Combination of microrna-21 and microrna-146a Attenuates Cardiac Dysfunction and Apoptosis During Acute Myocardial Infarction in Mice

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1 Cittion: Moleculr Therpy Nucleic Acids (16) 5, e96; doi:1.138/mtn.16.1 Officil journl of the Americn Society of Gene & Cell Therpy All rights reserved /16 Combintion of microrna-1 nd microrna-16 Attenutes Crdic Dysfunction nd Apoptosis During Acute Myocrdil Infrction in Mice Wei Hung 1, Shn-Shn Tin 1, Peng-Zhou Hng 1, Chun Sun 1, Jing Guo 1 nd Zhi-Min Du 1, Recent studies hve reveled the cytoprotective roles of micrornas (mirnas) mir-1 nd mir-16 ginst ischemic crdic injuries. While these studies investigted ech of these mirnas s n independent individul fctor, our previous study hs suggested the possible interction between these two mirnas. The present study ws designed to investigte this possibility by evluting the effects of mir-1 nd mir-16 combintion on crdic ischemic injuries nd the underlying mechnisms. MiR-1 nd mir-16 synergisticlly decresed poptosis under ischemi/hypoxic conditions in crdiomyocytes compred with either mir-1 or mir-16 lone. Mice coinjected with gomir-1 nd gomir-16 hd decresed infrct size, incresed ejection frction (EF), nd frctionl shortening (FS). These effects were greter thn those induced by either of the two gomirs. Furthermore, greter decreses in p38 mitogen-ssocited protein kinse phosphoryltion (p-p38 MAPK) were observed with mir-1: mir-16 combintion s compred to ppliction of either of the mirnas. These dt suggest tht combintion of mir-1 nd mir-16 hs greter protective effect ginst crdic ischemi/hypoxi-induced poptosis s compred to these mirnas pplied individully. This synergistic ction is medited by enhnced potency of inhibition of crdiomyocyte poptosis by the mir-1 PTEN/AKT p-p38 cspse-3 nd mir-16 TRAF6 p-p38 cspse-3 signl pthwys. Moleculr Therpy Nucleic Acids (16) 5, e96; doi:1.138/mtn.16.1; published online 15 Mrch 16 Subject Ctegory: shrnas, siras nd MiRNAs; Therpeutic proof of concept Introduction Acute myocrdil infrction (A) is leding cuse of deth worldwide nd it is chrcterized by inflmmtion, crdiomyocyte poptosis, nd crdic fibrosis nd cn led to left ventriculr dilttion nd hert filure. 1, Experimentl nd clinicl studies hve shown tht crdiomyocyte poptosis following A is cused by oxidtive stress, ischemi, nd hypoxic injury nd reperfusion; 3 mitigting this process is fesible wy of preventing nd treting A. However, the mechnisms underlying ischemi-induced poptosis in crdiomyocytes re poorly understood. MicroRNAs (mirnas) ply criticl role in the pthogenesis of crdiovsculr diseses nd ischemi-induced poptosis. 5,6 MiR-1 ws shown to prevent myocrdil poptosis ginst ischemi/reperfusion (I/R)- nd H O -induced crdiomyocyte injury vi regulting its trget genes progrmmed cell deth (PDCD) nd ctivtor protein 1 (AP1) pthwy, 7,8 nd reduced cell poptosis nd myocrdil infrct size t n erly stge of A. 9 On the other hnd, mir-1 expression is induced by AKT nd it in turn produces n ntipoptotic effect by suppressing FsL nd ctivting AKT through inhibiting PTEN, constituting feed forwrd loop. 1 Furthermore, mir-1 inhibition decreses p38 MAPK levels in A-98 cells. 11 Moreover, Wng et l. 1 identified the protective effect of mir-16 ginst myocrdil I/R injury which cn ttenute ctivtion of nucler fctor κb (NF-κB) by repressing expression of interleukin-1 receptor-ssocited kinse1 (IRAK1) nd tumor necrosis fctor (TNF) receptor- ssocited fctor 6 (TRAF6). In ddition, recent study showed tht mir-16 together with IRAK1, TRAF6 nd p-p38 constitutes negtive feed-forwrd loop tht cn disrupt cytokine protein synthesis in humn THP-1 monocytes. 13,1 MiRNAs regulte the expression of multiple genes by binding to trget trnscripts through imperfect sequence complementrity. While the modultion of single trget by n individul mirna my sometime hve only subtle effects, simultneous repression of multiple genes cn hve significnt impcts on cells. 15 This hs been confirmed for mir-1 nd its trget genes PDCD, PTEN, sprouty 1 (SPRY1), nd SPRY, s well s for mir-16 nd its trgets TRAF6 nd IRAK1 in crdiovsculr disese. 1,16 The cpcity of mirnas to trget multiple genes mkes them useful therpeutic tools tht cn be more potent thn gents tht ct on single gene. Moreover, recent studies from our group 17 nd others 15,18 hve reveled new mechnism of mirna ction whereby mirna pirs ct synergisticlly on trget genes, leding to potentition of their biologicl effects. Synergistic interctions cn increse the efficcy of therpeutics while reducing their side effects nd slowing the development of drug resistnce. In previous study, we developed topologicl prmeter synergy score to evlute the globl synergistic mirna regultion of poptosis under pthophysiologicl conditions nd identified number of ntipoptotic mirna pirs, including The first two uthors contributed eqully to this work. 1 Institute of Clinicl Phrmcology of the Second Affilited Hospitl, Hrbin Medicl University, Hrbin, Chin; The University Key Lbortory of Drug Reserch, Heilongjing Province, Hrbin, Chin. Correspondence: Zhi-Min Du, Institute of Clinicl Phrmcology of the Second Affilited Hospitl, Hrbin Medicl University, Xuefu Rod 6#, Nngng District, Hrbin 1586, Chin. E-mil: dzm1956@16.com Keywords: poptosis; myocrdil infrction; mir-1; mir-16 Received 13 Jnury 16; ccepted 8 Jnury 16; published online 15 Mrch 16. doi:1.138/mtn.16.1

2 Crdioprotective Role of mir-1 nd mir-16 Pirs the mir-1: mir-16 pir. 17 We conjectured tht combined tretment with mir-1 nd mir-16 would protect crdiomyocytes ginst cell deth induced by ischemi to greter extent thn either of these two mirnas. To test this hypothesis, we compred the ntipoptotic effects of mir-1 or mir-16 lone nd in combintion of the two in cultured neontl crdiomyocytes nd mouse model of A. Results Cytoprotective effect of mir-1: mir-16 mirna pir ginst hypoxi-induced crdiomyocyte poptosis We evluted the effects of cotrnsfected mir-16 nd mir-1 on hypoxi-induced cellulr injury in primry neontl rt crdiomyocytes. MiR-16 nd mir-1 levels were incresed when trnsfected with either one or both mirnas (Figure 1,b). To determine whether cotrnsfection produces synergistic effect on cell vibility, dose response curves nd combintion indices were clculted from cell vibility dt obtined from crdiomyocytes cultured under hypoxic conditions. The lowest combintion index ws.3 when cell vibility ws.7, indicting synergistic effect (Figure 1c). Under these conditions, the concentrtion of mir-1 nd mir-16 ws + nmol/l for totl concentrtion of 8 nmol/l. We compred the vibility of crdiomyocytes trnsfected with different mir- 1/miR-16 rtios (/, /6, or 6/ nmol/l) under hypoxi. Cell vibility ws reduced under hypoxic conditions; cotrnsfection of mir-1 nd mir-16 brogted this reduction (Figure 1d). A rtio of / nmol/l hd more pronounced effect in terms of restoring cell vibility thn rtios of 6/ or /6. Therefore, / nmol/l combintion ws used for subsequent experiments. The hypoxi-induced decrese in cell vibility ws prtilly restored by tretment with mir-1 or mir-16 lone or in combintion (Figure 1e). Notbly, cotrnsfection of mir-1 nd mir-16 produced the gretest effect thn either mir- 1 or mir-16 lone (P <.1), which ws reversed by ppliction of mir-1 nd mir-16 inhibitors (Figure 1e). In order to determine whether the decrese in cell vibility induced by hypoxi could be scribed to poptotic cell deth nd the rescuing effect of mir-1: mir-16 pir could be explined by their potentil ntipoptotic ction, we first used TUNEL ssy to detect DNA frgmenttion for poptosis. We found tht hypoxi incresed TUNELpositive cells indicting poptosis s compred to normoxic conditions (3.9 ± 1.8 versus 6.7 ±.7%; P <.1) (Figure,b). The number of poptotic cells ws mrkedly decresed in the presence of mir-1 or mir-16, nd this decrese ws more pronounced with the presence of both mirnas. These effects were ll reversed by inhibitor tretment (Figure,b). Next, we mesured the chnges of expression nd ctivities of cspse-3, known key downstrem protese tht executes the poptotic cscde. 1, As illustrted in Figure c, d, hypoxi incresed the level of cspse-3 mrna nd ctivity s compred to normoxic controls. These propoptotic chnges were blocked by trnsfection of mir-1 nd mir-16 either individully or in combintion; greter effect ws observed in the ltter instnce. Cytoprotective effect of mir-1: mir-16 mirna pir ginst ischemi-induced crdiomyocyte poptosis We then tried to clrify whether the ntipoptotic effects of mir-1 nd mir-16 seen in cultured cells under hypoxi conditions lso exist under in vivo conditions in A. Figure 3, b shows tht A significntly incresed crdiomyocyte poptosis, nd tretment with mir-1 nd mir- 16, whether in combintion or individully, drsticlly decresed this ischemic poptosis compred with the shmtreted mice. Moreover, in ccordnce with our in vitro experiments under hypoxic conditions, the mir-1: mir-16 pir lso produced remrkbly greter mgnitude of llevition of poptosis thn tretment with the individul mirnas. Furthermore, ischemi incresed cspse-3 mrna levels by ~5.5 fold (Figure 3c) nd ctivity by ~3. fold (Figure 3d), compred with the shm control. As expected, the mir-1: mir-16 pir produced greter effects on ischemi-induced ctivtion of cspse-3 thn mir-1 or mir-16 lone. MiR-1: mir-16 mirna pir decreses infrct size nd improves crdic function Rel-time PCR performed hours fter injection confirmed the successful delivery of mir-1 nd mir-16 into the myocrdium s evidenced by.9- nd 5.3-fold increses in mir-1 nd mir-16 levels, respectively (Figure,b). We found tht A incresed serum lctte dehydrogense (LDH) ctivity ( mrker for crdic injury) by 3.-fold compred to the control group, which ws significntly ttenuted by gomir-1/gomir-16. No significnt difference in LDH ctivity ws observed between gomir-1 nd gomir-16 being injected in combintion or individully (Figure c). We next detected the functionl role of gomir-1/ gomir-16 in infrct hert, nd found tht gomir-1/ gomir-16 significntly reduced the infrct size in (Figure d,e). Moreover, echocrdiogrphic dt indicted tht ejection frction (EF) nd frctionl shortening (FS) were significntly reduced in A, but gomir-1/gomir- 16 incresed these prmeters indicting improved crdic function by these mirnas (Figure g,h). The protective effects of mir-1/mir-16 cotrnsfection were efficiently blocked by ntgomir-1/ntgomir-16. No significnt difference in intrventriculr septum thickness in distole, intrventriculr septum thickness in systole (IVSS), left ventricle posterior wll dimeter in end-distole, nd left ventricle posterior wll dimeter in end-systole were observed between gomir-1 nd gomir-16 being injected in combintion or individully (Supplementry Tble S1). Notbly, in greement with our in vitro results, mir-1/ mir-16 combintionl tretment produced significntly greter protective effect on the ischemic myocrdium thn single-mirna tretment. MiR-1: mir-16 mirna pir produces beneficil effects by incresing AKT ctivity nd decresing p38 ctivity PTEN nd TRAF6 re the direct trgets of mir-1 nd mir- 16, respectively. 1,16 We therefore exmined these two proteins in ischemic herts. As depicted in Figure 5,b, mir-1 or mir-16 lone downregulted the protein levels of PTEN nd TRAF6, respectively, in ischemic herts nd similr effect ws observed with the mir-1: mir-16 pir. Moleculr Therpy Nucleic Acids

3 Crdioprotective Role of mir-1 nd mir-16 Pirs 3 Reltive mir-1 level 6 b Reltive mir-16 level 6 c d e OD vlue (9 nm) nmol/l OD vlue Cl mir-1 mir-16 mir-1/ OD vlue (9 nm) / 16 / 16 (/6 nmol/l) / 16 (6/ nmol/l) +Inh +Inh Figure 1 Prosurviving effects of mir-1: mir-16 mirna pir ginst hypoxi-induced crdiomyocyte deth in cultured neontl rt ventriculr myocytes (NRVMs). Cells were trnsfected with mir-mimics for 36 hours, nd then treted with hypoxi for 1 hours. (,b) Chnges of expression of mir-1 nd mir-16 determined by rel-time PCR. n = /group. (c) Dose response curves (mir-1 or mir-16: 1,,, 6, nd 8 nmol/l; mir-1/-16: 1/1, /, /, 6/6, 8/8 nmol/l). n = 6/group. (d) Cell vibility in the rtios of mir-1/- 16 under hypoxi. n = 6/group. (e) Cell vibility determined by MTT ssy. n = 6/group. Dt re shown s men ± stndrd error of the men, P <.1, one-wy nlysis of vrince. OD vlue (9 nm) MiR-1 hs been shown to prticipte in ischemic postconditioning-medited crdic protection ginst I/R injury nd crdic dysfunction through the PTEN/AKT signling pthwy. 3 To see if the sme pthwy ws lso involved in our model, we determined the expression of the phosphorylted or ctivted form of AKT (p-akt), which directly reflects the ctivity of the AKT signling pthwy. Ischemi did not ffect the level of totl AKT (Figure 5c); however, this cused significnt decrese (6.5%) in p-akt compred to the shm group (Figure 5c). Coinjection of gomir-1 nd gomir- 16 or gomir-1 lone either significntly incresed the p-akt in ischemic crdiomyocytes (Figure 5c). P38 MAPK is known to trnsduce poptotic or deth signl in crdiomyocytes, nd enhnced ctivtion (or phosphoryltion) of p38 MAPK contributes to myocrdil poptosis. We thus investigted whether gomir-1/gomir-16 ffect p38 MAPK ctivities (p-p38) in our model. Three dys post-, ischemi did not ffect the expression level of totl p38 MAPK (Figure 5d), but cused significnt increse in p38 MAPK phosphoryltion with.3-fold increse of p-p38 MAPK expression compred with the shm group (Figure 5d). Furthermore, coinjection of gomir-1 nd gomir-16 significntly inhibited the ctivtion of p38 MAPK in ischemic myocrdium to greter extent thn gomir-1 or gomir-16 lone (Figure 5d). We used sirna to silence TRAF6 nd PTEN expression nd then determined p-p38 MAPK protein expression. The efficiency of TRAF6 nd PTEN knock-down by the sirnas ws verified t both mrna nd protein levels, which were otherwise significntly decresed in crdiomyocytes reltive to sirna-nc (Figure 6 c,e). Next, we exmined totl p-38 nd p-p38 MAPK expression in cells trnsfected with TRAF6 nd PTEN sirnas. Our results showed tht TRAF6 or PTEN sirna did not ffect the expression of totl p38 MAPK (lower pnel, Figure 6c,f), but cused significnt decrese in p38 MAPK phosphoryltion compred with the control group (upper pnel, Figure 6c,f). Our western blot dt showed tht the rtio of p-akt to totl AKT incresed in PTEN knockdown crdiomyocytes (Figure 6g), consistent with the view tht PTEN is negtive regultor of AKT, which cn cuse cell deth. 5 To vlidte tht mir-1 PTEN/p-AKT regultes p-p38, expression of p-akt nd p-p38 ws detected in cells treted with mir-1 mimic or PI3K inhibitor LY9 (Figure 6h,i). MiR-1 significntly bolished hypoxi-induced inctivtion of AKT nd ctivtion of p38, nd these effects were reversed by LY9 (Figure 6h,i). Inhibition of p38 MAPK by SD358 decresed hypoxi-induced poptosis nd cspse-3 ctivity (Supplementry Figure S1). In conclusion, mir-1/mir-16 synergisticlly inhibited p-p38/ cspse-3 ginst myocrdil poptosis (Figure 6J).

4 Crdioprotective Role of mir-1 nd mir-16 Pirs +Inh +Inh Merge Tunel Hoechst b c d 3 3 Tunel positive cells (%) 1 +Inh +Inh Reltive mrna level of Cspse-3 1 +Inh +Inh Figure Anti-poptotic effect of mir-1: mir-16 mirna pir ginst hypoxi-induced crdiomyocyte poptosis in NRVMs. () Effects of mir-1/mir-16 on crdiomyocytes poptosis ssessed by TUNEL stining. Scle br = 1 µm. (b) TUNEL-positive cell (%). n = 6/group. (c) Cspse-3 mrna level determined by rel-time PCR. n = /group. (d) Cspse-3 ctivity determined by colorimetric ssy. n = 6/group. Dt re shown s mens ± stndrd error of the men, P <.5, P <.1, one-wy nlysis of vrince. Reltive Cspse-3 ctivity 3 1 +Inh +Inh Discussion It hs been well ccepted tht mirnas ply importnt roles in regulting every spects of myocrdil injuries nd crdic function in the setting cute myocrdil infrction. While the published studies primrily focus on the role of individul mirnas, the possibly interctions between mirnas remined scrcely investigted despite tht such interctions re being incresingly recognized. It is cler tht multiple mirnas coexist in the crdic cells nd their ctions re not isolted from one nother if mirna-mirna interctions re in plce. The present study shed light on this issue. The min findings of this study include: (i) mir- 1/miR-16 synergisticlly reduced ischemi/hypoxiinduced crdiomyocyte poptosis both in vivo nd in vitro; (ii) mir-1/mir-16 synergisticlly reduced infrct size nd meliorted the impired crdic function of infrct mice in vivo; nd (iii) mir-1/mir-16 synergisticlly inhibits crdiomyocyte poptosis through the mir-1 PTEN/AKT p-p38 cspse-3 nd mir-16 TRAF6 p-p38 cspse-3 signling pthwys. The synergistic ctions confer n enhnced cytoprotective power fforded by the mirnas ginst ischemic poptotic cell deth leding to meliorted myocrdil injuries nd improved crdic function in infrct herts. Our study thus unrveled novel mode of mirna ctions t the lyer of two-mirna combintion tht offers synergistic regultion on bsic cell biology process nd the ssocited conditions such s ischemic crdic disese. Recent studies found two mirnas combintion synergisticlly therpy of some diseses. MiR-3 nd let-7 combintion synergisticlly suppressed non-smll-cell lung cncer growth. 15 MiRNA-13 nd mirna-15 synergisticlly inhibit the growth of humn bldder cncer cells. 18 Coinhibition of mirna-1b nd mirna-1 exerts synergistic inhibition on the prolifertion nd invsion of humn gliom cells. 19 However, mirna combintion ws seldom investigted in hert disese, for instnce combintion of mirna-99 nd mirna-133 exerts synergic effect on crdic differentition. MiRNAs re known to ply importnt roles in the pthologicl conditions involving poptosis, including A nd hert filure. 6 Among these poptosis-regulting mir- NAs, mir-1 nd mir-16 hve both been documented to elicit ntipoptotic effects thereby beneficil effects on ischemic myocrdil injury; 7 1,1 however, whether there exists synergistic ction between these two mirnas similr to those reported for two mirnas combintion ws unknown. Here, we used n in vitro hypoxi model nd in vivo ischemic model to demonstrte the ntipoptotic effects nd the cellulr signling mechnisms of the mir-1: mir-16 pir. Our results showed tht ischemi/hypoxi induced significnt myocrdil injury, s reflected by incresed crdiomyocyte poptosis, incresed infrct size, elevted LDH ctivity, nd decresed EF nd FS. All these nomlies were llevited Moleculr Therpy Nucleic Acids

5 Crdioprotective Role of mir-1 nd mir-16 Pirs 5 Tunel-positive cells (%) Merge Tunel Hoechst b c d Reltive mrna level of Cspse-3 Cspse-3 ctivity Figure 3 Cytoprotective effect of the mir-1: mir-16 mirna pir ginst ischemi-induced crdiomyocyte poptosis in mouse model of cute myocrdil infrction (A). () Effects of mir-1/mir-16 on crdic poptosis evluted by TUNEL stining. Scle br = 1 µm. (b) TUNEL-positive cell (%). n = 8/group. (c) Cspse-3 mrna level determined by rel-time PCR. n = /group. (d) Cspse-3 ctivity determined by colorimetric ssy. n = 8/group (P <.5, P <.1, one-wy nlysis of vrince). by mir-1/mir-16 combintion tretment, confirming the crdio-protective property of these mirnas. Moreover, mir- 1/miR-16 combintion produced greter benefit effects thn ech of these mirnas seprtely. We hve lso identified the signling pthwy responsible for the ntipoptotic effects of mir-1/mir-16 ginst ischemi-/hypoxi-induced myocrdil injuries. In this study, we observed n ctivtion of cspse-3, s the key protese executing the poptotic cscde in the intrinsic nd extrinsic poptotic pthwys, 7 9 in crdiomyocytes during ischemi nd hypoxi. MiR-1 nd mir-16 reversed the ischemi-/ hypoxi-induced bnorml enhncement of cspse-3 ctivity. Notbly, the combintion of the two mirnas consistently produced stronger effects thn ech mirna individully. Mny reports hve demonstrted tht p38 MAPK plys n importnt role in ischemi-induced poptosis with its ctivtion by phosphoryltion contributing to crdic injury,,3 nd tretment with phrmcologicl inhibitor of p38 MAPK SB358 cn prevent crdiomyocyte deth. 31,3 Recent studies found mir-16 by directly trgeting IRAK1 nd TRAF6 limits p38 MAPK ctivtion. 13,1,33,3 Whether mir-16 medited TRAF6-p-p38 MAPK signling pthwy contributes to ischemi/hypoxi-induced poptosis in crdiomyocytes hs not been previously studied. In greement with previous studies, we found tht mir-16 downregulted TRAF6 in ischemic crdiomyocytes ccounting prtilly for the ntipoptotic efficcy of this mirna, consistent with our observtion tht direct silencing of TRAF6 by sirna decresed the level of p-p38 MAPK in neontl rt crdiomyocytes. Overexpression of mir-1 in trnsgenic mouse model resulted in suppression of ischemi-induced upregultion of PTEN expression, 35 increse in p-akt expression, reduction of infrct size, nd meliortion of hert filure. PTEN is mjor negtive regultor of AKT 36 whose ctivity is modulted by its bundnce, oxidtion, or phosphoryltion. 37 Regultion of the p38 pthwy by AKT nd the impct on cell survivl hve been reported in severl other models, including endothelil cells 38 nd β-cell, 39 nd in E1A-induced poptosis. Whether mir-1 elicited PTEN/AKT-p-p38 MAPK signling contributes to ischemi-/hypoxi-induced poptosis in crdiomyocytes hs not been previously studied. As further evidence, we showed tht mir-1 suppressed PTEN nd upregulted p-akt in ischemic crdiomyocytes. Moreover, PTEN sirna decresed the level of p-p38 MAPK but incresed p-akt in neontl rt crdiomyocytes. Furthermore, PI3K inhibitor LY9 incresed the level of p-p38 MAPK nd decresed p-akt in hypoxiinduced neontl rt crdiomyocytes. Additionlly, inhibiting p38 MAPK ttenuted the hypoxi-induced increses in crdiomyocytes poptosis nd cspse-3 ctivity. On the bsis of these findings, we proposed tht suppression of the p38 MAPK/cspse-3 cscde my be n underlying mechnism of mir-1 nd mir-16 ginst ischemic injury in A herts. It should be noted tht inhibition of the p38 MAPK/cspse-3 signl pthwy my merely be one of the protective mechnisms of mir-1/ mir-16 combintion in ischemic crdiomyocytes. While other signling pthwys my lso contribute to the ntipoptotic property

6 6 Crdioprotective Role of mir-1 nd mir-16 Pirs d Reltive mir-1 level 6 b Reltive mir-16 level 6 c, e IA/AAR (%) 6 LDH ctivity (U/ml) 1,5 1, 5 f g h EF (%) 6 FS (%) Figure Reduction of infrct size nd improvement of crdic function by the mir-1: mir-16 pir in A mice. (,b) Trnscript levels of mir-1 nd mir-16 determined by rel-time PCR. n = /group. (c) Serum lctte dehydrogense ctivity in nd gomir-1/ gomir-16 mice. n = 8/group. (d) Representtive imges showing infrct res in cross section slices. (e) Sttisticl nlysis of IA/AAR rtio. AAR, re t risk; IA, infrct re. n = 8/group. (f) Representtive photogrphs of hert function. IVSD, intrventriculr septum thickness in distole; IVSS, intrventriculr septum thickness in systole; LVPWD, left ventricle posterior wll dimeter in end-distole; LVPWS, left ventricle posterior wll dimeter in end-systole. (g) Ejection frctions. n = 8/group. (h) Frctionl shortening. n = 8/group (P <.5, P <.1, one-wy nlysis of vrince). of these mirnas, our results do not in ny wy exclude this possibility. As whole, our study reveled tht the combintion of mir-1 nd mir-16 offers superior effect ginst crdic ischemi-/hypoxi-induced poptosis compred with either mir-1 or mir-16 lone. The synergistic ction my be medited by the mir-1 PTEN/AKT p-p38 cspse-3 nd mir-16 TRAF6 p-p38 cspse-3 signling pthwys. Our study implies tht mir-1/mir-16 combintion my be new therpeutic cndidte in the tretment of ischemic hert diseses. Mterils nd methods Animls. Helthy dult mle Kunming mice (5 3 g) were used in this study. Mice were mintined with food nd wter under stndrd niml room conditions (temperture 3 ± 1 C; humidity 55 ± 5%). All experimentl protocols were in ccordnce with nd pproved by the Experimentl Animl Ethic Committee of Hrbin Medicl University, Chin. Use of nimls complied with the Guide for the Cre nd Use of Lbortory Animls published by the US Ntionl Institutes of Helth (NIH Publiction, 8th Edition, 11). Moleculr Therpy Nucleic Acids

7 Crdioprotective Role of mir-1 nd mir-16 Pirs 7 b c d Figure 5 Chnges of protein levels of PTEN, TRAF6, p-akt, nd p-p38 in A mice. () Representtive western blot bnds of PTEN protein. n = 5/group. (b) Representtive western blot bnds of TRAF6 protein. n = /group. (c) Representtive western blot bnds of p-akt nd AKT proteins. n = /group. (d) Representtive western blot bnds of p-p38 nd p38 proteins. Vlues given were normlized to the bnd intensity of β-ctin s n internl control. n = /group (P <.5, P <.1, one-wy nlysis of vrince). Administrtion of gomir-1/-mir-16 nd their ntgomirs. The gomir-1 nd gomir-16 (Ribo-bio, Gungzhou, Chin) re double-strnded RNA nlogues identicl to the mture mmu-mir-1-5p (5 -UAGCUUAUCAG ACUGAUGUUGA-3 ) nd mmu-mir-16-5p (5 -UGAGAAC UGAAUUCCAUGGGUU-3 ). MiRNA negtive control (mir- NC) sequence is (5 -UUUGUACUACACAAAAGUACUG-3 ). All these constructs were chemiclly modified nd conjugted with cholesterol moiety for in vivo pplictions with long-lsting stbility nd enhnced trget specificity nd ffinity. The mice were rndomly divided into the following groups:,, + NC, gomir-1 (1 nmol kg 1 ), gomir-16 (1 nmol kg 1 ), gomir-1/mir-16 (5/5 nmol kg 1 ), ntgomir-1/mir-16 (1/1 nmol kg 1 ), gomir-1/mir-16 (5/5 nmol kg 1 ), nd ntgomir-1/ mir-16 (1/1 nmol kg 1 ). AgomiR-1 nd gomir- 16 were dministered by intrmusculr injection into the left ventricle myocrdium t multiple sites t dosge of 1 nmol kg 1 in. ml sline hours prior to. 1 The dosge of gomir ws determined ccording to the published studies. 1, For mice, n equl volume of sline ws given. Mouse model of nd mesurement of infrct size. Mice were subjected to left nterior descending (LAD) coronry rtery ligtion to induce s described previously. 3 Remote tissues of ischemic res in the left ventricles were quickly dissected for subsequent nlysis. The mesurement of re t risk nd infrct size ws ccording to previous study. 3 Three dys fter,.3 ml Evns blue dye (%, Sigm Aldrich St. Louis, MO) ws injected retrogrdely into the ven cv to delinete the region of myocrdil perfusion. After wshing out remining blood nd trimming out the right ventricle, the left ventricle ws cut into -mm thick slices nd stined with 1% triphenyltetrzolium chloride t 37 C for 1 minutes, nd the infrct re ws stinless while the live re turned red. The re t risk (ischemic re) nd infrct re were clculted using Imge ProPlus 5. softwre (Medi Cybernetics, Wokinghm, UK). For further study, the tissues in ischemic re of the herts were collected nd stored t 8 C. Echocrdiogrphic mesurements. Three dys post-, the chnges in left ventriculr function were evluted by trnsthorcic echocrdiogrphy with n ultrsound mchine (Pnoview β15, Cold Spring Biotech, Tiwn, Chin) equipped with 3-MHz phsed-rry trnsducer. Left ventriculr EF nd FS were clculted from M-mode recording. Menwhile, intrventriculr septum thickness in distole, intrventriculr septum thickness in systole, left ventricle

8 8 Crdioprotective Role of mir-1 nd mir-16 Pirs b TRAF6 c p-p38 β-ctin p38 d e f g PTEN p-p38 β-ctin p38 P-AKT AKT h i j p-akt p-p38 AKT p38 Figure 6 Synergistic suppression of p-p38 MAPK by the mir-1: mir-16 pir in neontl rt ventriculr myocytes. (,b) Verifiction of the efficcy nd specificity of TRAF6 sirna in silencing TRAF6 t both mrna nd protein levels. n = /group. (c) Effect of TRAF6 sirna on the protein level of p-p38. n = /group. (d,e) Verifiction of the efficcy nd specificity of PTEN sirna in silencing PTEN t both mrna nd protein levels. n = /group. (f,g) Chnges of p-p38 nd p-akt levels in PTEN sirna trnsfected cells. n = /group. (h,i) Effects of PI3K inhibitor LY9 on p-akt nd p-p38 levels. Cells were preincubted with LY9 (1 μmol/l) for hours, then trnsfected with mir-1 for 36 hours, nd finlly treted with hypoxi for 1 hours. n = /group (P <.5, P <.1, one-wy nlysis of vrince). (j) The digrm illustrting the synergistic mechnism of mir-1: mir-16 pir ttenutes poptosis. posterior wll dimeter in end-distole, nd left ventricle posterior wll dimeter in end-systole were mesured. Neontl rt ventriculr myocytes culture nd trnsfection. The procedures to culture neontl rt ventriculr myocytes were the sme s described previously. Briefly, the herts of neontl Wistr rts were rpidly removed, minced in serumfree Dulbecco's Modified Egle Medi (DMEM, HyClone, Logn, UT), nd then digested in.5% trypsin solution. Dispersed cells were suspended in Dulbecco's Modified Egle Medi (DMEM, HyClone) contining 1% fetl bovine serum nd centrifuged. The isolted cells were plted into culture flsks (noncoted) nd.1 mmol/l bromodeoxyuridine ws dded into the medium to deplete nonmyocytes. Crdiomyocytes were cultured under condition of 5% CO t 37 C. MiR-1-mimic, mir-16-mimic, mir-1 inhibitor, mir- 16 inhibitor, nd NC were synthesized by Gungzhou RiboBio (Gungzhou, Chin). PTEN sirna nd TRAF6 sirna were purchsed from Snt Cruz Biotechnology (Snt Cruz, CA). Crdiomyocytes (1 1 5 per well) were strved in serum-free medium for hours before trnsfection with X-treme GENE sirna trnsfection regent (Roche, Penzberg Germny) ccording to the mnufcturer s instructions. The dosge of mir-mimic ws determined bsed Moleculr Therpy Nucleic Acids

9 Crdioprotective Role of mir-1 nd mir-16 Pirs 9 upon our previous study. 17 Thirty-six hours fter trnsfection, neontl rt ventriculr myocytes were treted with hypoxi under 1% O, 5% CO, nd 9% N for 1 hours in modulr incubtor. Cspse-3-specific inhibitor z-devd-fmk ( μmol/l) ws purchsed from Merck, Drmstdt, Germny. PI3K inhibitor LY9 (1 μmol/l) nd p38mapk inhibitor SB358 ( μmol/l) were purchsed from Sigm. Quntittive rel-time reverse trnscription-pcr. Totl RNA from cultured neontl crdiomyocytes fter different tretments or hert tissue ws extrcted using TRIZOL regent (Invitrogen, Crlsbd, CA) ccording to the mnufcturer s protocols. The levels of cspse-3, PTEN, TRAF6, mir-1, nd mir-16 mrna were determined using SYBR Green incorportion on Roche Light- Cycler8 Rel Time PCR system (Roche, Germny), with U6 s n internl control for mir-1 nd mir-16 nd GAPDH for cspse-3, PTEN, nd TRAF6. The sequences of primers were: mir-1 forwrd: 5 -GGGGTAGCTTATCAG ATCG-3 nd reverse: 5 -TGGAGTCGGCAATTGCACTG-3 ; mir-16 forwrd: 5 -GGGTGAGAACTGAATTCCAT-3 nd reverse: 5 -TCGGCAATTGCACTGGATAC-3 ; U6 forwrd:5 - GCTTCGGCACATATACTAAAAT-3 nd reverse: 5 -CGCTTC ACGAATTTGCGTGTCAT-3 ; GAPDH forwrd: 5 -AAGAAGGT GGTGAAGCAGGC-3 nd reverse: 5 -TCCACCACCCAGTT GCTGTA-3 ; PTEN forwrd: 5 -TGGATTCGACTTAGACTTGA CCT-3 nd reverse: 5 -GCGGTGTCATAATGTCTCTCAG-3 ; TRAF6 forwrd: 5 -AAAGCGAGAGATTCTTTCCCTG-3 nd reverse: 5 -ACTGGGGACAATTCACTAGAGC-3 nd cspse- 3 forwrd: 5 -CTCGCTCTGGTACGGATGTG-3 nd reverse: 5 -TCCCATAAATGACCCCTTCATCA-3. The mount of trget ( ΔΔ CT ) ws obtined by normlizing to endogenous reference nd reltive to clibrtor (verge of the control smples). Serum LDH ssy. The blood smples were collected from mice 7 hours fter occlusion of the coronry rtery nd the ctivity of myocrdil specific enzyme LDH ws mesured with the colorimetric method ccording to the mnufcture s protocols (Nnjing Jincheng Bioengineering Institute, Nnjing, Chin). Cspse-3 ctivity ssy. Myocrdil cspse-3 ctivity ws determined by use of colorimetric ssy kits (Beyotime Institute of Biotechnology, Jingsu, Chin) ccording to the mnufcturer s instructions. After experimentl tretments, crdiomyocytes were centrifuged (6 g, C, 5 minutes) nd wshed with ice-cold PBS. Cells were then lysed in icecold cell lysis buffer for 15 minutes. Smples were centrifuged t, g for 1 minutes t C, nd 3 μl superntnt smples were incubted with 1 μl of substrte ( mmol/l Ac- DEVD-pNA) in 6 μl of ssy buffer t 37 C for hours. The bsorbnce ws mesured t 5 nm. Terminl deoxynucleotidyl trnsferse dutp nick end lbeling (TUNEL). Apoptosis of crdiomyocyte ws detected by stining ventriculr specimens nd neontl rt ventriculr myocytes with the In situ Cell Deth Detection Kit (TUNEL fluorescence FITC kit, Roche) ccording to the mnufcturer s instruction. After TUNEL stining, the ventriculr specimens (3 dys post-) or crdiomyocytes (1 hours fter hypoxi) were immerged into the Hoechst 333 (Sigm-Aldrich) solution to stin nuclei of living nd poptotic cells. Fluorescence stining ws viewed under lser scnning confocl microscope (Olympus, Fluoview1, Tokyo, Jpn). Numbers of poptotic crdiomyocytes re presented s the percentge of totl cells counted. MTT ssy nd combintion index clcultion. Cells ( 1 cells/well) were seeded in 96-well culture plte. Cell vibility ws mesured by 3-(,5-dimethylthizol--yl)-,5- diphenyltetrzolium bromide (MTT) ssy ccording to the mnufcturer s instructions. The bsorbnce ws mesured t 9 nm. The combintion index vlue ws clculted by using the Clcusyn softwre (Biosoft, Cmbridge, UK) bsed on the medin effect eqution s previously reported in detils. 5 A combintion index vlue < 1, =1, or >1 denotes synergism, dditive effect, or ntgonism, respectively. Western blot nlysis. Totl protein smples were extrcted from peri-infrct region of left ventriculr myocrdium for immunoblotting nlysis. 1 Protein smple (7 µg) ws frctionted by SDS-PAGE (1 % polycrylmide gels) nd trnsferred to nitrocellulose membrne. The membrnes were blocked in 5% nonft milk PBS for hours nd then incubted t C overnight with the following primry ntibodies: p38 MAPK (Cell Signling Technology, Dnvers, MA), p-p38mapk (Cell Signling Technology, Dnvers, MA), TRAF6 (Snt Cruz Biotechnology, Snt Cruz, CA), PTEN (Proteintech Group, Chicgo, IL), p-akt (Cell Signling Technology, Dnvers, MA), AKT (Cell Signling Technology, Dnvers, MA), nd β-ctin (ZSJZ-Bio, Beijing, Chin). After wshing, the membrne ws incubted with secondry ntibody for 1 hour. Imges were cptured on the Odyssey CLx Infrred Imging System (LI-COR Biosciences, Lincoln, NE). Western blot bnds were quntified using Odyssey CLx v.1 softwre (LI-COR Biosciences, Lincoln, NE). The dt were normlized to β-ctin s n internl control. Dt nlysis. Group dt re expressed s men ± stndrd error of the men. Sttisticl comprisons between two groups were performed by t-test, nd mong multiple groups were performed by one-wy nlysis of vrince followed by Tukey s multiple comprison tests. Differences were considered to be sttisticlly significnt when P <.5. Dt were nlyzed using the GrphPd Prism 5. (GrphPd Softwre, L Joll, CA) nd SPSS 1. (SPSS, Chicgo, IL). Supplementry mteril Tble S1. Ventriculr thickness (mm) obtined from the echocrdiogrphic nlysis of the mice three dys fter A. Figure S1. Inhibition of p38 MAPK by SD358 decresed hypoxi-induced poptosis nd cspse-3 ctivity. Acknowledgments This work ws supported in prt by the Mjor Progrm of Ntionl Nturl Science Foundtion of Chin (81381), Specilized Reserch Fund for the Doctorl Progrm of Higher Eduction of Chin (113713). All uthors declre tht they hve no competing interests for this study.

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