438

Cardiac Myocytes Express Multiple Gap Junction Proteins H. Lee Kanter, Jeffrey E. Saffitz, and Eric C. Beyer Electrical propagation in the normal heart occurs via intercellular transfer of current at gap junctions. Alterations in intercellular coupling in the diseased heart are critical in the pathogenesis of reentrant ventricular arrhythmias. Until recently only a single gap junction protein was known to couple cardiac myocytes. We have now identified and sequenced two additional distinct gap junction proteins (connexins) expressed in the mammalian heart. The sequences differ in their predicted cytoplasmic regulatory domains. Expression of all three connexins by canine ventricular myocytes has been confirmed by Northern blotting and by immunohistochemistry with connexin-specific antisera. Immunoelectron microscopy confirmed that all three connexins are localized to myocyte gap junctions. The presence of multiple connexins in myocyte gap junctions suggests novel mechanisms for regulating cardiac electrical coupling. (Circulation Research 1992;70:438-444)

Sudden cardiac death in patients with healed myocardial infarcts is often precipitated by spontaneous ventricular tachycardia thought to occur by reentry.1,2 The pathogenesis of these malignant arrhythmias depends on a region of slow, heterogeneous electrical conduction.1-4 Although alterations in active membrane properties of cardiac myocytes undoubtedly contribute to arrhythmogenesis, recent observations implicate derangements in intercellular current transfer at gap junctions as a critical determinant of slow conduction leading to reentry.5-8 To understand the regulation of coupling in normal and disease states, we have sought to determine the molecular composition of cardiac gap junctions. Until recently, only a single gap junction protein was known to couple cardiac myocytes.9-12 We have now identified two additional protein components of myocyte gap junctions. Gap junctional channels are formed by members of a family of proteins called connexins, which share homologous extracellular and transmembrane regions but contain unique cytoplasmic domains that likely constitute physiological gating structures.13 Different From the Departments of Pediatrics (E.C.B.), Medicine (H.L.K., J.E.S., E.C.B.), Pathology (J.E.S.), and Cell Biology (E.C.B.), Washington University School of Medicine, St. Louis, Mo. Supported by National Institutes of Health grants HL-45466,

HL-17646, and HL-36773, a fellowship from the North American Society for Pacing and Electrophysiology (H.L.K.), American Heart Association Established Investigator (J.E.S.) and ClinicianScientist (E.C.B.) Awards, and a grant from the McDonnell Foundation. Address for correspondence: Dr. Eric C. Beyer, Division of Hematology, Box 8125, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Received September 3, 1991; accepted November 20, 1991.

connexins form channels with differing biophysical properties.14-16 Double whole-cell patch-clamp studies of heart cell pairs from neonatal rat17-19 or chick embryo20.21 have identified multiple, physiologically distinct gap junctional channels. We have hypothesized that these multiple channels might each be composed of different connexins. Several years ago, we cloned a rat cDNA encoding a cardiac gap junction protein, connexin43 (Cx43).22 Cx43 is highly conserved in other mammalian and vertebrate species.16,23-26We have since identified three different connexin sequences expressed in the chick embryo heart: Cx43, connexin42 (Cx42), and connexin45 (Cx45).27 In the present study, we have identified three homologous gap junction proteins in the mammalian heart and have demonstrated that all three proteins are expressed in cardiac myocyte gap junctions. Materials and Methods DNA Cloning and Sequencing Canine genomic DNA was obtained from Clontech, Palo Alto, Calif. The canine Cx45 sequence was amplified from genomic DNA using the polymerase chain reaction2, with primers corresponding to the amino terminus (TGAGATCTATGAGTTGGAGCTTTCTG) and carboxyl terminus (AGAGATCTTTAAATCCAGACAGAGTT) of chick Cx45. A portion of the canine homologue of chick Cx42 was amplified by polymerase chain reaction using genomic DNA and a degenerate/consensus primer corresponding to a conserved sequence in the first connexin extracellular loop (CTGCAGCCTGGCTGTA/GAG/C/ AAACGTCTGCTA/TGAC) and an antisense primer corresponding to the carboxyl terminus of chick Cx42

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Kanter et

al Multiple Connexins in Cardiac Myocytes

ATG AGT TGG AGC TTT CTG ACT CGC CTG CTA GAG GAG ATC CAC AAC CAT TCC ACA TTT GTG F I N L L V S F S T W S L T R R

60

GGG AAG ATC TGG CTC ACT GTA TTG ATT GCC TTT CGG ATT GCC CTT ACA GCT GTA GGA GGG I F I W L V L I A A L A V R T G G T G V V S

120

21

GAG TCC ATC TAT TAC GAT GAG CAA AGC AAA TTT GTA TGT AAC ACA GAG CAG CCA GGC TGT K C N F V I C S Y D E Q S T E Q P G Y B

180

41

GAA AAT GTC TGC TAT GAT GCT TTT GCA CCC CTC TCC CAC GTG CGC TTC TGG GTA TTC CAG F S V F Q A F A P L R W V N D Y V C E

240

61

ATC ATC CTG GTG GCA ACC CCC TCA GTG ATG TAC CTG GGT TAT GCC ATT CAT AAG ATT GCC A I K S V L A I G Y I M Y I L V A T P

300

81

101

AAA ATG K M R

---

-

V

GAG CAT GGA GAA GCA GAC AAG AAG GCA GCT CGG AGC AAA CCG TAT GCA ATG CGT K K A A K A E A D R S P Y M R E G Z R R F S S D V F S T

121

TGG AAA CAG CAC CGG GCT CTT GAA GAA ACA GAA GAG GAC CAT GAA GAG GAT CCC ATG ATG L E E Q T D D P E E M M A R W D A G

141

TAT CCA GAA ATG GAA TTG GAA AGT GAA AAA GAA AAT AAG GAG CAG AAC CAA CCT S N Q N Q P E L E P E M E Y 0 P R I

---

-

A

AAA CCC K P A

360

420

FIGURE 1. Sequence of canine connexin45. The complete coding 540 sequence of nucleotides is shown in light type (three-letter groups) with

161

AAG CAT GAT GGC CGA CGG CGG ATT CGG GAG GAT GGG CTT ATG AAA ATC TAC GTG CTG CAG E L I V L Q I Y R D G M G R R R D R

TTG CTT GCA AGG ACC GTA TTT GAG GTG GGT TTT CTG ATA GGG CAG TAT TTT CTG TAT GGC L V L F V F I G Y Y G F G Q L A T L R L A T V

600

181

TTC CAA GTC CAC CCA TTT TAT GTG TGC AGC AGA CTT CCT TGC CCT CAT AAA ATA GAC TGC S L P C P I D C F R F Q V P V C Y K V F S E

660

201

TTT ATT TCT AGA CCT ACT GAA AAG ACC ATC TTC CTT CTA ATA ATG TAT GGT GTT ACA GGC L K T L V I F I T G Y G F I S P T E R S C

720

221

241

CTT TGC CTG TTG CTT AAC ATT TGG GAA ATG CTC CAT TTA GGG TTT GGG ACA ATT CGA GAC E L I W L D I F G G R L N T E M L C L L V M

261

TCA CTA AAC AGT AAA AGG AGG GAA CTG GAA GAT CCG GGT GCT TAT AAT TAT CCT TTC ACT D P A N P S L F Y Y T N E G S R R E L T K S N T

840

TGG AAT ACA CCA TCT GCT CCC CCT GGC TAT AAC ATT GCC GTC AAA CCA GAT CAG ATC CAG V S A P P N I A P D Q I Q G Y N P T W

900

281

439

80

residues numbered on the right. The

derived amino acid sequence is shown in boldface (beneath each three-letter groups). The positions of amino acids in chick connexin45 that differ are shown beneath their counterparts in the canine clone. Dashes represent nucleotide codons or amino acids that are present in only one species but not the other.

M TAC ACC GAA CTG TCC AAT GCT AAG ATT GCC TAC AAG CAA AAC AAG GCC AAC ATC GCC CAG I A Q N A N A Q K S N A I Y L T Y M

960

301

GAA CAA CAG TAT GGC AGC CAC GAG GAG AAT CTC CCC GCT GAC CTG GAG ACT CTG CAG CGG A D L L N L P R S E T E E Q G Y Q Q N I N

1020

321

GAG ATT AAA ATG GTT CAG GAA CGC TTG GAT CTA GCA ATC CAG GCC TAC AGT CAC CAA AAC N A Q A S I I K R L L Y V D Q E M E Q N N M V A

1080

341

361

AAC CCC P N

---

-

G

381

CAC GGA CCC CGG GAA AAA AAA GCC AAA GTG GGG TCC AAA GCT GGG TCC AAC AAG A S N A V S K G P X G R E X G S S S

AGC AGT GCT AGT AGC AAA TCA GGA GAT GGG AAG AAC TCT GTC TGG ATT TAA V N S I S S W G D G S A S S

(CTAAGAGAATTCTCACACC/TGACAA/GA /GTCA/

GTCTGACCT). Restriction sites

were

incorporated

into all primers to facilitate subcloning into Bluescript vectors (Stratagene Inc., La Jolla, Calif.). The DNA

fragment encoding the canine Cx42 homologue was used to screen a canine genomic DNA library (Stratagene) by hybridization according to Beyer et a122 to isolate clones containing the full coding sequence. DNA sequencing was performed using plasmid templates, Sequenase enzyme (USB, Cleveland, Ohio), and oligonucleotide primers as previously described.27 RNA Blots Isolated canine left ventricular myocytes were prepared as described previously.29 These preparations contain virtually no other cell type. Total cellular RNA was prepared from canine left ventricle or from the isolated myocytes, separated on formaldehyde/

1140

1191

agarose gels, and prepared for Northern blotting as previously described.22 Blots were then hybridized with specific 32P-labeled DNA probes corresponding to carboxyl-terminal portions of the coding regions of rat Cx43, dog connexin40 (Cx40), and dog Cx45. In some control experiments, blots were rehybridized with the cDNA for rat cardiac troponin 1,30 which corresponds to an mRNA of -850 bp.

Antiserum Production and Immunocytochemistry Preparation and characterization of the anti-Cx43 antisera have been described previously.9'29 Cx4Oand Cx45-specific antisera were prepared by immunizing rabbits with synthetic peptides conjugated to keyhole limpet hemocyanin, according to methods used previously.9 For Cx45, the synthetic peptide immunogen was from residues 285-298 of dog Cx45, CSAPPGYNIAVKPDQ, a sequence that is identical

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Circulation Research Vol 70, No 2 February 1992

440

ATG GGT GAC TGG AGC TTC CTG GGC GAG TTC CTG GAG GAA GTA CAC AAG CAC TCC ACA GTG V T K H L E V S G H F L F E E S W D G M

60

ATA GGC AAG GTC TGG CTC ACT GTC CTT TTC ATA TTC CGC ATG CTG GTG CTG GGC ACA GCT A T G M L V L R L F F V I L T G K V W I V

120

21

GCC GAG TCA TCC TGG GGG GAT GAG CAG GCA GAT TTC CAG TGT GAT ACC ATG CAG CCT GGC G T M P Q D Q C A D F D W E Q S G S A E M S Q L G P

180

41

TGT GGG AAT GTC TGC TAC GAC CAA GCC TTC CCC ATC TCT CAC ATC CGC TAC TGG GTG CTG L E W V R Y S Q A F P I I Y D N V C G C F V K E

240

61

CAG ATC ATC TTC GTG TCC ACA CCA TCT TTG GTG TAC ATG GGC CAC GCA ATG CAC ACG GTG V T H M A K H G S L V Y P T I V S I F 0

300

AAA GAG E K N

348

GAG TAC CCA GTG GCC GAG AAG GCA GAG CTG TCC TGC S C V L K K Y A A P E E T C K

402

1

81

lOl

CGC ATG CAG GAG AAG CGG AAC GTG CGG AAG GCT GAG AGG K A R V R K R E Q N E M R K E K M E

117

GCG GGC GCT GGC TCT TAC --S Y G G A A Q Y T D S

---

-

0

---

-

GCC A

E

---

---

---

Q

-

E

M

135

TGG GAA GAA GTG AAT GGA AGG ATT GTC CTC CAG GGC ACT CTG CTC AAC ACT TAT GTC TGC Y V C L V L N T G T I L G R Q V N E W E Y S R I S K G D

155

AGT ATC CTG ATC CGC ACT ACC ATG GAG GTG GCC TTC ATT GTG GGC CAG TAC CTT CTC TAT P L I Y L Y F V G Q V A I I R L M E T S I

I

A

GGG ATC TTC CTG GAC ACA CTG CAT GTC TGC CGC AGG AGT CCC TGC CCC CAC CCC GTC AAC N H V P P H R P C V C R S F L L D T 175 G I A I Y 0 E

195

TGT TAT GTA TCC CGG CCC ACA GAG AAG AAT GTC TTC ATT GTC TTC ATG CTG GCT GTG GCT L A V A T V F K I V F M N E S R P Y V C I

215

GCA CTA TCC CTC TTC CTC AGC CTG GCT GAA CTC TAC CAC CTG GGC TGG AAG --L H W K L Y L G S A L E S L F A L K V

---

AAG

-

K

A

CAG CGC TTT GTC AAG TCT GGG CAG GGC ATG GCT GAG TGT CAG CTT --- CCT Q L P K S G G A C Q R V M E Q F P R R E S T S P S P A Y S

462 FIGURE 2. Sequence of canine connexin40. The complete coding sequence 522 of nucleotides is shown in light type (three-lettergroups) with residues numbered on the right. The derived amino 582 acid sequence is shown in boldface (beneath each three-letter group). The 642 positions of amino acids in chick connexin42 that differ are shown beneath 696 their counterparts in the canine connexin40 clone. Dashes represent resi750 dues that are present in only one species.

233

CTC AGA R L E

GGA CCC TCG GCT GGC ATA GTC CAG AAC TGC ACA CCA CCC CCT GAC TTC AAT CAG TGT CTG P F C L P N Q D P I T C Q N P A V G S G Y V 0 R A M E A

810

251

AAA AAT GGC CCT GGG GGG AAA TTC TTC AAT CCC TTC AGT AAC AAG ATG GCT TCC CAG CAG K Q S K A P S N N Q F P K N K F G P G G S I S A N

870

271

AAC ACA GAC AAT CTG GCC ACT GAG CAG GTG CAA GGC CAG GAG CCA ATT CCT GGG GAG GGT P I V P G G E G P N Q Q T B Q L A E D N D A A R H S F A

930

291

TTC ATT AAT ATC CGT TAT GCC CAG AAG CCT GAG GTA --- CCT AAT GGA GCC TCC CCA A S P P G K P V N A N E I I Y Q F R C A A S E S M E K S S M

984

---

311

-

P

329

C

GGT CAC CGC CTT CCC CAT GGC TAT CAG AGC GAC AAG CGC CGT CTC AGC AAG K R L S H Y D R H S G X Q L P G R -

346

---

-

A

E

S

F

N

E

F

GCC AGC AGC AAG GCC AGG TCA GAT GAT CTG TCA GTG TGA K S V D L R S S A D S A

in chick and dog Cx45. For dog Cx4O/chick Cx42, the peptide used was from residues 260-279 of chick Cx42, SAPQVERAQMYTPPPDFNQC; 11 residues in this sequence including the last nine are identical in chick Cx42 and dog Cx4O. Antisera were initially characterized by enzyme-linked immunosorbent assay against peptide and then screened for reactivity by immunohistochemistry. Light-microscopic immunohistochemistry. Isolated canine ventricular myocytes were incubated with anti-peptide antisera or with nonimmune rabbit serum, followed by peroxidase-conjugated secondary reagents (Vector Laboratories, Inc., Burlingame, Calif.) according to the methods of Hsu et al.31 In some control experiments, specificity of antibody reaction was confirmed by coincubation of the immune serum with the corresponding peptide immunogen (100 ,ug/ml).

---

---

---

-

-

-

A

S

R

1035

1074

Immunoelectron microscopy. Isolated canine ventricular myocytes were fixed briefly in 1% paraformaldehyde in phosphate-buffered saline (immediately after isolation), rinsed, and incubated with connexispecific antisera or nonimmune rabbit serum, followed by colloidal gold-conjugated secondary antisera (EY Laboratories, San Mateo, Calif.). Immunostained cells were then fixed with 2% glutaraldehyde and processed for electron microscopy as described previously.29 Results Cloning of Cardiac Connexins All connexins studied to date have single copy genes that lack introns in their coding sequences. We took advantage of these characteristics and used the polymerase chain reaction and genomic cloning to isolate homologues of chick Cx42 and chick Cx45 from canine genomic DNA. Using oligonucleotide

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Kanter et al Multiple Connexins in Cardiac Myocytes MGDWSaLGkLLdkVqaySTagGKVWLsVLFIFRILLLGTAvESaWGDEQSaFrCNTqQPGCENVCYDksFPISHV

Dog Cx40 Dog Cx45

MGDWSFLGefLEEVHkHSTviGKVWLTVLFIFRmLvLGTAaESsWGDEQadFqCdTmQPGCgNVCYDqaFPISHi M-sWSFLtrLLEEiHnHSTfvGKiWLTV7LiaFRIaLtavggESiyyDEQSkFvCNTeQPGCENVCYD-faP1SHV

75 75 73

Rat Cx43 Dog Cx40 Dog Cx45

RFWVLQIIFV--- SVPtLlYlaHvfyvMRkeE --- KlN-kK--eEelK--vA-qtdGvnvE ---MhlkqiEiKkf RyWVLQIIFV---StPsLvYmgHamhtvRmqE---KrNvrK--AERaK--eA--gaG-syE- YPvaE-KaE RFWVfQII1VatpSVmyLgYaiHkiakMehgEadkKaarsKpyAmRwKqhrAleeteedhEedpMmYPemElesE

135 131 148

Rat Cx43 Dog Cx40 Dog Cx45

GLLrTYiiSILfksVFEVAFLliQwyiYGFsLsavYtCkRdPCPHqVDCF1SR KygiEE-hGKvK-rG lscwEEvnrGrivlqG --tLLnTYVcSILiRTtmEVAFivGQY1LYGifLdtlhVCrRsPCPHpVnCyvSR KenkEqnqpKpKhdGrrriredGLmkiYVlqlLaRTVFEVgFLiGQYfLYGFqvhpfYVCsRlPCPHkiDCFiSR

203 199 223

Rat Cx43 Dog Cx40 Dog Cx45

PTEKTIFIiFMLvVslvSLaLNIiE-LfyvFfKgvkdR-VKgrSdpyhAttgplsPSkdcGspkyaytNgcssPt PTEKnvFIvFMLaVaaLSLfLs1aE-LyhlgwKklrqRfVK--SgqgmAecqlpgPS--aG----ivqNctppPd PTEKTIF1liMygVtgLcLlLNIwEmLhlgFgtirdslnsK--rreledpgaynyPftwntpsappgyNiavkP-

276

Rat Cx43 Dog Cx40 Dog Cx45

aplspmspPGyKlvtgdrNnsScrnyNKqASeQNwaNysaE--QnrmGQ-~-aG-stlsnshAQ-pfdfPddNqn fnqalkngPGgK---- ffNpfS----NKmASqQNtdNlatE--Q-vqGQepipGegfIniryAQ-kPevP- -N-g dqiqytelsnaK-iaykqNkaniageqqygSheenlpadlEtlQreikmvqerldlaIqayshQnnPhgPrekk-

344 325 369

Rat Cx43

AKkVaaG-HeLqplaivDqRpSSrASSrASsrpRpDDLeI

382 357 396

Rat Cx43

A---spG-HrLphGyqSDKRrlSkASSKA----RsDDLsv

Dog Cx40 Dog Cx45

AK-V--Gska---G--SnK--SS-ASSK-Sgdgknsvw-I

441

265 296

FIGURE 3. Amino acid sequences of three mammalian cardiac gap junction proteins. The amino acid sequences denived from rat connexin43 (Cx43) cDNA22 and the amplified dog connexin40 (Cx40) and connexin45 (Cx45) genomic sequences are shown with optimal alignment. Identical amino acids are illustrated with uppercase letters, and nonidentical residues are illustrated with lowercase letters. Dashes represent spaces added to optimize alignment. Peptide sequences used as synthetic peptide immunogens

for connexin-specific antibody production are shown in bold italics.

primers corresponding to the amino and carboxyl termini of chick Cx45, we amplified its canine homologue. Dog Cx45 has a predicted molecular mass of 45,538 d, and 85% of amino acids are identical to chick Cx45 (Figure 1). We were unable to amplify the entire coding sequence of the canine homologue of chick Cx42 using a similar strategy, probably because of nucleotide divergence of the amino-terminal sequence. However, we were successful at amplifying a partial canine sequence using a primer corresponding to the carboxyl terminus of chick Cx42 and a degenerate/ consensus primer corresponding to the first extracellular loop conserved in all connexins. This 860-bp

V ",,-10-W

DNA sequence, corresponding to bases 205-1074 in Figure 2, was subsequently used as a probe to screen a dog genomic library by hybridization to isolate a genomic clone that contained the full coding sequence of the canine homologue of chick Cx42. Because this canine homologue has a predicted molecular mass of 39,937 d, it is therefore referred to as dog Cx40 (Figure 2). In dog Cx40, 70% of amino acids are identical to chick Cx42. Figure 3 shows the derived amino acid sequences of the canine homologues, dog Cx40 and dog Cx45, in comparison with rat Cx43 (which is >97% identical in all mammalian species studied).16'24-26 The matching residues in these mammalian gap junction proteins fall

M V

M

V

a".AA

C''

A FIGURE 4. Northem blots demonstrating expression of multiple gap junction mRNAs in canine ventricle and isolated cardiac myocytes. Total cellular RNA was prepared from dog ventricle (V) or from isolated myocytes (M), separated on formaldehyde! agarose gels, and prepared for Northern blotting as described in "Materials and Methods. " Blots were then incubated under high-stringency conditions with specific 32P-labeled DNA probes corresponding to carboxyl-terminal portions of the coding regions ofdog connexin40 (panel A), rat connexin43 (panel B), and dog connexin45 (panel C) or a probe for rat cardiac troponin I (panel D). RNAs from both ventricle and isolated ventricular myocytes hybridized to all four probes and had similar relative intensities. The exposure times for the different probes were 6 days, 18 hours, 6 days, and 2 hours for panels A-D, respectively. Arrowheads indicate the migration of 18S and 28S rRNAs. Downloaded from http://circres.ahajournals.org/ by guest on May 9, 2015

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1. 'W:,

4:..

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probing of these blots with a rat cardiac troponin I probe produced bands of similar relative intensities in ventricle and myocytes (Figure 4D).

.,

49

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D FIGURE 5. Immunohistochemical localization of three connexins in isolated myocytes. Isolated canine ventricular myocytes were incubated with anti-peptide antisera specific for connexin40 (panel A), connexin43 (panel B), or connexin45 (panel C) or with nonimmune rabbit serum (panel D), followed by peroxidase-conjugated secondary reagents. An identical pattern of staining is observed with each anticonnexin reagent and corresponds to the known distribution of gap junctions in the intercalated disks. Preimmune or nonimmune sera ( panel D) or antisera preincubated with the peptide immunogen showed no specific reaction. Bar, 20 pn.

within the predicted transmembrane and extracellular regions conserved in other connexins.13 The Cx40, Cx43, and Cx45 polypeptide sequences diverge markedly in their predicted cytoplasmic domains, which likely would endow channels composed of these connexins with unique biophysical properties.'4 16

Expression of Cardiac Connexin mRNAs Although we have previously shown that whole chick heart contains mRNAs for multiple gap junction proteins, the exact cell type expressing each connexin was not determined. We have now addressed this question using RNA blots and immunohistochemistry. We extracted RNA from canine ventricular myocardium and from pure populations of isolated canine ventricular myocytes and performed Northern blot analysis. Specific probes for Cx40, Cx43, and Cx45 all hybridized at high stringency with total RNA from both whole tissue and isolated myocytes (Figure 4). Cx40 hybridizes to an mRNA of -2.6 kb, Cx43 hybridizes to a 3.4-kb mRNA, and Cx45 hybridizes to a 2.1-kb mRNA. Since the derived connexin protein sequences are of similar size, the differences in mRNA length must represent differences in untranslated sequences, as in the chick homologues.27 Re-

Immunolocalization of Cardiac Connexins We used immunohistochemistry to confirm that the connexin proteins were expressed by cardiac myocytes. We have previously prepared and characterized specific anti-Cx43 antisera.929 We prepared additional antisera directed against synthetic peptides representing unique amino acid sequences for dog Cx40/chick Cx42 and dog/chick Cx45 (identified in Figure 3). Each of these antisera demonstrated specific labeling of isolated canine ventricular myocytes in a pattern consistent with the known distribution of cardiac gap junctions in intercalated disks (Figure 5). Nonimmune or preimmune sera showed no specific reactivity. The reactivity of each anticonnexin serum with myocytes was abolished by preincubation with its corresponding peptide immunogen, whereas incubation with an unrelated peptide had no effect. To confirm that the immunohistochemical staining of cardiac myocytes at intercalated disk regions did indeed represent labeling of gap junctions, we performed immunoelectron microscopy using isolated canine ventricular myocytes. Figure 6 demonstrates gold-conjugated antibodies specifically bound to ultrastructurally recognized gap junctions after incubation with primary antisera specific for each connexin. The gold particles decorate only the single gap junctional surface accessible to antibodies in the isolated myocytes. The circular appearance of some junctions shown does not represent internalization but may represent the tendency of the structures to sequester hydrophobic regions away from the aqueous environment after disaggregation. The appearance of these gap junctions is somewhat reminiscent of the structures observed by Mazet et aP32 in dissociated adult rat cardiac cells. Discussion Results of the present study indicate that gap junctions interconnecting cardiac myocytes contain three different gap junction proteins. Our current analysis does not provide exact quantitation of the amounts of the different connexins, but the blots were hybridized with identical amounts of probes labeled to similar specific activities. Therefore, based on the relative exposure times necessary for production of the Northern blots (see Figure 4), it appears that Cx43 is the most abundant connexin mRNA in the adult canine myocytes. In contrast, Cx42 appears to be much more abundant in the chick heart.27 Although Cx45 is largely an embryonic isoform in the chick,27 it is clearly also present in the adult canine

heart. This study demonstrates that Cx40, Cx43, and Cx45 are all expressed in cardiac myocytes. The relative intensities of the hybridizing bands produced by these probes in the ventricle and isolated myocytes

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Kanter et al Multiple Connexins in Cardiac Myocytes .)W

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A), connexin43 (panel B), or connexin45 (panel C) or nonimmune rabbit serum (panel D), followed by colloidal gold-conjulgated secondary antisera as described in "Materials and Methods. " The specific labeling of ultrastructurally identifiable gap junctions by all three anti-connexin reagents confirmed the presence of all three proteins within cardiac myocyte gap junctions. Bar, 0.5 mn.

W.'

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similar to that produced by a probe for the contractile protein cardiac troponin I (Figure 4). Thus, it appears that most of the mRNA in the whole tissue derives from myocytes. However, this does not exclude additional expression of these connexins by other cell types. Indeed, we have evidence that Cx40 is expressed by a number of cell types, including vascular smooth muscle cells (authors' unpublished observations). We have previously demonstrated that chick Cx42 and chick Cx45 are expressed in several different organs in the chick are

myocyte

embryo.27

Previous studies have implicated alterations in the number, size, and distribution of myocyte gap junctions as potential determinants of lethal arrhythmias.5-8,33 Results of the present study identifying multiple cardiac connexins suggest novel mechanisms for regulating electrical coupling. In parallel studies, we have been studying the properties of gap junctional channels derived from chick Cx42, Cx43, and Cx45 by stable transfection of a communicationdeficient cell line. Our preliminary data suggest that chick Cx42, Cx43, and Cx45 form channels with different unitary conductances and different sensitivities to transjunctional voltage.34 Differential expression of individual connexins or differential responses of their channels to second messengers might provide multiple mechanisms for modulation of cardiac coupling under both normal and pathophysiological conditions. For example, it has recently been reported that specialized myocytes of the atrioventricular node and the His-Purkinje system of the adult rat heart express much less Cx43 than ventricular myocytes, despite their extensive interconnections by gap june-

FIGURE 6. Immunoelectron microscopic localization of three connexins in canine ventricular myocyte gap junctions. Isolated canine ventricular myocytes were stained with connexin-specific antisera for connexin40 (panel

tions.35 Therefore, expression of other connexins may account for the specialized electrical properties of these components of the cardiac conduction system. Similarly, connexin expression may be altered in diseased myocardium, analogous to the protein isoform switches of members of other cardiac multigene families in response to myocardial hypertrophy or failure.36 Finally, the discovery that myocytes are coupled by multiple connexins provides an impetus for the development of connexin-specific pharmacological agents. Interestingly, Wit37 has suggested that the selective modulation of intercellular coupling in the heart might be an appropriate target for a new class of antiarrhythmic agents.

Acknowledgments We should like to thank Dr. Peter B. Corr for providing the isolated cardiac myocytes used in these studies and for helpful and enthusiastic discussions. We would also like to thank Dr. Anne Murphy for the kind gift of the rat cardiac troponin I probe. Eileen Westphale, Charlene Croker, Karen Green, and William Kraft provided invaluable technical assistance. The nucleotide sequences for dog connexin40 and dog connexin45 have been submitted to the Genbank and EMBL data bases with respective accession numbers M81347 and M81348. References Wellens HJJ, Durrer DR, Kie KI: Observations on mechanisms of ventricular tachycardia in man. Circulation 1976;54: 237-246 2. Janse MJ, Wit AL: Electrophysiological mechanisms of ventricular arrhythmias resulting from myocardial ischemia and infarction. Physiol Rev 1989;69:1049-1169 1.

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KEY WORDS * gap junctions * intercellular communication E connexins * electrical conduction * ion channels * intercellular junctions * molecular cloning

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Cardiac myocytes express multiple gap junction proteins. H L Kanter, J E Saffitz and E C Beyer Circ Res. 1992;70:438-444 doi: 10.1161/01.RES.70.2.438 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1992 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571

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Cardiac myocytes express multiple gap junction proteins.

Electrical propagation in the normal heart occurs via intercellular transfer of current at gap junctions. Alterations in intercellular coupling in the...
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