those used in micestudies when normalized by body surface area [12 14] The

experiments were approved by the ethics Committees of SunYat-sen University of medical Science

22 Echocardiographic Studies andHistological Analysis Ratswere anesthetized with 2 isoflurane allowing spontaneousbreathing The Vevo 2100 scanner equipped with a 16MHzProbe (Visual Sonics Toronto Canada) was used to per-form echocardiography analysis After a good-quality two-dimensional image was obtained M-mode images of leftventricles were recorded Left ventricular anterior and poste-rior wall thickness (LVAWand LVPW) fractional shortening(LVFS) and ejection fraction (LVEF) were measured andanalyzed

Once all rats were sacrificed the hearts were washedwith PBS and dried to weigh and fixed within 4 paraformovernight Paraffin-embedded sections (7 120583m) were stainedwith hematoxylin and eosin Slides were examined withbrightfield microscopy and times400 images were capturedThe mean myocyte diameter was calculated by randomlymeasuring 100 cardiomyocytes in 10 randomly chosen fieldsaccording to method and criteria reported [15 16]

23 Transmission Electron Microscopy Analysis Cardiac tis-sues were cut into 1mm cubes immediately after sacri-fice and fixed in 25 glutaraldehyde and then postfixedin 1 osmium tetroxide After acetone dehydration andembedding the ultrathin sections were stained as methodsdescribed [17] Images were taken at 120KV with JEM1400electron microscopy equipped with a high-performanceCCD camera

24Mitochondrial Oxygen Consumption Hearts were freshlyexcised and the apex of left ventricle (200mg) was obtainedfrom hearts in three experimental groups Then freshmitochondria were isolated using mitochondria extrac-tion and isolation kit (GenMed Scientifics) according tothe manufacturerrsquos instruction Clark-type oxygen electrode(Strathkelvin Scotland) was used to determine the rate ofmitochondrial respiration (25∘C) The respiration mediumwas prepared asmethod reported [18] Following the additionof 1mM adenosine diphosphate (ADP) the state III respira-tion was recorded After the complete phosphorylation of theadded ADP the state IV respiration was recorded as wellTherespiration control rate (RCR) which reflects the function ofmitochondria was represented by the ratio of rate of state IIIrespiration to the rate of state IV respiration Mitochondrialprotein concentration was determined by BCA protein assaykit (Thermo Scientifics) The rates of oxygen consumptionwere expressed as nmol O

2minmg

25 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) According to the manufacturerrsquos instruction myocar-dial RNA was extracted from snap-frozen tissues with Trizolreagent (Invitrogen) The SYBR-Green Quantitative PCR kit(Takara) was used to determine the mRNA expression levelsof each target gene by iCycler iQ system (Bio-Rad) AllPCR reactions were performed in triplicate Specific primer












































3 Results









0

1

2

3

4

LVW

BW

(mg

g)


lowastlowast

lowastlowast

0

1

2

3

4H

WB

W (m

gg)

(a)


H an

d E

(400

x)

(b)

0

10

20

30

Mea

n m

yocy

te d

iam

eter

(120583m)


lowastlowastlowast

(c)

0

1

2

3

4

LVAWsLVPWs

(mm

)

lowast lowast

lowastlowastlowast


20

40

60

80

100

()

LVEFLVFS

(d)

Figure 1 Continued


0

1

2

3

4

5

ANFBNP

(fold

chan

ge)

Relat

ive m

RNA

leve

ls


120573-MHC


(e)









(a)

0

100

200

300

400

500


lowast

(nm

olm

inminus1

mgminus

1)


(b)

0

2

4

6

8RCR

Ratio


lowastlowast

(c)

0

20

40

60

80

100ATP


(nm

ol120583

g)

lowast lowast

(d)

00

05

10

15

20

MCADLCAD CPT2


(fold

chan

ge)

CPT1-120573


lowastlowast lowast

lowast

(e)

p65

Histone H3

Cyto Nuc

Con

trol

Mod

el+

Rapa

Mod

el

Con

trol

Mod

el+

Rapa

Mod

el

120572-Tubulin

(f)

Figure 2 Continued


p65

00

05

10

15

20

25

Cyto

Nuc

Rela

tive p

rote

in le

vels

(fold

ove

r con

trol)


lowastlowast

lowastlowast

(g)

Con

trol

Mod

el+

Rapa

Mod

el

I120581B-120572

120572-Tubulin

(h)

00

05

10

15

Rela

tive p

rote

in le

vels

(fold

ove

r con

trol)



lowast

(i)






0

50

100

150

200

250

IL-1120573

(ng

mL)

lowastlowastlowast

lowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa +

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 005

(a)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

0

20

40

60

IL-2

(ng

mL)

lowastlowastlowast

lowastlowast

P lt 0001

P lt 0001

Mod

el+

Rapa

(b)

100

80

60

40

20

0

Con

trol

Mod

el

TNF-120572

(ng

mL)

Mod

el+

Rapa

+

sip6

5

lowastlowast

Mod

el+

Rapa

(c)

6

4

2

0

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

P lt 005

P lt 005

P lt 0001

P lt 0001

P lt 0001

P lt 001

IL-1120573IL-2TNF-120572

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5


lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Mod

el+

Rapa

(d)






0

1

2

3

Relat

ive c

ell-s

urfa

ce ar

ea

P lt 0001P lt 0001


Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

(a)

0

1

2

3

4

ANFBNP

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

120573-MHC

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 001


lowastlowastlowast

(b)

0

05

10

15

Rela

tive T

MRE

fluor

esce

nce

P lt 001P lt 005

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65


(c)

0

10

20

30

40

50

ATP

conc

entr

atio

n(120583

Mg

cell

prot

ein)

Con

trol

Mod

elM

odel+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

P lt 0001

P lt 0001


(d)






4 Discussion







-Tubulin

Con

trol

p-mTOR

Total m-TOR

Isop

rote

reno

l

Iso+

Rapa

Iso+

Rapa

+TN

F-120572

Iso+

Rapa

+si

p65

PPAR120572

PGC1120572

120572

(a)

0

1

2

3

p-m

TOR

tota

l mTO

R(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowast

Mod

el+

Rapa

(b)

00

05

10

15

Relat

ive p

rote

in le

vels

of P

PAR120572

(fo

ld ch

ange

)

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(c)

00

05

10

15Re

lativ

e pro

tein

leve

ls of

PG

C-1120572

(fo

ld ch

ange

)

P lt 001

P lt 001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(d)

00

05

10

15

Rela

tive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5



PPAR120572

PGC-1120572

P lt 0001

P lt 001

P lt 001

P lt 001

Mod

el+

Rapa

(e)

00

05

10

15

MCADLCAD

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 001

P lt 001

P lt 001



(f)

00

05

10

15

CPT2

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

CPT1-120573

lowast lowast

lowast

lowastlowastlowast

Mod

el+

Rapa

P lt 001

P lt 001

P lt 005

P lt 005

(g)









Abbreviations






Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























































3 Results









0

1

2

3

4

LVW

BW

(mg

g)


lowastlowast

lowastlowast

0

1

2

3

4H

WB

W (m

gg)

(a)


H an

d E

(400

x)

(b)

0

10

20

30

Mea

n m

yocy

te d

iam

eter

(120583m)


lowastlowastlowast

(c)

0

1

2

3

4

LVAWsLVPWs

(mm

)

lowast lowast

lowastlowastlowast


20

40

60

80

100

()

LVEFLVFS

(d)

Figure 1 Continued


0

1

2

3

4

5

ANFBNP

(fold

chan

ge)

Relat

ive m

RNA

leve

ls


120573-MHC


(e)









(a)

0

100

200

300

400

500


lowast

(nm

olm

inminus1

mgminus

1)


(b)

0

2

4

6

8RCR

Ratio


lowastlowast

(c)

0

20

40

60

80

100ATP


(nm

ol120583

g)

lowast lowast

(d)

00

05

10

15

20

MCADLCAD CPT2


(fold

chan

ge)

CPT1-120573


lowastlowast lowast

lowast

(e)

p65

Histone H3

Cyto Nuc

Con

trol

Mod

el+

Rapa

Mod

el

Con

trol

Mod

el+

Rapa

Mod

el

120572-Tubulin

(f)

Figure 2 Continued


p65

00

05

10

15

20

25

Cyto

Nuc

Rela

tive p

rote

in le

vels

(fold

ove

r con

trol)


lowastlowast

lowastlowast

(g)

Con

trol

Mod

el+

Rapa

Mod

el

I120581B-120572

120572-Tubulin

(h)

00

05

10

15

Rela

tive p

rote

in le

vels

(fold

ove

r con

trol)



lowast

(i)






0

50

100

150

200

250

IL-1120573

(ng

mL)

lowastlowastlowast

lowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa +

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 005

(a)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

0

20

40

60

IL-2

(ng

mL)

lowastlowastlowast

lowastlowast

P lt 0001

P lt 0001

Mod

el+

Rapa

(b)

100

80

60

40

20

0

Con

trol

Mod

el

TNF-120572

(ng

mL)

Mod

el+

Rapa

+

sip6

5

lowastlowast

Mod

el+

Rapa

(c)

6

4

2

0

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

P lt 005

P lt 005

P lt 0001

P lt 0001

P lt 0001

P lt 001

IL-1120573IL-2TNF-120572

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5


lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Mod

el+

Rapa

(d)






0

1

2

3

Relat

ive c

ell-s

urfa

ce ar

ea

P lt 0001P lt 0001


Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

(a)

0

1

2

3

4

ANFBNP

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

120573-MHC

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 001


lowastlowastlowast

(b)

0

05

10

15

Rela

tive T

MRE

fluor

esce

nce

P lt 001P lt 005

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65


(c)

0

10

20

30

40

50

ATP

conc

entr

atio

n(120583

Mg

cell

prot

ein)

Con

trol

Mod

elM

odel+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

P lt 0001

P lt 0001


(d)






4 Discussion







-Tubulin

Con

trol

p-mTOR

Total m-TOR

Isop

rote

reno

l

Iso+

Rapa

Iso+

Rapa

+TN

F-120572

Iso+

Rapa

+si

p65

PPAR120572

PGC1120572

120572

(a)

0

1

2

3

p-m

TOR

tota

l mTO

R(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowast

Mod

el+

Rapa

(b)

00

05

10

15

Relat

ive p

rote

in le

vels

of P

PAR120572

(fo

ld ch

ange

)

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(c)

00

05

10

15Re

lativ

e pro

tein

leve

ls of

PG

C-1120572

(fo

ld ch

ange

)

P lt 001

P lt 001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(d)

00

05

10

15

Rela

tive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5



PPAR120572

PGC-1120572

P lt 0001

P lt 001

P lt 001

P lt 001

Mod

el+

Rapa

(e)

00

05

10

15

MCADLCAD

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 001

P lt 001

P lt 001



(f)

00

05

10

15

CPT2

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

CPT1-120573

lowast lowast

lowast

lowastlowastlowast

Mod

el+

Rapa

P lt 001

P lt 001

P lt 005

P lt 005

(g)









Abbreviations






Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

LVW

BW

(mg

g)


lowastlowast

lowastlowast

0

1

2

3

4H

WB

W (m

gg)

(a)


H an

d E

(400

x)

(b)

0

10

20

30

Mea

n m

yocy

te d

iam

eter

(120583m)


lowastlowastlowast

(c)

0

1

2

3

4

LVAWsLVPWs

(mm

)

lowast lowast

lowastlowastlowast


20

40

60

80

100

()

LVEFLVFS

(d)

Figure 1 Continued


0

1

2

3

4

5

ANFBNP

(fold

chan

ge)

Relat

ive m

RNA

leve

ls


120573-MHC


(e)









(a)

0

100

200

300

400

500


lowast

(nm

olm

inminus1

mgminus

1)


(b)

0

2

4

6

8RCR

Ratio


lowastlowast

(c)

0

20

40

60

80

100ATP


(nm

ol120583

g)

lowast lowast

(d)

00

05

10

15

20

MCADLCAD CPT2


(fold

chan

ge)

CPT1-120573


lowastlowast lowast

lowast

(e)

p65

Histone H3

Cyto Nuc

Con

trol

Mod

el+

Rapa

Mod

el

Con

trol

Mod

el+

Rapa

Mod

el

120572-Tubulin

(f)

Figure 2 Continued


p65

00

05

10

15

20

25

Cyto

Nuc

Rela

tive p

rote

in le

vels

(fold

ove

r con

trol)


lowastlowast

lowastlowast

(g)

Con

trol

Mod

el+

Rapa

Mod

el

I120581B-120572

120572-Tubulin

(h)

00

05

10

15

Rela

tive p

rote

in le

vels

(fold

ove

r con

trol)



lowast

(i)






0

50

100

150

200

250

IL-1120573

(ng

mL)

lowastlowastlowast

lowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa +

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 005

(a)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

0

20

40

60

IL-2

(ng

mL)

lowastlowastlowast

lowastlowast

P lt 0001

P lt 0001

Mod

el+

Rapa

(b)

100

80

60

40

20

0

Con

trol

Mod

el

TNF-120572

(ng

mL)

Mod

el+

Rapa

+

sip6

5

lowastlowast

Mod

el+

Rapa

(c)

6

4

2

0

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

P lt 005

P lt 005

P lt 0001

P lt 0001

P lt 0001

P lt 001

IL-1120573IL-2TNF-120572

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5


lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Mod

el+

Rapa

(d)






0

1

2

3

Relat

ive c

ell-s

urfa

ce ar

ea

P lt 0001P lt 0001


Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

(a)

0

1

2

3

4

ANFBNP

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

120573-MHC

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 001


lowastlowastlowast

(b)

0

05

10

15

Rela

tive T

MRE

fluor

esce

nce

P lt 001P lt 005

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65


(c)

0

10

20

30

40

50

ATP

conc

entr

atio

n(120583

Mg

cell

prot

ein)

Con

trol

Mod

elM

odel+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

P lt 0001

P lt 0001


(d)






4 Discussion







-Tubulin

Con

trol

p-mTOR

Total m-TOR

Isop

rote

reno

l

Iso+

Rapa

Iso+

Rapa

+TN

F-120572

Iso+

Rapa

+si

p65

PPAR120572

PGC1120572

120572

(a)

0

1

2

3

p-m

TOR

tota

l mTO

R(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowast

Mod

el+

Rapa

(b)

00

05

10

15

Relat

ive p

rote

in le

vels

of P

PAR120572

(fo

ld ch

ange

)

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(c)

00

05

10

15Re

lativ

e pro

tein

leve

ls of

PG

C-1120572

(fo

ld ch

ange

)

P lt 001

P lt 001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(d)

00

05

10

15

Rela

tive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5



PPAR120572

PGC-1120572

P lt 0001

P lt 001

P lt 001

P lt 001

Mod

el+

Rapa

(e)

00

05

10

15

MCADLCAD

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 001

P lt 001

P lt 001



(f)

00

05

10

15

CPT2

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

CPT1-120573

lowast lowast

lowast

lowastlowastlowast

Mod

el+

Rapa

P lt 001

P lt 001

P lt 005

P lt 005

(g)









Abbreviations






Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

5

ANFBNP

(fold

chan

ge)

Relat

ive m

RNA

leve

ls


120573-MHC


(e)









(a)

0

100

200

300

400

500


lowast

(nm

olm

inminus1

mgminus

1)


(b)

0

2

4

6

8RCR

Ratio


lowastlowast

(c)

0

20

40

60

80

100ATP


(nm

ol120583

g)

lowast lowast

(d)

00

05

10

15

20

MCADLCAD CPT2


(fold

chan

ge)

CPT1-120573


lowastlowast lowast

lowast

(e)

p65

Histone H3

Cyto Nuc

Con

trol

Mod

el+

Rapa

Mod

el

Con

trol

Mod

el+

Rapa

Mod

el

120572-Tubulin

(f)

Figure 2 Continued


p65

00

05

10

15

20

25

Cyto

Nuc

Rela

tive p

rote

in le

vels

(fold

ove

r con

trol)


lowastlowast

lowastlowast

(g)

Con

trol

Mod

el+

Rapa

Mod

el

I120581B-120572

120572-Tubulin

(h)

00

05

10

15

Rela

tive p

rote

in le

vels

(fold

ove

r con

trol)



lowast

(i)






0

50

100

150

200

250

IL-1120573

(ng

mL)

lowastlowastlowast

lowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa +

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 005

(a)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

0

20

40

60

IL-2

(ng

mL)

lowastlowastlowast

lowastlowast

P lt 0001

P lt 0001

Mod

el+

Rapa

(b)

100

80

60

40

20

0

Con

trol

Mod

el

TNF-120572

(ng

mL)

Mod

el+

Rapa

+

sip6

5

lowastlowast

Mod

el+

Rapa

(c)

6

4

2

0

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

P lt 005

P lt 005

P lt 0001

P lt 0001

P lt 0001

P lt 001

IL-1120573IL-2TNF-120572

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5


lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Mod

el+

Rapa

(d)






0

1

2

3

Relat

ive c

ell-s

urfa

ce ar

ea

P lt 0001P lt 0001


Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

(a)

0

1

2

3

4

ANFBNP

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

120573-MHC

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 001


lowastlowastlowast

(b)

0

05

10

15

Rela

tive T

MRE

fluor

esce

nce

P lt 001P lt 005

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65


(c)

0

10

20

30

40

50

ATP

conc

entr

atio

n(120583

Mg

cell

prot

ein)

Con

trol

Mod

elM

odel+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

P lt 0001

P lt 0001


(d)






4 Discussion







-Tubulin

Con

trol

p-mTOR

Total m-TOR

Isop

rote

reno

l

Iso+

Rapa

Iso+

Rapa

+TN

F-120572

Iso+

Rapa

+si

p65

PPAR120572

PGC1120572

120572

(a)

0

1

2

3

p-m

TOR

tota

l mTO

R(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowast

Mod

el+

Rapa

(b)

00

05

10

15

Relat

ive p

rote

in le

vels

of P

PAR120572

(fo

ld ch

ange

)

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(c)

00

05

10

15Re

lativ

e pro

tein

leve

ls of

PG

C-1120572

(fo

ld ch

ange

)

P lt 001

P lt 001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(d)

00

05

10

15

Rela

tive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5



PPAR120572

PGC-1120572

P lt 0001

P lt 001

P lt 001

P lt 001

Mod

el+

Rapa

(e)

00

05

10

15

MCADLCAD

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 001

P lt 001

P lt 001



(f)

00

05

10

15

CPT2

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

CPT1-120573

lowast lowast

lowast

lowastlowastlowast

Mod

el+

Rapa

P lt 001

P lt 001

P lt 005

P lt 005

(g)









Abbreviations






Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(a)

0

100

200

300

400

500


lowast

(nm

olm

inminus1

mgminus

1)


(b)

0

2

4

6

8RCR

Ratio


lowastlowast

(c)

0

20

40

60

80

100ATP


(nm

ol120583

g)

lowast lowast

(d)

00

05

10

15

20

MCADLCAD CPT2


(fold

chan

ge)

CPT1-120573


lowastlowast lowast

lowast

(e)

p65

Histone H3

Cyto Nuc

Con

trol

Mod

el+

Rapa

Mod

el

Con

trol

Mod

el+

Rapa

Mod

el

120572-Tubulin

(f)

Figure 2 Continued


p65

00

05

10

15

20

25

Cyto

Nuc

Rela

tive p

rote

in le

vels

(fold

ove

r con

trol)


lowastlowast

lowastlowast

(g)

Con

trol

Mod

el+

Rapa

Mod

el

I120581B-120572

120572-Tubulin

(h)

00

05

10

15

Rela

tive p

rote

in le

vels

(fold

ove

r con

trol)



lowast

(i)






0

50

100

150

200

250

IL-1120573

(ng

mL)

lowastlowastlowast

lowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa +

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 005

(a)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

0

20

40

60

IL-2

(ng

mL)

lowastlowastlowast

lowastlowast

P lt 0001

P lt 0001

Mod

el+

Rapa

(b)

100

80

60

40

20

0

Con

trol

Mod

el

TNF-120572

(ng

mL)

Mod

el+

Rapa

+

sip6

5

lowastlowast

Mod

el+

Rapa

(c)

6

4

2

0

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

P lt 005

P lt 005

P lt 0001

P lt 0001

P lt 0001

P lt 001

IL-1120573IL-2TNF-120572

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5


lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Mod

el+

Rapa

(d)






0

1

2

3

Relat

ive c

ell-s

urfa

ce ar

ea

P lt 0001P lt 0001


Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

(a)

0

1

2

3

4

ANFBNP

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

120573-MHC

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 001


lowastlowastlowast

(b)

0

05

10

15

Rela

tive T

MRE

fluor

esce

nce

P lt 001P lt 005

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65


(c)

0

10

20

30

40

50

ATP

conc

entr

atio

n(120583

Mg

cell

prot

ein)

Con

trol

Mod

elM

odel+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

P lt 0001

P lt 0001


(d)






4 Discussion







-Tubulin

Con

trol

p-mTOR

Total m-TOR

Isop

rote

reno

l

Iso+

Rapa

Iso+

Rapa

+TN

F-120572

Iso+

Rapa

+si

p65

PPAR120572

PGC1120572

120572

(a)

0

1

2

3

p-m

TOR

tota

l mTO

R(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowast

Mod

el+

Rapa

(b)

00

05

10

15

Relat

ive p

rote

in le

vels

of P

PAR120572

(fo

ld ch

ange

)

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(c)

00

05

10

15Re

lativ

e pro

tein

leve

ls of

PG

C-1120572

(fo

ld ch

ange

)

P lt 001

P lt 001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(d)

00

05

10

15

Rela

tive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5



PPAR120572

PGC-1120572

P lt 0001

P lt 001

P lt 001

P lt 001

Mod

el+

Rapa

(e)

00

05

10

15

MCADLCAD

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 001

P lt 001

P lt 001



(f)

00

05

10

15

CPT2

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

CPT1-120573

lowast lowast

lowast

lowastlowastlowast

Mod

el+

Rapa

P lt 001

P lt 001

P lt 005

P lt 005

(g)









Abbreviations






Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















p65

00

05

10

15

20

25

Cyto

Nuc

Rela

tive p

rote

in le

vels

(fold

ove

r con

trol)


lowastlowast

lowastlowast

(g)

Con

trol

Mod

el+

Rapa

Mod

el

I120581B-120572

120572-Tubulin

(h)

00

05

10

15

Rela

tive p

rote

in le

vels

(fold

ove

r con

trol)



lowast

(i)






0

50

100

150

200

250

IL-1120573

(ng

mL)

lowastlowastlowast

lowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa +

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 005

(a)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

0

20

40

60

IL-2

(ng

mL)

lowastlowastlowast

lowastlowast

P lt 0001

P lt 0001

Mod

el+

Rapa

(b)

100

80

60

40

20

0

Con

trol

Mod

el

TNF-120572

(ng

mL)

Mod

el+

Rapa

+

sip6

5

lowastlowast

Mod

el+

Rapa

(c)

6

4

2

0

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

P lt 005

P lt 005

P lt 0001

P lt 0001

P lt 0001

P lt 001

IL-1120573IL-2TNF-120572

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5


lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Mod

el+

Rapa

(d)






0

1

2

3

Relat

ive c

ell-s

urfa

ce ar

ea

P lt 0001P lt 0001


Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

(a)

0

1

2

3

4

ANFBNP

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

120573-MHC

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 001


lowastlowastlowast

(b)

0

05

10

15

Rela

tive T

MRE

fluor

esce

nce

P lt 001P lt 005

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65


(c)

0

10

20

30

40

50

ATP

conc

entr

atio

n(120583

Mg

cell

prot

ein)

Con

trol

Mod

elM

odel+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

P lt 0001

P lt 0001


(d)






4 Discussion







-Tubulin

Con

trol

p-mTOR

Total m-TOR

Isop

rote

reno

l

Iso+

Rapa

Iso+

Rapa

+TN

F-120572

Iso+

Rapa

+si

p65

PPAR120572

PGC1120572

120572

(a)

0

1

2

3

p-m

TOR

tota

l mTO

R(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowast

Mod

el+

Rapa

(b)

00

05

10

15

Relat

ive p

rote

in le

vels

of P

PAR120572

(fo

ld ch

ange

)

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(c)

00

05

10

15Re

lativ

e pro

tein

leve

ls of

PG

C-1120572

(fo

ld ch

ange

)

P lt 001

P lt 001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(d)

00

05

10

15

Rela

tive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5



PPAR120572

PGC-1120572

P lt 0001

P lt 001

P lt 001

P lt 001

Mod

el+

Rapa

(e)

00

05

10

15

MCADLCAD

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 001

P lt 001

P lt 001



(f)

00

05

10

15

CPT2

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

CPT1-120573

lowast lowast

lowast

lowastlowastlowast

Mod

el+

Rapa

P lt 001

P lt 001

P lt 005

P lt 005

(g)









Abbreviations






Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

250

IL-1120573

(ng

mL)

lowastlowastlowast

lowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa +

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 005

(a)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

0

20

40

60

IL-2

(ng

mL)

lowastlowastlowast

lowastlowast

P lt 0001

P lt 0001

Mod

el+

Rapa

(b)

100

80

60

40

20

0

Con

trol

Mod

el

TNF-120572

(ng

mL)

Mod

el+

Rapa

+

sip6

5

lowastlowast

Mod

el+

Rapa

(c)

6

4

2

0

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

P lt 005

P lt 005

P lt 0001

P lt 0001

P lt 0001

P lt 001

IL-1120573IL-2TNF-120572

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5


lowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Mod

el+

Rapa

(d)






0

1

2

3

Relat

ive c

ell-s

urfa

ce ar

ea

P lt 0001P lt 0001


Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

(a)

0

1

2

3

4

ANFBNP

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

120573-MHC

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 001


lowastlowastlowast

(b)

0

05

10

15

Rela

tive T

MRE

fluor

esce

nce

P lt 001P lt 005

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65


(c)

0

10

20

30

40

50

ATP

conc

entr

atio

n(120583

Mg

cell

prot

ein)

Con

trol

Mod

elM

odel+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

P lt 0001

P lt 0001


(d)






4 Discussion







-Tubulin

Con

trol

p-mTOR

Total m-TOR

Isop

rote

reno

l

Iso+

Rapa

Iso+

Rapa

+TN

F-120572

Iso+

Rapa

+si

p65

PPAR120572

PGC1120572

120572

(a)

0

1

2

3

p-m

TOR

tota

l mTO

R(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowast

Mod

el+

Rapa

(b)

00

05

10

15

Relat

ive p

rote

in le

vels

of P

PAR120572

(fo

ld ch

ange

)

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(c)

00

05

10

15Re

lativ

e pro

tein

leve

ls of

PG

C-1120572

(fo

ld ch

ange

)

P lt 001

P lt 001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(d)

00

05

10

15

Rela

tive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5



PPAR120572

PGC-1120572

P lt 0001

P lt 001

P lt 001

P lt 001

Mod

el+

Rapa

(e)

00

05

10

15

MCADLCAD

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 001

P lt 001

P lt 001



(f)

00

05

10

15

CPT2

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

CPT1-120573

lowast lowast

lowast

lowastlowastlowast

Mod

el+

Rapa

P lt 001

P lt 001

P lt 005

P lt 005

(g)









Abbreviations






Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

Relat

ive c

ell-s

urfa

ce ar

ea

P lt 0001P lt 0001


Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

(a)

0

1

2

3

4

ANFBNP

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

120573-MHC

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 001


lowastlowastlowast

(b)

0

05

10

15

Rela

tive T

MRE

fluor

esce

nce

P lt 001P lt 005

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65


(c)

0

10

20

30

40

50

ATP

conc

entr

atio

n(120583

Mg

cell

prot

ein)

Con

trol

Mod

elM

odel+

Rapa

Mod

el+

Rapa

+TN

F-120572

Mod

el+

Rapa

+si

p65

P lt 0001

P lt 0001


(d)






4 Discussion







-Tubulin

Con

trol

p-mTOR

Total m-TOR

Isop

rote

reno

l

Iso+

Rapa

Iso+

Rapa

+TN

F-120572

Iso+

Rapa

+si

p65

PPAR120572

PGC1120572

120572

(a)

0

1

2

3

p-m

TOR

tota

l mTO

R(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowast

Mod

el+

Rapa

(b)

00

05

10

15

Relat

ive p

rote

in le

vels

of P

PAR120572

(fo

ld ch

ange

)

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(c)

00

05

10

15Re

lativ

e pro

tein

leve

ls of

PG

C-1120572

(fo

ld ch

ange

)

P lt 001

P lt 001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(d)

00

05

10

15

Rela

tive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5



PPAR120572

PGC-1120572

P lt 0001

P lt 001

P lt 001

P lt 001

Mod

el+

Rapa

(e)

00

05

10

15

MCADLCAD

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 001

P lt 001

P lt 001



(f)

00

05

10

15

CPT2

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

CPT1-120573

lowast lowast

lowast

lowastlowastlowast

Mod

el+

Rapa

P lt 001

P lt 001

P lt 005

P lt 005

(g)









Abbreviations






Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















4 Discussion







-Tubulin

Con

trol

p-mTOR

Total m-TOR

Isop

rote

reno

l

Iso+

Rapa

Iso+

Rapa

+TN

F-120572

Iso+

Rapa

+si

p65

PPAR120572

PGC1120572

120572

(a)

0

1

2

3

p-m

TOR

tota

l mTO

R(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowast

Mod

el+

Rapa

(b)

00

05

10

15

Relat

ive p

rote

in le

vels

of P

PAR120572

(fo

ld ch

ange

)

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(c)

00

05

10

15Re

lativ

e pro

tein

leve

ls of

PG

C-1120572

(fo

ld ch

ange

)

P lt 001

P lt 001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(d)

00

05

10

15

Rela

tive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5



PPAR120572

PGC-1120572

P lt 0001

P lt 001

P lt 001

P lt 001

Mod

el+

Rapa

(e)

00

05

10

15

MCADLCAD

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 001

P lt 001

P lt 001



(f)

00

05

10

15

CPT2

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

CPT1-120573

lowast lowast

lowast

lowastlowastlowast

Mod

el+

Rapa

P lt 001

P lt 001

P lt 005

P lt 005

(g)









Abbreviations






Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















-Tubulin

Con

trol

p-mTOR

Total m-TOR

Isop

rote

reno

l

Iso+

Rapa

Iso+

Rapa

+TN

F-120572

Iso+

Rapa

+si

p65

PPAR120572

PGC1120572

120572

(a)

0

1

2

3

p-m

TOR

tota

l mTO

R(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowast

Mod

el+

Rapa

(b)

00

05

10

15

Relat

ive p

rote

in le

vels

of P

PAR120572

(fo

ld ch

ange

)

P lt 0001

P lt 0001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(c)

00

05

10

15Re

lativ

e pro

tein

leve

ls of

PG

C-1120572

(fo

ld ch

ange

)

P lt 001

P lt 001

lowastlowastlowast

lowastlowastlowast

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

Mod

el+

Rapa

(d)

00

05

10

15

Rela

tive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5



PPAR120572

PGC-1120572

P lt 0001

P lt 001

P lt 001

P lt 001

Mod

el+

Rapa

(e)

00

05

10

15

MCADLCAD

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

P lt 005

P lt 001

P lt 001

P lt 001



(f)

00

05

10

15

CPT2

Relat

ive m

RNA

leve

ls(fo

ld ch

ange

)

Con

trol

Mod

el

Mod

el+

Rapa

+

TNF-120572

Mod

el+

Rapa

+

sip6

5

CPT1-120573

lowast lowast

lowast

lowastlowastlowast

Mod

el+

Rapa

P lt 001

P lt 001

P lt 005

P lt 005

(g)









Abbreviations






Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Abbreviations






Acknowledgments


References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article rapamycin attenuated cardiac hypertrophy...

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