Aging Influences Cardiac Mitochondrial Gene Expression and ...€¦ · 542 | JIAN ET AL. | MOL MED 17(5-6)542-549, MAY-JUNE 2011 INTRODUCTION Aging is a significant factor that con-tributes

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INTRODUCTIONAging is a significant factor that con-

tributes to mitochondrial damage anddysfunction (1–3). The progression toheart failure (independent of cause) isassociated with a progressive decline inthe activity of mitochondrial respiratorypathways which leads to diminished ca-pacity for adenoside triphosphate (ATP)production (4). Mitochondrial oxidativephosphorylation is the primary energysource in the myocardial cell. The mo-lecular mechanism of mitochondrialdamage and dysfunction to cardiomy-

ocytes and its contribution to the out-come of T-H in relation to aging are notfully known. The information concern-ing the molecular changes occurring inmitochondria after trauma will be valu-able in both understanding the patho-physiology associated with trauma inrelation to aging, and designing betterintervention strategies to reduce mortal-ity and organ dysfunction.

In a recent study using a T-H model inthe rat, both the expression and the enzy-matic activity of cytochrome c oxidase, acomponent of the mitochondrial respira-

tory complex, was found to be reducedsignificantly, along with an increase incytosolic cytochrome c levels, decrease inmitochondrial ATP levels and increasedcardiomyocyte apoptosis (5). It has beenshown that cardiac output, left ventricu-lar performance and organ blood flow inthe liver, small intestine and kidney de-creased significantly following T-H andresuscitation (6,7). A prolonged depres-sion of cardiovascular function followingT-H, despite fluid resuscitation was alsoreported by other investigators (8–10).

We recently developed a custom ro-dent mitochondrial gene chip with probesets representing genes on the nuclearand mitochondrial DNA relevant to thestructure and function of mitochondria.Our initial experiments demonstrated atotal of 483 signature genes that were al-tered by hypoxia in the neonatal mouse

Aging Influences Cardiac Mitochondrial Gene Expressionand Cardiovascular Function following Hemorrhage Injury

Bixi Jian,1 Shaolong Yang,1 Dongquan Chen,2 Luyun Zou,3 John C Chatham,3 Irshad Chaudry,1,4 andRaghavan Raju1,4,

1Center for Surgical Research, Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama, United States ofAmerica; 2Clinical Translational Science Institute and Center for Neurosciences, West Virginia University, Morgantown, West Virginia,United States of America; 3Department of Medicine, Division of Cardiovascular Disease, University of Alabama at Birmingham,Birmingham, Alabama, United States of America; and 4Department of Microbiology, University of Alabama at Birmingham,Birmingham, Alabama, United States of America

Cardiac dysfunction and mortality associated with trauma and sepsis increase with age. Mitochondria play a critical role in theenergy demand of cardiac muscles, and thereby on the function of the heart. Specific molecular pathways responsible for mito-chondrial functional alterations after injury in relation to aging are largely unknown. To further investigate this, 6- and 22-month-old rats were subjected to trauma-hemorrhage (T-H) or sham operation and euthanized following resuscitation. Left ventricular tis-sue was profiled using our custom rodent mitochondrial gene chip (RoMitochip). Our experiments demonstrated a declined leftventricular performance and decreased alteration in mitochondrial gene expression with age following T-H and we have identi-fied c-Myc, a pleotropic transcription factor, to be the most upregulated gene in 6- and 22-month-old rats after T-H. Following T-H,while 142 probe sets were altered significantly (39 up and 103 down) in 6-month-old rats, only 66 were altered (30 up and 36 down)in 22-month-old rats; 36 probe sets (11 up and 25 down) showed the same trend in both groups. The expression of c-Myc and car-diac death promoting gene Bnip3 were increased, and Pgc1-α and Ppar-α a decreased following T-H. Eleven tRNA transcripts onmtDNA were upregulated following T-H in the aged animals, compared with the sham group. Our observations suggest a c-myc–regulated mitochondrial dysfunction following T-H injury and marked decrease in age-dependent changes in the transcrip-tional profile of mitochondrial genes following T-H, possibly indicating cellular senescence. To our knowledge, this is the first reporton mitochondrial gene expression profile following T-H in relation to aging.© 2011 The Feinstein Institute for Medical Research, www.feinsteininstitute.orgOnline address: http://www.molmed.orgdoi: 10.2119/molmed.2010.00195

Address correspondence and reprint requests to Raghavan Raju, Department of Surgery,

VH-G094, Volker Hall, 1670 University Blvd, Birmingham, AL 35294. Phone: 205-975-9710; Fax:

205-975-9715; E-mail: [email protected].

Submitted October 7, 2010; Accepted for publication December 21, 2010; Epub

(www.molmed.org) ahead of print December 22, 2010.

R E S E A R C H A R T I C L E

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cardiac myocytes (11). This included sev-eral transcripts on mtDNA. Pathwayanalysis demonstrated predominantchanges in the expression of genes in-volved in oxidative phosphorylation,glucose and fatty acid metabolism andapoptosis. The most upregulated genesfollowing 24-h hypoxia included HIF-1αinducible genes Bnip3, Pdk1 and Aldoc.While Bnip3 is important in the car-diomyocyte death pathway, Pdk1 en-zyme is critical in conserving mitochon-drial function by diverting metabolicintermediates to glycolysis.

In this manuscript, we report our find-ings on mitochondrial gene expressionchanges in rat cardiomyocytes subjectedto in vitro hypoxia and age-dependentcardiac mitochondrial gene expressionchanges in an in vivo model of T-H. Wesubjected 6- and 22-month-old rats to T-H, and mitochondrial gene expressionchanges were studied using our customgene chip, RoMitochip. Our findingsidentify possible new pathways thatmight regulate mitochondrial functionfollowing hemorrhage and its relation-ship to aging.

MATERIALS AND METHODS

AnimalsPregnant (E19) Fisher 344 rats were

purchased from Charles River Laborato-ries (Wilmington, MA, USA) and 2-day-old pups were used for neonatal car-diomyocytes isolation (12). For T-H study,6- and 22-month-old Fisher 344 rats wereobtained through the National Instituteof Aging (Bethesda, MD, USA). Severalstudies have characterized the cardiovas-cular system in the aged rat (13,14). Specifically, previous histological charac-terizations of aged rat heart have demon-strated fibrotic infiltration of left ventricleand decreased volume density of car-diomyocytes (15). All animal experimentswere carried out in accordance with theprotocol approved by the InstitutionalAnimal Care and Use Committee of theUniversity of Alabama at Birminghamand were consistent with the guidelinesof the National Institutes of Health.

Neonatal Cardiomyocyte Isolationand Hypoxic Exposure

Cardiomyocytes were isolated fromneonatal Fisher 344 rats (Harlan Labora-tories, CT, USA) as reported previously(12). After 4 d in culture, the cells wereexposed to normoxia or 1% hypoxia in aCO-48 incubator (New Brunswick, NJ,USA) for 24 h. RNA was isolated andgene expression profiles were determinedusing the RoMitochip developed in ourlaboratory as described previously (11).

Trauma-Hemorrhage ProcedureThis procedure was performed as de-

scribed earlier (6). For surgery, the ratswere fasted for 16 h prior to the proce-dure but allowed water ad libitum, anes-thetized with isoflurane (Minrad, Bethle-hem, PA, USA) and restrained in a supineposition. A 5-cm midline laparotomy wasperformed, which was closed ascepticallyin two layers with sutures (Ethilon 6/0,Ethicon, Somerville, NJ, USA). Bothfemoral arteries and the right femoral veinwere asceptically cannulated with poly-ethylene-10 tubing (Becton Dickinson,Sparks, MD, USA). The blood pressurewas monitored constantly by attachingone of the catheters to a blood pressureanalyzer (Digi-Med BPA-190, Micro-MedInc., Louisville, KY, USA). Upon awaken-ing, animals were bled rapidly throughthe other arterial catheter to a mean arte-rial blood pressure of 35 ±5 mmHgwithin 10 min. The bleeding was contin-ued till 50% blood volume was removedin approximately 45 min and the lowblood pressure was maintained for an-other 45 min by giving 40% of the shedblood volume as Ringer’s lactate in smallvolumes. At the end of the shock period,the rats were resuscitated with four timesthe shed blood volume in the form of lac-tated Ringer’s solution over 60 min.Bipuvacaine was applied to the incisionsites, catheters removed and incisionswere closed with sutures. The same sur-gical procedures were conducted onsham-operated animals, but they did notundergo hemorrhage or resuscitation.Two h following resuscitation, left ven-tricular performance was assessed, ani-

mals euthanized and left ventricles re-moved. The left ventricular performancewas evaluated by anesthetizing the ratswith pentobarbital sodium (~30mg/kgbody wt), inserting a PE-50 catheter intothe left ventricle via right carotid artery.Left ventricular contractility parameters,maximal rate of left ventricular pressureincrease (+dP/dtmax) and decrease(–dP/dtmax) were determined (6). Four tofive rats were used in each group.

MicroarrayThe RoMitochip had probe sets repre-

senting mitochondrial genes from bothrat and mouse. The mouse probe setswere validated previously (11). Mito-chondrial genome consists of a circularDNA (mtDNA) with about 16,000 basesand 37 transcripts (16). Of the 37 tran-scripts, 13 code for proteins, 2 code forribosomal RNAs (12 and 16S), and 22code for tRNAs. The 13 proteins on themitochondrial DNA are: seven subunitsof NADH dehydrogenase, cytochrome b,three cytochrome c oxidase subunits (1, 2and 3), and ATP synthase 6 and 8. Thegene sequences representing the rat mi-tochondrial genome included in the genechip encompassed published mtDNA se-quences of ten different inbred strains:WKY/NCrl (Wistar Kyoto; Accession #DQ673907); BN/NHsdMcwi (BrownNorway; Accession # AC_000022);F344/NHsd (Fisher 344; Accession #DQ673909); ACI/Eur (August × Copen-hagen Irish; Accession # DQ673908);FHH/Eur (fawn hooded hypertensive;Accession # DQ673910); GK/Swe (Acces-sion # DQ673913); GK/Far (Accession #DQ673912); T2DN/Mcwi (Accession #DQ673915); GH/OmrMcwi (Accession #DQ673911) and SS/JrHsdMcwi (DahlSalt-sensitive; Accession # DQ673914)(17). These specific strains were chosenbecause complete sequences of mtDNAwere available for each of them. Thegene transcripts from these ten differentstrains were aligned and 48 differentprobe sets were created to represent allthe 37 transcripts from these ten strains.The chip (11 micron, Affymetrix plat-form) contained a total of 419 probe sets


A G I N G A N D I N J U R Y A L T E R M I T O C H O N D R I A L G E N E E X P R E S S I O N

representing genes from mitochondrialand nuclear DNA from the rat.

Microarray ExperimentationThe microarray experiments were per-

formed as described before (11,18).Briefly, RNA was isolated from snapfrozen cardiomyocytes or snap frozenleft ventricles and RNA integrity waschecked by resolving on an Agilent 2100Bioanalyzer (Agilent, Santa Clara, CA,USA). One hundred ng of total RNA wasamplified from each RNA sample and la-beled using the Affymetrix Whole-Tran-script Assay (WTA) Sense Target Label-ing Protocol (Affymetrix, Santa Clara,CA, USA). Eleven μg of labeled senseDNA was used for hybridization. Ribo-somal RNA was not removed. The hy-bridized chips were washed using theAffymetrix fluidics station 450, stained,and scanned on the 3000 7G scanner asdescribed by the manufacturer(Affymetrix). GeneChip hybridizationswere carried out in the Gene ExpressionShared Facility of the ComprehensiveCancer Center at the University of Ala-bama at Birmingham. To study the geneexpression profile following in vitro hy-poxia or normoxia, four chips were usedper group. For in vivo experiments, onechip was used per animal and four chipswere used in each group.

Data AnalysisGene expression data was normalized

using RMA (19) and quantile normaliza-tion methods. After normalization, anal-ysis of variance (ANOVA) was applied tocompare gene expression level changes.P values for pairwise comparisons wereadjusted using the Tukey method inanalysis of variance (ANOVA). We usedstatistical software R and SAS v9.13 fordata analysis. For hierarchical clustering,raw data were quantile normalized, log2transformed, and pairwise compared byt test. Additional intensity filter was ap-plied. The selected gene lists were clus-tered using hierarchical clustering meth-ods and visualized using normalizedlog2-transformed intensities. P value wascorrected for False Discover Rate (FDR)

with Benjamini-Hochberg methods usingsoftware package GeneSpring (Agilent,CA, USA).

Real-time PCRReal-time PCR was carried out using

FAM-labeled Taqmann real-time PCRprimers for c-myc and β-actin (ABI, Fos-ter City, CA, USA). The template cDNAwas prepared by random priming fromRNA isolated from the tissues. The re-sults were expressed in relation to β-actinexpression. The PCR reaction was carriedout in an ABI 7500 thermal cycler (ABI).

Western BlotProtein expression of c-myc, Bnip3 and

Pgc1-α were analyzed by Western blot asdescribed (20). Briefly, total proteins intissue lysates were resolved using 4% to12% Nupage gel (Invitrogen, Carlsbad,CA, USA) and transferred to PVDF mem-branes. The membranes were saturatedwith blocking buffer (10 mmol/L Tris,150 mmol/L NaCl, and 0.05% Tween-20supplemented with 5% dry milk) for 1 hat room temperature and incubated withthe respective primary antibodies: Bnip3(Abcam Inc, Cambridge, MA, USA),Pgc1-α (Cell Singling Technology, Bev-erly, CA, USA), or β-actin (Abcam). Themembranes then were washed 5× withTBST (Tris-buffered saline supplementedwith 0.05% Tween-20) followed by incu-bation with an appropriate secondary an-tibody (Santa Cruz Biotechnology, SantaCruz, CA, USA) conjugated with horse-radish peroxidase for 1 h at room temper-ature. The membranes were againwashed 5× with TBST and probed usingECL (Amersham, Piscataway, NJ, USA),and autoradiographed.

All supplementary materials are availableonline at www.molmed.org.

RESULTS

Cardiac Function Decreases withAging and following Trauma-Hemorrhage Injury

Blood loss following hemorrhage proce-dure was confirmed by the blood hemato-

crit level (data not shown) followingreperfusion. Additionally, T-H resulted inincreased plasma lactate and decreasedmean arterial pressure as compared withsham controls in both age groups (datanot shown). Two h following T-H and re-suscitation, heart performance was testedby measuring +dP/dt and –dP/dt. Asseen in Figure 1, there was a significantdecrease in left ventricular function (±dP/dt) in the 22-month-old sham- operated rats when compared with6-month-old rats demonstrating an age-dependent decline in cardiac perform-ance. Furthermore, there was a markeddecrease in cardiac performance of theaged animals (Figure 1A) subjected to T-Has compared with the younger ones, dem-onstrating the age-dependent effect of he-morrhagic injury on heart performance.

Mitochondrial Gene Expression inCardiomyocytes following Hypoxia

Mitochondrial energetics is critical tocardiac function and, to investigate mo-

Figure 1. Left ventricular performance de-creases with aging and T-H. Six- and 22-month-old rats were subjected to sham orT-H procedure and left ventricular per-formance (+ and –dP/dt) was measured.dP/dt values expressed as mean > SE.Panel A, >dP/dt and panel B, –dP/dt. See“Materials and Methods” section for ex-perimental details.



lecular changes that may have con-tributed to altered mitochondrial func-tion following T-H and in the aged, ourobjective was to profile the mitochondr-ial transcriptome in the left ventriculartissue of 6- and 22-month-old rats. TheRoMitochip was designed to study mito-chondrial gene expression in the rats andmice, and we previously tested themouse probe sets using an in vitro hy-poxia model (11). In this study, prior tousing the RoMitochip in the in vivo T-Hmodel, we tested the rat probe sets usingRNA isolated from hypoxic or nomoxicneonatal rats. The objective of this studywas mainly to test the mitochondrialgene chip. This experiment was not in-tended to infer hypoxic molecular path-ways in vivo. The isolated neonatal car-diomyocytes were subjected to 24-hhypoxia (1% oxygen) and microarray ex-periments were performed in quadrupli-cate. Of the 419 rat mitochondrial probesets on RoMitochip, 208 demonstratedsignificant alteration as defined by P <0.05 and 163 probe sets demonstratedmore than 1.2-fold with significance (P <0.05). Among the probe sets that demon-strated most upregulation were Bnip3(10-fold), Egln3 (7.5-fold), HK2, Myc (1.9-fold), Mdm2 (1.8-fold) and Vdac1 (1.5-fold) (Supplementary Table 1A). Allthese genes (except Egln3) also werefound to be upregulated in the mousecardiomyocytes in the previous study(11). The expression of Pla2g2a (phospholi-pase A2, group IIA) was the most down-regulated (29-fold). Among the othergenes that were downregulated follow-ing hypoxia were Bcl2 (2.7-fold), Mtch2(2.2-fold) Casp7 (2.1-fold), Casp3 (2.9-fold)and Bid (1.7-fold) (Supplementary Table1B). Among the 163 probe sets thatdemonstrated significant change beyond1.2-fold, 100 were downregulated and 63were upregulated following 24-h hy-poxia (Supplementary Table S1).

Influence of Age on Gene ExpressionFollowing T-H

Following T-H, there were moredownregulated genes than upregulatedin both 6-month as well as in 22-month

groups (Supplementary Table 2). Morestrikingly, the total number of genes al-tered (upregulated or downregulated)was markedly less in the aged groupand it was more prominent in the case ofdownregulated genes. Overall, when ex-pression of 142 mitochondrial probe setsdemonstrated significant change follow-ing T-H in the 6-month group, the ex-pression of only 66 were altered in theaged group (Supplementary Table 2).Among the upregulated genes, the ma-jority of them were upregulateduniquely in each age group—in contrastto the downregulated genes, amongwhich most of the downregulated genesin the 22-month group also werechanged similarly in the 6-month group,demonstrating a clear difference in mito-chondrial gene expression profile follow-ing injury in relation to aging.

Age-Independent Expression ofMitochondrial Genes following T-H

There were 11 genes upregulated and25 downregulated following T-H, com-mon to both age groups (SupplementaryTable 3). Myc, Jak2, glutaredoxin (Glrx1),hexokinase 2 (Hk2), HIF-1α and Sod2 wereamong the genes significantly upregu-lated in both 6- and 22-month-old ani-mals. Among these, Myc was the mostupregulated (6-month: 6.2-fold; 22-month:3.8-fold) followed by Jak2 (6-month: 3.9-fold; 22-month: 2.3-fold) (Figure 2).Whereas, the expression of genes carnitinepalmitoyltransferase 1 (Cpta), glycerol 3-phosphate acyltransferase (Gpam), uncou-pling protein (Ucp) 3 and isocitrate dehydro-genase 1 (Idh1) were downregulated inboth age groups (see Figure 2). Thoughexpression of these genes were alteredfollowing T-H irrespective of the age ofthe animal, the amplitude of expressionwas less in the aged than in the younger(see Figure 2, Supplementary Table 3).

Mitochondrial Gene Expression in 6-Month-Old Animals following T-H

The expression of a total of 36 probesets were increased and that of 103 probesets were decreased in 6-month-old rats,after T-H and resuscitation. When the

subset of genes that were similarly alteredin the aged rats was excluded, theuniquely increased or decreased probesets were 28 and 78 respectively (Supple-mentary Table S2). Among the genes thatdemonstrated a significant upregulationfollowing T-H only in the 6-month agegroup were: tissue-type transglutaminase(Tgm2), caspase 3 (Casp3), Mdm2, caspase 12(casp12), Bnip3, Tp53 and Tfam. Ppar-α,Pgc-α, Casp9 and Ogt expression were sig-nificantly downregulated following T-Hin the 6-month-old rats. The Western blotexperiments confirmed that the increasedgene expression of Bnip3 and decreasedexpression of Pgc1-α were consistent withtheir protein expression (Figure 3A, B).

Mitochondrial Gene Expression in 22-Month-Old Animals following T-H

The gene expression profile of 22-month-old rats demonstrated 30 probesets to be upregulated and 36 downregu-lated following T-H. However, 11 of the 30upregulated and 25 of the 36 downregu-lated genes also were altered in the 6-month-old rats. Therefore expression ofonly a smaller number of genes was ob-served as unique to the aged group (Sup-plementary Table 4). Among the uniquelyupregulated transcripts in the 22-monthage group, 12 of the 19 probe sets repre-

Figure 2. Mitochondrial gene expressionchanges following T-H. Changes in expres-sion of a subset of mitochondrial genesfollowing T-H in two different age groups(6- and 22- months). Upregulated (A) anddownregulated (B) genes in each groupare shown as bars. Bars represent meanfold change with a P < 0.05.



sented mtRNA transcripts. The 11uniquely downregulated genes included,alcohol dehydrogenase (aldh) 1 and 2, caveolin1 (Cav1) α and nitric oxide synthase (Nos) 1.

mtDNA Transcript Alteration followingT-H

The RoMitochip had 48 probe sets rep-resenting mtDNA transcripts, none ofthese were found to be significantly al-tered in 6-month-old rats. However, 13 ofthese probe sets were upregulated signifi-cantly in the 22-month-old rats. Twelve ofthe 13 probe sets represented tRNA tran-scripts: t-RNA to threonine, aspartate,isoleucine, phenylalanine, alanine, glycine,arginine, glutamate and serine (Figure 4).We had previously reported similar in-creased expression of tRNA transcripts fol-lowing hypoxia in cardiomyocytes (11).

Myc Expression following T-H in 6- and 22-Month-Old Rats

As shown in Figure 2, the most upregu-lated gene following T-H in both agegroups was c-Myc. The increase in mRNAlevels of Myc was consistent with its pro-tein expression (Figure 5A–C). The in-crease in c-Myc expression was associatedwith decreased expression of Pgc-1α gene

in the 6-month-old rats, which is also con-firmed by Western blot (Figure 3A).

DISCUSSIONA prolonged depression of cardiovascu-

lar function occurs following hemorrhagicshock despite fluid resuscitation. Agingalso is found to be a contributing factor toposttraumatic complications and mortal-ity (21). Impaired mitochondrial functionand decreased mitochondrial ATP havebeen observed following T-H (22).

Hemorrhagic shock causes a wholebody hypoxia/reperfusion (H/R) injury,leading to dysregulation of biochemicalpathways and multiple organ dysfunction

syndrome (6,23–27). Cardiac output,stroke volume, and cardiac contractilitydecrease significantly following T-H(7,28–30). Experimental evidences indicatethat reactive oxygen species (ROS) playsignificant role in cardiac ischemia/reper-fusion (I/R) injury and mtDNA has beenshown to be a major target of ROS- mediated cellular injury (31,32). Also, mi-tochondrial calcium overload plays an im-portant part in I/R injury (33,34). DuringI/R, ischemia induced by the deprivationof coronary blood flow and subsequentreoxygenation by reperfusion results in adramatic elevation of mitochondrial ROSgeneration (35,36). This may lead to either

Figure 3. Pgc-1α and Bnip3 protein expres-sion changes following T-H. Left ventriculartissues from 6-month-old rats subjected toT-H or sham operation were tested forPgc-1α (A) and Bnip3 (B) by Western blot.A representative result from two sham andtwo T-H rats are given. The bar graph rep-resents mean and standard error of threeexperiments using tissues from four shamand four T-H rats.

Figure 4. Alteration of tRNA transcripts on mtDNA following T-H. Transcripts of tRNAs thatdemonstrated a significant upregulation in 22-month-old rats following T-H. Expressionchanges in each animal are represented as a heat map (see “Materials and Methods”for details).

Figure 5. Upregulation of c-Myc following T-H. Left ventricular tissues from rats subjectedto T-H or sham operation were tested by Western blot (A and B) or real time PCR (C) forc-Myc expression. The bands were quantified by densitometry (C).



programmed cell death or a precondition-ing response that may prevent cell deathafter a subsequent, more severe ischemia.The studies from our group and othershave demonstrated hepatic and cardiacdysfunction following T-H (37–39). T-Hcauses tissue hypoxia and the oxygen de-ficiency has been demonstrated to havedirect effects on sarcolemma and on mito-chondria (40). Furthermore, when anoverwhelming amount of evidence linksmitochondrial dysfunction with cellularsenescence and aging, it has been ob-served that subsarcolemmal and interfib-rillar mitochondria experience functionaldecline during aging with a selective re-duction in oxidative phosphorylation (41).These functional correlates might explainage- dependent effect of T-H on contractil-ity and relaxation. Additionally, our experiments demonstrated enhanced en-doplasmic reticulum (ER) stress in meta-bolically active organs following T-H(20,42). Previous studies have successfullydemonstrated the usefulness of focusedmicroarrays in profiling mitochondrialgene expression (11,43,44). One of the fac-tors involved in T-H injury that causes ERstress is hypoxia, and our recent in vitrostudies have identified over 400 signaturemitochondrial genes that were altered fol-lowing hypoxia in cardiomyocytes (11).Along with decreased ATP levels, we alsoobserved augmented expression of genescorresponding to glycolysis (for example,hexokinase) as well as those involved inpreventing the entry of pyruvate to mito-chondria (for example, Pdk1) (11), consis-tent with the well-known Warburg effect.One of the most upregulated genes inmouse cardiomyocytes subjected to hy-poxia was Bnip3. Similarly, when rat car-diomyocytes were subjected to hypoxiaand gene expression profiled using RoMi-tochip, as reported in this communication,Bnip3 was the most profoundly expressedgene (Supplementary Table 1A). In addi-tion to this, the results from rat experi-ments shared altered expression of severalgenes with the mouse cardiomyocytesthat were subjected to 24-h hypoxia. Inter-estingly, as shown in this paper, many ofthese genes (such as Bnip3, caspase 3 and

hexokinase) also were altered following T-H in an in vivo model. Apart from iden-tifying augmentation of the cardiac deathpromoting gene in this in vivo injurymodel, the study also unraveled the in-creased expression of a gene less knownin injury models, c-Myc.

The results of the study using 6- and22-month-old rats indicate that there werefewer genes altered following T-H in theaged (22-month) group as compared theyounger (6-month). In addition, the extentof change for individual genes thatdemonstrated altered expression was lessin the aged group as compared with the6-month-old animals (see Figure 2). Theseobservations are important in the contextof cellular senescence induced by theaging process (Supplementary Table 1)

Interestingly, there was an upregula-tion of 10 tRNAs located in mtDNA inthe old (22-month) rats (see Figure 4).This increased level of tRNAs was con-sistent with the observation in in vitrohypoxia experiments using cardiomy-ocytes (11). This might imply that there isa transcriptional-to-translational switchin the aged-to-conserved energy. As seenin Figure 2, we observed a significantdownregulation of Pgc1-α, Ppar-α andthe purine receptor Adora1 and upregula-tion of Bnip3, caspase 3, caspase 12 and p53in 6-month-old rats. However, thesegenes remained unaltered in 22-month-old rats indicating an age- dependent de-cline or inability in their expression. Ad-ditionally, among the most upregulatedgenes following T-H in both age groupswere c-Myc (Figure 5), Janus kinase (Jak) 2,hexokinase (Hk) 2 and Hif-1α; and amongthe most downregulated genes in bothage groups included carnitine palmitoyl-transferase I (Cpt1) and glycerol 3-phos-phate acyltransferase (Gpam). Amongthese, c-Myc mRNA expression was themost augmented. These observationsconfirm a reduced utilization of fattyacid oxidation products in the mitochon-dria (downregulation of Cpt1) and aswitch to glycolysis following T-H, akinto hypoxic condition.

Our results are quite consistent withthe previous reports that c-Myc activa-

tion leads to downregulation of Pgc-1αas well as the downstream target Cpt-1implicated in fatty acid oxidation (45),though we observed an age-related dif-ference in the expression pattern of thesegenes. Using Western blot, we confirmedthat the increase in gene expression ofc-Myc was followed by marked increasein its protein expression (see Figure 5).Pgc-1α coactivates the coordinated ex-pression of genes involved in mitochon-drial biogenesis, energy production anddefense systems against reactive oxygenspecies (ROS), such as the genes encod-ing Tfam, ATP synthase, and uncouplingproteins. Pgc-1α acts with transcriptionfactors such as Ppar-γ, retinoid receptor(RXR)-α and nuclear respiratory factor-1(Nrf-1). It has been shown previouslythat Ppar expression after T-H decreases,and its augmented expression reducesliver apoptosis following T-H (46). Uponevents such as oxidative or cold stress,expression of Pgc-1α is upregulated bytranscriptional activities and down-stream target genes are activated (47).Pgc-1 family of transcriptional coactiva-tor control fatty acid oxidation and mito-

Figure 6. Regulation of Pgc-1α and mito-chondrial function by c-Myc, T-H andaging. Aging and T-H are known to reducePgc-1α activity. Our results indicate that c-Myc expression increases following T-H inboth age groups and this transcriptionfactor might be the mediator for de-creased Pgc-1α following injury, resulting indiminished mitochondrial biogenesis andfunction.



chondrial biogenesis in the adult heart.In ischemic and hypoxic conditions,Pgc-1 is downregulated to promote glu-cose metabolism and suppress fatty acidoxidation (48). However, c-Myc, apleiotropic transcription factor that en-hances the glycolytic pathway, also is re-ported to enhance mitochondrial activityby producing biosynthetic substrates.c-Myc was increased significantly in bothage groups (6.3- and 3.8-fold), thoughmore at 6 months (see Figure 5). HIF, thetranscript of which was also increasedsignificantly at both age groups (1.5- and1.4-fold, at 6 and 22 months respectively)(see Figure 2), specifically blocks accessof glycolytic end products to mitochon-dria. Hif-1 is an oxygen-sensing tran-scription factor that initiates transcrip-tion of an array of genes during hypoxicinsult. Decreased cellular oxygen resultsin decreased breakdown and stable cyto-plasmic level of Hif-1α. This allows thedimerization of Hif-1α with Hif-1β andbinding of the dimer to hypoxia responseelement (HRE) to recruit transcriptionalcoactivators (49). The effect of hypoxia inpromoting glycolysis while reducingATP production through decreased mito-chondrial oxidation in cardiomyocytessubjected to hypoxia was observed previ-ously in in vitro experiments (11). The en-hancement of c-Myc following T-H issuggestive of its role in promoting gly-colytic process in injury. Furthermore, c-Myc activates p53—promoting apoptosis.The increased expression of c-Myc is alsoimportant in T-cell activation and inproinflammatory mediators, as it in-structs a complex inflammatory pro-gram, including induction of IL-1β(50,51). HIF and c-Myc act on multipletargets to regulate carbon metabolismand act in concert to fine tune adaptiveresponses to hypoxic environment (52).In pathological stress, c-Myc regulatesthe increased metabolic energy demand,then mitochondrial biogenesis (45). Ad-ditionally, the c-Myc protein level hasbeen shown to increase markedly afterhypoxia and ischemia (53). The upregu-lation of c-Myc was consistent with theobserved decreased expression of genes

negatively regulated by c-Myc (Cpt1[–2.3-fold] and Gpam [–2.1-fold]). Thecritical function of c-Myc in regulatingcardiac energy metabolism during stressand its marked upregulation after T-H inboth age groups give substantial cre-dence to its significance in energy bal-ance in response to T-H injury and ensu-ing cell death (20).

The reason for the lack of change inPgc-1α or Bnip3 transcripts in the oldage (22 months), to be consistent with anincrease in c-Myc and Hif-1α, remains tobe elucidated and we speculate that thisis characteristic of cellular senescenceconcomitant with aging. In this study,we examined the mitochondrial gene ex-pression changes at 2 hours followingT-H. In further studies, we also mayhave to test at 24 hours and 72 hours fol-lowing T-H to determine the influence ofaging and injury on temporal gene ex-pression changes. It also remains to be ex-plored whether, in aging, c-Myc-inducedmitochondrial biogenesis involves path-ways independent of Pgc-1α and itsdownstream effectors. Therefore, thegenes induced by c-Myc following T-Hin the different age groups remain to beidentified, and this is expected to bringattention to the observed age-specificgene expression profile.

Aging is associated with a reducedaerobic activity in humans and declinedmitochondrial function (54). A reductionin maximal mitochondrial ATP produc-tion rate and mitochondrial DNA abun-dance occurs with age in associationwith muscle weakness and reduced en-durance in elderly people (55,56). A pro-gressive decline in muscle mitochondrialDNA abundance and protein synthesiswith age also has been reported by oth-ers (57,58). The identification of c-Myc asa potential target gene in injury andaging and its relationship to mitochondr-ial function may unravel novel pathwaysin injury and its resolution in relation toaging (Figure 6). Our future studiesusing clinical specimens will further as-certain the significance of c-Myc-inducedmitochondrial functional alteration andits role in energy balance.

ACKNOWLEDGMENTSThe study was supported by NIH

grants AG 031440 (R Raju), GM 39519 (I Chaudry), HL101192, HL079364 andHL67464 (JC Chatham) and the UABHSF GEF Scholar Award (R Raju). Themicroarray experiments were carried outin the Heflin Center for Genomic Scienceby Michael Crowley and supported bythe UAB Comprehensive Cancer CenterCore Grant 5P30 CA13148-37.

DISCLOSURERoMitochip is a custom chip made

with funding from the National Instituteof Aging. None of the authors had finan-cial profit from this chip in this study.

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Aging Influences Cardiac Mitochondrial Gene Expression and ...€¦ · 542 | JIAN ET AL. | MOL MED 17(5-6)542-549, MAY-JUNE 2011 INTRODUCTION Aging is a significant factor that con-tributes