ISSN 100727626 CN 1123870ΠQ 2007 8 Chinese Journal of Biochemistry and Molecular Biology 23 (8) :660 665 MLP CalcineurinΠNFAT,, 3, (, 100083), LIM (muscle LIM protein,mlp) MLP, (phenylephrine, PE), RNAi MLP, MLP (calcineurin)π T (nuclear factor of activated T2cells, NFAT), PE, ANP BNP, MLP RNAi MLP PE BNP, NFAT, MLP, calcineurinπnfat ; LIM ;calcineurinπnfat Q593 + 3 Involvement of MLP in Cardiac Hypertrophy is Related to CalcineurinΠNFAT Signaling Pathway GUI You2Jing, AN Guo2Shun, NI Ju2Hua 3, J IA Hong2Ti ( Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Beijing 100083, China) Abstract In response to numerous pathologic stimuli, the cardiomyocytes undergo a hypertrophic response characterized by increased myocardial cell size and activation of fetal cardiac genes The abnormal expression of myocardium developing factor muscle LIM protein (MLP) contributes to this process To investigate the mechanism of MLP participates in cardiac hypertrophy, we used phenylephrine ( PE) to induce hypertrophic response in cultured rat neonatal ventricular myocytes and down2regulated expression of MLP with RNA interference The relation between MLP and calcineurinπnuclear factor of activated T2cells (NFAT) signaling pathway was analyzed Increased cell area and expression of ANP, BNP was observed in PE stimulated cardiomyocytes Down2regulation of expression of MLP by RNA interference significantly inhibited PE induced hypertrophic response Further more, activation of calcineurinπnfat signaling pathway was reduced in MLP RNA interfered myocytes after PE stimulation These results indicate that MLP is intimately involved the process of cardiac hypertrophy and possibly regulates this process through calcineurinπnfat signaling pathway Key words cardiac hypertrophy ; muscle LIM protein ; calcineurinπnfat signaling pathway :2007204205, :2007206211 (No 30200131) 3 Tel : 010282801622, E2mail : juhuani @bjmu edu cn Received : April 5,2007 ; Accepted :June 11, 2007 Supported by National Natural Science Foundation of China (No 30200131) 3 Corresponding author Tel : 010282801622, E2mail : juhuani @bjmu edu cn
8 :MLP CalcineurinΠNFAT 661, MLP calcineurin,, MLP,,,, (cardiac hypertrophy) (MyoD ) (myogenin), MLP, [13 (AChR) ] [1,2 ],MLP,, MLP,, MLP, c2fos c2myc PE [3 ] ( Ca 2 + ) RNA (RNA interference, RNAi) MLP (calcineurin)πnfat, MLP calcineurinπnfat,,mlp PE Ca 2 + BNP, ANP, (angiotensin,ang ), (endothelin21, ET21), Ca 2 + Calcineurin Π, Ca 2 + (calmodulin) T (nuclear factor of activated T cells, NFAT) calcineurin NFAT calcineurin, GATA4, Calcineurin NFAT 011 mgπml (Sigma) 10 % FCS( Gibco) 100 IUΠml [4 ] calcineurin 2actinin calsarcin21, Z [5 ] LIM (muscle LIM protein,mlp) BrdU DMEMΠM 199 LIM, 2 LIM MLP,MLP 112, MLP cdna, MLP ;,MLP Z, RNAi, DNA : 5 2GAT CCC [6 ] MLP CAG AGA CCA CCG GAA ATG AA T TCA AGA GAT [7, ] TCA TTT CCG GTG GTC TCT TTT TTA23 5 2AGC MLP TTA AAA AAG AGA CCA CCG GAA ATG AAT CTC [8 10 ] TTG AAT TCA TTT CCG GTG GTC TCT GGG23, [11 ] MLP, MLP T4 Bgl Hind psuper,, MLP ( phenylephrine, PE), 1 calcineurin2nfat, 1 111 [14 ], Sprague2 Dawley, 1 10 5 Πcm 2 100 IUΠml 011 mmolπl BrdU(Sigma) DMEMΠ medium 199 (4 1) 2 d psuper2mlp RNAi p9 NFAT,MLP GL3 9 NFAT Z (luciferase), Joerg Heineke zyxin 2actinin N2RAP telethonin ( T2cap) psuper psuper MLP RNAi [12,8 ] MLP,MLP Transfectamine2000 ( Invitrogen) Z, MLP 3 5 h,,24 h PE p9 NFAT GL3 prl CMV psuper MLP
662 23 RNAi psuper, 212 MLP PE ( ) 113 RNA RT2PCR TRIzol ( Invitrogen) 24 h PE(40 molπl) 24 h RNA, M2MLV ( Sigma) RNA,RT2PCR cdna, PCR 115 % ANP MLP Fig12A,PE BNP MLP calcineurin A 114 215 10 5 Π 24, ( lane2 lane4) 24 h, PE 24 h, PBS, 4 % 10 min, 015 % Triton X2 100 ( Sigma) 10 % NGS, 2 actinin (,Sigma) 4, RNAi MLP 2 d TBS TRITC 37 1 h, (Fig12C) PBS 3,, RNAi MLP PBS 2 211 PE MLP 213 MLP PE calcineurinπ NFAT, PE 24 h,psuper2mlp RNAi ANP BNP,RT2PCR ANP BNP ( Fig12A), [15, 2actinin ANP BNP ] BNP,ANP BNP PE NFAT, calcineurinπnfat [16 (Fig11A), 40 molπl PE ], (Fig11B), PE MLP calcineurinπnfat MLP, PE (Fig11A) MLP cdna psuper2mlp RNAi, MLP ( lane2) ; psuper2mlp RNAi PE MLP, PE ( ) ; psuper2mlp RNAi PE (Fig12B), ANP BNP Fig12A, MLP PE BNP, ANP psuper2mlp RNAi 24 h, 40 molπl PE Fig 1 Cardiac hypertrophy induced by PE (A) Rat neonatal ventricular myocytes in serum2free medium were treated with 0, 10, 40, 100 molπl PE and total RNA was extracted 24 hours later Expression of MLP, ANP, BNP and 2actin were analyzed by RT2PCR (B) Culture cardiac myocytes were untreated (left) or treated (right) with 40 molπl PE for 24 hours Then cells were immunostained against 2actinin to indicate cardiomyocytes
8 :MLP CalcineurinΠNFAT 663 Fig 2 RNAi2mediated inhibition of PE induced hypertrophy (A) Rat neonatal ventricular myocytes were transiently transfected with empty psuper RNAi vector (lanes 1, 2) or with RNAi construct targeting MLP (lanes 3, 4) Then cells were kept in serum2free medium with (lanes 2, 4) or without (lanes 1, 3) presence of PE (40 molπl) for 24 hours Total RNA was extracted and mrna expression levels were determined by RT2PCR (B) Cardiomyocytes were transfected and stimulated as described above Images were obtained by contrast phase microscope (C) Average cell size of each group were analyzed by Leica confocal software Approximately 30250 myocytes were analyzed per group 24 h, RNA,RT2PCR calcineurin calcineurin, NFAT, NFAT psuper2mlp RNAi MLP PE calcineurin NFAT, Fig13, PE PE,calcineurinΠNFAT Fig 3 Effect of MLP RNAi on the calcineurin expression and NFAT transcription activity changes induced by PE in rat neonatal ventricular myocytes (A) The myocytes were transiently transfected with empty psuper RNAi vetor (control) or with psuper2mlp RNAi vector (MLP RNAi), then stimulated by PE (40 molπl) for 24 hours Calcineurin and MLP RNA levels were measured by RT2PCR (B) All cells were cotransfected with a luciferase reporter plasmid driven by nine NFAT consensus binding sites and cotransfected with psuper RNAi vetor (control) or with psuper2mlp RNAi vector (MLP RNAi) NFAT transcription activity was measured by Luc system after stimulation with or without 40 molπl PE for another 24 hours
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