Forkhead Transcription Factors Coordinate Expression of Myocardial KATP Channel Subunits and Energy...

Montessuit and Alex J. BaertschiPierre Philip-Couderc, Nadia Isidoro Tavares, Angela Roatti, René Lerch, Christophe

Subunits and Energy MetabolismForkhead Transcription Factors Coordinate Expression of Myocardial KATP Channel

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Forkhead Transcription Factors Coordinate Expressionof Myocardial KATP Channel Subunits and

Energy MetabolismPierre Philip-Couderc, Nadia Isidoro Tavares, Angela Roatti,

Rene Lerch, Christophe Montessuit, Alex J. Baertschi

Abstract—Coordinate adaptation of myocyte metabolism and function is fundamental to survival of the stressed heart, butthe mechanisms for this coordination remain unclear. Bioinformatics led us to discover that Foxs are key transcriptionfactors involved. We performed experiments on the mouse atrial cell line HL-1, neonate rat heart myocytes, and an adultrat model of myocardial infarction. In electrophoretic mobility-shift assays, FoxO1 binds to the FoxO concensus site ofthe KATP channel subunit KIR6.1 promoter. In primary atrial culture, targeting FoxO1 and FoxO3 with siRNAspecifically reduces mRNA expression of FoxO1 and -O3 and KIR6.1. Western blots, confocal immunofluorescence,and quantitative RT-PCR was applied for measuring expression of 10 Fox, 6 KATP channel subunits, and 12 metabolicgenes. FoxF2, -O1, and -O3 strongly associate with expression of KATP channel subunits (in particular, KIR6.1,SUR1A and SUR2B) in different heart tissues and in the periinfarct zone of the left ventricle. Patch-clamp recordingsdemonstrate that molecular plasticity of these channels is matched by pharmacological plasticity and increasedsensitivity to a metabolic challenge mimicked by the protonophore CCCP. A balance of FoxF2 and FoxO also regulatesexpression of at least 9 metabolic genes involved in setting the balance of glycolysis and �-oxidation. Bioinformaticsshows that the transcriptional mechanisms are highly conserved among chicken, mouse, rat, and human, and Fox areintimately linked to other metabolic sensors. Thus, FoxF2 and -O are key transcription factors coordinating expressionof KATP channels and energy metabolism. (Circ Res. 2008;102:e20-e35.)

Key Words: Fox transcription factors � KATP channels � metabolic genes � myocardial infarction

Metabolic adaptation is vitally important for cellularfunction, stress adaptation, and survival. This is partic-

ularly true in metabolically highly active tissues. In heart,1

brain,2 kidney,3 adipose tissue,4 muscle,5 and blood vessels,6

the ATP-dependent potassium channels (KATP channels)emerge as the most important sensors of cellular energystatus.7 During metabolic stress, these heterooctameric chan-nels open in response to an increased cytoplasmic ADP/ATPratio, thereby hyperpolarizing the cell and reducing thecalcium influx and metabolic demand.1 KATP channelsprotect cardiac myocytes in pathophysiological situations,8 aswell as during vigorous exercise. For example, mice withknockout of the pore-forming KATP channel subunit KIR6.2,or mice overexpressing dominant negative KIR6.1, tolerateonly half of the workload compared with wild-type mice, andhalf of them die.9,10

KATP channels are, however, amazingly diverse, varyingin their subunit composition from one tissue to the next. Thepotassium channel pore consists of 4 KIR6.2 subunits in leftventricle,11 pancreatic �-cells,12 and vascular endothelium13

and mainly of 4 KIR6.1 subunits in vascular smooth mus-cle.14 The brain2 and skeletal muscle15 express both KIR6.1and KIR6.2. The same cardiac tissue may change its subunitcomposition: subunit Kir6.1 is increasingly expressed in leftventricle following ischemia,16 exercise,17 or treatment withKATP channel agonist.18 Moreover, differences in subunitcomposition of cardiac KATP channels arise during develop-ment, aging, and between male and female.19–21 The varyingexpression of the regulatory subunit sulfonylurea receptor(SUR) adds additional complexity. SUR2A is expressed inleft ventricle,22 SUR1A in pancreatic �-cells, and SUR2B invascular smooth muscle.23

The puzzling molecular diversity of these metabolic sen-sors raises 2 unresolved questions. What mechanisms causethe varying expression of the KATP channel subunits? Isaltered composition of KATP channels coordinated withaltered metabolic control? We previously found that atria andventricles strikingly differ in KATP channel properties andsensitivity to ADP24,25 and sought to determine whethercorresponding differences existed in transcriptional mecha-

Original received June 12, 2007; resubmission received October 25, 2007; revised resubmission received December 5, 2007; accepted January 3, 2008.From the Department of Neuroscience (P.P.-C., A.R., A.J.B.) and Division of Cardiology (N.I.T., R.L., C.M.), Hopitaux Universitaires de Geneve,

Centre Medical Universitaire, Geneva, Switzerland.Correspondence to Dr Alex J. Baertschi, Department of Neuroscience, CMU, 1 rue Michel Servet, 1211 Geneva 4, Switzerland. E-mail

[email protected]© 2008 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.107.166744

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nisms. Accordingly, our bioinformatics approach reveals thatForkhead transcription factors (Foxs) would be prime candi-dates mediating KATP channel expression (Figure 1 andTables 1 and 2). Foxs have been known since 1990. Theirspecific DNA-binding domain is highly conserved throughanimal evolution. They are emerging as key players indevelopmental and metabolic processes and could act astransactivators or transrepressors.26 Their powerful role isattested by the fact that some Foxs, such as FoxA, can actindependently of acetylation or compaction of the nucleo-some. In pancreatic �-cells, FoxA2 causes expression of a

cluster of genes including SUR1-KIR6.2, GLUT2, glucoki-nase, hexokinases, and L-pyruvate kinase.27 The number ofFox members seems to correlate with anatomical and func-tional complexity: there are 4 Fox genes in Saccharomyces,15 in Caenorhabditis, 20 in Drosophila, and 39 in Homosapiens.26 Potential binding sites for Foxs also exist on manygenes encoding glucose and fatty acid transporters andenzymes of the glycolytic and �-oxidation pathway (Table 3).Thus Foxs possibly coordinate the expression of KATPchannels and enzymes and transporters of metabolic path-ways. The experiments described below support thishypothesis.

Materials and MethodsCell CultureHearts from 2- to 3-day-old rats were carefully microdissected toseparately collect right atrial appendages, left atrial appendages, leftventricles, and right ventricles. The tissues were incubated for 4hours at 4°C in trypsin (2.5%) and dissociated for 15 minutes at37°C. After preplating at a density of 130 000 cells/cm2 on plastic toremove a majority of fibroblasts, the suspended cells were plated andcultured for 24 hours on plastic dishes for the extractions of mRNAand protein. Cells were also plated at low density (10 000 to 50 000cells/dish) on fibronectin–gelatin coated 22-mm glass slides andcultured pending immunocytochemistry or patch-clamp recordings,as described previously.28,29 Medium was DMEM-F12 with 10%FBS, insulin–selenium–transferrin, penicillin–streptomycin, andascorbic acid. To prevent fibroblast proliferation, 20 �mol/L cyto-sine arabinonucleoside was added to the culture medium. Contami-nation with vascular smooth muscle cells or myofibroblasts was9.1�2.2% after 48 hours (mean�SEM, N�10), as ascertained byimmunofluorescence for �-smooth muscle actin (antibody wasprovided by Dr M.-L. Piallat, Centre Medical Universitaire, Geneva,Switzerland).

The HL-1 mouse atrial cell line30 was a generous gift from DrWilliam C. Claycomb (LSU Health Sciences Center, New Orleans,La). HL-1 cells were cultured under a 5% CO2 atmosphere inClaycomb medium (JRL Bioscience) supplemented with 10% FBS,4 mmol/L L-glutamine, 0.1 mmol/L norepinephrine, 100 U/mLpenicillin, and 100 �g/mL streptomycin. The medium was replacedevery 24 to 48 hours. Cells were grown in T75 culture flasks or on22-mm glass coverslips that were precoated overnight with Dulbec-co’s PBS containing 0.012 mg/mL fibronectin and 0.2 mg/mLgelatin.

Myocardial InfarctionThis investigation conforms to the NIH Guide for the Care and Useof Laboratory Animals. Myocardial infarction was induced in 200- to225-g male rats by ligature of the left coronary artery as de-scribed.31,32 Control rats underwent a sham operation. One, 7, 28,and 140 days after coronary occlusion, the heart was excised under

Table 1. Forkhead Transcription Factors With Potential Binding Sites on Promoters of Both Channel andMarker Genes

SUR2 Promoter KIR6.1 Promoter

47–50 kb 30–31 kb 19–25 kb 19–19.3 kb 12–12.3 kb

MLC2a D3 F1 F2 I1 J1 J2* A1 A2 A1 A2 P1 P1 A1 A2 C1 D3 F1 F2 I1 J1 J2 L1*

proANP Q1 P1 A1 A2 C1 D3 F1 F2 I1 J1 J2 L1*

MLC2v A1 A2 D3 I1 J2 P1 P1

proBNP A1 A2 D3 I1 J2* P1

Pairwise gene sequence alignment reveals an association between Forkhead transcription factors, channel genes, and atrial andventricular markers. Note 5 clusters of putative Fox-binding sites in the SUR2–KIR6.1 gene. Sequences are numbered in kilobases(kb) 5� relative to first nucleotide of transcript. *Forkhead consensus site.

Figure 1. Predicted putative binding sites for transcription fac-tors on KATP channel genes. A, SUR2-KIR6.1 gene on chromo-some 12p (human) and 4 (rat). B, SUR1-KIR6.2 gene on chro-mosome 11p (human) and 1 (rat). Criteria for selection oftranscription factors were: 90% sequence identity between spe-cies (human, rat, mouse, and chicken), exclusion of commonlyencountered members of the transcription machinery, andknown expression in the cardiovascular system. Note the promi-nent role of the Forkhead family of transcription factors (boldlettering). See the section Bioinformatics under Materials andMethods and the section Abundance of Fox-Binding Sites onSUR-KIR6 and Metabolic Genes under Results.

Philip-Couderc et al Foxs Coordinate KATP Channels and Metabolism e21




deep pentobarbital anesthesia (150 mg/kg IP). The infarct borderzone, defined as the myocardium 2 mm around the infarct scar, wascarefully dissected out. Cardiomyocytes were isolated by incubationin collagenase solution, followed by stepwise recovery to physiolog-ical calcium levels.

RNA Extraction, Reverse Transcription, andReal-Time PCRTotal RNA was extracted by disrupting cells in TRIzol reagent(Invitrogen) and prepared according to recommended procedures.The quality of RNA was controlled in agarose gels by the 18S/28Sratio. RNA concentration was measured by spectrophotometry at 260nm, and their quality was controlled by the 280/260 nm emissionratio. RNA was subjected to DNase I to remove all genomic DNAcontamination with DNA-free (Ambion) as indicated by the manu-facturer. Reverse transcription was performed on 1 �g of total RNAwith Thermoscript (Invitrogen), random primers, and dNTP asindicated by the manufacturer. Real-time PCR was performed oncDNA with SYBR Premix ExTaq Takara in an I-Cycler thermocy-cler (Bio-Rad). Oligonucleotides were designed with the softwarePrimer express 2. Specific TaqMan probes were synthesized byApplied Biosystems and primers by Microsynth (for primers andprobes, see Table 4). The standard curve method was used forrelative quantification. The mRNA values were normalized by thecorresponding 18S rRNA and cyclophilin mRNA. The differentnormalization procedures yielded similar results.

Electrophoretic Mobility-Shift AssaysNuclear extracts from cell cultures were prepared according to themethod of Schreiber et al.33 Hela cells were used as positive control

of the method, using probes for SP-1. Transfected HL-1 cells andnontransfected primary cultured atrial myocytes were used for thetests. Positive controls for Fox binding were made with HL-1 cellstransfected with FoxO1 plasmid. Oligonucleotides were obtainedfrom Microsynth: ATTCGATCGGGGCGGGGCGAG for the tran-scription factor SP-1 as a positive control; TGATGAGTGTTTGTT-TATGAG (FoxO probe; Figure 4A) and GATGAGTGTTTAT-TATATGAG (FoxO1 probe; Figure IC and ID in the online datasupplement) for the consensus sequence (mouse, rat, human con-served) found in the KIR6.1 promoter. Oligonucleotides werelabeled by the T4 polynucleotide kinase with [�-32P]ATP andpurified on spin columns (NucleoSpin extract II [MN]). The labeledprobes (80 000 to 100 000 cpm) were incubated with, or as a controlwithout, the nuclear extracts in gel shift–binding buffer (20%glycerol, 5 mmol/L MgCl2, 2.5 mmol/L EDTA, 2.5 mmol/L dithio-threitol, 250 mmol/L NaCl, 50 mmol/L Tris-HCl (pH7.5), 0.25mg/mL poly(dI-dC). Cold probe was coincubated for displacing theradioactive probe. Electrophoresis of DNA–protein complexes wasperformed on 4% polyacrylamide gels (60:1 acrylamide:bisacryl-amide). Gels were dried and placed for at least 5 days against anautoradiographic film with an amplification screen at �80°C.

Western BlotCell cultures were disrupted with lysis buffer (25 mmol/L Tris,150 mmol/L NaCl, 5 mmol/L EDTA, 1% [vol/vol] Triton X-100, pH7.5, supplemented with a cocktail of protease inhibitors [Roche]).Fifty micrograms of protein were loaded on a polyacrylamide–sodium dodecyl sulfate gel that was blotted on a 0.45-mm nitrocel-lulose membrane BA85 (Schleicher et Schuell), as described

Table 2. Forkhead Transcription Factors With Potential Binding Sites on Promoters of BothChannel and Marker Genes

SUR1 Promoter KIR6.2 Promoter

11–11.2 kb 6.2–6.4 kb 2.9–3.8 kb 4.2–4.5 kb 0.3–0.4 kb

MLC2a P1

proANP P1

MLC2v P1 P1

proBNP A1 A2 D3 F1 I1 J1 J2 P1* P1

Pairwise gene sequence alignment reveals an association between Forkhead transcription factors, channel genes,and atrial and ventricular markers. Note 5 clusters of putative Fox-binding sites in the SUR1–KIR6.2 gene. Sequencesare numbered in kilobases (kb) 5� relative to first nucleotide of transcript. *Forkhead consensus site.

Table 3. Putative Binding Sites on Promoters of Metabolic Genes

Metabolic Gene Protein Binding to Promoter Region

Abbreviation Name I II III

CD36 Fatty acid transporter 36 Fox O1O4 A1 A2 J2 D1 M1 F1 F2 C1 C2 SREBP PPAR ROR HIF

LPL Lipoprotein lipase Fox A1 A2 D3 O1 C1 C2 F1 F2 O4 O3 J2 M1 PPAR HIF

FABP3 Fatty acid binding protein Fox A1 A2 C1 C2 F1 F2 D1 O1 O3 SREBP HIF

MCAD Medium chain acyl CoA dehydrogenase Fox O3 O1 O4 HIF SREBP

ACC� Acetyl CoA carboxylase � Fox A1 A2 C1 C2 D3 F2 I1 J1 O1 O4 SREBP Nkx2.5

MCD Malonyl Co decarboxylase Fox A1 D3 I1 L1 M1 SREBP HIF PPAR

GLUT4 Glucose transporter 4 SREBP PPAR Fox F2 A1 A2 J2 C1 C2O3 O4

GLUT1 Glucose transporter 1 OCT GATA Nkx2.5 PPAR SREBP

PFK2 Phospho-fructokinase 2 SREBP HIF PPAR

GAPDH Glyceraldehyde phosphate dehydrogenase HIF PPAR NFKB SREBP

PKM Pyruvate kinase-muscle SREBP PPAR ROR Fox A1 A2 C1 C2 O4 I1HIF

SREBP1 Sterol responsive element binding protein 1 SREBP Fox A1 A2 D3 F1 F2 I1 L1

Note: Promoter regions I, II, and III are 0–3 kb, 3–10 kb, and 10–20 kb upstream of the translation initiation site, respectively.

e22 Circulation Research February 1, 2008




Table 4. Primers and Probes

Forward Primers Reverse Primers Probes

Normalization genes

18S TCGAGGCCCTGTAATTGGAA CCCTCCAATGGATCCTCGTT AGTCCACTTTAAATCCTT

Cyclophilin GGTCCTGGCATCTTGTCCAT CTCAGTCTTGGCAGTGCAGATAA

K� channel genes

KIR 6.1 Mm TTGAACCACACGGGTCATCAT GCAGGTGCTATTTGCTTGGTTAG

KIR 6.1 Rn AATTCATGACTCCAGAAGGAAACC GGGACTCAGTGAGTGGCTGTCT CCCATCAGAATCATG

KIR 6.2 Mm CACGGCGGGATAAGTCTACCT ATCATTTGCCCCCTTCTTGTC

KIR 6.2 Rn CCTTGATGGTCCCGTTCAGA GCAGCAGTCCCCACTCTCTCT TCCTCTACCCTTGACTCC

SUR1 ATGACGGCTGGGCAGATCT GAGGTTTGGGCATAAGAAGAAAAA

SUR1A Rn AGCATGCGTGGTACTCATC GCTGAGAGCTCCCTGTGTAG CTGCCACCTCCATCTCCAACTCC

SUR1B Rn TGCCTCTCTCTTCCTCACA GTTGGATCTCCATGTCTGC GAAGTCCGCATGGTCTCCAA

SUR2A Mm TTTTCTGAGGGTATTTTAGTGGAGTGT CACCAAAGTAGAAAAGAGGCCATT

SUR2A Rn CGGGCCTTGTTTTAGTCTT AAAGAGGCCATTCTTGTGC TGCGATACTGGTCCAAACCTGCT

SUR2B Mm ACATGGCCACGGAAAACATT CCCGATGAGCTATGGTTACGA

SUR2B Rn GGGTTCACACCATTCTGACT CGAACACTCCATCCTCCTG TTGGAATACGACACCCCGGAAAG

KIR2.1 Rn TCACGGCTGCCTTCCTCTT TCGGGCACTCGTCTGTAACA

KvLQT1 Rn GAAACACTTCAACCGGCAGATC TCAGGATTCTCCGCAGCATA

Forkhead genes

FoxA2 Rn GAGAAAGGACAAAAATGCATCCA GCACGCAGAAACCATAAATTAAATC

FoxC2 Rn TCCACGGGCGCAAGAG CAAGAACAAAATAGCCTGGTTACAAA

FoxD3 Rn TCCGTACTACCGGGAGAAGTTC ATCTTGACGAAGCAGTCGTTGA

FOXF2 Hs GCGTTCCCCAATCTGAATACC CCTTACGCATGGCACATCTTC

FoxF2 Rn ACTCCCAGTCCCCCAACACT ACTAGACTATCGGTTCTTCATTTCTTCA

FoxJ2 Rn CCTGGTGCCAATGAGGAGAT CCTTGGGCTGATGTCAACTGT

FoxM1 Rn ACTTGGAATCACAGCAGAAACGA GTGCGCCTAGTGGGAGTTCA

FOXO1 Hs TCTTCCTCCTGAGTCTGGGTAATT CAGAGAGCTACCAAGGATTCATGA

FoxO1 Rn TGTTCTGCTACAGTGAGAAGTCATGA CAGAGAGCTACCAAGGATTCATGA

FOXO3 Hs AAGCCAGCTACCTTCTCTTCCA GTGGCTAAGTGAGTCCGAAGTGA

FoxO3 Rn TGCCGATGGGTTGGATTT CCAGTGAAGTTCCCCACGTT

FoxO4 Rn GAGGTTCGACCCCACGTATG AGTCGGGATCGAGGTCTAGGA

FoxP1 Rn ATGCACACGGTGGGACCTAT GTTCTGCGCAATATCTGCTGAA

Metabolic genes

ACC� Mm TGTGGACGTTGAATTGATTTACG GTGGCAGCCGTTCATGATG

ACC� Rn GAATTTGTCACCCGCTTTGG TGGAGCGCATCCACTTGA

AK1 Mm ACACGGTGCTGGACATGCT TCGGGTAGCCGTCGATCA

AK1 Rn ACCCGAGGGAGGTGAAACAG GGCCTGCGTCCACATACAGT

AMPK�1 Mm TCAGGCACCCTCACATCATC CAGAGACATATTCCATCACCATGAA

AMPK�1 Rn CAAGCCCACCCGATTCTTT TTTCGGCAACCAAGAATGG

AMPK�2 Mm TCCTGTCTGCCGTGGATTACT GGGCATCCAGCAGCACAT

AMPK�2 Rn AGTCCTCCAACGGGTTCCTT TGGCATCCTTTCAGGATGTG

CD36 Mm AATTAGTAGAACCGGGCCACGTA GAATCAAGGGAGAGCACTGGTTT

CD36 Rn AACAACAAGGCCAGGTATCACA TCATGGCGAGGAACAGAACA

CK Mm CCTCTACAATAAGCTTCGCGATAAG GGGTTGTCCACCCCAGTCT

CK Rn GCAACACCCACAACAAGTTCA AGCACCTTGGCCATGTGATT

GLUT4 Mm ATGGCTGTCGCTGGTTTCTC ACCCATAGCATCCGCAACAT

GLUT4 Rn TGACAGGCTGGGCTGATGT ACAGTGGACGCTCTCTTTCCA

LPL Rn GGCATACAGGTGCAATTCCA CCCACGTTGTTACAGCGATTC

MCAD Mm TTTCTTGTTGGCGCGTTCTA GTGTCGGCTTCCACAATGAA

MCAD Rn AAGCAGGAGCCGGGACTAG GGCAAACTTCCGAGCAATTG

(Continued )





previously.33a Loading controls were performed by red Ponceaustaining. Hybridizations were revealed with chemiluminescent sub-strate according to the protocol of the manufacturer (Roche).Quantification of the detected proteins was performed by scanningand with Metamorph software (Universal Imaging). Primary anti-bodies were Goat anti-Kir6.1 (sc-11224, Santa Cruz Biotechnology),rabbit anti-Kir6.2 (APC-020, Alomone), rabbit anti-SUR (sc-5791,Santa Cruz Biotechnology), rabbit anti-SUR2B (sc-5793 Santa CruzBiotechnology), rabbit anti-FoxF2 (ab23306, Abcam), goat anti-

FoxO3 (ab17026 Abcam), rabbit anti–green fluorescent protein(GFP) (Synaptic Systems).

PlasmidsFoxF2 expression plasmids were a generous gift from Dr P.Carlsson, Goteborg University (Goteborg, Sweden).34 Wild-typeFoxO1-GFP expression plasmids were a generous gift from Dr T.G.Unterman (McGill University, Montreal, Canada).35 FoxO3a expres-sion plasmids were ordered at ADGENE and are the original

Figure 2. Fox-induced expression ofKATP channel subunit and metabolicgenes in the HL-1 mouse atrial cell line.Each mean and SEM stems from 3 to 8separate cultures and transfectionseries. *P�0.05 relative to no transfec-tion and empty vector; �P�0.05 relativeto F2, O3. mRNA levels were determinedtwice for each culture. For abbreviations,see Table 3. Note predominant effect ofFoxF2 and FoxO on KIR6.1, inhibition ofglycolytic genes by FoxF2, and stimula-tion of �-oxidation genes by FoxO.

Table 4. Continued

Forward Primers Reverse Primers Probes

MCD Mm CAAGGAGCTGCAGAAGGAATTT AGCAGCCACTTGGTGAATCC

MCD Rn CCCTGGTTGTTCTGCATGTG CAGACGGAGGGCACTCTTTC

PFK2 Mm AGCCATGAGAGTCCGAAAACA AATCTGTCCACCTTCCTTTGTCA

PFK2 Rn GCAACGAAGATGCCTTTGGA CACAGGATCTGGGCAACGA

PPAR� Mm GCAGTGCCCTGAACATCGA TCGCCGAAAGAAGCCCTTA

PPAR� Mm CAGGCCGAGAAGGAGAAGCT GGCTCGCAGATCAGCAGACT

PKM Mm AGGAGATGATTAAGTCTGGAATGAATG TGATGGTCTCTGCATGGTACTCA

PKM Rn GAGCAGGACGTGGACATGGT CTCTCCCAGGACCTTCCTAACC

SREBP1a Rn/Mm TGCCATGGGCAAGTACACA TGGATATAGCATCTCCTGCACACT





construct from Dr M.E. Greenberg (Harvard Medical School, Bos-ton, Mass).36

Small Interfering RNASmall interfering (si)RNAs were obtained from Invitrogen to knockdown mouse FoxO1 and -O3 and were successfully tested in HL-1mouse atrial myocytes. Because the design was optimized to alsoknock down rat Fox, 1 of the siRNA for FoxO1, and 2 of the siRNAsfor FoxO3 also worked in rat. siRNAs with scrambled sequenceserved as control. A fluorescent siRNA was used as a control forsuccessful electroporation. For each culture well, 1 electroporation(see below) was performed immediately after cell dissociation on 2million preplated cells. Up to 6 wells were pooled for RNAextraction from the cultures 24 hours later. Each pooled sampleserved as 1 single data point, and 5 to 7 data points per gene wereaveraged for the statistical analysis.

Cell Transfection and ElectroporationLipofections of HL-1 cells were performed with Lipofectamine 2000(Invitrogen) as recommended by the manufacturer. Cells were lysed48 hours posttransfection. For single-cell studies, primary culturedmyocytes were electroporated with a Bio-Rad Genpulse electropo-rator as described.28 For nucleoporation of primary cultured atrial

myocytes with siRNAs, protocol no. 09 of Amaxa AG (Koln,Germany) was applied, as recommended by the manufacturer.

Immunocytochemistry, Confocal Microscopy, andQuantitative ImagingMyocytes were fixed in 2% paraformaldehyde and stained asdescribed,28 using antibodies listed previously. Image stacks ofimmunostained cultures were acquired on a confocal microscope ata fixed laser setting and analyzed.28 For each cell, the meanbackground-subtracted fluorescence of regions of interest (nucleus,cytoplasm) was determined with Metamorph software at 3z-positions, normalized by exposure time, and averaged; means ofaverages�SEM were obtained for groups of 10 to 16 cells. Forexample, red fluorescence of KIR6.1/Texas Red was plotted asfunction of cytoplasmic or nuclear green fluorescence of FoxO1–enhanced GFP (EGFP).

BioinformaticsPromoter analysis was performed online at the Lawrence LivermoreInstitute Laboratory (http://www.dcode.org) for the following genes:SUR1-KIR6.2, SUR2-KIR6.1, MLC2a, MLC2v, proANP, proBNP,and the metabolic genes listed in Tables 1 and 2. The analysis of thepromoter across species was performed for chicken, mouse, rat, and

Figure 3. A and B, Effects of no, sham,or Fox transfection on FoxO3 mRNA andFoxO3, FoxF2, and KIR6.1 protein inHL-1 cells. Note that FoxF2 overexpres-sion stimulates FoxO3 and KIR6.1,whereas FoxO overexpression inhibitsFoxF2. C, Effects of FoxO3 transfectionon FoxO3 and KIR6.1 in quantitativeimmunocytochemistry (HL-1). D throughF, Three separate experiments showingeffects of transfections of FoxF2, -O3,and -O1 on KIR6.1 and KIR6.2 proteinquantified from Western blots (HL-1). G,Expression of FoxO1-EGFP stimulatesexpression of KIR6.1 in primary culturedatrial myocytes. Significant positive cor-relation (P�0.003) between wt FoxO1-EGFP fluorescence and immunoreactiveKIR6.1 (arbitrary units) for 11 atrial myo-cytes. Another 11 myocytes withoutgreen fluorescence were analyzed in thesame cultures (for all 22 myocytes,R�0.71, P�0.0002). Scale bar�5 �m.O1-E indicates FoxO1-EGFP; Combi,overlay of DAPI (6-diamidino-2-phenylindole), KIR6.1, and FoxO1-EGFP.





human with the ECR Browser software (http://ecrbrowser.dcode.org). The alignment between pairs of promoter regions was per-formed with blastz software and designed with zpicture (http://zpicture.dcode.org). This alignment was submitted to Rvista2 toidentify the putative transcription factor binding sites at http://rvista.dcode.org. The putative promoter and transcription factorbinding regions were confirmed with Genomatix ElDorado andGene2Promoter software.

ElectrophysiologyWhole cell patch-clamp recordings of the KATP current wereobtained as described previously.24,25,29 From a holding potential of�40 mV, voltage ramps were imposed every 30 seconds from �80to �90 mV over a 10-second period. This resulted in quasi–steady-state current–voltage curves. Membrane potential was measured incurrent-clamp mode at 0 pA at the end of each ramp. The pipettesolution contained (in mmol/L) 120 KCl, 1.3 CaCl2, 1.3 MgCl2, 10Hepes, 10 glucose, 10 BAPTA, plus 1 mmol/L K-ATP and

10 �mol/L K-ADP. The pH was adjusted to 7.3, and osmolality to290 milliosmol/kg. The bath solution contained (in mmol/L): 5 KCl,1 CaCl2, 1 MgCl2, 118 NaCl, 10 Hepes, and 10 glucose. The pH wasadjusted to 7.4, and osmolality was adjusted to 290 milliosmol/kgwith sucrose. Drugs were administrated to 23 rat cardiomyocytes inthe perfusion system in the sequence control, diazoxide (100 �mol/L),pinacidil P-1075 (100 �mol/L), glibenclamide (1 �mol/L), with 2 to4 minute washes between drug applications. Metabolic sensitivitywas tested in a separate series of 24 rat cardiomyocytes by applica-tion of low concentrations (20 nmol/L) of the protonophore CCCP tomimic metabolic stimulation.25

Statistical AnalysisThe statistical analysis was performed on Sigmastat 2 SPSS. ThemRNA and protein levels were compared by ANOVA with a posthoc Newman–Keuls test. Time varying expressions of Foxs incardiac infarction studies were compared with a Dunnett test. Thecorrelation analysis was performed with a linear regression and acovariance test, and confirmed by Spearman correlation. Multipleregression analysis was performed with SPSS software.

ResultsAbundance of Fox-Binding Sites on SUR-KIR6and Metabolic GenesAlignments of chicken, mouse, rat, and human genes codingfor KATP channel subunits and metabolic enzymes allowedus to identify the most highly conserved sequences in the 5�upstream region and the presumed transcription factor bind-ing sites (Figure 1; detailed charts are available on request).In sequences 5� upstream of the Kir6.1 coding region,Rvista2.0 finds a high redundancy of binding sites for FoxO1,-O3, -O4, -F2 (FREAC2), -C1 (FREAC3), -D1 (FREAC4),-L1 (FREAC7), and a common Forkhead consensus site.Immediately upstream of the SUR2 coding region, Rvista2.0finds almost the same sites: FoxO1, -O3, -F2, -D1, -D3, -J2,-P1, and a common Forkhead consensus site. For the Kir6.2gene, Rvista finds only 1 putative site for FoxC1 (FREAC3)and high redundancy for MEF2, OCT, SREBP1a, SP1, AP2,and NF-�B.

Presumed promoter sequences of KATP channel subunitswere also aligned with presumed promoter sequences ofproANP, proBNP, MLC2a, and MLC2v (Tables 1 and 2;detailed charts available on request). There are 15 highlyconserved sequences 5� upstream of the Kir6.1 gene (morethan 90% sequence identity between species) but only 2sequences 5� upstream of the SUR2 gene. Alignments of theKIR6.1 promoter with putative atrial markers (MLC2a andproANP promoters) yield striking clusters of binding sites forFoxA1, -A2, -C1, -D3, -F1, -F2, -I1, -J1, -J2, and -L1 (Tables1 and 2). Alignments with the MLC2v and proBNP promoterregions yield negative results, except for FoxP1.

A similar analysis on metabolic genes yields clusters ofFox binding sites on the CD36, LPL, FABP3, MCAD,ACC�, and MCD promoters for the �-oxidation pathway andon the GLUT4 and PKM promoters for the glycolyticpathway (Table 3). Interestingly, a Foxs cluster is alsoobserved for SREBP1a, as are binding sites for SREBP1a,HIF1�, PPAR, and Nkx2.5. Thus in silico, Foxs emerge froma library of 430 transcription factors as the most prevalenttranscriptional regulators of KATP channel subunits andmetabolic pathways.

Figure 4. Electrophoretic mobility-shift assays (EMSA) andsiRNAs prove cause-and-effect between FoxO and KIR6.1expression. A1, Nuclear extracts of HL-1 overexpressingFoxO1-EGFP slow down migration of P32-labeled probe withFoxO-binding DNA sequence of the KIR6.1 gene promoter (lane2). Coadded cold probe almost abolishes this shift in migration(lane 3). Lane 1 shows free probe alone. Also see supplementalFigure IC and ID. A2, In positive control, nuclear extract of Helacells slow down P32-labeled probe with SP-1–binding DNAsequence (lane 2), and cold probe almost erases shift (lane 3).B, siRNAs directed against FoxO1 and FoxO3 significantlyreduce expression of KIR6.1 in primary cultured neonatal ratatrial myocytes. Data are means�SEM of mRNA normalized bycyclophilin mRNA in 5 to 7 pooled cultures. *P�0.05, **P�0.01.SCR indicates control, siRNA with scrambled sequence.





FoxO1, -O3, and -F2 Cause Increased GeneExpression of KIR6, SUR, MetabolicTransporters, and Enzymes in Atrial MyocytesFoxO1, -O3, and -F2 were selected from a group of 10 Foxsfrom ongoing experiments on rat hearts. The transfection ofHL-1 cells with a FoxO3 plasmid36 induces a 15-fold increasein FoxO3 mRNA, a 3-fold increase in KIR6.1 mRNA, a tightpositive correlation between FoxO3 and KIR6.1 protein inquantitative double-immunocytochemistry, and a 5- to 26-fold increase of KIR6.1 protein in Western blots (Figures 2and 3). Moreover, FoxO3 induces a 2-fold increase in KIR6.2and a 4-fold increase in SUR2A mRNA (Figure 2). Wild-typeFoxO1-EGFP35 induces green fluorescence and increasedKIR6.1 expression (supplemental Figure IA and IB; R�0.66,P�0.01, n�22 cells), and a 6-fold increase in KIR6.1 protein(Figure 3). Interestingly, transfection with FoxO1-EGFP (orFoxO3) represses FoxF2 protein, whereas human FoxF2plasmid34 increases FoxO1/O3 mRNA and protein 10-fold

(Figure 3B). FoxF2 also increases SUR2A, KIR6.2, andKIR6.1 mRNA (Figure 2). In primary rat atrial myocytes,electroporation with FoxO1-EGFP plasmid induces greenfluorescence and increased expression of immunoreactiveKIR6.1 (Figure 3G); electroporation with FoxF2 plasmidincreases nuclear FoxF2 in inverse correlation with nuclearFoxO1-EGFP (R��0.51, P�0.05, N�16), supporting theresults from HL-1 cells.

Foxs potently modulate the expression of genes coding forfree fatty acid and glucose transport and metabolic enzymes(Figure 2). Many of these findings are new. FoxF2 down-regulates, whereas FoxO1 induces phospho-fructokinase 2, inHL-1 cells, possibly via downregulation and induction ofSREBP1a, respectively.37–39 Overexpression of FoxF2 down-regulates gene expression throughout the glycolytic pathway.FoxO1 and -O3 induce increased expression of malonyl-CoAdecarboxylase (MCD), which inactivates malonyl CoA, aninhibitor of carnitine palmitoyltransferase-1 (CPT-1), and

Figure 5. Differential expression of Foxsin the 4 chambers of the rat heart andtheir link to KATP channel expression.A, mRNA levels were quantified by real-time PCR (I-Cycler, Bio-Rad) with spe-cific primers (see Table 6) and normal-ized by the 18S ribosomal RNA level.Each mean and SEM is from 8 differentmyocyte cultures. The myocytes weredissociated from the tissue, purified bypreplating, and cultured in cytosinearabinonucleoside. Contamination byfibroblasts or vascular smooth musclecells was �9%. Individual measureswere determined twice on extracts ofeach culture. Note differential distributionof Foxs, with high levels of FoxA2, -C2,and -F2 in the left ventricle (LV), highlevels of FoxO1, -O3, -O4, and -P1 inright and left atria (RA and LA), and highlevels of FoxJ2 in right heart (RA andRV). *P�0.05 relative to ventricles,�P�0.05 relative to left heart, #P�0.05relative to all others. B, Differentialexpression of Foxs results in differentialexpression of KATP channel subunits(shown in Figure 6). Associationsbetween Fox and channel subunits aremarked in black (significant, positive),gray (significant, negative), and white(nonsignificant). For details, see Table 4.Note predominant associations withFoxF2 and FoxO.





FoxO3 increases expression of medium chain acyl CoAdehydrogenase (MCAD). The metabolic sensor AMPK�1 isalso upregulated by FoxO1 and -O3. Additional findingsconfirm previous studies on other cell types40 and LPL41;gluconeogenesis, glycolysis, and lipid gene expression.39

Putative Fox binding sites also exist on peroxisomeproliferator-activated receptors (PPARs), which positivelyimpact on fatty acid oxidation, as confirmed by experimentson HL-1 cells. The results thus suggest cause-and-effectbetween Foxs and expression of KATP channels and meta-bolic genes.

Causal Relationships Between FoxO and ChannelSubunit Expression Are Demonstrated byElectrophoretic Mobility-Shift Assays and siRNAsNuclear extracts from mouse atrial HL-1 myocytes overex-pressing FoxO1 slow down the gel migration of a P32-labeledoligonucleotide with FoxO consensus sequence that is presentin the KIR6.1 gene promoter. Cold probe erases this shift(Figure 4A, top). Nuclear extracts from nontransfected pri-mary rat cardiomyocyte cultures also cause a shift of theradioactive FoxO probe, and the cold probe decreases theintensity of the shifted band by �90% (supplement Figure ICand ID). Cause-and-effect is further indicated by a 76% to83% decrease in expression of KIR6.1 mRNA, 24 hoursfollowing electroporation of primary cultured rat atrial myo-

cytes with anti-FoxO siRNAs (Figure 4B). Remarkably,anti-FoxO1 siRNA affects primarily FoxO1 and less soFoxO3 mRNA, and vice versa, but both types of siRNAsinhibit KIR6.1 expression relative to the control with scram-bled sequence.

Coordination of Tissue-Specific Expression of Fox,Channel Subunits, and Metabolic Genes inNeonate Rat CardiomyocytesAtria and ventricles differ in their development and mechan-ical stress; right and left heart are exposed to different oxygentension. Would they differ in Fox, channel subunits, andmetabolic genes? This is indeed the case. FoxA2, -C2, and-F2 display the highest mRNA levels in the left ventricle,whereas FoxO1, -O3, -O4, and -P1 are highly expressed inthe atria (Figure 5A). FoxM1 is expressed at the same level inall cardiac compartments (supplemental Figure II). There is aright/left heart difference in expression for FoxJ2. At theprotein level, FoxO3 protein is expressed more strongly inright atrium and FoxF2 in left ventricle. This is indicated byWestern blots (Figure 6) and quantitative immunocytochem-istry (supplemental Figure IIIA). FoxO3 is mainly localizedin the nucleus. FoxF2 resides in both the nucleus andperinuclear zone (Figure 6A2 and 6B2).

Significant associations exist between Fox and KATPchannel subunit mRNA: both positive (in black: FoxJ2, -O1,-O3) and negative (in gray: FoxD3, -F2) (Figures 5B, 6A3,

Figure 6. Endogenous Foxs and KIR6.1in primary myocyte cultures from neo-nate rat heart. A1 and B1, High levels ofFoxO3 protein in right atrium, and FoxF2in left ventricle. A2, Immunolocalizationof FoxO3 in myocytes from right atriumand left ventricle. B2, Immunolocalizationof FoxF2 in right and left ventricle. Scalebar�10 �m. A3, Positive correlationbetween FoxO3 and KIR6.1 mRNAs(P�0.015; R�0.66). B3, Negative corre-lation between FoxF2 and KIR6.1mRNAs (P�0.001; R��0.94). Extractsfor protein (A1 and B1) and mRNA (A3and B3) were made from cultured myo-cytes as indicated for Figure 5. Note sig-nificant positive association of KIR6.1with FoxO3 mRNA and negative associa-tion with FoxF2.





and 6B3; details in Table 5). Atria express 4- to 12-foldhigher levels of KIR6.1 and SUR1A mRNA than ventricles,and right heart expresses up to 6-fold higher levels of SUR2BmRNA than left heart (Figure 7A). SUR2A, KIR6.2,l andSUR1B mRNAs are evenly distributed throughout the cardiacchambers. Significant associations also exist between Foxand metabolic enzyme mRNAs (Table 5). In general, ventri-cles express higher mRNA levels for all enzymes andtransporters studied (Figure 8); many of these findings arenew, and others extend previous results.42,43

Protein levels generally conform to the mRNA measure-ments (Figure 7B). Kir6.1 is significantly (2-fold) higher inatrial than ventricular myocytes, whereas KIR6.2 is evenlydistributed throughout. Immunocytochemistry shows thepresence of KIR6.1 protein in myocytes, displaying thetypical striated pattern in left ventricular myocytes (Figure7C), a patchy pattern in atrial myocytes, and a mixed patternin right ventricle. These most probably reflect the differentamounts of contractile proteins present in neonatal myocytes.SUR2B expression is highest in atria and very low in leftventricle (supplemental Figure IIIB).

High Expression of Foxs FollowingMyocardial InfarctionResults so far indicate a tissue-specific, Fox-related expres-sion of KATP channel subunit and metabolic genes, possiblycaused by the varying metabolic challenges. Would ventric-ular tissue, when exposed to stress, acquire new Fox-relatedcharacteristics? Because FoxO1 and -F2 knockout mice arenot viable, we tested this hypothesis in a rat model ofmyocardial infarction,31 where KIR6.1 and SUR mRNAs arehighly expressed in the infarct border zone.44 This zone isexposed to low PO2 and high mechanical stress.45 We tested inthese same rats the time-varying patterns of Fox expression.Myocardial infarction elicits waves of Fox expression, start-ing with FoxC2 and -O1 at 7 days, continuing with FoxO3and -J2 at 56 days, and FoxF2, -O3, and -J2 at 20 weeks (opencircles, Figure 9A). Overexpression of genes in the periin-

farcted zone reaches mRNA copy numbers found in theFox-transfected HL-1 cell line. No significant changes occurin FoxA2, -O4, -D3, -P1 (Figure 9A) and -M1 (supplementalFigure IVA). In shams (closed circles), all Fox mRNA levelsremain stable over the 20 week period.

Changes in Foxs parallel changes in KATP channel subunitmRNA. The most striking results are the strong positiveassociations between FoxF2 and KIR6.1, SUR1A, SUR2A,and SUR2B (Figure 9B and Table 6), and associations withFoxO1, -O3, -J2, and -C2. These represent almost the sameset of transcription factors revealed in studies on neonate ratmyocytes. Note that at these high levels of FoxF2 expression,there is a high expression of KIR6.1, as in HL-1 cells.

Other potassium channels, such as IK1 (KIR2.1, KIR2.3),46

Ito (Kv1.4),47 IKs (KvLQT1),48 and IKAch (KIR3.1),49 alsodisplay a regional expression in the heart. However, Rvistabioinformatics analysis of their 5� promoter/enhancer regionsfails to suggest a clear implication of Foxs. Indeed, noassociation was found between Foxs and KIR2.1 or KvLQT1mRNA after myocardial infarction (supplemental Figure IVBand IVC).

Functional Plasticity Matches Molecular Plasticityof KATP ChannelsA remarkable molecular plasticity thus exists for Foxs andchannel subunits, indicating a pronounced expression ofsubunits KIR6.1, SUR1A, and SUR2B in regions (rightatrium, left ventricle infarct border zone) exposed to low PO2.Atria are already known to display increased diazoxidesensitivity in correlation with increased metabolic sensitivityof the KATP channels,25 but right/left differences had not yetbeen examined. Right atrial myocytes from neonatal ratsreadily responded to 100 �mol/L diazoxide by generatingwithin 3 to 4 minutes an inwardly rectifying potassiumcurrent of 250 to 550 pA (eg, Figure 10A1). Subsequent100 �mol/L pinacidil (P-1075) only slightly increased thecurrent by 200 pA. The potassium current was totally abol-ished by 1 �mol/L glibenclamide, as expected. Diazoxide

Table 5. Correlations Between Expression of Fox and KATP Channel Subunit mRNA in the Neonate Rat Heart

FoxA2(R; P )

FoxC2(R; P )

FoxD3(R; P )

FoxF2(R; P )

FoxJ2(R; P )

FoxM1(R; P )

FoxO1(R; P )

FoxO3(R; P )

FoxO4(R; P )

FoxP1(R; P )

KIR6.1 �0.56; 0.06 �0.44; 0.15 0.22; 0.38 �0.94; <0.01* 0.19; 0.55 0.10; 0.74 0.67; 0.02* 0.65; 0.01* 0.53; 0.07 0.55; 0.06KIR6.2 �0.27; 0.39 �0.57; 0.06 0.49; 0.10 �0.30; 0.33 0.13; 0.68 0.36; 0.24 0.17; 0.60 0.16; 0.61 0.24; 0.44 0.31; 0.31SUR1A �0.48; 0.11 �0.30; 0.33 0.36; 0.25 �0.72; <0.01* 0.14; 0.65 �0.17; 0.59 0.42; 0.17 0.74; <0.01* 0.44; 0.15 0.47; 0.12SUR1B 0.16; 0.60 0.42; 0.17 0.32; 0.30 0.11; 0.71 0.43; 0.15 0.26; 0.40 0.28; 0.37 0.49; 0.11 0.05; 0.86 0.23; 0.46SUR2A 0.03; 0.92 0.15; 0.63 �0.75; <0.01* 0.45; 0.14 0.18; 0.58 0.29; 0.36 0.48; 0.11 0.49; 0.10 �0.01; 1 0.41; 0.18SUR2B 0.17; 0.59 0.02; 0.95 �0.68; 0.14 0.36; 0.24 0.70; 0.05* �0.50; 0.10 0.13; 0.69 0.04; 0.90 0.30; 0.35 0.20; 0.52CD36 0.46; 0.13 0.17; 0.59 0.44; 0.15 0.73; <0.01* 0.56; 0.06 �0.28; 0.38 �0.89; <0.01* �0.89; <0.01* �0.68; 0.01* �0.56; 0.056LPL 0.01; 0.95 �0.30; 0.34 0.90; <0.01* 0.37; 0.23 0.03; 0.92 0.24; 0.45 �0.57; 0.052 �0.54; 0.069 �0.11; 0.72 �0.40; 0.19ACC� 0.59; 0.04* 0.35; 0.26 0.42; 0.17 0.74; <0.01* 0.43; 0.16 �0.14; 0.67 �0.80; <0.01* �0.84; <0.01* �0.42; 0.17 �0.67; 0.01*MCD 0.41; 0.18 0.20; 0.52 0.59; 0.04* 0.55; 0.06 0.15; 0.64 0.16; 0.62 �0.52; 0.08 �0.68; 0.01* �0.42; 0.17 �0.68; 0.01*CK 0.62; 0.03* 0.35; 0.27 0.33; 0.29 0.72; <0.01* 0.56; 0.06 �0.32; 0.31 �0.85; <0.01* �0.82; <0.01* �0.77; <0.01* �0.63; 0.03*AK1 0.31; 0.33 �0.29; 0.36 �0.58; 0.05* �0.58; 0.05* �0.20; 0.95 �0.26; 0.41 �0.54; 0.07 �0.51; 0.09 �0.38; 0.22 �0.39; 0.21GLUT4 0.55; 0.06 0.21; 0.51 0.24; 0.46 0.62; 0.02* 0.70; 0.01* �0.45; 0.14 �0.77; <0.01* �0.85; <0.01* �0.80; <0.01* �0.58; 0.06PFK2 �0.01; 0.97 �0.06; 0.83 0.12; 0.70 �0.40; 0.90 0.38; 0.22 �0.13; 0.68 �0.13; 0.70 �0.23; 0.46 �0.41; 0.18 �0.13; 0.68PKM 0.37; 0.39 0.05; 0.86 0.31; 0.31 0.17; 0.59 0.42; 0.17 �0.09; 0.77 �0.37; 0.23 �0.53; 0.07 �0.29; 0.35 �0.28; 0.38AMPK�1 0.21; 0.95 �0.25; 0.43 0.77; <0.01* 0.37; 0.23 0.15; 0.63 0.13; 0.67 �0.56; 0.06 �0.65; 0.02* �0.11; 0.73 �0.63; 0.27AMPK�2 �0.42; 0.17 �0.42; 0.17 0.29; 0.35 �0.58; 0.05* �0.36; 0.35 0.45; 0.14 0.51; 0.09 0.24; 0.45 0.68; 0.01* �0.09; 0.77

Significant correlations are in boldface, correlations with P�0.1 are italic, and correlations with P�0.1 are in lightface. Note prominent correlations for FoxF2 andFoxO.





(100 �mol/L) hardly induced any current in left ventricularmyocytes (eg, Figure 10A2), whereas pinacidil induced acurrent comprised between 500 to 1000 pA (positive control).In Figure 10A4, the current densities clearly indicate a highsensitivity to diazoxide in right and left atrium and rightventricle, and a low sensitivity in left ventricle. In contradis-tinction, the current densities in response to pinacidil werestatistically identical in all tissues (Figure 10A5). The myo-cyte KATP current in the highly stressed infarct border zoneof the left ventricle is also highly sensitive to diazoxide.44 Tobetter characterize the functional implications, another seriesof experiments was performed on 24 cardiomyocytes bymimicking a metabolic challenge with a very low concentra-tion (20 nmol/L) of CCCP (Figure 10B). Brisk responseswere obtained in right and left atrial and right ventricularmyocytes, and a very small response in left ventricularmyocytes. Thus normal left ventricular myocytes stand out bytheir minimal responses to diazoxide and CCCP. Comparedwith left ventricular myocytes, right ventricular myocytesexpress as much mRNA for creatine kinase, adenylate kinase,and pyruvate kinase (Figure 8); thus increased ATP-ADP

buffering in left ventricular myocytes seems not to accountfor their lack of response to CCCP.

DiscussionThe capability of adapting cellular function and energymetabolism to varying physiological and pathological condi-tions is vitally important in animal cells. In the present work,we show that the Fox family may play a central role inexpression of both molecular sensors of energy status and keyregulatory genes of energy metabolism. First, in atrial cells,the expression of FoxO1, -O3, and -F2 cause increasedexpression of KATP channel subunits (the quintessentialmetabolic sensors7) and selective up- and downregulation ofspecific metabolic genes. A causal relationship betweenFoxO and KIR6.1 expression is demonstrated by electro-phoretic mobility-shift assay and by experiments withsiRNAs. Second, FoxO1, -O3, -F2, and -J2 are distributedunevenly within different cardiac chambers of the neonatalrat, in association with channel subunits KIR6.1, SUR1A, andSUR2B and 9 metabolic genes.42,43,50 Third, the periinfarctedzone of the rat left ventricle reveals an impressive plasticity of

Figure 7. Differential expression of KATPchannel subunits in myocytes from neo-nate rat heart. A, Quantitative RT-PCR ofKATP channel subunits. The mRNA lev-els were quantified by real-time PCR(I-Cycler, Bio-Rad) with specific primersand TaqMan probes for each subunit(see Table 6) and normalized by the 18SRNA level. For n and symbols, see thelegend of Figure 4. Note even distribu-tion of KIR6.2/SUR2A, high levels ofKIR6.1 and SUR1A in atria, and high lev-els of SUR2B in right heart. B, Proteinlevels of KIR6.1 and KIR6.2 in myocytesderived from the 4 chambers of the ratheart by immunoblotting from 3 separateculture series; below, quantification ofgel scans. *P�0.05 relative to ventricles.Note higher levels of KIR6.1 protein inatria and even distribution of KIR6.2. Elu-tion of KIR6.2 at an apparent molecularweight of 37 kDa is mentioned by anti-body vendor (Alomone) and in manypublications. C, Immunocytochemistryfor KIR6.1 in cardiomyocytes from differ-ent cardiac chambers. Scalebar�10 �m. Extracts for mRNA (A) andprotein (B) were made with myocyte cul-tures derived from each cardiacchamber.





FoxO1, -O3, -F2, and -J2, associated with the increasedexpression of KATP channel subunits (KIR6.1 and all SUR)and with the known remodeling of metabolic pathways.31

These findings support the hypothesis that Foxs mediate thetissue-specific gene expression in the face of different me-chanical and hypoxic challenges.

Although our study demonstrates a Fox-mediated expres-sion of KATP channel subunits, it does not resolve theirquantitative subcellular distribution in mitochondria, sarco-plasmic reticulum, and plasma membrane. In confocal mi-croscopy, immunofluorescence signals from subunits that areendogenously expressed in mitochondria and reticulum over-whelm signals emitted from the adjacent plasma membrane.Thus definite proof is still lacking that sensitivity of atrial andright ventricular myocytes to diazoxide or CCCP is explainedsolely by increased plasmalemmal KIR6.1, SUR1A, orSUR2B.

Would plasticity in KATP channel expression and compo-sition be advantageous to the cell? The total absence ofcardiac KIR6.2 expression or inactivation by dominant neg-ative KIR6.1 is clearly detrimental to the tolerance of intenseexercise in mice.9,10 Thus increased expression and function

of KATP channels carry vital benefits. The increasedexpression of KIR6.1 (a channel subunit of lower conduc-tance than KIR6.251) and the expression of SUR1A andSUR2B may serve to limit the maximal KATP currentwhile increasing the sensitivity to ADP52 and to a meta-bolic challenge. One may anticipate that following myo-cardial infarction, the periinfarcted zone will readily adaptto hypoxia. The risks of reentry arrhythmia are minimized,because the low conductance KIR6.1 subunit prevents anexcessive shortening of the action potential.8,25,53 Interest-ingly, recent studies unrelated to KATP channels haveshown that myocardial infarction,54 myocardial reperfu-sion injury,55 heart failure,56,57 or myocyte hypertrophy58

induce an overexpression of FoxO. Other studies unrelatedto FoxO have shown increased expression of KIR6.1following ischemia16 and exercise.17 Moreover, recentwork suggests that FoxO and KATP channels reducecardiac ageing and electrical instability in drosophila.59

Our results thus link tissue stress and expression of Foxand KATP channels into a coherent framework.

Other sensors of energy status impact on the expressionand function of Foxs. AMP kinase (AMPK)60–62 may signalmetabolic stress (AMP/ATP ratio) to Fox.63–65 We show thatFoxO1 and -O3 strongly increase the expression of AMPK�1in atrial cells; in hepatocytes, AMPK stimulation directsFoxO1 to the proteolytic degradation pathway,60 possibly in anegative-feedback arrangement. Sirtuins66 are activated bythe NAD�/NADH ratio and trap FoxO1 in the nucleus ofhepatocytes.67 The low-oxygen sensor HIF1�68,69 is a poten-tial transcription factor for FoxC2, -F2, -O1, and -O3 (sup-plemental Figure V). Homologs of SREBP1a act as oxygensensors in fission yeast70; SREBP1a is a potential transcrip-tion factor for expression of the SUR2-KIR6.1 and KIR6.2genes (Figure 1). Although not exhaustive, this list of energyand low-oxygen sensors demonstrates their potential, intimaterelationship with Foxs.

Based on present experiments and previous studies,27,71–73

we consider that each cardiac chamber is chronically exposedto a different workload, metabolic demand, and metabolicsupply. This leads through as yet unidentified signal trans-ducers to different expression profiles of Foxs and thus ofKATP channel subunits and key enzymes for �-oxidation orglycolysis. In response to an increased workload, eachcardiac chamber adjusts its local excitability, force ofcontraction, and mobilization of metabolic pathways, thusoptimizing the overall adaptation of the heart to stress. Inpathological situations, for example, after myocardial in-farction, the chronic stretch45 or possibly recurrent ische-mia in the infarct border zone remodels the left ventricularwall, changes the KATP channel profile, and modifies theexpression level of key enzymes of �-oxidation andglycolysis. Controversial results regarding the switch fromfatty acid oxidation to glycolysis32,74 –77 may result from avariable interplay between FoxO3, -O1, -F2, -C2 andPPAR expression.

In conclusion, FoxF2, -O1, -O3, and most likely -C2 and-J2 are intimately involved in both metabolic sensitivity of

Figure 8. Expression of metabolic genes in primary culturedcardiomyocytes. Each mean and SEM is from 3 separate cul-tures and transfection series. *P�0.05 relative to RA, �P�0.05relative to left ventricle. mRNA levels were determined twice foreach culture extract. Note a generally higher gene expression inboth right and left ventricle.





KATP channels and transcriptional control of energy metab-olism. They direct a tissue- and stress-dependent expressionof metabolically sensitive potassium channels and the mobi-lization of additional or alternative energy supplies. The

Fox-dependent coordination of metabolic responses appearsto be of vital importance in metabolically highly activetissues. Identification of the exact signaling pathways fromstress to Foxs remains a challenging task of the future.

A

B

Figure 9. Increased expressions ofFoxO, -J2, -F2, and -C2 in periinfarctedzone of the adult rat left ventricle inassociation with KATP channel subunits.A, mRNA levels were quantified inextracts of myocytes isolated from theperiinfarcted zone 1, 7, 56, and 140 dayspostsurgery. Each mean and SEM isfrom 4 rats with myocardial infarction(open circles) and 4 sham controls(closed circles). Note waves of overex-pression for FoxO1, -C2, -O3, -J2, and-F2 in infarcted rats relative to shams(double-sided error bars, P�0.05), andrelative to day 1 (*P�0.05) (ANOVA forrepeated measures). For other Fox, seetext and supplemental Figure IIIA. B, Dif-ferential expression of Foxs results indifferential expression of KATP channelsubunits. Associations are marked inblack (significant, positive), gray (signifi-cant, negative), and white (nonsignifi-cant). For details, see Table 6. Note sig-nificant associations with FoxF2 andFoxO.

Table 6. Correlations Between Expression of Fox and KATP Channel Subunit mRNA in the Periinfarcted Zone of the Rat LeftVentricle

FoxA2 FoxC2 FoxD3 FoxF2 FoxJ2 FoxM1 FoxO1 FoxO3 FoxO4 FoxP1

KIR6.1 �0.27; 0.31 �0.54; 0.03* 0.01; 0.96 0.67; <0.01* 0.39; 0.13 0.43; 0.09 0.04; 0.88 0.55; 0.02* 0.27; 0.31 0.06; 0.80

KIR6.2 0.06; 0.80 0.14; 0.61 0.30; 0.26 0.27; 0.30 0.32; 0.18 0.14; 0.60 0.57; 0.02* 0.10; 0.71 0.02; 0.93 0.27; 0.30

SUR1A 0.11; 0.68 0.44; 0.21 0.27; 0.31 0.58; 0.02* 0.59; 0.02* 0.31; 0.27 0.18; 0.49 0.26; 0.32 0.21; 0.43 0.09; 0.73

SUR1B 0.09; 0.72 0.32; 0.13 0.03; 0.89 0.32; 0.23 0.44; 0.08 0.41; 0.11 0.13; 0.63 0.36; 0.18 0.03; 0.91 0.09; 0.75

SUR2A 0.01; 0.95 0.38; 0.14 0.04; 0.86 0.59; 0.01* 0.44; 0.08 0.51; 0.06 0.15; 0.58 0.24; 0.37 0.18; 0.50 10�3; 0.99

SUR2B 0.02; 0.78 0.29; 0.27 0.06; 0.82 0.61; 0.01* 0.45; 0.07 0.48; 0.06 0.09; 0.72 0.25; 0.35 0.19; 0.47 0.01; 0.96

Significant correlations are in boldface, correlations with P�0.1 are italic, and correlations with P�0.1 are in lightface. Note strong correlations of FoxF2 or FoxOmainly with KIR6.1, SUR1A, and SUR2.





AcknowledgmentsWe thank Irene Papageorgiou for excellent assistance in studies onmyocardial infarction and Mauro Serafin for advice inpatch clamping.

Sources of FundingSupported by the Swiss National Science Foundation, Swiss Univer-sity Conference project “Heart remodelling in Health and Disease,”Swiss Heart Foundation, Novartis Foundation, Societe Academiquede Geneve, and the Gustave and Simone Prevot Foundation.

DisclosuresNone.

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Circ Res No 166744R – Supplement – Philip-Couderc et al – Page 1

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Figure S1 A, B Green fluorescence of FoxO1-EGFP transfected HL-1 cell (A) and associated increased KIR6.1 staining (B). Note lack of green fluorescence in 3 other cells and corresponding low immunostaining of KIR6.1. Nuclei are colored blue (Dapi).

Figure S1 C Electro-mobility shift of Fox O1 probe upon addition of nuclear extract of primary cultured right atrial myocytes (Lane 2). Cold probe erases shift (Lane 3). Lane 1: free probe without extract. Granular precipitate in lower right is an artefact.

Figure S1 D Electro-mobility shift of FoxO1 probe upon addition of nuclear extract of primary cultured right (RA) or left atrial (LA), or right (RV) or left ventricular (LV) myocytes (Lanes 2). Cold probe erases shift (Lanes 3). Lane 1: free probe. Note prominent shift for atria.

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Figure S1 A, B Green fluorescence of FoxO1-EGFP transfected HL-1 cell (A) and associated increased KIR6.1 staining (B). Note lack of green fluorescence in 3 other cells and corresponding low immunostaining of KIR6.1. Nuclei are colored blue (Dapi).

Figure S1 C Electro-mobility shift of Fox O1 probe upon addition of nuclear extract of primary cultured right atrial myocytes (Lane 2). Cold probe erases shift (Lane 3). Lane 1: free probe without extract. Granular precipitate in lower right is an artefact.

Figure S1 D Electro-mobility shift of FoxO1 probe upon addition of nuclear extract of primary cultured right (RA) or left atrial (LA), or right (RV) or left ventricular (LV) myocytes (Lanes 2). Cold probe erases shift (Lanes 3). Lane 1: free probe. Note prominent shift for atria.

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Days post-infarction ( ) or sham ( )•º

1 7 56 140

5

10

15

20

25

FoxM

1 m

RN

A / 1

8 S

rRN

AK

vLQ

T1 m

RN

A /

18 S

rRN

A

0

10

20

30

40

KvL

QT1

mR

NA

/ 18

S rR

NA

0

10

20

30

40

KIR

2.1

mR

NA

/ 18

S rR

NA

0

10

20

30

40

50

60

KIR

2.1

mR

NA

/ 18

S rR

NA

0

10

20

30

40

50

60

Figure S4 Time course of FoxM1, KIR2.1, and KvLQT1 mRNA in myocytes isolated from the peri-infarct tissue of left ventricle after myocardial infarction in adult rats, and from the corresponding tissue in shams. Changes over time and differences between groups were not significant. Means ± S.E.M. are from 4 rats in each group and at each time point.


A

B

C

5

10

15

20

25

FoxM

1 m

RN

A / 1

8 S

rRN

A


1 7 56 140


1 7 56 140

5

10

15

20

25

FoxM

1 m

RN

A / 1

8 S

rRN

AK

vLQ

T1 m

RN

A /

18 S

rRN

A

0

10

20

30

40

KvL

QT1

mR

NA

/ 18

S rR

NA

0

10

20

30

40

KvL

QT1

mR

NA

/ 18

S rR

NA

0

10

20

30

40

KvL

QT1

mR

NA

/ 18

S rR

NA

0

10

20

30

40

KIR

2.1

mR

NA

/ 18

S rR

NA

0

10

20

30

40

50

60

KIR

2.1

mR

NA

/ 18

S rR

NA

0

10

20

30

40

50

60

KIR

2.1

mR

NA

/ 18

S rR

NA

0

10

20

30

40

50

60

KIR

2.1

mR

NA

/ 18

S rR

NA

0

10

20

30

40

50

60

Figure S4 Time course of FoxM1, KIR2.1, and KvLQT1 mRNA in myocytes isolated from the peri-infarct tissue of left ventricle after myocardial infarction in adult rats, and from the corresponding tissue in shams. Changes over time and differences between groups were not significant. Means ± S.E.M. are from 4 rats in each group and at each time point.



Figure S5 Data charts showing > 90% identity between species (human, rat, mouse) of promoter sequences of FoxC2, FoxF2, FoxO1, and FoxO3 with putative binding sites for HIF1α. Small vertical bars in pink lane show site of action of HIF1α. Software used was R-vista (see Methods).

Forkhead Transcription Factors Coordinate Expression of Myocardial KATP Channel Subunits and Energy...

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