Acta Pharmacologica Sinica (2018) 39: 1197–1207 © 2018 CPS and SIMM All rights reserved 1671-4083/18 www.nature.com/aps
Article
Inverse changes in plasma tetranectin and titin levels in patients with type 2 diabetes mellitus: a potential predictor of acute myocardial infarction?
Mohd Aizat Abdul RAHIM1, Zubaidah Haji Abdul RAHIM2, Wan Azman WAN AHMAD3, Marina Mohd BAKRI2, Muhammad ']DÀU,60$,/32QQ+DML+$6+,04, 5, *
1Centre of Preclinical Science Studies, Faculty of Dentistry, Universiti Teknologi MARA, 47000 Sungai Buloh, Selangor, Malaysia; 2Department of Oral and Craniofacial Sciences, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia; 3Department of Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia; 4Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia; 5University of Malaya Centre for Proteomics Research, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
Abstract An early intervention using biomarkers to predict acute myocardial infarction (AMI) will effectively reduce global heart attack incidence, particularly among high-risk patients with type 2 diabetes mellitus (T2DM). This study attempted to identify potential biomarkers by detecting changes in the levels of plasma proteins in T2DM patients following onset of AMI in comparison with those without AMI. Volunteer T2DM patients without AMI (control; n=10) and T2DM patients with AMI (n=10) were recruited. Plasma samples from these patients were evaluated via two-dimensional gel electrophoresis (2DE) to screen for proteins with level changes between the two JURXSV7KHDEXQGDQFHRIVSRWVRQJHOLPDJHVZDVDQDO\]HGXVLQJ3URJHQHVLV6DPH6SRWVDQGVXEMHFWHGWRIDOVHGLVFRYHU\UDWH )'5 DQDO\VLV3URWHLQVSRWVZLWKVWDWLVWLFDOO\VLJQLÀFDQWFKDQJHVRIDWOHDVWIROGZHUHVHOHFWHGIRUPDVVVSHFWURPHWU\ 06 DQDO\VLV 'XHWRVWURQJFDUGLDFFRQQHFWLRQVWHWUDQHFWLQDQGWLWLQZHUHHYDOXDWHGE\HQ]\PHOLQNHGLPPXQRVRUEHQWDVVD\ (/,6$ 7KHDGMXVWHG PYDOXHVDQGIROGFKDQJHVEHWZHHQWKHWZRJURXSVUHVXOWHGLQLGHQWLÀFDWLRQRISURWHLQVSRWVZLWKVLJQLÀFDQWO\DOWHUHGDEXQGDQFH 8SRQ06DQDO\VLVSODVPDSURWHLQVZHUHLGHQWLÀHG WHWUDQHFWLQWLWLQFOXVWHULQKDSWRJORELQP\RVLQ]LQFIQJHUSURWHLQ'1$ repair protein RAD50, serum albumin, apolipoprotein A-IV, caspase-6, aminoacyl tRNA synthase complex-interacting multifunctional protein 1, serotransferrin, retinol-binding protein 4, transthyretin, alpha-1-antitrypsin, apolipoprotein A-I and serum amyloid A. &RPSDUDEOHSDWWHUQVRIFKDQJHVLQWHWUDQHFWLQDQGWLWLQEHWZHHQWKHFRQWURODQG$0,JURXSVZHUHFRQÀUPHGXVLQJ(/,6$,QVXPPDU\ tetranectin and titin in plasma appeared to be closely associated with the onset of AMI among T2DM patients and can be used as potential biomarkers for prediction of a cardiac event, though this requires validation in a prospective cohort study.
Keywords: tetranectin; titin; acute myocardial infarction; coronary disease; diabetes mellitus; proteomics; 2D gel electrophoresis; enzyme-linked immunosorbent assay
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Introduction 8&0BB$UWLFOHMVS ,QRQHVWXG\DGXOWVZLWK7'0 Acute myocardial infarction (AMI) was responsible for the were shown to have a two to four times higher risk of CVD deaths of at least seven million patients in 2012[1]. According FRPSDUHGZLWKDGXOWVZLWKRXWWKHGLVRUGHUDQGZHUHDKLJK to a statistical analysis, type 2 diabetes mellitus (T2DM) is an risk group for AMI[2],WLVHVWLPDWHGWKDWE\WKH\HDU LQÁDPPDWRU\LOOQHVVZLWKDFOHDUFXWUHODWLRQVKLSZLWKFDUGLR million people globally, predominantly in developing coun vascular disease (CVD) (American Heart Association, 2017; tries, will have metabolic syndrome>@, which is linked with the http://www.heart.org/HEARTORG/Conditions/Diabetes/ cardiac condition. :K\'LDEHWHV0DWWHUV&DUGLRYDVFXODU'LVHDVH'LDEHWHVB Through proteomics, many researchers have been studying the use of biomarkers due to their ability to monitor suscep *To whom correspondence should be addressed. tibility to as well as progression and resolution of diseases, E-mail [email protected] KHDOWKFRQGLWLRQVWUHDWPHQWRXWFRPHVDQGSRVWPRUWHP 5HFHLYHG$FFHSWHG analysis[4]. Proteomics has been widely used as a means of iden www.nature.com/aps 1198 Rahim MAA et al
tifying candidate biomarkers and immunochemistry markers for WKH8QLYHUVLW\RI0DOD\D0HGLFDO&HQWUH 800& 5()12 various illnesses, particularly infectious and neoplastic diseases>@. DQG)DFXOW\RI'HQWLVWU\8QLYHUVLW\RI0DOD\D ') ,QOLJKWRIWKLVDSSURDFKWKH+XPDQ3URWHRPH3URMHFWDLPVWR 2% / ([SHULPHQWDOVDPSOHVXVHGLQWKHVWXG\ PDSDOOKXPDQSURWHLQVE\FKDUDFWHUL]LQJDOOJHQHVRIWKH were collected from patients admitted to the UMMC Cardiol known genome>@. Together with the availability of the entire ogy and Coronary Care Wards, and control samples were col KXPDQFRGLQJVHTXHQFHLQWKH+XPDQ*HQRPH3URMHFWWKHSUR lected from patients attending the outpatients’ clinic at UMMC WHRPHSURMHFWPDUNVWKHEHJLQQLQJRIDQHZHUDIRUJHQHUDWLQJ Medical Clinic. Samples from admitted patients were col a deeper understanding of both human genes and proteins. lected within three days following onset of AMI, depending on ,QDSURWHRPLFVZRUNIORZWZRGLPHQVLRQDOJHOHOHFWUR the patients’ medical status. Each volunteer was interviewed SKRUHVLV '( GHOLYHUVDPDSRILQWDFWSURWHLQVWKDWUHÁHFWV and requested to complete a questionnaire concerning per FKDQJHVLQWKHSURWHLQDEXQGDQFHOHYHOLVRIRUPVRUSRVW sonal and health information, including smoking and alcohol translational modifications (PTMs) based on the isoelectric GULQNLQJKDELWVKHDOWKDQGGHQWDOSUREOHPVUDGLDWLRQH[SR point, relative molecular mass, solubility, and relative abun VXUHPDMRUVXUJLFDOSURFHGXUHVDQGDQWLELRWLFSUHVFULSWLRQV dance of proteins[7]. These properties notwithstanding, 2DE Informed written consent was obtained from every patient allows for the isolation of proteins in microgram amounts for prior to plasma collection. Blood samples were obtained by further structural analysis by mass spectrometry (MS) or pro peripheral venous puncture and collected into plastic whole WHLQVHTXHQFLQJ06KDVDVVXPHGDNH\UROHIRULGHQWLÀFDWLRQ EORRGWXEHVZLWKVSUD\FRDWHGGLSRWDVVLXP('7$:LWKLQ DQGSUHFLVHTXDQWLÀFDWLRQRISURWHLQVIURPFRPSOH[VDPSOHV KRIFROOHFWLRQWKHEORRGVDPSOHVZHUHFHQWULIXJHGDWðg WKHUHE\HQDEOLQJODUJHVFDOHDQGKLJKWKURXJKSXWFKDUDFWHU IRUPLQDW&7KHUHVXOWLQJVXSHUQDWDQWRUSODVPD ization of the human proteome> @. Because of its simplicity, was immediately divided into 100 μL aliquots and stored at DFFXUDF\KLJKUHVROXWLRQDQGVHQVLWLYLW\PDWUL[DVVLVWHG &)ROORZLQJPRQWKVRIVXEMHFWHQUROOPHQWDGPLW ODVHUGHVRUSWLRQLRQL]DWLRQWLPHRIÁLJKWPDVVVSHFWURPHWU\ WHGSDWLHQWVDQGRXWSDWLHQWVZHUHUHFUXLWHGIRUWKLVVWXG\ 0$/',72)06 LVXVHIXOIRUWKHLGHQWLÀFDWLRQRISURWHLQVE\ 7KHH[FOXVLRQFULWHULDIRUUHFUXLWHGVXEMHFWVZHUHSUHJQDQF\ SHSWLGHPDVVÀQJHUSULQWLQJ smoking during the previous three months, antibiotic use dur &OLQLFDOO\KXPDQVHUXPSURWHLQVKDYHEHHQH[WHQVLYHO\ LQJWKHSUHYLRXVWKUHHPRQWKVDQGSULRUPDMRUVXUJHULHV,Q used as biomarkers for human disease detection, including in DFFRUGDQFHZLWKWKHVHH[FOXVLRQFULWHULDSODVPDVDPSOHVIURP various cancers, neurodegenerative diseases, and hepatic cir VXEMHFWVZHUHILQDOO\FKRVHQIRUSURWHRPLFDQDO\VLV7KH rhosis. Because human plasma contains an enormously com GHPRJUDSKLFVDQGFOLQLFDOFKDUDFWHULVWLFVRIVXEMHFWVLQYROYHG SOH[PL[WXUHRIRYHUSURWHLQVLQFOXGLQJFORWWLQJIDFWRUV in the study are shown in Table 1. plasma has recently been further investigated and has shown potential for diagnosing Alzheimer’s and atherosclerotic dis Separation of proteins by two-dimensional gel electrophoresis eases[10, 11],QDFOLQLFDOVHWWLQJQDWULXUHWLFSHSWLGHV&UHDFWLYH ,QLWLDOO\Ǎ/ DSSUR[LPDWHO\JRISURWHLQ RISODVPD protein, creatine kinase, and cardiac troponin are already used ZDVLQFXEDWHGLQVDPSOHEXIIHU PRO/XUHDPPRO/ as biomarkers in acute cardiac care[12]. Therefore, the human DTT, 2% v/v,3*EXIIHUS+²v/v7ULWRQ; IRU plasma proteome has the potential to predict the onset of PLQDQGWKHQLQUHK\GUDWLRQEXIIHU PRO/XUHDv/v AMI. Because acute AMI contributes to a large percentage of ,3*EXIIHUS+²v/v7ULWRQ;w/v Orange mortality worldwide, early intervention using biomarkers that * IRUDQRWKHUPLQDWURRPWHPSHUDWXUHZLWKDÀQDOYRO FDQSUHGLFW$0,SDUWLFXODUO\LQKLJKULVN7'0SDWLHQWVPD\ XPHRI/7KHÀQDOUDWLRRIVDPSOHEXIIHUWRUHK\GUDWLRQ help reduce the global heart attack incidence. EXIIHUZDV 2QFHWKHPL[WXUHZDVFRPSOHWHO\GLVVROYHG In this study, plasma samples were collected from T2DM one Immobiline DryStrip Gel (pH 4–7 linear, 11 cm, GE volunteers with AMI three days after admission, and T2DM sub Healthcare, Uppsala, Sweden) was passively rehydrated with MHFWVZLWKRXW$0,ZHUHWKHFRQWUROJURXS%RWKVDPSOHVZHUH WKHPL[WXUHDQGNHSWLQDVHDOHGHQYLURQPHQWIRUDPLQLPXP VXEMHFWHGWR'(DQGSURWHLQVZHUHVHSDUDWHGDFFRUGLQJWRWKHLU RIKDWURRPWHPSHUDWXUH,VRHOHFWULFIRFXVLQJ ,() ZDV isoelectric points and relative molecular masses. Proteins with SHUIRUPHGRQWKHRWKHUVWULSVZLWKDQ(WWDQ,3*SKRU,VR changes in abundance were subsequently identified using HOHFWULF)RFXVLQJ8QLW *(+HDOWKFDUH8SSVDOD6ZHGHQ DQG 0$/',72)72)DQDO\VLVDQGTXDQWLWDWLYHO\YDOLGDWHGZLWK WKH\ZHUHPDLQWDLQHGDW&:LWKDPD[LPXPFXUUHQWRI HQ]\PHOLQNHGLPPXQRVRUEHQWDVVD\ (/,6$ 7KHSULPDU\DLP Ǎ$SHUVWULSWKHUHK\GUDWHG,3*JHOVWULSVZHUHHOHFWURSKR of this study was to use proteomic analysis to identify plasma UHVHGDW9IRUK VWHSDQGKROG 9IRUK JUDGLHQW proteins with altered abundance among T2DM patients follow 9IRUK JUDGLHQW DQG9IRUPLQ VWHSDQG ing the onset of AMI compared with patients without AMI. hold). 3ULRUWRVRGLXPGRGHF\OVXOIDWHSRO\DFU\ODPLGHJHOHOHFWUR Materials and methods SKRUHVLV 6'63$*( RQH²JUDGLHQW6'63$*(JHO Recruitment of subjects ZLWKDWRWDOYROXPHRIDSSUR[LPDWHO\P/ZDVSUHSDUHGIRU This study was conducted among diabetic Malaysian sub HDFKVWULS8SRQFRPSOHWLRQRI,()WKHIRFXVHGVWULSVZHUH MHFWVDFFRUGLQJWRWKH'HFODUDWLRQRI+HOVLQNLDQGDSSURYHG HTXLOLEUDWHGIRUPLQLQ6'6HTXLOLEUDWLRQEXIIHU mmol/L by the Ethical Committee (Institutional Review Board) of 7ULVS+PRO/XUHD v/v glycerol, 2% w/v SDS)
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7DEOHDemographics and clinical characteristics of subjects. ORFDWLRQ$IWHUFDOFXODWLQJWKHEDFNJURXQGFRUUHFWHGDEXQ dance, the normalized volume was calculated for each pro Parameter T2DM without AMI T2DM with AMI tein spot that was detected by the software. The volumes for (n=10) (n=10) both groups were compared to determine the respective fold changes and PYDOXHV XQSDLUHGtWHVW RIWKHSURWHLQVSRWVDV 6H[ 0 )1 calculated by the image analysis software. Each protein spot (WKQLFLW\ 0 & ,2 RIDOWHUHGDEXQGDQFHZDVH[SUHVVHGDVDYHUDJHGQRUPDOL]HG volume±standard error of the mean (SEM). The PYDOXHVODWHU Age (year)3 XQGHUZHQWIDOVHGLVFRYHU\UDWH )'5 DQDO\VLVEDVHGRQWKH [14] HbA1c (%)3 PHWKRGGHVFULEHGE\%HQMDPLQL+RFKEHUJ . This statistical analysis was performed with the help of a statistician, and 10 )²0DOH )HPDOHRIWKH0DOD\VLDQVXEMHFWV WKHIRUPXODZDVSUHSDUHGLQD0LFURVRIW([FHOÀOHDWDQDOSKD 2 0 & ,²0DOD\ &KLQHVH ,QGLDQRIWKH0DOD\VLDQVXEMHFWV YDOXHRI(DFKRIWKHKLJKO\UHVROYHGSODVPDSURWHLQ 39DOXHVDUHH[SUHVVHGLQPHDQ6' VSRWVWKDWZDVVWDWLVWLFDOO\VLJQLÀFDQW DGMXVWHGP ZLWK IROGFKDQJHVRIDWOHDVWZDVVXEVHTXHQWO\VHOHFWHGIRU06 containing 1% w/v DDT on an orbital shaker. The solution DQDO\VLV$VDUHVXOWSODVPDVSRWVZHUHVHOHFWHGIRUVXEVH ZDVWKHQUHSODFHGE\HTXLOLEUDWLRQEXIIHUFRQWDLQLQJw/v TXHQWH[SHULPHQWV7KHSURWHLQVSRWVUDQJLQJIURPWRPP LRGRDFHWDPLGHIRUDQRWKHUPLQZLWKJHQWOHVKDNLQJ6XE LQGLDPHWHUZLWKWKHGHVLUHGVLJQLÀFDQFHOHYHOZHUHPDQXDOO\ VHTXHQWO\6'63$*(ZDVSHUIRUPHGXVLQJDQ6(5XE\ H[FLVHGIURPWKHVLOYHUVWDLQHG'(JHOVDQGSRROHGIURPDOO Electrophoresis System (GE Healthcare, Uppsala, Sweden) VLPLODUJHOVWRPD[LPL]HWKHFRQILGHQW06UHVXOWV7KHJHO ÀOOHGZLWKDQRGHEXIIHUS+ PRO/7ULV DW&RQ SOXJVZHUHWKHQNHSWK\GUDWHGLQGRXEOHGLVWLOOHGZDWHUIRU JUDGLHQWJHOVFRQWDLQLQJ6'6ZLWKWKHVWULSVVHDOHGLQ IXUWKHUSURWHLQLGHQWLÀFDWLRQ SODFHZLWKw/vDJDURVHLQ6'6HOHFWURSKRUHVLVEXIIHU PPRO/7ULV PPRO/JO\FLQHw/v6'6 6'63$*( ,GHQWLÀFDWLRQRISURWHLQVE\0$/',72)72) ZDVSHUIRUPHGDW9IRUKIROORZHGE\9IRUK )RULQJHOWU\SWLFGLJHVWLRQWKHJHOSOXJVZHUHGHVWDLQHG 7KHPD[LPXPFXUUHQWDQGSRZHUZHUHP$DQG:SHU ZLWKPPRO/SRWDVVLXPIHUULF\DQLGHPPRO/VRGLXP gel, respectively. WKLRVXOIDWHIRUPLQZLWKVKDNLQJXQWLOWKH\WXUQHGWUDQVSDU $Q06FRPSDWLEOHVLOYHUVWDLQLQJSURWRFROZDVSHUIRUPHG ent. The gel plugs were then reduced in 10 mmol/L DTT/100 according to the method previously described, with slight PPRO/DPPRQLXPELFDUERQDWHIRUPLQDW&IROORZHG PRGLÀFDWLRQV>@. The procedure was carried out on an orbital E\DON\ODWLRQLQPPRO/LRGRDFHWDPLGHPPRO/ shaker, as all of the steps in the staining technique required ammonium bicarbonate for 20 min in the dark at room tem gentle shaking. After gel electrophoresis, the gel slabs were SHUDWXUH7KHSOXJVZHUHWKHQZDVKHGWKUHHWLPHVZLWK VRDNHGLQÀ[LQJVROXWLRQ v/v ethanol, 10% v/v acetic acid) acetonitrile/100 mmol/L ammonium bicarbonate for 20 min IRUDWOHDVWPLQ7KHJHOVZHUHWKHQLQFXEDWHGLQVHQVLWL] with shaking, followed by dehydration with 100% acetonitrile LQJVROXWLRQ v/v HWKDQROPRO/VRGLXPDFHWDWHWUL IRUPLQZLWKVKDNLQJDQGFHQWULIXJDWLRQLQDVSHHGYDFXXP K\GUDWHPPRO/VRGLXPWKLRVXOIDWH IRUDWOHDVWDQRWKHU at low speed at ambient temperature until the gel plugs were PLQ)ROORZLQJWKUHHFRQVHFXWLYHPLQZDVKHVZLWKGRXEOH FRPSOHWHO\GU\7KHSOXJVZHUHGLJHVWHGLQQJǍ/WU\SVLQ distilled water, the solution was substituted with silver solu LQPPRO/DPPRQLXPELFDUERQDWHDW&2QWKHIRO tion (14.72 mmol/L silver nitrate) and left to shake for 20 ORZLQJGD\WKHSHSWLGHPL[WXUHVZHUHH[WUDFWHGWZLFHZLWK min. Subsequently, the gel slabs were quickly washed twice DQGDFHWRQLWULOHVHTXHQWLDOO\DQGFRQFHQWUDWHGLQD DQGLPPHUVHGLQGHYHORSLQJVROXWLRQ PPRO/VRGLXP speed vacuum. The dried peptides were subsequently recon carbonate, 0.2% v/v formaldehyde). The protein spots were VWLWXWHGZLWKIRUPLFDFLGDQGGHVDOWHGXVLQJ=LS7LS& GHYHORSHGXQWLODVXIÀFLHQWUHVROXWLRQZDVUHDFKHG7KHVROX Micropipette Tips. WLRQZDVWKHQUHSODFHGZLWKVWRSVROXWLRQ PPRO/('7$ 3HSWLGHPL[WXUHVZHUHDQDO\]HGXVLQJD0$/',72) disodium dihydrate) to prevent overstaining. After washing 72)$QDO\]HU $%6&,(;86$ 7KHWU\SWLFGLJHVWHGSHS ZLWKGRXEOHGLVWLOOHGZDWHUWKUHHWLPHVWKHGHYHORSHGJHO WLGHVZHUHFU\VWDOOL]HGZLWKDQǂF\DQRK\GUR[\FLQQDPLF VODEVZHUHWKHQVWRUHGDW& DFLGPDWUL[VROXWLRQ PJP/ǂF\DQRK\GUR[\FLQQDPLF acid, 70% acetonitrile, 0.1% v/v7)$DTXHRXVVROXWLRQ DQG Analysis of gel images VSRWWHGRQWRDZHOO0$/',WDUJHWSODWH7KH06UHVXOWV $IWHUVWDLQLQJWKH'(JHOVZHUHVFDQQHGDWGSLXVLQJ ZHUHDXWRPDWLFDOO\DFTXLUHGZLWKDWU\SVLQDXWRGLJHVWH[FOX ImageScanner III (GE Healthcare, Uppsala, Sweden). Image sion list, and the 20 most intense precursor ions were selected analysis was performed using Progenesis SameSpots (ver IRU0606DQDO\VLVZLWKDPLQLPXP61RIDWOHDVW06 VLRQ1RQOLQHDU'\QDPLFV8. 86$ 7HQJHOLPDJHV and MS/MS acquisition and interpretation were carried out with the best resolution were chosen to represent each group XVLQJ72)72)6HULHV([SORUHU6RIWZDUH YHUVLRQ$% and used for further analysis. In brief, the gel images were SCIEX, USA). The spectra were then processed and analyzed DOLJQHGWRDGMXVWDOORIWKHVSRWVDFFXUDWHO\LQWKHH[DFWVDPH XVLQJ3URWHLQ3LORW6RIWZDUH YHUVLRQ$%6&,(;86$
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DQGWKHLQKRXVH0$6&273URJUDP 0DWUL[6FLHQFH8. WR WZRJURXSVZHUHHQODUJHGDVVKRZQLQ)LJXUH%7KHVSRWV VHDUFKIRUSHSWLGHPDVVÀQJHUSULQWVDQG0606GDWD%RWK were obtained from the software and tabulated according to combined MS and MS/MS searches were conducted against the respective protein labels. By using Progenesis SameSpots WKH8QL3URWGDWDEDVH /DVWXSGDWHG -XO\ ZLWKWKHIRO DQG)'5DQDO\VLVDWRWDORIKLJKO\UHVROYHGSURWHLQVSRWV lowing search parameters: Homo sapiens; trypsin enzyme; one ZHUHGLIIHUHQWLDOO\UHJXODWHG DGMXVWHGP 'HQVLWRP missed cleavage; peptide mass tolerance at 100 ppm; fragment etry analysis of the highly resolved spots was conducted PDVVWROHUDQFHDW'D IL[HGDQGYDULDEOHPRGLILFDWLRQV in terms of the average normalized volumes, PYDOXHVDQG including cysteine carbamidomethylation and methionine IROGFKDQJHV3URWHLQVSRWVWKDWZHUHVWDWLVWLFDOO\VLJQLÀFDQW R[LGDWLRQUHVSHFWLYHO\ DQGLQFOXVLRQRIPRQRLVRWRSLFPDVVHV DGMXVWHGP ZLWKDPLQLPXPIROGFKDQJHVRIXSRQ According to the MASCOT search results, protein scores )'5DQDO\VLVDUHUHFRUGHGLQ7DEOH2IWKHVHSURWHLQVSRWV JUHDWHUWKDQZHUHFRQVLGHUHGVLJQLÀFDQW P 10 were significantly upregulated and 24 were significantly downregulated in T2DM patients with AMI compared with 9DOLGDWLRQRISURWHLQFRQFHQWUDWLRQE\(/,6$ 7'0SDWLHQWVZLWKRXW$0,7KHVHSURWHLQVSRWVRI $QDO\VLVRIWKHLGHQWLÀHGSURWHLQVDQGUHVSHFWLYHFDUGLDFDVVR altered abundance comprised a total of 17 plasma protein FLDWLRQVIURPDWH[WPLQLQJGDWDEDVH>@ led to the further selec clusters. tion of plasma tetranectin and titin for analysis by competitive ELISA. This immunoassay employed the quantitative sand wich enzyme immunoassay technique. Based on the manu facturer’s instructions, protein determination was performed 7DEOHAverage normalised volumes of plasma protein spots of T2DM patients. RQWZRDQWLERGLHVVSHFLÀFWRWHWUDQHFWLQDQGWLWLQ,QSULQFLSOH WKHPLFURWLWHUSODWHSURYLGHGE\WKHPDQXIDFWXUHUZDVSUH Protein Average Normalised Volume×10 $GMXVWHG )ROG FRDWHGEHIRUHKDQGZLWKDVSHFLÀFDQWLERG\)RUHDFKSURWHLQ 6SRW 6(0ï) P-value Change* ten samples and the standard were pipetted into the appropri /DEHO ZLWKRXW$0, ZLWK$0, P ate microtiter plate wells. In theory, any desired analyte pres ent in the samples was bound by the immobilized antibody. ² 8SRQUHPRYLQJDQ\XQERXQGVXEVWDQFHVDELRWLQFRQMXJDWHG ² DQWLERG\VSHFLÀFWRLWVDQDO\WHZDVDGGHG$IWHUZDVKLQJDYL ² GLQFRQMXJDWHGWRKRUVHUDGLVKSHUR[LGDVHZDVDGGHGWRHDFK ² PLFURSODWHZHOODQGLQFXEDWHG)ROORZLQJDZDVKWRUHPRYH ² DQ\XQERXQGDYLGLQHQ]\PHUHDJHQW··WHWUDPHWK\O ² benzidine substrate was added to the wells. Only those wells ² FRQWDLQLQJWKHGHVLUHGDQDO\WHELRWLQFRQMXJDWHGDQWLERG\ DQGHQ]\PHFRQMXJDWHGDYLGLQH[KLELWHGDFKDQJHLQFRORU The intensity of the developed color was proportional to the amount of analyte bound in the initial step. The color devel RSPHQWRIWKHHQ]\PHVXEVWUDWHUHDFWLRQZDVWHUPLQDWHGE\ the addition of sulfuric acid solution, and the color intensity ZDVPHDVXUHGVSHFWURSKRWRPHWULFDOO\DWDZDYHOHQJWKRI ² nm. The concentration of analyte in the samples was then ² determined by comparing the optical density of the samples to ² the standard curve generated for each antigen. ² ² ² Results ² Analysis of plasma proteins ² Profiling of human plasma samples by 2DE separated hun ² dreds of protein spots based on different isoelectric points and relative molecular masses. A typical representative 2DE ² profile of plasma within the range of pH 4–7 with the rela ² tive molecular mass declining from 200 kDa to 10 kDa was REWDLQHGDVGHPRQVWUDWHGLQ)LJXUH$:KHQ'(ZDV ² performed on plasma samples from diabetic patients with ² AMI, the profiles obtained were similar to those of the con ² WUROV7KH'(SODVPDSURWHLQSURÀOHVUHVROYHGLQWKHSUHVHQW ² study were also generally comparable with those published >@ ² LQWKH6ZLVV'3$*(GDWDEDVH )RUHQKDQFHGYLVXDOL]D ² tion and comparison, individual protein spots between the
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,GHQWLÀFDWLRQRISODVPDSURWHLQVRIDOWHUHGDEXQGDQFH The results for plasma proteins were enumerated in terms of 7KHLGHQWLWLHVRIWKHSURWHLQVSRWFOXVWHUVZHUHFRQÀUPHGE\ WKHLU8QL3URW.%DFFHVVLRQQXPEHUWKHRUHWLFDOS,WKHRUHWL MS and a database search. Every spot with altered abundance cal mass, MASCOT score, number of peptides matched, and LQWKH'(JHOVODEVZDVVXEMHFWHGWR06DQDO\VLVIRULGHQWL VHTXHQFHFRYHUDJH 7DEOH 06ZDVDEOHWRLGHQWLI\SUR fication. According to the MASCOT search results, protein WHLQVZLWKQRWDEOHFKDQJHVEHWZHHQWKHH[SHULPHQWDODQGFRQ VFRUHVJUHDWHUWKDQZHUHFRQVLGHUHGVLJQLILFDQW P trol groups, including clusterin (4 protein species), haptoglo
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*3URWHLQVSRWZDVQRWVLJQLÀFDQWO\LGHQWLÀHGLQ06DQDO\VLV 0$6&276FRUH **Aminoacyl tRNA synthase complex-interacting multifunctional protein 1.
ELQ SURWHLQVSHFLHV '1$UHSDLUSURWHLQ5$' SURWHLQ P\RVLQYDOXHVPD\EHGXHWRSRVWWUDQVODWLRQDOPRGLÀFD species), serum albumin (4 protein species), apolipoprotein tions and/or fragmentation of the proteins. $,9 SURWHLQVSHFLHV VHURWUDQVIHUULQ SURWHLQVSHFLHV WHWUDQHFWLQ SURWHLQVSHFLHV WLWLQ SURWHLQVSHFLHV UHWLQRO 9DOLGDWLRQRIWKHSODVPDSURWHLQVRILQWHUHVWXVLQJ(/,6$ binding protein 4 (2 protein species), transthyretin (1 protein When the plasma proteins of altered abundance were sub VSHFLHV DOSKDDQWLWU\SVLQ SURWHLQVSHFLHV DQGDSROLSR MHFWHGWRWH[WPLQLQJXVLQJWKHPHWKRGRI3OHWVFKHU)UDQNLOG SURWHLQ$, SURWHLQVSHFLHV (LJKWSURWHLQVSRWVKRZHYHU et al>@, relevant clinical associations were generated (Table 4). ZHUHQRWVWDWLVWLFDOO\VLJQLILFDQW FOXVWHULQP\RVLQ]LQF According to the literature, most of the identified plasma ILQJHUSURWHLQDPLQRDF\OW51$V\QWKDVHFRPSOH[LQWHU proteins of interest had already been associated with cardiac DFWLQJPXOWLIXQFWLRQDOSURWHLQFDVSDVHDQGVHUXPDP\ORLG FRURQDU\DUWHU\GLVHDVHV &$'V RUDWKHURVFOHURVLV)URPWKLV A, and this is most likely due to the limited gel plugs and low list, tetranectin and titin were further selected for quantitative SURWHLQFRQFHQWUDWLRQ:LWKWKHH[FHSWLRQVRIKDSWRJORELQ validation by sandwich ELISA. While tetranectin was chosen DQGP\RVLQWKHWKHRUHWLFDOS,DQGPDVVYDOXHVRIPRVWRI PDLQO\EHFDXVHRILWVDVVRFLDWLRQZLWKÀEULQRO\VLVWKHVHOHFWLRQ the protein spots of interest were apparently comparable with of titin was based on its relationship with cardiac and muscle the values detected from their resolved positions in the 2DE diseases and because it is a resident cardiac protein. The regu SURÀOHVVKRZQLQ)LJXUH9DULDWLRQVLQWKHKDSWRJORELQDQG ODWLRQSDWWHUQVDQGVLJQLÀFDQFHOHYHOVRIWHWUDQHFWLQDQGWLWLQ
Acta Pharmacologica Sinica www.chinaphar.com Rahim MAA et al 1203
7DEOH/LVWRIVLJQLÀFDQWSODVPDSURWHLQVDVVRFLDWHGZLWKUHVSHFWLYHFOLQLFDOGLVHDVHVPLQHGIURPOLWHUDWXUH[16].
Protein Diseases*
Clusterin $O]KHLPHU·VGLVHDVHOLPELVFKHPLDFDQFHUGHPHQWLDWUDQVLHQWQHRQDWDOQHXWURSHQLDNLGQH\GLVHDVH)XFK·VHQGRWKHOLDO dystrophy, intermittent claudication, atherosclerosis, diabetes mellitus, peripheral vascular disease, and coronary artery disease. Haptoglobin Anaemia, autoimmune haemolytic anaemia, mastitis, vascular disease, glucose-6-phosphate dehydrogenase deficiency, cancer, kidney disease, malaria, endometritis, liver disease, thrombocytopenia, sleeping sickness, diabetes mellitus, arthritis, tuberculosis, vaginal discharge, pneumonia, thrombotic thrombocytopenic purpura, thalassaemia, bilirubin metabolic disorder, LQÁDPPDWRU\ERZHOGLVHDVHVZLQHLQÁXHQ]DREHVLW\KHSDWLWLV%O\PSKDWLFV\VWHPGLVHDVHKHDUWGLVHDVHGLDUUKHDVFXUY\ hepatitis C, rheumatic fever, leukopenia, and endometriosis. Myosin-13 None. =LQFÀQJHU None. protein 445 DNA repair Nijmegen breakage syndrome, ataxia telangiectasia, and microcephaly. protein RAD50 6HUXPDOEXPLQ Kidney disease, diabetes mellitus, liver disease, hypertension, cancer, lung disease, protein-energy malnutrition, peritonitis, anemia, protein-losing enteropathy, coronary artery disease, brain disease, portal hypertension, cerebrovascular disease, hepatitis B, hypersensitivity reaction type II disease, Alzheimer’s disease, hyperthyroidism, pneumocystosis, cholera, sarcoidosis, schistosomiasis, periodontal disease, uveitis, fatty liver disease, myopathy, leprosy, meningoencephalitis, dermatitis, gallbladder disease, eosinophilia, diphtheria, exanthem, atherosclerosis, diarrhohea, bilirubin metabolic disorder, hyperglycaemia, diabetic retinopathy, heart disease, inflammatory bowel disease, cholestasis, obesity, esophageal varix, hypersensitivity reaction type I disease, arthritis, decubitus ulcer, metabolic acidosis, peripheral vascular disease, neuropathy, varicose veins, tuberculosis, ovarian hyperstimulation syndrome, metabolic syndrome X, asthma, hyperthyroxinaemia, lipid metabolism disorder, hyperparathyroidism, leukopenia, pancreatitis, thrombocytopenia, hepatitis C, autonomic neuropathy, meningitis, rhinitis, amyloidosis, lymphatic system disease, arthus reaction, multiple sclerosis, pulmonary embolism, neonatal jaundice, hypercalcemia, bronchitis, cholangitis, intestinal obstruction, acquired immunodeficiency syndrome, urticaria, dementia, K\SRJO\FDHPLDDFXWHF\VWLWLVF\VWLFÀEURVLVK\SRWK\URLGLVPVKRUWERZHOV\QGURPHWHWDQXVPDODULDSRUWDOYHLQWKURPERVLV hyperhomocysteinaemia, mastitis, hyperphosphatemia, vasculitis, osteoporosis, hyperuricemia, hypertensive retinopathy, thyrotoxicosis, bronchiectasis, tropical sprue, pyuria, otitis media, hyperinsulinism, substance abuse, polycystic kidney GLVHDVHDWULDOÀEULOODWLRQSRO\QHXURSDWK\DOFRKROGHSHQGHQFHFDSLOODU\OHDNV\QGURPHSHSWLFXOFHUGLVHDVHFRQMXQFWLYLWLV visceral leishmaniasis, bacteriuria, cataract, pneumothorax, macroglobulinaemia, disseminated intravascular coagulation, PHDVOHVWR[LFHQFHSKDORSDWK\SULPDU\LPPXQRGHÀFLHQF\GLVHDVHVWHDWRUUKRHDKHSDWLWLV$LQIHUWLOLW\WR[LFPHJDFRORQDRUWLF aneurysm, pericarditis, hypokalemia, influenza, peripheral artery disease, compartment syndrome, calcinosis, stomatitis, pericardial effusion, cryoglobulinaemia, intermittent claudication, anorexia nervosa, alopecia, dengue disease, porphyria, hypoparathyroidism, candidiasis, pain agnosia, gastroenteritis, hepatic vein thrombosis, macular retinal edema, synovitis, rabies, constipation, syphilis, fascioliasis, pertussis, mediastinitis, limb ischaemia, and goiter. Apolipoprotein A-IV Carotenaemia, obesity, coronary artery disease, atherosclerosis, lattice corneal dystrophy, diabetes mellitus, Guillain-Barre syndrome, and lipid metabolism disorder. Aa tRNA Neuritis and cancer. synthase P1** Caspase-6 Huntington’s disease, cancer, Alzheimer’s disease, and acute hemorrhagic conjunctivitis. 6HURWUDQVIHUULQ Haemochromatosis, anaemia, congenital disorder of glycosylation, substance abuse, kidney disease, liver disease, cancer, alcohol dependence, protein-energy malnutrition, thalassaemia, diabetes mellitus, haemosiderosis, atransferrinemia, cholera, lung disease, restless legs syndrome, porphyria cutanea tarda, aceruloplasminemia, hepatitis C, protein-losing enteropathy,
alzheimer’s disease, arthritis, hypertension, meningitis, diphteria, malaria, vitamin B12 deficiency, diarrhea, brain disease, REHVLW\H\HGLVHDVHFRURQDU\DUWHU\GLVHDVHLQÁDPPDWRU\ERZHOGLVHDVHDUWKURSDWK\3DUNLQVRQ·VGLVHDVHKHDUWGLVHDVH metabolic syndrome X, hepatitis B, fatty liver disease, cerebrovascular disease, atherosclerosis, male infertility, dementia, K\SHUVHQVLWLYLW\UHDFWLRQW\SH,,GLVHDVHSHULWRQLWLVVOHHSLQJVLFNQHVVZLWKGUDZDOGLVRUGHUDQRUH[LDQHUYRVDDQGF\VWLFÀEURVLV Tetranectin Inhalation anthrax, byssinosis, cancer, and vascular disease. Titin Myopathy, myasthaenia gravis, thymoma, heart disease, meconium aspiration syndrome, amyloidosis, respiratory failure, rippling PXVFOHGLVHDVH/DPEHUW(DWRQP\DVWKDHQLFV\QGURPHDVSK\[LDQHRQDWRUXPDQGSQHXPRWKRUD[ Retinol-binding Obesity, diabetes mellitus, metabolic syndrome X, fatty liver disease, Matthew-Wood syndrome, polycystic ovary syndrome, lipid protein 4 PHWDEROLVPGLVRUGHUK\SHUJO\FDHPLDDULERÁDYLQRVLVK\SHUYLWDPLQRVLV$K\SHUWHQVLRQK\SHULQVXOLQVLVPDWKHURVFOHURVLVDQG kidney disease.
(To be continued)
Acta Pharmacologica Sinica www.nature.com/aps 1204 Rahim MAA et al
Protein Diseases*
Transthyretin Amyloidosis, polyneuropathy, protein-energy malnutrition, carpal tunnel syndrome, hyperthyroxinaemia, neuropathy, autonomic neuropathy, atrial fibrillation, kidney disease, cancer, Alzheimer’s disease, liver disease, heart disease, diarrhea, diabetes mellitus, decubitus ulcer, choroid plexus papilloma, cerebrovascular disease, hypothyroidism, myopathy, dementia, eye disease, hypervitaminosis A, hyperthyroidism, anorexia nervosa, carotenemia, thyrotoxicosis, anemia, brain disease, pneumonia, hypertension, Crohn’s disease, cholestasis, short bowel syndrome, obesity, pancreatitis, phenylketonuria, blepharochalasis, and chronic obstructive pulmonary disease. 6HUXPDP\ORLG$ Amyloidosis, familial Mediterranean fever, anemia, splenic artery aneurysm, rheumatoid arthritis, atherosclerosis, cancer, kidney disease, and coronary artery disease. Alpha-1-antitrypsin Alpha-1-antitrypsin deficiency, chronic obstructive pulmonary disease, protein-losing enteropathy, liver disease, cancer, EURQFKLWLVF\VWLFÀEURVLVÀEURP\DOJLDEURQFKLHFWDVLVKLVWLRF\WRVLVSDQFUHDWLWLVYDVFXOLWLVVNLQGLVHDVHFKROHVWDVLV&URKQ·V disease, rheumatoid arthritis, asthma, adult respiratory distress syndrome, hypersensitivity reaction type II disease, diarrhoea, kidney disease, interstitial lung disease, colitis, pneumothorax, hepatitis B, constrictive pericarditis, pneumonia, steatorrhoea, disseminated intravascular coagulation, congenital disorder of glycosylation, otitis media, diabetes mellitus, haemochromatosis, coronary artery GLVHDVHSHULRGRQWDOGLVHDVHXYHLWLVJUDQXODUFHOOWXPRXUFXWDQHRXVÀEURXVKLVWLRF\WRPDDQGSRUWDOK\SHUWHQVLRQ Apolipoprotein A-I Atherosclerosis, coronary artery disease, lipid metabolism disorder, diabetes mellitus, metabolic syndrome X, obesity, amyloidosis, hypertension, cerebrovascular disease, kidney disease, liver disease, cancer, heart disease, arthritis, polycystic ovary syndrome, hyperinsulinism, hyper homocysteinemia, arcus senilis, peripheral vascular disease, Alzheimer’s disease, hyperlycaemia, dysbaric osteonecrosis, and hypothyroidism.
7KHFRPSOHWHOLVWRIDVVRFLDWHGGLVHDVHVUHODWHGWRSODVPDSURWHLQV7KH=VFRUHDQGFRQÀGHQFHOHYHOFRUUHVSRQGLQJWRHDFKSURWHLQZHUHGHWHUPLQHG XVLQJWKHWH[WPLQLQJGDWDEDVH,QVWDWLVWLFV=VFRUHGHQRWHVKRZPDQ\VWDQGDUGGHYLDWLRQVDQHOHPHQWRUGDWXPLVIURPWKHPHDQZKHUHDVFRQÀGHQFH level refers to the likelihood of all possible samples that can be expected to include the true population parameter. **Aminoacyl tRNA synthase complex-interacting multifunctional protein 1.
REWDLQHGE\(/,6$DQG'(ZHUHFRPSDUHG )LJXUH (/,6$ proteins or the specificity of the antibodies used for ELISA. was performed on ten samples from each group, and these The significance level may be improved by increasing the SURWHLQVZHUHFRQÀUPHGWREHSUHVHQWDWGLIIHUHQWFRQFHQWUD number of samples and/or using antibodies that are highly WLRQV)URPWKHVHFRQFHQWUDWLRQVWKHDEXQGDQFHSDWWHUQV VSHFLÀFWRIUDJPHQWVDQGLVRIRUPVIURPERWKWKHFRQWURODQG EHWZHHQERWKWKHFRQWURO ZLWKRXW$0, DQGH[SHULPHQWDO H[SHULPHQWDOJURXSV groups (with AMI) were calculated and found to be similar to 7HWUDQHFWLQLVD&W\SHOHFWLQDQGDQHQGRJHQRXVOLJDQG the initial 2DE data. Ultimately, the concentrations of the two that specifically binds to the plasminogen kringle 4 domain, SURWHLQVZHUHFRQÀUPHGWKURXJKWKLVPHWKRGDQGWKH(/,6$ thereby enhancing plasminogen activation[17]. It is primarily and 2DE approaches both demonstrated downregulation of involved in tissue remodeling and development>@. Based on WHWUDQHFWLQDQGXSUHJXODWLRQRIWLWLQLQGLDEHWLFVXEMHFWVZLWK WKHJHQHUDWHG'(SURÀOHWKHWHWUDQHFWLQGHWHFWHGZDVFORVHWR AMI. LWVWKHRUHWLFDOPDVV N'D ,WLVSRVVLEOHWKDWWKHGRZQUHJX ODWLRQRIWHWUDQHFWLQLQSODVPDLVGXHWRLWVÀEULQRO\WLFSURS Discussion erty in thrombus breakdown. Hence, tetranectin dissolves the Most of the identified proteins of altered abundance were thrombus blocking the coronary artery and promotes better linked with broad disease associations resulting from the blood circulation to cardiac muscles. This implied role of tet LQIODPPDWRU\UHVSRQVHEORRGFRDJXODWLRQDFXWHSKDVH UDQHFWLQLQWKHÀEULQRO\WLFSURFHVVVXJJHVWVWKDWLWKDVDIXQF response, cholesterol homeostasis, cardiac muscle tissue devel WLRQDOUROHLQ$0,SDWLHQWV)XUWKHUHYDOXDWLRQDQGFRUUHODWLYH opment, and other pathophysiological responses (Universal studies of cardiac tetranectin in the formation and dissolution Protein Resource, 2017; http://www.uniprot.org/). Some, RIÀEULQFORWVDUHKLJKO\UHFRPPHQGHG including tetranectin and titin, were reportedly associated Tetranectin, as a new regulator of fibrinolysis, has been with cardiovascular disease and diabetes. Due to their asso VWXGLHGE\.DPSHUet alXVLQJSODVPDIURP$0,SDWLHQWV ciations with cardiac metabolism pathways and presence in DQGKHDOWK\VXEMHFWVWRLQYHVWLJDWHLWVSRVVLEOHLQYROYHPHQW cardiac muscles, tetranectin and titin were further selected in the pathophysiology of AMI> @. This population study pro for concentration validation using ELISA. The results clearly YLGHVFOHDUHYLGHQFHRIDVLJQLÀFDQWGHFUHDVHRIWHWUDQHFWLQOHY showed regulation patterns similar to those found by the 2DE HOVLQ$0,SDWLHQWVFRPSDUHGZLWKKHDOWK\VXEMHFWV/LNHZLVH DQDO\VLVZLWKVOLJKWO\GLIIHUHQWIROGFKDQJHV7KHVLJQLÀFDQFH another MS study performed by Yin et al showed comparable level for tetranectin was, however, much lower than that cal results; the tetranectin levels were inversely correlated with culated from the 2DE analysis. The different P values may be the risk of atherosclerotic cardiovascular disease[20]. More attributable to the presence of isoforms and/or PTMs of these recently, Chen et alDOVRFRQÀUPHGWKDWWKHVHUXPWHWUDQHFWLQ
Acta Pharmacologica Sinica www.chinaphar.com Rahim MAA et al 1205
)LJXUH$QDO\VLVRISODVPDWHWUDQHFWLQDQGWLWLQE\'(DQG(/,6$6LPLODUSDWWHUQVRISODVPDWHWUDQHFWLQDQGWLWLQZHUHREWDLQHGXVLQJ'( $ DQG (/,6$ % +RZHYHUWKHPYDOXHIRUWHWUDQHFWLQZDVQRWDVVLJQLÀFDQWDVWKDWREWDLQHGWKURXJKWKH'(DSSURDFK 7'0SDWLHQWVZLWKRXW$0, T2DM patients with AMI. levels in patients suffering from stable CAD were decreased SURÀOHLVPRVWOLNHO\DWUXQFDWHGIUDJPHQWRIWKHQDWLYH compared with those of healthy controls[21]. These studies are kDa molecule. It is possible that the upregulation of titin in in agreement with the results obtained in this research. plasma is due to its dissociation from cardiac muscle when 7LWLQLVDODUJHSURWHLQWKDWLVORQJHUWKDQǍPLQOHQJWK myocardial infarction is caused by prolonged ischemia. When and the third most abundant muscle protein after myosin and tissue death occurs, the contraction of the striated cardiac mus actin. Also known as connectin, titin functions as a molecular cle tissue is altered. This pattern is in keeping with the clinical spring and is responsible for the passive elasticity of muscle[22]. evaluation performed by Vassiliadis et al>@. In their study, ,QIDFWWLWLQLVDPXVFOHVSHFLILFSURWHLQWKDWLVIRXQGLQFDU WKHLQYHVWLJDWRUVIRXQGWKDWDVWDWLVWLFDOO\VLJQLÀFDQWLQFUHDVH diac and skeletal muscles. It is essential for the contraction LQWKHOHYHORIDWLWLQIUDJPHQW 1979($5/,. ZDVGXHWRWKH of striated muscle tissues>@. Titin plays a crucial role in the GHJUDGDWLRQRIWLWLQE\003LQ$0,VXEMHFWV7LWLQLVWKH temporal and spatial control of the assembly and elasticity of first neoepitope biomarker that was specifically detected in the highly ordered sarcomeres of the striated muscle[24]. Titin serum and related to cardiac damage. In another serological limits the range of motion of the sarcomere in tension, thus VWXG\WKHSUHVHQFHRIWKH1%H[RQDFDUGLDFVSHFLILFWLWLQ contributing to the passive stiffness of muscle. By provid fragment, in blood has been suggested to be a sensitive marker ing connections at the level of individual microfilaments, IRUP\RFDUGLDOLQMXU\>@. Additionally, changes in titin phos titin contributes to the careful balance of forces between the phorylation and titin splicing occur in cardiac disease despite halves of a sarcomere and ultimately maintains sarcomere mutations in the titin gene[27], demanding further study of this integrity. Variations in the sequence of titin in different types SURWHLQWRHOXFLGDWHWKHVSHFLÀFPDFKLQHU\UHVSRQVLEOHIRUWLWLQ of muscles, including cardiac and skeletal muscles, have been PRGLÀFDWLRQV correlated with differences in the mechanical properties of The preliminary findings in this study have potential in these muscles. According to the UniProt database (Universal FOLQLFDODSSOLFDWLRQV7KHDERXWN'DSODVPDWLWLQIUDJPHQW Protein Resource, 2017), titin is robustly linked with diverse may be an additional diagnostic biomarker for AMI. Recently, cardiac processes, including blood coagulation, cardiac muscle troponin has become central to the definition of AMI in the FRQWUDFWLRQFDUGLDFPXVFOHÀEHUGHYHORSPHQWFDUGLDFPXVFOH consensus guidelines from the European Society of Cardiol hypertrophy, cardiac muscle tissue morphogenesis, cardiac ogy and the American College of Cardiology (Medscape, 2017; myofibril assembly, detection of muscle stretch, and many KWWS HPHGLFLQHPHGVFDSHFRPDUWLFOH RYHUYLHZ others. In general, a cardiac troponin (cTn) test is the most common 7KHDERXWN'DWLWLQVSRWWKDWZDVGHWHFWHGLQWKH'( test in a clinical setting, compared with tests of creatine kinase,
Acta Pharmacologica Sinica www.nature.com/aps 1206 Rahim MAA et al
lactate dehydrogenase, aspartate transaminase, myoglobin, SURMHFWLRQVIRU'LDEHWHV5HV&OLQ3UDFW ² LVFKHPLDPRGLÀHGDOEXPLQSUREUDLQQDWULXUHWLFSHSWLGHDQG 4 0RRQ$%KDOH16KLQGH*%6DKDUH37DOUHMD.3URWHLQELRPDUNHUV JO\FRJHQSKRVSKRU\ODVHLVRHQ]\PH%%2YHUWLPHLGHQWLÀFD LQOLIHDQGDIWHUOLIH-$GY0HG/LIH6FL ² tion of more proteins can lead to insight into the usefulness of 5 &HFLOLDQL)(FNHUVDOO'%XUFKPRUH5/HFFKL&3URWHRPLFVLQ YHWHULQDU\PHGLFLQH DSSOLFDWLRQVDQGWUHQGVLQGLVHDVHSDWKRJHQHVLV FRPELQDWRULDOELRPDUNHUVDVVRFLDWHGZLWK$0,)RULQVWDQFH DQGGLDJQRVWLFV9HW3DWKRO ² FRSHSWLQQRWRQO\LGHQWLÀHVSDWLHQWVDWULVNRIPRUWDOLW\EXWLWV 6 /HJUDLQ3$HEHUVROG5$UFKDNRY$%DLURFK$%DOD.%HUHWWD/et al. addition to cTn also improves sensitivity and rules out AMI. 7KHKXPDQSURWHRPHSURMHFW FXUUHQWVWDWHDQGIXWXUHGLUHFWLRQ0RO Thus, copeptin may help identify patients who may be safely &HOO3URWHRPLFV ² >@ discharged early from the emergency department . *RUJ$:HLVV:'XQQ0-&XUUHQWWZRGLPHQVLRQDOHOHFWURSKRUHVLV Apart from finding valuable diagnostic biomarkers, this WHFKQRORJ\IRUSURWHRPLFV3URWHRPLFV ² proteomic study aimed to identify possible biomarkers prior 8 Aebersold R, Mann M. Mass spectrometry-based proteomics. Nature to the onset of AMI. While the biological reasons for the ² FKDQJHVLQSURWHLQOHYHOVDUHQRWTXLWHXQGHUVWRRG$0, 9 (OHN*/DSLV.$SDWKRUDQHZURDGLQODERUDWRU\GLDJQRVWLFV" linked tetranectin and titin have the potential to be used in %LRORJLFDOPDVVVSHFWURPHWU\ IDFWVDQGSHUVSHFWLYHV3DWKRO2QFRO early screening for heart disease. In the future, a longitudinal 5HV ² 10 .ULVWHQVHQ/3/DUVHQ050LFNOH\+6DDE\/'LHGHULFKVHQ$& cohort study should be performed on similar patients at high /DPEUHFKWVHQ-et al3ODVPDSURWHRPHSURÀOLQJRIDWKHURVFOHURWLF risk for AMI to investigate whether tetranectin and titin can be disease manifestations reveals elevated levels of the cytoskeletal used as biomarkers to predict cardiac arrest. The tetranectin SURWHLQYLQFXOLQ-3URWHRPLFV ² level may indicate the status of cardiac thrombosis, whereas 11 7KDPELVHWW\0/RYHVWRQH6%ORRGEDVHGELRPDUNHUVRI$O]KHLPHU·V WKHWLWLQOHYHOPD\UHÁHFWWKHVHYHULW\RIFDUGLDFWLVVXHGHDWKLQ GLVHDVH FKDOOHQJLQJEXWIHDVLEOH%LRPDUN0HG ² SHRSOHZLWK$0,KLJKULVNIDFWRUV(DUO\VFUHHQLQJSDUWLFX 12 .RVVDLI\$*DUFLD$6XFFDU6,EUDKLP$0RXVVDOOHP1.RVVDLI\0 ODUO\DPRQJKLJKULVNSDWLHQWVFDQEHPDGHSRVVLEOHZLWKD et al. Perspectives on the value of biomarkers in acute cardiac care WHVWNLWGHVLJQHGWRGHWHFWWKHVHSURWHLQVLQDVSHFLÀHGUDQJHRI DQGLPSOLFDWLRQVIRUVWUDWHJLFPDQDJHPHQW%LRPDUN,QVLJKWV concentrations. The test kit will be suitable for use in diabetic ² clinics and hospitals if the proteins are found to be associated 13 Acta Pharmacologica Sinica www.chinaphar.com Rahim MAA et al 23 7VNKRYUHERYD/7ULQLFN-5ROHVRIWLWLQLQWKHVWUXFWXUHDQGHODVWLFLW\ /LSLQVNL0-(VFDUFHJD52' $VFHQ]R)0DJDOKDHV0$%DNHU1& RIWKHVDUFRPHUH-%LRPHG%LRWHFKQRO ² Torguson R, et al. A systematic review and collaborative meta-analysis 24 0D\DQV2YDQGHU9HQ3):LOP00XHV$ Acta Pharmacologica Sinica