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530 Human Nonmuscle Myosin Heavy Chains Are Encoded by Two Genes Located on Different Chromosomes Michael Simons, Mary Wang, 0. Wesley McBride, Sachiyo Kawamoto, Katsutoshi Yamakawa, David Gdula, Robert S. Adelstein, and Lawrence Weir Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 We report the cloning of cDNAs encoding two different human nonmuscle myosin heavy chains designated NMMHC-A and NMMHC-B. The mRNAs encoding NMMHC-A and NMMHC-B are both 7.5 kb in size but are shown to be the products of different genes, which are localized to chromosome 22q11.2 and chromosome 17p13, respectively. In agreement with previously reported results using avian tissues, we show that the mRNAs encoding the two myosin heavy chain isoforms are differentially expressed in rat nonmuscle and muscle tissues as well as in a number of human cell lines. The cDNA sequence encoding the 5' portion of the NMMHC-A isoform completes the previously published 3' cDNA sequence encoding a human myosin heavy chain, thus providing the cDNA sequence encoding the entire NMMHC-A amino acid sequence. Comparison of this sequence to cDNA clones encoding the amino-terminal one third of the NMMHC-B sequence (amino acids 58-718) shows them to be 89% identical at the amino acid level and 74% identical at the nucleotide level. (Circulation Research 1991;69:530-539) M yosin is a ubiquitous cytoskeletal protein present in all eukaryotic cells. Although best studied in tissues that manifest specialized contractile activity, such as cardiac and skeletal muscle, myosin in nonmuscle cells has been implicated in processes as diverse as cytokinesis, cell motility (for reviews, see References 1-3), secretion,4 and capping.5 There is now compelling genetic evidence that supports a role for nonmuscle myosin in cytokinesis, because cells that lack most of a particular myosin isoform lose their ability to divide.6,7 In vertebrates, the conventional myosin isoforms consist of a pair of heavy chains (200 kDa) and two pairs of light chains (15-28 kDa). In addition to the 480 kDa myosin isoforms, vertebrate intestinal brush border epithelial cells, similar to Acanthamoeba and Dictyostelium, contain a smaller myosin isoform (110 kDa) that preserves many of the functional properties of the globular amino terminal region of the From the Laboratory of Biochemistry (M.W., O.W.M.), National Cancer Institute, and the Laboratory of Molecular Cardiology (M.S., S.K, KY., D.G., R.S.A.), National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md.; and St. Elizabeth's Hospital (L.W.), Department of Cardiology, Boston, Mass. The sequences reported in this paper have been deposited in the GenBank data base (accession Nos. M69180 and M69181). Address for correspondence: Robert S. Adelstein, MD, Laboratory of Molecular Cardiology, National Institutes of Health, Building 10, Room 8N-202, Bethesda, MD 20892. Received January 8, 1991; accepted April 15, 1991. myosin heavy chain (MHC) but lacks the carboxyterminal rod region (for review, see Reference 8). Skeletal and cardiac MHCs exist as multigene families with a large number of isoforms encoded by different genes. The two different cardiac MIC isoforms manifest different rates of ATP hydrolysis9 and may serve different physiological functions. Less is known about the MHC isoforms present in vertebrate smooth muscle and nonmuscle cells. At present, two different smooth muscle MHC isoforms have been identified at the protein level,10 and the isolation of cDNA1112 and genomic clones'3 suggests that at least three MHC isoforms can be generated by alternative splicing of mRNA. For vertebrate nonmuscle MHCs (NMMHCs), the existence of isoforms first was suggested on the basis of peptide maps by Burridge and Bray.'4 Recently, the sequence for two cDNA clones (2.8 and 0.9 kb) encoding the same region of the NMMHC in chicken fibroblasts, but that showed differences throughout this sequence, was reported.15 These authors suggested that these two clones are encoded by different genes. The 0.9 kb clone is present in the nucleotide sequence of the cDNA encoding the entire NMMHC from chicken intestinal epithelial cells reported previously,16 which we refer to as NMMHC-A. The first mammalian NMMHC cDNA clones were obtained from a human macrophage and fibroblast library by Saez et al,'7 and comparison of these sequences with the avian sequence identified the human clones as the NMMHC-A isoform. Simons et al Nonmuscle Myosin Heavy Chains Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 One of the most interesting aspects of nonmuscle myosin function is the role it may play in smooth muscle cell proliferation. Proliferation of vascular smooth muscle is a cardinal feature of atheroscleroSiS.18 In addition, it has been implicated in the development of restenosis after angioplasty of vascular lesions,19 in the failure of venous bypass grafts,20 and in the vascular response to hypertension.21 Proliferating vascular smooth muscle undergoes a striking phenotypic change: instead of spindle-shaped cells capable of contractile activity, but incapable of division, one encounters polygonal actively secreting cells incapable of contraction, but capable of cytokinesis. On the biochemical level, a number of alterations occur as well. One of the most striking is an almost complete replacement of smooth muscle MHC with a nonmuscle isoform.22 This change in MHC phenotype has been observed in proliferating smooth muscle in culture22,23 as well as in vivo.24 Recently, we have demonstrated the expression of nonmuscle myosin in restenotic lesions in human coronary as well as peripheral arteries.25 The appearance of the nonmuscle isoform correlates with the newly acquired ability of smooth muscle cells to divide and disappears when cells return to their contractile phenotype. These observations suggest that nonmuscle myosins may play a pivotal role in smooth muscle proliferation and that understanding their role may shed light on the pathogenesis of a number of important vascular diseases. In this paper we present information on the cloning and sequence of two human NMMHC cDNAs. The sequence of one group of cDNA clones encodes the 5' portion of NMMHC-A. The second group of clones encodes a different NMMHC isoform, which we refer to as NMMHC-B. We show that these two isoforms are encoded by two different genes. Chromosomal mapping using hybrid panels and in situ hybridization showed that these genes are located on different chromosomes. The gene encoding NMMHC-A was localized by in situ hybridization to chromosome 22q11.2. The gene encoding NMMHC-B was localized to chromosome 17p13. This chromosome band contains at least one and possibly four genes encoding the skeletal muscle MHCs.26-30 Materials and Methods cDNA Cloning cDNA clones were isolated from a lambda gtlO library (Clontech Laboratories, Inc., Palo Alto, Calif.) and a lambda ZAP II library (Stratagene Inc., La Jolla, Calif.). Both libraries were constructed from human T lymphocyte (Jurkat) mRNA. The lambda gtlO library was screened initially with a 900 bp cDNA probe encoding the 5' region of a chicken NMMHC-A.16 Subsequently, a second NMMHC-A probe from the 5' end, as well as human probes isolated during the cloning process, were used to screen the library. The lambda ZAP II library was screened as outlined in "Results." Probes were la- 531 beled by random priming to a specific activity of 108 cpm/,ug using a kit (Prime Time C, United States Biochemical Corp., Cleveland, Ohio). Hybridization was carried out at 42°C in 40% formamide and 10% dextran sulfate. The stringency of the final wash using chicken cDNA probes was 0.2x SSC at 50°C and using human cDNA probes was 0.1 x SSC at 65°C. Phage inserts were subcloned either into pTZ19R (Pharmacia LKB Biotechnology, Piscataway, N.J.) or pGEM-3Zf(-) (Promega Corp., Madison, Wis.) vectors. In the case of clones obtained from the lambda ZAP II library, in vivo excision of clones was carried out as outlined by Stratagene. Sequence Analysis Double-stranded DNA was sequenced using a Sequenase kit (United States Biochemical Corp.). In some cases, restriction enzymes were used to make deletions in the original subclones that then were used as sequencing templates. Synthetic oligonucleotide primers were used to extend the nucleotide sequence. Sequence data was analyzed with the use of MICROGENIE software (Beckman Instruments, Inc., Fullerton, Calif.). mRNA Analysis Human RNA was prepared from cultured T lymphocytes by the method of McDonald et a131 or Chomczynski and Sacchi.32 Total rat RNA from a number of tissues was prepared by the latter method, as was total RNA from various human cell lines. Human cell lines, with the exception of Jurkat and A-431, were obtained from the American Type Culture Collection (ATCC) (Rockville, Md.), and cells were grown as per the instructions. A-431 cells were a gift from Dr. Stuart Aaronson (National Cancer Institute) and were grown in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Jurkat cells were obtained from Dr. Warren Leonard (National Institute of Child Health and Development) and were grown in RPMI-1640 medium and 10% fetal bovine serum. RNA samples were analyzed in a denaturing formaldehyde agarose (1%) gel according to a standard protocol,33 and capillary transfer to nylon membranes was carried out according to manufacturers' recommendations. Hybridization was carried out in the presence of 10% dextran sulfate and 40% formamide at 42°C for 12-16 hours with a final wash in 0.1 x SSC at 65°C or 0.2x SSC at 60°C for human samples. For analysis of rat mRNA, hybridization was carried out as above, but the stringency of the final wash was 0.5 x SSC or 0.2 x SSC at 60°C. Preparation of Probes for Chromosome Localization Cloned cDNA and genomic fragnents were used. The cDNA probes are described in this paper. A 350 bp genomic probe was obtained after EcoRI digestion of a 17 kb genomic clone obtained from a human placenta Charon 4A library (ATCC; Simons, Weir, Adelstein, unpublished data). This genomic clone was selected using a human lymphocyte cDNA probe, HL1.16 The 350 bp genomic intronic fragment is 5' to an 532 Circulation Research Vol 69, No 2 August 1991 836 ROD 1961 Ifa 11 AI HEAD (S1) a - i MHC-A 400 AA NT 225 -156 675 L AA NT ,__~~------721 1606 589 2163 (+707) 376 172 344 ¢ FIGURE 1. cDNA clonesofhumannonchain (NMMHC) fb302 and bZ801 and Z802 encode the human NMMHC-A and clones 0707, 756, 758, 759, and 760 encode the human NMMHC-B. Clone pNMHCM2 is from Saez et al.17Dashed lines represent areas that have not been sequenced. AA, amino acid; NT, nucleotide. t&.L -,.'.302pNMHCM2 ... muscle myosin heavy isofonns. Clones 2146 1676 58 MHC-B 715 H-|(+302) -- - 711 | ~~(+758) 710 (+759) 395 +,ao 718 (+756, +760) 2154 Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 DNA Isolation and Filter Hybridization DNA was isolated from hybrid cell lines, digested with EcoRI, and size-fractionated by agarose (0.7%) gel electrophoresis. After partial depurination, the fragments were transferred to positively charged nylon membranes in 0.4 M NaOH. The membranes were hybridized at high stringency (allowing 10% sequence divergence) at 42°C with 32P-labeled probes in 50% formamide containing 5x SSPE, 5x Denhardt's solution, 10% dextran sulfate, 0.2% sodium dodecyl sulfate, and sheared denatured herring sperm DNA at 200 ,ug/ml. Membranes were washed at 55°C in 0.1 x SSC containing 0.2% sodium dodecyl sulfate. The membranes were used repeatedly after removal of the probe in 0.4 M NaOH, neutralization, and prehybridization with carrier DNA. (0.05 ,ug/ml) present during the final 20 minutes. The cells were centrifuged, swollen, and fixed, and airdried metaphase spreads were prepared by standard procedures.38 After treatment with RNase A (100 ,ug/ml) for 1 hour at 37°C, the chromosomal DNA was denatured in 70% formamide and 2x SSC at 70°C or in 0.07N NaOH in 64% ethanol at 25°C for 3 minutes.39,40 Radiolabeled probes (specific activity, 3 x 107 cpm/,ug) were prepared by nick translation of recombinant plasmid DNAs with [3H]dTTP and [3H]dCTP. The probe was mixed with hybridization solution (50% formamide, 5% dextran sulfate, 2x Denhardt's solution, 2x SSC, 5 mM EDTA, 20 mM sodium phosphate, pH 6.4, and 200 ,g/ml sheared herring sperm carrier DNA), heat denatured, applied to slides (3 x 105 cpm/slide in 25 ,ul), and hybridized for 20 hours at 42°C. Nonspecifically bound probe was removed by washing in 50% formamide/2x SSC (pH 7.0) for 10 minutes at 42°C and in 2 x SSC at 42°C. The slides were coated with a 50% solution of NTB2 nuclear track emulsion (Eastman Kodak Co., Rochester, N.Y.) and stored dessicated at 4°C for 9 days before developing, staining (0.25% Wright stain), and photographing. The slides were destained and G-banded with 0.03% trypsin and 0.12% EDTA41 or replication banded37 by staining with 33258 Hoechst (150 ,ug/ml) for 30 minutes and exposure to UV illumination for 30 minutes after rinsing. The slides again were stained with Wright stain, and the same metaphase spreads were rephotographed. In Situ Hybridization Experiments were performed using peripheral blood lymphocytes from a normal male (46,XY) that were cultured for 72 hours at 37°C in RPMI-1640 supplemented with 15% fetal bovine serum, phytohemagglutinin (0.5 ,ug/ml), and antibiotics. Cultures were synchronized by addition of 100 ,ug/ml 5-bromo-2'-deoxyuridine37 or 10` M methotrexate38 for 17 hours before washing and resuspension in fresh medium containing 10-5 M thymidine and incubation for an additional 5.5 hours with colcemid Results cDNA Cloning and Sequencing A 900 bpAva I cDNA probe encoding the first 300 amino acids of the chicken intestinal epithelial cell NMMHC16 was used to obtain a single clone from a human T lymphocyte lambda gtlO library (0302, Figure 1). The clone contains a 1,356 bp insert that consists of 156 nucleotides of 5' untranslated cDNA and 1,200 nucleotides encoding the first 400 amino acids of a NMMHC. The location of the translation exon encoding the reactive thiols found in the myosin globular head. All fragments were purified by gel electrophoresis and labeled with dCTP (a-32P) by random oligonucleotide primed synthesis before use. Cell Hybrids The human and rodent parental cells, fusion procedure, and isolation and characterization of humanmouse and human-Chinese hamster somatic cell hybrids have been described.34-36 Briefly, hybrid cells were analyzed for the presence of all human chromosomes except Y by standard isoenzyme analyses, as well as by Southern analysis with probes from previously localized genes, and frequently by cytogenetic analysis. Simons et al Nonmuscle Myosin Heavy Chains A gtcctgcita ELA -19 533 LysLysGi.Ar*AuuTbrAspGi.AI SmrM.tPreAspAsmThrAlaAIaGInLysVu 1 374 1063 AAGAAGGAGCGGAACACTGACCAGGCGTCCATGCCCGACAACACAGCTGCCCAAAAGGTG A-A-T--- -T-A-T A-A--T-T-G-G-C-C - .Gi - Vat - - - Lu 377 1HU 1072 -A AA IHCA -6 MICA 52 HiMtAiiGiuGIiAIoAIaAspLysTTyrLiTyrValAsupLyuAnPhel Ie 17 taigtacicATGGCACAGCAAGCTGCCGATAAGTATCTCTATGTGGATAAAAACTTCATC AsiAuuPriLoiAlGlinAluAspTrpAliAlLyuLyuLueoVuITrpVaIPriSirAup 37 AACAATCCGCTGGCCCAGGCCGACTGGGCTGCCAAGAAGCTGGTATGGGTGCCTTCCGAC SerArHisLsuLeGlmlGIyeAsnVaIThrAspPhsTbrArgGIyIIluuTbrPrsArgllu 394 1123 TCCCATCTCTTGGGTATCAATGTGACCGATTTCACCAGAGGAATCCTCACCCCGCGCATC TC-T-G-G TG-G-T-TC-G-CC -G-T-C-6MU 1132 -6 CVs - - - - Met - - HMGtG1 - - - Ag - - - . - - - 397 nICA LyuVaIGIArgAspTyrVa1GInLysAIaGIuThrLysGIuGInAIaAspPheAIaI Ie 414 1193 AAGGTGGGACGGGATTACGTCCAGAAGGCGCAGACTAAAGAGCAGGCTGACTTT6CCATC C-C-A -C-T---A-A-C A A-TAG-A C - - - - - - - - - - - - - - - - - - - Val 417 ECB 1192 - IHGA 112 LysSirGi yPhuGluPrsAliaSerLunLyVGiGliVolGlt6iGArgGlNHiuVilGin 57 AAGAGTGGCTTTGAGCCAGCCAGCCTCAAGGAGGAGGTGGGCGAGAGAGGCCATGTGGAG MUCA 1243 GIuAI6LaLAIl,LsAlaThrTyrGIuArgH.tPhaArgTrpLemVaIleLArI ImAui 434 GAGGCCTTGGCCAAGGCGACCTATGABCBGATGTTCCGCTGGCTGBTGCTGCGCATCAAC MfU 1252 -A-A-A-A-T . HHA 172 LiiVaiGliAtnfllyLyVlLiVulAiALyiAsipAipl InGlnLynHietAiPri 77 A -CA A-CA-T-C 1312 -A - - MICA ECI 232 PriLyaPhuSerLytValGlmAspMiltAl iiLiuThrCyiLiiAsnoGlAlnSerVil 97 CtCAAGTTCTCCAAGGTGGAGGACATGGCAGAGCTCACGTGCCTCAACGAAGCCTCGGTG T T-C-T -T-T AT-G-A-T-G-T - - - - C-C-T Lw - - - - C-T-AT T - His - - 437 292 CTGCACAACCTCAAGGAGCG TTACTACTCAGGGCTCATCTACACCTATTCAGGCCTGTTC EC -T--A-A -T-T T-A-CT-A-T-T-G--T-C T-G - T-A A-A-T AC-T 352 MIHD CynVeiVl I IAuiPruTyrLyoAieLeLPrleITyrSurGnGiilileVaiGIiit 137 TGTGTGGTCATCAATCCTTACAAGAACCTGCCCATCTACTCTGAAGAGATTGTGGAAATG Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 GA-T-A-T -A a - lit T-T-A-T A-T-A-C .-- - 457 - - - - Gin - - - - - - - - - 412 MA MICA 472 E"U C-T-T - - - - T-A - - - - - - - - EUI 532 T-AT-T-AT-T-T - SerGhiSer - TyrArgSurMetMitiGliApArgGliAnpGhiSurl iLeuCynThrGlyGliSerGly 177 AlaGlyLysThrGiAuiThrLyiLysVallii GInTyrLouAiaTyrValAhiSurSir 197 - - - 592 ECU MICA y iLiiG iArgGI LeniLiiGi AAi 214 661 iG 1Gi CAGGGCGAGCTGGAGCGGCAGCTGCTGCAGGCC CACAAGAGCAAGAA6GAC -TT-A-A CATAATATT-CT-G-A-T-A -T-AG-A-GA - - G1VAr, - - NihiAuliPri - - - - - - - - - - 217 MIHC E - - - - Sir - - - - - - - - - - - - - - 703 TTCGGCAAATTCATTCGCATCAACTTTGATGTCAATGGCTACATTGTTGGAGCCAACATT 1: 712 -T A-C T-C T-6G 6G . MICA 763 MIC 7 MICA 623 ECU 832 MIC;A M3 ECU 832 - - - - - - - - - - Thr - - - - - - - - 257 GAGACTTATCTTTTGGAGAAATCTCGTGCTATCCGCCAAGCCAAGGAAGAACGGACCTTC -A-A-C-C-A-B G-T-T- A-A-T T-T-T - - - - - - - - - - Vol - - - - Asp - - - - m HNiuI11PhuTyrTyrLeuleuSerrGlyAIaulyBi6iuL nLysThrAspLuuLnLeun 294 CACATCTTCTATTATCTCCTGTCTG666CTGGAGAGCACCTGAAGACCGATCTCCTGTTG -A-A-A -T-T-CC-6T-BT-A A-T-T-T-G-TC-T - - -i - - - - - - - - - - Ser - - - - 297 BluPruTVrAuLyusTyrAr,PbeleLSurAuGIyNHiuVaiTbri IuPruGIVBiuGio 314 GAGCCGTACAACAAATACCGCTTCCTGTCCAAT6GACACGTCACCATCCCCGGGCAGCAG CT-TA-TC-T-T-G-A A -A6GA-TT-T-C-A-6-T-C - BlyPhi - Ass - - - - - - - Tyrl lPr - - - - - 317 EU 1792 EA 1B43 EOn 1952 497 - - - - 517 BiiiuHilyLuuLuoArVul IiSirGlrVuiLunGluLiuGiAsul iiViPhu 354 1003 GAGCAAATG6GCCTGCTGCGGGTCATCTCAGG6GTTCTTCAGCTCGGCAACATCGTCTTC r - - EC 577 - - - - W597 - 614 617 934 937 AIPhiLyiThrAr,LyoGlyMetPheArgThrVail yGtaLeuTyrLyoGi.GinLin 954 1993 GCCTTCAAGACGCGGAAGGGCATGTTCCGCACTGTGGGGCAGCTTTACAAGGAGCAGCTG 1912 -A-AT-A-CAA - Tyr - - Lys - - - - - - A-ATCT-C A-C T-T-C-T - - - - - - - Ser - 957 AAAliLsLiiHtAiiSorLoArgAsiThrAunPriAioPbiVilArgCyul ei limPri 674 1993 GCCAAGCTGATGGCATCGCTGAGGAACACGAACCCCAACTTTGTCCGCTGCATCATCCCC T 1972 A CC-CA-T-CC-A-C T-A T-T-T - Thr 677 AsnHi s61GlViLytAIaGlyLyslouAspPrtNiiLeuVIlLeuAspGinLuiArgCyu 694 Thy - - low 2323 AACCACGAGAAGAAGGCCGGCAAGCTGGACCCGCATCTCGTGCTGGACCAGCTGCGCTGC -T-T G-T-A-AT-T-A-C-A-C-A-T EU 2032 -T AspLuAupHutPiheGloGlThrHuetGiuAlaHetArgliiHietGlyloPruGIuGlu 334 ECU 1012 -ATTC-TCAA-TAAA-AG-A-TTCA-G-A-T-T-A-T-TTCT- l1LeuSerHlet - Lys - Val - Sir - - - Phi - - - Ser - 357 - - GliThrTyrLiLuuG1isLysSirArgAil IiArgGIiAiiLyuGiuGliAr,ThrPha 274 943 GACAAGGACATGTTCCAGGAGACCATGGAGGCCATGAGGATTATGGGCATCCCAGAAGAG A-T-AT A-A-CAC-A 52 MICA 93 T-T-CC-T-A - -- - - PhuSerHis - 337 At.Ies MICA - Anal IAI&TbhrLiuLnNHis6nB SrSirAspLysPhiVnlSmrGliLiuTrpLyoAip AACATCGCCACACTGCTCCACCAGTCCTCTGACAAGTTTGTCTCGGAGCTGTGGAAGGAT GB-A -T -AC-TT-G A-A-GA -G-B - VaI - - - - - - - - - Arg - - Ala - - - - VnlAnpArglili GlyLneuAspGiiVniAinGiyHMitSerGiThrAlnLeuProGly GTGGACCGCATCATCGGCCTGGACCAGGTGGCCGGCATGTCGGAGACCGCACTGCCCOGG T-G-G-T T-A-CA-T-T A-T-A-T T-TGG-TCC - - - - VaI - - Tr -Thr - - - PbhiGlSir - - MICA - 1723 GGCAASGTGGATTACAAAGCTGACGAGTGGCTGATGAAGAACATGGATCCCCTGAATGAC 1732 -G C-T-G-A-T T C 1783 237 PhiGlyLyVPhelliArglIeAuiPheAspValAsoGlyTyrl Va1GlVAIuAsulie 254 MIA - IAipTyrLyuAiaAup6i lTrpLimlMhlLyuAuiMitAspProLeuAsnAsp 594 61l1LGVy luLuiGi1AiuPheGlVAuiAloLysThrViaLyuAunAspAsnSerSurArg 234 643 AACCCCATCCTGGAGGCCTTCGGCAACGCCAAGACCGTGAAGAATGACAACTCCTCCCGC ECU 652 -T-A-T-C-AT-A-T-A-T-G-T A-T A-T-T - - - ElGB 1972 - - H isLysSirLyiLyiAp AsiPrl - 6111 GIGlylGiGTrpAuiPbelleAupPhiGlyLeumApLiiGliPriCyullAspLea 514 - MA 477 LysPbuhiGi1LysPriLys61iLiuLyuAspLysAlaAupPhiCyul lIeI I sTyrAla 574 1993 AAGTTCCAGAABCCCAAGCAGCTGAAGGACAAAGCTGATTTCTGCATTATCCACTATGCC T T A-T-A A-TCGA-AT-A-A GCTGGCAAGACGGAGAACACCAAGAAGGTCATCCAGTATCTGGCGTACGTGGCGTCCTCG A-T-TC-T-C C-T-T-T-T-A G- A-A-T-A -H - PruLVsAIiTbrAspLysSerPhiValGluLyuVailetGIiGimGnGl yTbrHiiPri 554 1903 CCCAAAGCCACCGACAA6AGCTTCGTGGAGAAGGTGATGCAGGAGCAGGGCACCCACCCC TA-T-A-C-T-T-A-AC-B-T-A--- A-TT UC4 1612 -T - - - LuiVil - - - - Ser - Ser 557 - Thr - TACAGGAGTATGATGCAAGACCGAGAAGATCAATCCATCTTGTGCACTGGTGAATCTGGA AT-C-C-T G6 G-A-T -T-T-G-C-G-A-TC-T - Cys LIe.-.------ . - MIC 1GiLysPriAi*G6yProProilyliiLuAlualeulemAipG1uGliCysTrpPhi 534 1543 ATTGAGAAGCCAGCAGGCCCCCCGGGCATTCTGGCCCTGCTGGACGAGGAGTGCTGGTTC 1552 -A- GA-T-GAA-T-T-TG-A TT-T-A-A - - Ar, - - Asm- - - Val - - - - - - - - - - 537 - IMIA - - 1483 GAGGGCATCGAGTGGAACTTCATCGACTTTGGCCTCGACCTGCAGCCCTGCATCGACCTC A A T-C-G-G-TEUC 1492 -A - TyrLynGlyLysLyiArr*6isGuMitPriPriHil hIITyrAliaiIThrAspThrAii 157 TACAAGGGCAABAABAGGCACGAGATBCCCCCTCACATCTATGCCATCACAGACACCGCC -GA-6 - Arp - ECU - MIA" LysLouGIuGiuL6ouPhmAuHi sThrMtPbe I LeoGIGu6InGuGiuTyrGInArg 494 1423 AAGCTGCAGCAGCTCTTCAACCACACCATGTTCATCCTGGAGCAGGAGGAGTACCAGCGC 6 T A MI4 1432 A-A-A- - A T tGlyPhBG6 l iPbuAspLuuAuuSurPhul.GhGiLuCyul IuAomTyrThrAumGlu 474 1383 GGCTTCGAGATCTTTGATCTGAACTCGTTTGAGCAGCTGTGCATCAATTACACCAATGAG A-A-T C C MUC 1372 -A-T-A-T-G - ElLA T - Ar. - - - - ICGA - LouHiiAuuLeuLyiGsiAr TyrTyrSerGlyL6el i1TyrThrTyrSurGluLniPhi 117 ElA - 1303 AAGGCTCTGGACAAGACCAAGAGGCAGGGCGCCTCCTTCATCGGGATCCTGGACATTGCC A T-T -A - - LyuAlhuLuAupLyuThrLyuArgG6luGyAlSurPhmullGlylhuLuuAspluAlo 454 CTGGTGGAGAATGGGAAGAAGGTGAAGGTGAACAAGGATGACATCCAGAAGATGAACCCG ECU L - - Arg - - - .97 AsiGlyViLiiGuGGlyl leArg II CyiArgGliGiyPhuPreAiiArgVnIVolPhe 714 2983 AACGGTGTTCTCGAGGGCATCCGTATCTGCCGCCAGGGCTTCCCCAACAGGGTGGTCTTC EU 2092 -T--C-G-A-G A T T-C-AA-A-T.__ 717 __-IIie E1LA Gin 2143 CAB MUC 2152 - FIGURE 2. Nucleotide and amino acid sequence comparison between two human nonmuscle myosin heavy chain (NMMHC) isoforns. Identical amino acids and nucleotides are indicated by dashes. Note that the extra three amino acids in NMMHC-B after amino acid 203 are responsible for the difference of three in the numbering of subsequent amino acids. The last amino acid for NMMHC-A (glutamine 715) is the first amino acid of the sequence reported by Saez et al.17 start codon (ATG) is indicated by a 1. The sequence of 19 nucleotides 5' to the ATG codon has been confirmed by sequence analysis of a genomic clone (Simons, Weir, Adelstein, unpublished data), and the cDNA sequence is shown in Figure 2. The ATG codon is preceded by an in-frame stop codon located six nucleotides upstream. The sequence around the 5' ATG start codon bears close similarity to the Kozak consensus sequences.42 To complete the nucleotide and amino acid sequence between that portion of the human NMMHC encoded by 4302 and the portion encoded by the cDNA clones that have been reported previously for a human NMMHC'7 (see Figure 1), we screened a Stratagene lambda ZAP T lymphocyte library with a cDNA probe from the 3' end of 4302 as well as a cDNA probe from the 5' end of clone HL-1. HL-1 is a 1.9 kb cDNA clone isolated from the same library 534 Circulation Research Vol 69, No 2 August 1991 4- (n m m W) tt cK I kb 9.5_ Human 7.5302 4.4- W V 9.5Human 707 7.5 -* 4.4- v- I 'i m -~~~~~~~~~~~~~~ ,, -;- - m 2~ ~ -8 kb 9.5Human 7.5302 4.4- S 0 0 E M c cn I m c e > - t~ w a, 0 , <-28S 9.57.5- -.0 Human 707 4.4- -28S '9 9~~~~~~~~~~~ -28S Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 Eth. Br. Eth. EBr. -18S <-18S as (4302 and encoding the same portion of the NMMHC as that encoded by the 5' portion of pNMHCM2.16 Two clones, (Z801 and ¢Z802, were isolated and partially analyzed to complete the missing sequence (see Figure 2). In the course of rescreening the human T lymphocyte lambda gtlO library with an 839 bp chicken intestinal epithelial cell NMMHC probe (encoding amino acids 310-590, see Reference 16), five cDNA clones ((707, 756, 758, 759, and 760, Figure 1) were isolated. Sequence analysis showed that these clones encoded a NMMHC isoform distinct from that represented by the clone 0302 and suggested that, similar to the findings of Katsuragawa et a115 for chicken NMMHCs, there existed at least two mRNAs encoding two different human NMMHCs. Figure 2 presents a comparison between the amino acid and nucleotide sequences of the two isoforms, which 0 * 28S <-28S FIGURE 3. RNA blot analysis of human cell lines. RNA blots were probed with clones 4302 (nonmuscle myosin heavy chain-A) and 0707 (nonmuscle myosin heavy chain-B). Forty micrograms of total RNA was electrophoresed, blotted, and hybridized with the indicated probes. Cell lines are listed on the top of each lane. Two different identically loaded gels are shown. The specific activity of the two different probes was approximately the same. The autoradiogram of the blot probed with clone 4707 (B probe) was exposed approximately five times longer than that probed with (0302 (A probe). Bottom panel shows a gel stained with ethidium bromide (Eth. Br.). The cell lines used, from left to right, were BUD-8 (fibroblast), Hs294T (melanoma), SK-MEL-2 (melanoma), SCC-15 (squamous cell carcinoma), A-431 (epidermoid carcinoma), HT-29 (adenocarcinoma), FHs74Int (intestinal epithelial cell), Jurkat (T-cell leukemia), HuT78 (T-cell lymphoma), SK-N-SH (neuroblastoma), U-138MG (glioblastoma), and Hs-683 (glioma). ; FIGURE 4. RNA blot analysis of rat tissues using human A and B probes. Approximately 30 pg total RNA was electrophoresed in each lane. The same blot was analyzed with each probe, indicated to the left of the blot. Bands seen below that at 7.5 kb most likely are due to cross-hybridization of the probes with smooth muscle (Intestine), cardiac (Heart), and skeletal muscle (Skeletal M.) mRNA encoding the respective muscle myosin heavy chains. Bottom panel shows the gel stained with ethidium bromide (Eth. Br.). have designated NMMHC-A and NMMHC-B. The identity of the amino acids between the two isoforms is 89% (amino acids 58-718) and 74% at the nucleotide level. The differences are spaced throughout the length of the sequence. Figure 3 is an RNA blot analysis of various human cell lines, including Jurkat cells, hybridized with a cDNA probe for NMMHC-A (4302) and NMMHC-B (0707). The stringency of the final wash (0.1 x SSC, 65°C) makes cross-hybridization negligible. The blot probed with clone (4707 was exposed approximately five times longer than the blot probed with clone 4302. In general, cultured cells contain more mRNA encoding the A isoform than the B isoform, though the amount of NMMHC mRNA varies from cell line to cell line. Both messages appear to be approximately 7.5 kb. Of note is the relatively weak signal seen in lane SK-N-SH (neuroblastoma) for the mRNA encoding NMMHC-A and the relatively weak signal seen in lane HT-29 (adenocarcinoma) for the mRNA encoding NMMHC-B (see "Discussion"). Figure 4 compares NMMHC mRNA levels detected in a number of nonmuscle as well as muscle tissues. Because human tissues were not readily available, we used total RNA prepared from rat tissues. The same blot was hybridized with the cDNA probe for NMMHC-A (4302) as well as a cDNA probe for NMMHC-B (4707), and the exposure time with (4707 we Simons et al Nonmuscle Myosin Heavy Chains TABLE 1. Segregation of Nonmuscle Myosin Heavy Chain-A Gene With Human Chromosome 22 Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 Gene/chromosome Human % chromosome -1Discordancy +/+ +/-/+ 1 16 11 16 52 28 2 13 14 14 54 29 3 18 9 18 50 28 4 22 5 38 30 45 5 18 9 8 60 18 6 23 4 26 42 32 7 12 15 28 40 45 8 17 10 20 32 48 9 19 8 13 55 22 12 10 8 24 15 60 11 16 11 12 24 56 12 18 9 22 46 33 13 10 17 26 42 45 14 13 14 30 38 46 17 15 10 29 39 41 16 12 15 25 43 42 17 18 9 40 28 52 18 18 9 33 35 44 19 16 11 13 55 25 20 20 7 21 47 29 21 21 6 42 26 51 0* 22 27 0 0 68 X 9 31 18 37 42 The nonmuscle myosin heavy chain-A gene (NMMHC-A) was detected as a 19 kb band in EcoRI digests of human-rodent somatic cell hybrid DNAs after Southern hybridization with a 350 bp intronic probe (data not shown). No cross-hybridizing rodent sequences were found under the hybridization conditions used. Detection of the human band is correlated with the presence or absence of each human chromosome in the group of somatic cell hybrids. Discordancy represents presence of the gene in the absence of the chromosome (+t-) or absence of the gene despite the presence of the chromosome (-/+); the sum of these numbers divided by total hybrids examined (x 100) represents percent discordancy. The human-hamster hybrids contained 28 primary clones and 14 subclones (19 positive of 42 total); the human-mouse hybrids represented 13 primary clones and 40 subclones (eight positive of 53 total). *Two independent human-hamster hybrids contained spontaneous breaks of chromosome 22 between IGLC (22q1l.1-q11.2) and PDGFB (22q12.3-q13.1). In one hybrid, the proximal long arm and IGLC were retained, but the myosin gene was lost, whereas the distal long arm was retained with NMMHC-A in the other hybrid that had lost IGLC. These results permit regional localization of the myosin gene to chromosome 22q11.1-qter. was only approximately 1.5 times longer than that for 4302. The results show that each probe hybridizes differently. Despite the fact that some cross-hybridization between isoforms A and B might be expected under these conditions of stringency (0.5 x SSC, 60°C), it still is apparent that the two mRNAs are differentially expressed in a tissue-dependent manner. Moreover, similar results to those shown in Figure 4 also were obtained at a stringency of 0.2x SSC, 600C. This is similar to the findings using avian tissues.15,43 Specifically, rat intestine and thymus appear to con- 535 tain more mRNA encoding NMMHC-A than mRNA encoding NMMHC-B, whereas rat brain and testis appear to be relatively enriched for the mRNA encoding NMMHC-B compared with NMMHC-A. Lung and kidney appear to contain relatively large amounts of both mRNAs. Of note are the mRNAs detected at 6.5-6.9 kb in intestine (smooth muscle), heart, and skeletal muscle cells; these bands presumably are due to cross-hybridization of the probes with the differentiated forms of the respective mRNAs for muscle myosins, which are more divergent from both NMMHC-A and NMMHC-B than are the A and B isoforms from each other. The detection of the muscle-specific mRNAs is dependent on both the reduced stringency of hybridization and the presence of these mRNAs in levels several orders of magnitude greater than the concentrations of nonmuscle myosin mRNAs in these tissues. It also should be noted that tissuespecific expression of NMMHC-A and NMMHC-B cannot be explained by cross-hybridization between these two probes but that the extent of tissue-specificity almost certainly is underestimated because of cross-hybridization. Mapping Nonmuscle Myosin Heavy Chain Genes Using Human-Rodent Somatic Cell Hybrids Both NMMHC genes were chromosomally mapped by Southern analysis of DNAs isolated from a panel of human-rodent somatic cell hybrids using 32P-labeled cDNA and genomic fragments as probes. A 350 bp genomic intronic probe of NMMHC-A identified a 19 kb band in EcoRI digests of human DNA. No crosshybridization with rodent sequences was found. Analysis of an entire series of human-rodent hybrid cell DNAs with the 350 bp genomic probe as well as a second genomic probe (data not shown) permitted unambiguous assignment of the NMMHC-A gene to human chromosome 22 (Table 1). The gene segregated discordantly (.18%) with all other human chromosomes. Examination of two hybrids containing spontaneous breaks involving chromosome 22 permitted regional localization of NMMHC-A to the region 22q11.1-qter. A 1.59 kb NMMHC-B cDNA probe (4707) detected six (2.9, 5.7, 8, 10, 16, and 22 kb) bands in EcoRI-digested human DNA as well as several crosshybridizing sequences in rodent DNAs (data not shown). In contrast to the NMMHC-A probe, the NMMHC-B cDNA probe identified hybridizing bands in an entirely different series of hybrid cell lines in the same panel, indicating localization of the gene on a different chromosome. The 2.9, 5.7, and 22 kb bands segregated concordantly (i.e., all present or all absent) in the hybrids, and they were localized to human chromosome 17 (Table 2), and there was greater than or equal to 21% discordancy with all other chromosomes. Moreover, the same panel of hybrids was analyzed with a p53 tumor antigen (TP53) probe, and there was precise correlation between hybrids retaining the TP53 gene and the NMMHC-B gene (data not shown). It was possible to 536 Circulation Research Vol 69, No 2 August 1991 TABLE 2. Segregation of Nonmuscle Myosin Heavy Chain-B Gene With Human Chromosome 17 Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 Gene/chromosome Human / chromosome Discordancy +1 -1+ +1+ I 39 7 17 27 27 43 3 2 23 21 27 33 13 3 22 22 39 20 26 4 32 12 42 5 42 21 4 23 30 6 29 17 30 14 34 7 27 17 8 28 38 33 13 8 23 21 38 39 9 25 19 7 29 41 10 14 30 5 39 11 37 18 26 9 39 12 38 28 16 8 27 43 13 3 16 28 21 14 14 37 30 9 26 15 28 16 15 31 34 32 16 20 24 14 42 1 17 43 1 45 2* 30 18 30 14 16 33 19 22 22 6 40 31 20 25 19 15 31 38 21 35 9 23 23 36 22 17 27 9 37 40 26 18 19 27 41 X The nonmuscle myosin heavy chain-B gene (NMMHC-B) was detected as 2.9, 5.7, 8, 10, 16, and 22 kb bands in EcoRI digests of human-rodent somatic cell hybrid DNAs after hybridization with the 1.59 kb NMMHC-B cDNA clone 4707. The 2.9, 5.7, and 22 kb bands cosegregated in all hybrids, and these bands could be resolved from cross-hybridizing rodent sequences (data not shown). The human-hamster hybrids consisted of 28 primary clones and 13 subclones (16 positive of 41 total); the human-mouse hybrids contained 19 primary clones and 30 subclones (28 positive of 49 total). *The two discordancies represent failure of both TP53 and NMMHC-B to segregate with other chromosome 17 markers in two independent human-hamster hybrids. In addition, TP53 and NMMHC-B segregated discordantly with all other chromosome 17 markers in eight independent hybrid lines all isolated from one series of human-mouse hybrids involving a single human parental (VA2) cell line (data not shown). These results strongly suggest that the human parental HPRT- SV40 transformed WI 18 line VA244 used in preparing this single group of somatic cell hybrids35 contains one copy of chromosome 17 with a break involving 17pl13, because eight independent hybrids from this series retained chromosome 17 in the absence of both TP53 and NMMHC-B, whereas four other hybrids retained all chromosome 17 markers including TP53 and NMMHC-B. localize the gene regionally by examining another series of hybrids isolated after fusing human parental cells containing a reciprocal chromosome 17;22 (p13;qll) translocation with mouse fibroblasts.36 Four independent hybrids retained the 17p13-qter translocation chromosome, and human TP53 and NMMHC-B were absent from all four hybrids. Thus, NMMHC-B can be assigned to band 17pl3, which is also the locus for TP53.36 t I _w W j. 4~ _44A i~~~~ .e p *. - * #3** . 4 .A * § *s s ~~* ' f Ar 21 4} :9% * * 22 t v FIGURE 5. In situ hybridization with nonmuscle myosin heavy chain A cDNA probe. Top panel: Representative metaphase spread containing a grain on chromosome 22 (lower arrow) and one on another chromosome to indicate background (see bottom panel). Middle panel: Same metaphase spread after Giemsa replication banding. Bottom panel: Distribution of grains on chromosome 22 and background grains on chromosomes 21 and Y in 61 metaphases. Regional Localization by In Situ Hybridization The presence of NMMHC-A on chromosome 22 was confirmed and the gene was regionally localized to 22q11.2 by in situ hybridization of metaphase Simons et al Nonmuscle Myosin Heavy Chains TABLE 3. Comparison of Chicken and Human Myosin Heavy Chain Amino Acids (% Identity) HNMB CNMB HNMA 88 CNMA 88 92 CNMB ... 98 79 HNMA 89 ... ... CNMA, chicken nonmuscle myosin heavy chain (MHC)-A.16 Entire amino acid sequence used; CNMB, chicken nonmuscle MHCGB15; HNMA, human nonmuscle MHC-A (see Figure 2); HNMB, human nonmuscle MHC-B (see Figure 2). Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 spreads with a 3H-labeled NMMHC-A cDNA probe (4302, Figure 5). Metaphase spreads containing a grain on a small (G group) chromosome were chosen for analysis. Thirty-eight grains (26% of total grains) of the total 146 grains were found on chromosome 22, and 12 of the grains (32%) were specifically localized to band qll.2. The other grains were randomly distributed on chromosomes 21,Y and other chromosomes (Figure 5C). No clustering of grains was detected on chromosome 17. Discussion The results presented here show that two distinct 200 kDa MHC isoforms exist in human cells and that these isoforms are encoded by genes located on different chromosomes. The two isoforms show considerable sequence similarity. Sequence comparison of human and chicken NMMHC isoforms leads us to postulate that human NMMHC-A is a homologue of chicken FMHA, and human NMMHC-B is a homologue of chicken FMHC (Table 3, Reference 15). Note that both NMMHC isoforms are highly conserved between the species. Indeed, the sequence identity between the human and chicken NMMHC-A (92%) and human and chicken NMMHC-B (98%) is greater than the identity between either the human or chicken NMMHC-A and NMMHC-B (89% or 88%, see Table 3). Thus, the conservation of sequence between the species is greater than conservation of sequence between the isoforms, which suggests a distinct function for each isoform. These distinct functions might be expected to be reflected by the differential expression of each isoform in different tissues or cell types. To investigate this, we performed the series of Northern blot analyses shown in Figures 3 and 4. In general, the mRNA encoding NMMHC-A is more abundant than that encoding NMMHC-B in human cell lines. However, in agreement with recent studies using avian tissues, which reported an increase in the mRNA encoding NMMHC-B in brain,'1543 we show that the mRNA for B is more abundant than that for A in human neuroblastoma cells (SK-N-SH). However, the finding that glioma (Hs-683) and glioblastoma (U138MG) cells contain relatively more mRNA encoding NMMHC-A rather than -B suggests significant differences in the distribution of these isoforms within brain tissues. 537 Our results with rat tissues, though not quantitative, support the findings from avian tissues which were that the mRNA encoding NMMHC-A was relatively more abundant than the mRNA for NMMHC-B in spleen and intestinal epithelial cells, whereas the mRNA encoding NMMHC-B was relatively more abundant in brain and testis. Kidney contains approximately equal amounts of both mRNAs. Combining the sequence presented here with that of Saez et al17 gives a protein sequence for NMMHC-A of 1,961 amino acids, which is two amino acids more than the 1,959 amino acids reported by Shohet et al16 for the chicken NMMHC-A. This discrepancy results from an insertion of an extra glutamine residue within the run of five glutamine residues at amino acids 1,3461,35016 and an insertion of alanine and serine for a single valine residue at amino acid 1,390.16 Also, all NMMHCs sequenced to date have a deletion in the area of amino acid 202 compared with the smooth muscle MHC,45 the significance of which is presently unknown. NMMHC-A has a deletion of 10 amino acids, and NMMHC-B has seven amino acids deleted. Cloned cDNA and genomic probes of NMMHC-A and NMMHC-B have been used to localize these genes to human chromosomes 22 and 17, respectively, by Southern analysis of DNAs from a panel of human-rodent somatic cell hybrids. Furthermore, the gene for NMMHC-A has been localized to a region 22q11.2 by in situ hybridization of metaphase chromosome spreads. The NMMHC-B gene was localized further to a region 17p13 by analysis of hybrids constructed from parental cell lines containing a translocation involving this telomeric band. The extent of cross-hybridization between NMMHC-A and NMMHC-B, under conditions of high stringency, was negligible and therefore allowed localization of the NMMHC-B gene using the corresponding cDNA probe. The localization of the NMMHC-A gene to chromosome 22 is in agreement with the findings of Saez et al.17 No subchromosomal localization was reported in that study. The gene for NMMHC-B was localized to a region of chromosome 17 known to carry three sarcomeric (skeletal) MHC genes (MYH 1, 2, 4)26-30 and a fetal skeletal MHC gene (MYH 3).27 A gene for the atrial (cardiac) myosin light chain (MYL 4)46 also is present on that chromosome. A fifth skeletal MHC gene (MYH 5) has been localized provisionally on chromosome 7 by in situ hybridization with a mouse conserved 3' cDNA coding sequence.47 Two cardiac MHC genes are located on chromosome 14.48 49The significance of the localization of one of the NMMHC genes to the same area of chromosome 17 that contains skeletal MHC genes is unclear, because these represent members of two multigene families that diverged before speciation, and more closely related MHC genes of each type also are found on entirely different chromosomes. Nevertheless, the probability of random association of two genes in a region representing no more than 0.5% of the human genome is not high. 538 Circulation Research Vol 69, No 2 August 1991 It is safe to assume that the nonmuscle isoforms are the oldest myosins, whereas more specialized proteins such as skeletal and cardiac myosins developed later in the course of evolution. Furthermore, a very significant sequence conservation between cor- responding chicken and human NMMHCs implies that two nonmuscle myosins arose relatively early. The localization of the NMMHC-B gene to the same area of chromosome 17 as skeletal MHC genes suggests that this gene may have been the ancestor of skeletal muscle myosin genes. The availability of cDNA clones encoding human NMMHC isoforms will allow detailed study of their function and regulation and should help shed light on the role played by nonmuscle myosins in various biological processes, such as smooth muscle proliferation. Acknowledgments Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 The authors gratefully acknowledge the expert technical assistance of Yvette A. Preston and Judy Yu and the expert manuscript preparation of Catherine Magruder. References 1. Kiehart DP: Molecular genetic dissection of myosin heavy chain function. Cell 1990;60:347-350 2. 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Saez LJ, Gianola KM, McNally EM, Feghali R, Eddy R, Shows TB, Leinwand LA: Human cardiac myosin heavy chain genes and their linkage in the genome. Nucleic Acids Res 1987;15:5443-5459 KEY WORDS * gene localization * cDNA cloning * expression gene Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017 Human nonmuscle myosin heavy chains are encoded by two genes located on different chromosomes. M Simons, M Wang, O W McBride, S Kawamoto, K Yamakawa, D Gdula, R S Adelstein and L Weir Circ Res. 1991;69:530-539 doi: 10.1161/01.RES.69.2.530 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1991 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/69/2/530 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/