Basic Res Cardiol (2014) 109:423 DOI 10.1007/s00395-014-0423-z

ORIGINAL CONTRIBUTION

MicroRNA-144 is a circulating effector of remote ischemic preconditioning Jing Li • Sagar Rohailla • Nitai Gelber • James Rutka • Nesrin Sabah • Rachel A. Gladstone • Can Wei • Pingzhao Hu • Rajesh K. Kharbanda Andrew N. Redington

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Received: 8 April 2014 / Revised: 11 June 2014 / Accepted: 23 June 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Remote ischemic preconditioning (rIPC) induced by cycles of transient limb ischemia and reperfusion is a powerful cardioprotective strategy with additional pleiotropic effects. However, our understanding of its underlying mediators and mechanisms remains incomplete. We examined the role of miR-144 in the cardioprotection induced by rIPC. Microarray studies first established that rIPC increases, and IR injury decreases miR-144 levels in mouse myocardium, the latter being rescued by both rIPC and intravenous administration of miR-144. Going along with this systemic treatment with miR-144 increased P-Akt, P-GSK3b and P-p44/42 MAPK, decreased p-mTOR level To this original contribution an invited editorial is available at doi:10.1007/s00395-014-0429-6.

Electronic supplementary material The online version of this article (doi:10.1007/s00395-014-0423-z) contains supplementary material, which is available to authorized users.

and induced autophagy signaling, and induced early and delayed cardioprotection with improved functional recovery and reduction in infarct size similar to that achieved by rIPC. Conversely, systemic administration of a specific antisense oligonucleotide reduced myocardial levels of miR-144 and abrogated cardioprotection by rIPC. We then showed that rIPC increases plasma miR-144 levels in mice and humans, but there was no change in plasma microparticle (50–400 nM) numbers or their miR-144 content. However, there was an almost fourfold increase in miR-144 precursor in the exosome pellet, and a significant increase in miR-144 levels in exosome-poor serum which, in turn, was associated with increased levels of the miR carriage protein Argonaute2. Systemic release of microRNA 144 plays a pivotal role in the cardioprotection induced by rIPC. Future studies should assess the potential for plasma miR-144 as a biomarker of the effectiveness of rIPC induced by limb ischemia, and whether miR-144 itself may represent a novel therapy to reduce clinical ischemia–reperfusion injury.

J. Li  S. Rohailla  N. Gelber  C. Wei  A. N. Redington (&) Division of Cardiology, Labatt Family Heart Center, Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X 8, Canada e-mail: [email protected]

Keywords miR-144  Remote ischemic preconditioning  Ischemia/reperfusion injury  mTOR  Exosomes  Argonaute-2

J. Rutka  N. Sabah Arthur and Sonia Labatt Brain Tumour Research Centre, The Hospital for Sick Children, Toronto, ON, Canada

Introduction

R. A. Gladstone Human Developmental and Regenerative Biology, Harvard University, Cambridge, USA P. Hu Genetic and Genomic Biology, Hospital for Sick Children (P.H.), Toronto, Canada R. K. Kharbanda The John Radcliffe Hospital, Oxford, UK

Remote ischemic preconditioning (rIPC), induced by brief periods of limb ischemia and reperfusion using a blood pressure cuff or tourniquet [23], has rapidly translated from animal models [22, 27, 34] to proof-of-principle clinical trials (RCTs) many, but not all, showing decreased myocardial injury and improved long-term outcomes early after cardiovascular surgery and coronary angioplasty [16], and several recent studies showing improved long-term

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outcomes in cardiac surgical, elective and emergency coronary angioplasty and stroke patients [6, 30, 36, 39]. Despite these promising clinical findings, our understanding of the underlying mechanisms of cardioprotection by rIPC remains incomplete. Neural stimulation is a key element of the ‘upstream’ signaling [8, 34, 37], and we and others [20, 28, 33, 34] have shown that this induces release of a circulating humoral factor(s) that can induce cardioprotection downstream in distant organs via stimulation of G-protein coupled receptors [34, 38, 47] and induction of intracellular kinase signature similar to that of local preconditioning [27, 34]. More recently, STAT5 signaling has been implicated in the human myocardial phenotype [17]. Identification of the exact nature of the circulating factor(s) has proven elusive although recently a chemokine, stromal cell-derived factor-1, was shown to increase in the plasma after rIPC, increase myocardial expression of its receptor protein chemokine receptor 4 (CXCR4), and induce cardioprotection [5]. However, its cardioprotection was only partially abrogated by a blocker, AMD3100, suggesting that additional pathways may be at play [32]. Furthermore, the effects of rIPC appear to extend beyond traditional cardioprotective pathways. We have previously described the important effects of rIPC on gene expression in mouse myocardium [25] and human neutrophils [24], and chronic administration of the rIPC stimulus after the ischemic period has been shown to modify myocardial remodeling following experimental MI [43] and improve the rate and extent of recovery in patients after stroke [30] suggesting effects beyond immediate cytoprotective mechanisms. Given this pleiotropy, we hypothesized that microRNAs (miRNA) may have a role in rIPC-induced myocardial protection. miRNAs are a class of endogenous, small, noncoding RNAs that regulate gene expression posttranscriptionally [26, 44]. The recent discovery that miRNAs circulate in a stable form in blood [41, 45], suggest that circulating miRNAs can serve as biomarkers, as well as function as mediators of disease, and protection from disease. The role of miRNAs in rIPC-induced myocardial protection has not been studied extensively. In one study, limited to analysis of two miRs (miR-1 and miR-21), there was differential expression depending on the type of stimulus (local vs. remote) [10]. In a more extensive analysis of microRNAs expression in humans undergoing cardiac surgery, miR-1 expression appeared to be reduced and miR-388-p increased by rIPC, although clearly the associated cardiac disease and its treatment in patients, and the use of atrial biopsies, limits the interpretation of these data [35]. We therefore conducted this study to examine miRNA changes in the heart after rIPC using microarray analysis, and subsequently examined the role of miR-144 in rIPC-induced cardioprotection.

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Methods For a detailed description of all methods, see Supplementary material online. Key methods are described briefly below. All animal protocols were approved by the Animal Care and Use Committee of the Hospital for Sick Children in Toronto and conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication No. 85-23, revised 1996). Studies on human volunteers were approved by the research ethics board of the hospital for Sick Children, Toronto. Induction of remote ischemic preconditioning (rIPC) Remote ischemic preconditioning was induced by four cycles of 5 min of limb ischemia (by tourniquet in mice, BP cuff inflated to 200 mmHg in humans) followed by 5-min reperfusion as previously described [23, 25]. MicroRNA stem loop RT-PCR Total RNA was extracted from left ventricular tissue using TRIzol Reagent (Invitrogen), according to the manufacturer’s instructions. RT-PCR was performed using TagMan MicroRNA assay kit (ABI). Data were normalized by evaluating RNA U6 (RNU6B, ABI) expression. Mouse Langendorff preparation and global ischemia/ reperfusion model In order to examine the myocardial effects of the in vivo interventions, without the potential confounding effects on other systems, isolated mouse hearts were mounted on the Langendorff perfusion apparatus as previously described [27], and perfused under non-recirculating conditions at a constant pressure of 80 mmHg with 37 °C Krebs–Henseleit buffer (KHB). After a 20-min stabilization period, hearts were subjected to 30 min of no-flow global ischemia followed by 60-min reperfusion. Measurement of infarct size Infarct size was assessed via 1.25 % 2,3,5-triphenyltetrazolium chloride (TTC, Sigma) staining as described previously [23, 24]. Antisense oligonucleotide preparation and delivery Single-stranded RNAs were synthesized by VBC Biotech (Vienna), antagomiR-144 (50 -agUACAUCAUCUAUACugua-Chol-30 ); and a scrambled (mutated) miRNA as a

Basic Res Cardiol (2014) 109:423 Fig. 1 a The study design for effects of antagomiR-144 on rIPC-induced cardioprotection in mouse ischemia–reperfusion model. b The study design for systemic delivery of miR-144

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Preparation and administration of miR-144 The mature miR-144 sequence used was 50 -uaCAGUAUAGAUGAUGUAcuag-Chol-30 . miR-144 and control oligonucleotides were dissolved in PBS before administration. C57BL/6 mice received miR-144, or miRCo (8 mg/kg body weight in 200 ll) or a comparable volume of PBS (200 ll) via tail vein injections. C57BL/6

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control (AntagomiR-Co/miR-Co: 50 -aaGGCAAGCUGACCCUGAaguu-Chol-30 ). Each oligonucleotide was deprotected, desalted, and purified by high-performance liquid chromatography. Antagomir and control oligonucleotides were dissolved in PBS before administration. C57BL/6 mice received antagomiR-144, or antagomiRCo (8 mg/kg body weight in 200 ll, per day) or a comparable volume of PBS (200 ll) through three consecutive daily tail vein injections. The dose was used based on an established protocol used by Dimmeler et al. [2]. Mice were divided into five groups: Group 1 (PBS, n = 7), Group 2 (PBS ? rIPC, n = 6), Group 3 (AntimiRCo ? rIPC, n = 5), Group 4 (AntimiR-144 ? rIPC, n = 5), Group 5 (AntagomiR-144 alone, n = 5) (Fig. 1a for study design).

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mice were divided into four groups: Group 1 (PBS, n = 6), mice received intravenous PBS and 60 min later, hearts were isolated and mounted on Langendorff preparation for global ischemia/reperfusion experiments. Group 2 (miRCo, n = 6), mice received miR-Co (200 ll, 8 mg/kg) followed by ischemia reperfusion; Group 3 (miR-144 Day1, n = 8) mice received miR-144 (200 ll, 8 mg/kg), followed by ischemia/reperfusion after 60-min injection; Group 4 (miR-144 Day3, n = 4), mice received miR-144 through three consecutive daily tail vein injections, global ischemia/reperfusion was performed on the next day after final injection (Fig. 1b for study design). Immunoblotting Western blotting was conducted according to standard protocols. Phospho-Akt (Ser473), phospho-p44/42 MAP Kinase (Thr202/Tyr204), phospho-GSK3b (Ser9), phospho-mTOR (Ser2481), Atg5, SQSTM1/p62 (cell signaling), LC3 antibody (Novus), Cathepsin L (R&D Systems) and anti-CD63 antibody (System Biosciences Inc.) was used as primary antibodies. Immunoblots were scanned using an Odyssey LI-COR and quantified using Image Studio (Ver 2.1).

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Mouse and human plasma preparation Remote ischemic preconditioning was performed and blood was collected 15 min later in K2 EDTA tubes (Beckton Dickinson) and processed within 5 min for plasma preparation. Blood samples were first centrifuged at 1,500g for 15 min at 4 °C. The supernatant was collected and transferred to nuclease-free tubes, centrifuged again at 14,000g for 15 min at 4 °C. The supernatant was processed further for total RNA extraction. Human blood samples were collected at pre (baseline) and post rIPC. RNA isolation A miRNeasy Mini Kit (Qiagen) was used to isolate total RNA from mouse and human plasma according to the manufacturer’s instructions with cel-miR-39 (Qiagen) spiked for normalization of the RNA preparation. Exosome isolation and measurement of exosome numbers Exosomes were isolated from mouse serum using ExoQuick (System Biosciences) according to the manufacturer’s instructions. Exosome quantification and characterization of microparticles between 50 and 400 nm was performed using the NanoSight LM10-B system (NanoSight Ltd.). Isolation of RNAs from mouse serum exosomes and exosome-poor supernatants Isolation of exosomal and supernatant RNAs was performed using the miRNeasy Mini Kit. Exosome or supernatant was diluted with 1 ml of QIAzol Solution according to the manufacturer’s instructions with cel-miR-39 spiked for normalization of the RNA preparation. The levels of miR-144 were determined by MicroRNA stem loop RTPCR, as described above. Precursor miR-144 level in mouse serum exosomes was measured using miScript Precursor Assays and miScript II RT Kit (Qiagen). Argonaute-2 co-immunoprecipitation and RNA extraction Using exosome-poor supernatant (250 ll), we performed immunoprecipitation experiments to determine whether miR-144 co-fractionates with an Argonaute-2 (Ago2) protein complex. We combined 2 lg of Ago2 rabbit monoclonal antibody (Cell Signalling Inc.) or normal rabbit IgG antibody (Santa Cruz Inc.) with 250 ll of supernatant (prepared as described above). After overnight incubation at 4 °C, the analysate was added to 20 ll of

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Resin Slurry (Pierce Classic IP kit) and incubated for 2 h at 4 °C with constant shaking. The resin was then washed three times with cold IP lysis/wash buffer, and the sample was eluted in 1-ml QIAzol and processed for RNA isolation. Statistical analysis For all comparisons, statistical significance was determined using one-way ANOVA, followed by post hoc testing (Newman–Keuls) where appropriate. Values of p B 0.05 were considered statistically significant. Data are shown as mean ± SE (standard error).

Results The role of miR-144 in the cardioprotection induced by rIPC Initial myocardial miRNA microarray expression profiling in C57BL/6 mice showed that 22 of 655 miRNAs were significantly modified by rIPC (16 upregulated, 6 downregulated—Supplementary Table 1). Of note, miR 451 was not significantly altered by rIPC. Four miRNAs (miR-144, miR-451, miR-27a-5p, miR-489) were selected for validation by quantitative miRNA stem loop RT-PCR, confirming a significant increase in miR-144 level after rIPC (Fig. 2a, Supplementary figure 3). Furthermore, we found that IR injury alone led to a marked reduction in myocardial miR-144 levels (Fig. 2b). As a result of these findings and the previous implication of miR-144 in recovery from IR injury [48], we then chose to test directly the role of miR-144 in the cardioprotection induced by rIPC by studying the effects of intravenous administration of antagomiR-144. As shown in Fig. 3a, miR-144 expression was reduced by 60 % in heart at 24 h after the last of three daily intravenous injections of antagomiR-144. In contrast, the mutated antagomiR control had no effect on miR-144 expression level compared with the PBS treatment. We also examined the miR-144 expression level in liver after injection of miR-144 and antamiR-144, antamiR-144 decreased and miR-144 increased miR-144 expression at day 3, but unchanged at 1 h after injection (Supplementary figure 4). In order to delineate the cardiac-specific effects of miR144, we used a Langendorff isolated heart model of global IR injury. There were no statistically significant differences in baseline hemodynamic functional parameters among all groups (data not shown). As shown in Fig. 3, prior in vivo rIPC improved the post-ischemic cardiac performance in isolated perfused hearts. On the basis of LV developed pressure, recovery of post-ischemic contractile function

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Fig. 2 Cardiac miR-144 expression in rIPC. a Mouse myocardial miR-144 level was determined by MicroRNA stem loop RT-PCR, miR-144 was normalized to expression of the internal control (RNU6B) (n = 6). b Myocardial miR-144 levels significantly

decrease compared to controls after ischemia–reperfusion injury (n = 4). Data are shown as mean ± SEM. Statistical significance is shown as *p \ 0.05, §p \ 0.01 vs. control

was greater in PBS ? rIPC, miR-Co ? rIPC hearts than in PBS sham hearts (Fig. 3b). By the end of the 60-min reperfusion period, a significantly greater functional recovery was observed in PBS ? rIPC (91.3 ± 2.5 % of pre-ischemic value), miR-Co ? rIPC (94.3 ± 3.2 %) compared with PBS alone (77.7 ± 1.3 % of pre-ischemic value, p \ 0.01, Fig. 3b). Diastolic recovery was also improved by rIPC, at the end of the reperfusion period, LVEDP was significantly lower in PBS ? rIPC (15.9 ± 2.5 mmHg), miR-Co ? rIPC (16.2 ± 1.1 mmHg) than in PBS-treated hearts (24.2 ± 2.7 mmHg, p = 0.046 and p = 0.037, Fig. 3c). In the group treated with antagomiR-144 prior to rIPC (antagomiR-144 ? rIPC), no significant differences in LVDP, or LVEDP, were seen relative to the PBS group. Myocardial infarct size was assessed by TTC staining (Fig. 3d). Consistent with the improved functional recovery, infarct size was significantly reduced by PBS ? rIPC (27.5 ± 5 %) and miR-Co ? rIPC (30.4 ± 2 %) compared to PBS alone (44.7 ± 3 %, p = 0.02 and p = 0.004), and this effect was abrogated by injection of antagomiR-144 (antagomiR-144 ? rIPC: 49 ± 4 %, p = ns compared with PBS). AntagomiR-144 injection alone did not result in a significant difference in infarct size compared to PBS-treated hearts.

three consecutive daily injections (miR-144 Day3). miR-144 levels were increased over twofold, compared to PBS control, both after 1-h injection, and 1 day after 3 days of miR144 injection (Fig. 4a). We then showed that the reduction in myocardial miR-144 levels (Fig. 2b) induced by IR injury was rescued both by pretreatment with intravenous miR-144 (Fig. 4b) and rIPC (Fig. 4c). Going along with these observations, as shown in Fig. 4, hearts harvested from animals pre-treated with intravenous miR-144 or rIPC were equally protected against lethal IR injury at both 60 min (early window) and 24 h (delayed window) after miR-144 treatment, as manifested by improved functional recovery (Fig. 5a–d) and a significant reduction in infarct size (Fig. 5e, f). By the end of the 60-min reperfusion period, a significantly greater functional recovery was observed in miR-144 Day1 (91.8 ± 1.7 %), compared with PBS alone (72.9 ± 2.3 % of pre-ischemic value, p \ 0.01, Fig. 5a) and miR-144 Day 3 (94.9 ± 1.2 %), compared with PBS Day 3 (77.7 ± 1.3 % of pre-ischemic value, p \ 0.01, Fig. 5c). Diastolic recovery was also improved by miR-144, at the end of the reperfusion period, LVEDP was significantly lower in miR-144 Day 1 (18.4 ± 2.0 mmHg), miR-144 Day 3 hearts (17.1 ± 3.2 mmHg) than in PBS-treated hearts (26.7 ± 3.2 mmHg, 24.2 ± 2.7 mmHg, p = 0.04 and p = 0.079, Fig. 4b, d). Infarct size was significantly reduced by miR-144 Day 1 (25.9 ± 4 %) compared to PBS alone (39.0 ± 2 %, p = 0.015, Fig. 5e). miR-144 Day 3 (30.1 ± 3 %) compared to PBS Day 3 (44.7 ± 3 %, p = 0.012, Fig. 5f).

Systemic administration of miR-144 induces early and delayed cardioprotection in mouse ischemia– reperfusion model To examine if systemically delivered miR-144 can induce early or delayed cardioprotection, intravenous miR-144 was administered by tail vein injection. Assessment of ischemia– reperfusion injury (Langendorff) was performed immediately after a single injection (miR-144 Day1), or 1 day after

Target gene analysis: miR-144 regulation of autophagy pathways through downregulation of mTOR The mammalian target of rapamycin (mTOR) is key negative regulator of autophagy, and a known target gene of miR-144

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Fig. 3 AntagomiR-144 reverses rIPC-induced cardioprotection. a Myocardial miR-144 levels after receiving either antagomiR-144 or miR-Co via three consecutive daily tail vein injections (3 9 8 mg/ kg). Injection of PBS or scrambled oligonucleotide (miR-Co) produced no change in miR-144 levels, but there was a decrease in mIR-144 with antagomiR-144. n = 3–4 per group. b The recovery of left ventricular-developed pressure (LVDP) was reduced in antagomir alone or antagomir ? rIPC groups. c Diastolic recovery (LVEDP left ventricular end-diastolic pressure). Asterisk denotes a statistically

significant difference between PBS ? rIPC and miR-Co ? rIPC groups vs. PBS (*p \ 0.05) after 60 min of reperfusion. d Myocardial infarct size was measured with triphenyltetrazolium chloride (TTC) staining. A representative basal left ventricular section is presented for each group. Prior treatment with AntagomiR-144, but not scrambled Antagomir control (miR-Co), abrogates the effect of rIPC to reduce infarct size. n = 5–7 per group. Data are shown as mean ± SEM. Statistical significance is shown as *p \ 0.05 vs. PBS, § p \ 0.01 vs. PBS

[19]. We found miR-144 downregulated phospho-mTOR and total mTOR levels in the mouse heart after miR-144 injection and in turn was associated with increased autophagy signaling. Levels of Atg5 and Cathepsin L were significantly increased in the hearts from miR-144 injected mice (p \ 0.01 and p \ 0.05) (Fig. 6a, b). LC3II/I ratio also increased in miR-144 injected heart (p [ 0.05 vs. PBS group).

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One hour after miR-144 injection, we showed increased levels of phospho-Akt, phospho-GSK3b and phospho-p44/42 MAP Kinase in the myocardium (Fig. 6c, d), suggesting that miR144 recapitulates the early protective kinase response characteristic of the preconditioned phenotype [27].

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Circulating miR-144 after rIPC and possible mechanisms of transport Because of prior observations that rIPC releases a humoral cardioprotective factor into the bloodstream, we then examined the effects of rIPC on circulating levels of miR144. Fifteen minutes after completion of rIPC, there was an approximate twofold increase in plasma miR-144 levels in both rIPC-treated mice and human volunteers as shown in Fig. 7a, b. Plasma transport of miR-144 Given the known role of exosomes and microvesicles as important transport vehicles of miRNA’s in the circulation [7], we subsequently examined plasma microparticle (MP) responses to rIPC. Following separation and re-suspension, there was no difference in MP (50–400 nm) numbers as analysed by NanoSight (Fig. 8) and, using stem loop RTPCR analysis, there was a non-significant increase in miR144 (Fig. 9a). However, there was an almost fourfold increase in miR-144 precursor (Fig. 9b) in the exosome pellet, and a significant increase in miR-144 levels in exosome-poor serum (Fig. 9c). To further investigate the binding of extracellular miRNA to Argonaute-2 (Ago2), a known extracellular miRNA carrier, anti-Ago2 immunoprecipitates were subjected to TaqMan miRNA Assay. Our

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Fig. 4 Myocardial miR-144 level after systemic administration of miR-144. a Myocardial miR-144 level after 60-min injection, and 1 day after 3 days of miR-144 intravenous administration. miR-144 levels were increased over twofold, compared to PBS control. n = 5 per group (b, c), mouse cardiac miR-144 levels after IR injury was higher in mouse heart both by pretreatment with intravenous miR-144 (b, n = 3–6 per group) and rIPC (c, n = 3–4 per group). Data are shown as mean ± SEM. Statistical significance is shown as *p \ 0.05 vs. PBS

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results show that Ago2-bound miR-144 levels increase following rIPC (Fig. 9d), suggesting that Ago2 also plays a role in extracellular miR-144 transport.

Discussion To our knowledge, this is the first study to examine the role of miR-144 after remote ischemic preconditioning (rIPC). Our results show that miR-144 plays a central role in the cardioprotection afforded by rIPC. First, we showed that rIPC was associated with increased myocardial expression of miR-144 and that prior rIPC and pretreatment with intravenous miR-144 homologue oligonucleotide rescued the fall in miR-144 levels seen with ischemia–reperfusion (IR) injury. We then showed, as expected, that rIPC reduced infarct size and improved functional recovery in isolated hearts subjected to IR injury, and that these effects were completely abrogated by pretreatment of the donor animals with an antisense oligonucleotide against miR-144. Importantly, the effects of rIPC were recapitulated by intravenous administration of miR-144 homologue oligonucleotide, there being an early window (associated with induced autophagy and increased phospho-Akt, phosphoGSK and phospho-p44/42 MAPK signal) within 60 min of administration and a delayed window of cardioprotection demonstrable 24 h after three daily injections of miR-144.

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Having subsequently demonstrated that plasma miR-144 levels are increased in mouse and humans subjected to rIPC, we then examined the possible mechanisms of plasma transport of miR-144 as theoretically free microRNA in the plasma should be digested by plasma RNase. Previous studies have shown that cardioprotective microRNA’s are carried in extracellular fluids within exosomes and microparticles [3]; however, we were unable to demonstrate that rIPC was associated with increased number or miR-144 levels in plasma-borne microparticles

b Fig. 5 Intravenous miR-144 provides early and delayed cardiopro-

tection. a, c The recovery of left ventricular-developed pressure (LVDP) was improved in miR-144 Day 1 and miR-144 Day 3 groups. b, d Diastolic recovery (LVEDP left ventricular end-diastolic pressure). Asterisk denotes a statistically significant difference between miR-144 Day 1 and miR-144 Day 3 groups vs. PBS (*p \ 0.05) after 60-min reperfusion. e, f Myocardial infarct size was measured by TTC staining. A representative basal left ventricular section is presented for each group. There was significant cardioprotection 60 min after a single injection, and 1 day after three daily injections of miR-144. n = 4–8 per group. Data are shown as mean ± SEM. Statistical significance is shown as *p \ 0.05 vs. PBS

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GAPDH 37KDa Fig. 6 miR-144 regulation of the autophagy pathway. a Representative western blot and b quantification of P-mTOR, mTOR, Atg5, LC3 II, Cathepsin L, and p62 protein expression in the mouse heart 1 h after miR-144 injection. c Representative western blot and

d quantification of P-Akt, P-GSK,and P-p44/42 MAPK protein expression in the myocardium 1 h after miR-144 injection. n = 5 per group. Data are shown as mean ± SEM. Statistical significance is shown as *p \ 0.05 vs. PBS. §p \ 0.01 vs. PBS

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Fig. 7 The effects of rIPC on circulating human and mouse miR-144 levels. Plasma was collected from mice and human to measure miR144 levels using MicroRNA Stem-Loop RT-PCR. a Plasma miR-144 levels in mice subjected to rIPC (4 9 5 min cycles of limb ischemia/ 5 min reperfusion), there was a twofold increase in circulating miR144 levels. [*p \ 0.05, control (n = 9), rIPC (n = 8)]. b Circulating

miR-144 levels before and after rIPC in eight human volunteers. rIPC was administered using a blood pressure cuff around the upper arm. Blood was collected before and after rIPC. There was a 1.6-fold increase in miR-144 levels following rIPC. Data are shown as mean ± SEM. Statistical significance is shown as *p \ 0.05 vs. control, §p \ 0.01 vs. pre-rIPC

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Exosome Size (nm) Fig. 8 Exosomes were isolated from serum of rIPC-treated and control animals using ExoQuick precipitation solution. The exosome extract was diluted 1:20 for analysis with the NanoSight. Data collection were performed using NanoSight software (V. 2.3) with the detection threshold set at 6 to maximize sensitivity while minimizing

noise. Duplicate measurements were made for each sample (n = 5 per group). Overall, there was no difference in the absolute numbers of circulating exosomes following rIPC. The inset panel shows a representative EM image of the exosome sample, and western blotting shows positive binding with the exosomal membrane marker CD63

(50–400 nm). Instead, we showed that there was a fourfold increase in hairpin miR-144 precursor in the exosomal fraction, and a marked increase in miR-144 levels in the exosome-free plasma supernatant, suggesting an alternate plasma carriage mechanism. We therefore examined a relatively recently described mechanism of miR-transport, carriage within complexes of the protein Argonaute-2 (Ago-2) [4]. In subsequent experiments, we were able to demonstrate co-immunoprecipitation of plasma miR-144 and Ago-2 suggesting this as the mechanism of plasma transport of miR-144 into heart after rIPC, and possibly after intravenous administration. These novel observations

establish a pivotal role for miR-144 in the cardioprotection associated with rIPC, and raise the possibility of miR-144 as a potential cardioprotective therapy. MicroRNA-144 was originally identified as an erythroid-specific miRNA, which is required for subsequent survival and maturation of the erythroid lineage [9, 11]. dos Santos et al. [46] observed that miR-144/451 null erythrocytes were more sensitive to oxidative stress, and a subsequent study showed that overexpression of microRNA-144/451 enhances nuclear FOXO3a activity, which protects erythroid cells against oxidant stress. In cancer cells, miR-144 has been shown to reduce TRAIL-induced

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B precursor miR -144 level in mouse serum exosome (compare t o cont rol)

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Fig. 9 The levels of miR-144 in circulating serum exosomes. Exosomes were isolated from mouse serum using ExoQuick. a miR-144 level in mouse serum exosome was measured using stem loop RT-PCR. n = 9–10 per group. b Precursor miR-144 levels in serum exosomes was determined by miScript Precursor Assay. n = 8–10 per group. c Following exosome isolation, miR-144 levels were measured in the supernatant. miR-144 levels were significantly

increased in exosome-poor supernatant after rIPC. n = 5–6 per group. d To elucidate a potential extracellular miRNA transport mechanism, the binding of miR-144 to Ago2 protein in blood serum by subjecting anti-Ago2 immunoprecipitates to TaqMan miRNA assay was performed. Ago2-bound miR-144 levels were increased following rIPC. n = 5 per group. Data are shown as mean ± SEM. Statistical significance is shown as *p \ 0.05 vs. control

apoptosis by targeting caspase-3 [31]. These studies have helped establish that miR-144 is a cytoprotective miRNA. There have only been two studies evaluating the effects of miR-144 in the myocardium. Using gain-of-function and loss-of-function approaches, Zhang et al. [48] determined the functional role of the miR-144/451 cluster (miR-144 and 451 are co-expressed and processed from a single gene locus) in cardiomyocyte death under simulated ischemic conditions. They observed that overexpression of either miR-144 or miR-451 augmented in vitro cardiomyocyte survival in response to stimulated ischemia–reperfusion injury. However, in a subsequent study, the same group observed that local IPC-induced myocardial functional recovery was impaired in antagomiR-451 treated, but not in antagomiR-144 treated hearts using a murine IPC model and suggested that, in local IPC at least, miR-144 was not an important regulator of cytoprotection in local

preconditioning [42]. Interestingly, in a recent study of the miR expression in response to local preconditioning in rats, neither miR-144 nor miR-451 appeared to play a role in the responses, perhaps suggesting species-specific differences [40]. Our data suggest the role of the miR-144/451 cluster may be quite different in remote preconditioning. Our array and PCR data showed that miR-451 was not significantly altered in the myocardium after rIPC. Conversely, miR-144 was significantly increased in the myocardium after rIPC induced by transient limb ischemia, and was associated with changes in the expression of known downstream targets. In this regard, we examined the effects of miR-144 on mTOR expression. mTOR is a negative regulator of autophagy, which in turn is a key regulator of cellular survival. There are two specific binding sites in the mTOR 30 UTR region with perfect Watson–Crick matches at

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miRNA positions 1–7 and 2–8 [19]. The interaction of miR-144 and mTOR have been examined in human cancer biology, those tumors with greatest miR-144 expression having the lowest potential for proliferation, and improved clinical prognosis [19]. This beneficial effect of miR-144 to promote autophagy signaling may also be relevant to cardioprotection. Increased autophagy signaling via suppression of mTOR is associated with improved cardiomyocyte survival [1], and in a recent study in mice, the cardioprotection induced by remote limb ischemia was associated with increased autophagy signaling, although mTOR was not specifically examined [14]. Our data are compatible with these prior data, and suggest that miR-144 may be a specific mediator of these previous observations. Increased autophagy may also be expected to modify chronic remodeling responses to myocardial infarction and, while speculative, may contribute to the benefits on myocardial recovery previously shown with ‘chronic conditioning’ when rIPC is delivered daily for 28 days after MI [43]. These potential effects of miR-144, and rIPC, are potentially very important in many ways, and should form the basis of further studies. We also showed that miR-144 induced a pro-survival signaling kinase signature (increased P-Akt, P-GSK,and P-p44/42 MAPK protein expression—Fig. 5c), characteristic of that described in local preconditioning by the Yellon group [15], and our findings in rIPC [27, 34]. Examination of the mechanisms of induction of this kinase response was beyond the scope of the current study, but the rapid nature of the response (within 1 h) would suggest that it may not be a post-transcriptional effect. In this regard, it is interesting to note that it has recently been shown that autophagy-associated proteins themselves (e.g., ATG5, LC3, increased in response to miR-144 in our studies) may increase phosphorylation of ERK [29]; however, the mechanism for this un-anticipated rapid induction of pro-survival kinases by miR-144 clearly requires future study. These important systemic roles of miR-144 in the cardioprotection afforded by rIPC were confirmed by studies using intravenous miR-144 homologue and antagomir. Intravenous administration of antagomiR-144 led to reduced myocardial levels of miR-144 and abrogated the cardioprotection induced by limb ischemia rIPC. Conversely, intravenous miR-144 recapitulated the degree of cardioprotection observed with rIPC. Going along with this, we showed that circulating miR-144 levels were augmented after rIPC in mice and humans, suggesting that it may represent a ‘humoral’ factor previously identified, but not characterized in studies of rIPC induced by transient limb ischemia and other peripheral stimuli [33, 34, 37]. Nonetheless, it is possible that there will be more than one humoral factor associated with the rIPC stimulus. Davidson et al. [5] recently showed that the rIPC-induced

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cardioprotection was associated with increased circulating levels of the chemokine, stromal cell-derived factor-1 (SDF-1), and increased myocardial expression of its receptor protein CXCR4. However, its cardioprotection was only partially abrogated by the CXCR4 blocker, AMD3100, suggesting that SDF-1-CXCR4 pathway may have a limited or secondary role [5]. Our data strongly indicate a primary role of miR-144 as a circulating effector of rIPC-induced cardioprotection. Interestingly, increased circulating levels of miR-144 have previously been demonstrated in human volunteers subjected to mental stress. Katsuura et al. [21] showed significant elevation of miR144/144* and miR-16 levels in medical students immediately, and for several weeks, after participating in a national examination. The possible mechanisms of plasma transport were not examined in the Katsuura study, but clearly ‘free’ microRNA (and in our studies, plasma-borne mirR-144 after rIPC, or after intravenous administration) would be expected to be rapidly digested by circulating RNase in the plasma. We therefore examined two possible protective carriage mechanisms. Several studies have shown that small microvesicles, exosomes and microparticles, frequently carry miR’s in extracellular fluids and plasma. Intriguingly, Giricz et al. [12] have recently demonstrated that extracellular vesicles in the effluent from hearts subjected to local IPC can induce cardioprotection in naı¨ve myocardium, although the active constituent of these exosomes was not examined. Interestingly, we were neither able to show an increase in circulating microparticle numbers (over a range of 50–400 nm—see Fig. 6), nor were we able to show a significant increase in miR-144 in the exosomal fraction after rIPC. That said, our method of exosome isolation, purification, and included microparticle size was different to those described by Giricz et al. Furthermore, because we used whole plasma samples (perhaps reducing our signal-to-noise ratio) rather than crystalloid effluent, it is possible that we failed to demonstrate a change in exosome number because of these methodologic differences, and while the effect of anticoagulant (e.g., EDTA versus citrate) on our ability accurately to measure exosome numbers remains to be determined, there may be an advantage to the use of citrate in this regard [13]. However, we were able to show a fourfold increase in miR144 precursor hairpin oligonucleotide in the exosomes, and increased miR-144 in the exosome-poor serum supernatant. We then showed that miR-144 immunoprecipitates with the argonaute protein, AGO-2. Argonaute proteins are evolutionarily conserved, ubiquitously expressed, and bind to siRNAs or miRNAs to guide post-transcriptional gene silencing either by destabilization of the mRNA or by translational repression [18]. Taken together, our results suggest that rIPC increases miR-144 precursor in circulating exosomes, which liberates miR-144 into the serum,

Basic Res Cardiol (2014) 109:423

which then binds to AGO-2 protein for carriage in circulation. Consequently, while further studies are clearly required, we speculate that the endogenous release of miR144 may act as a biomarker of preconditioning, facilitating the study of the effectiveness, dose–response, and abrogation of rIPC in clinical trials. Furthermore, the systemic administration of miR-144, or agents to increase its expression or activity, may represent a novel therapeutic avenue for protection against ischemia–reperfusion injury in cardiovascular disease such as myocardial infarction and stroke. There are several other limitations to our study. Firstly, there are several hundred potential targets of miR-144, and we studied only cardioprotective pathways, and did not directly assess other targets or RNA silencing. Consequently, additional and potential adverse effects of miR144 cannot be estimated from our studies. We did not perform a dose–response study of miR-144. Instead, we extrapolated the dose used from Dimmeler’s study [2]. As a result, our data may not represent the optimal cardioprotection that could be achieved by systemic administration of miR-144, and further studies will be needed to establish optimal dosing and pharmacokinetics, now that its role in remote cardioprotection is established. Similarly, in our studies of systemic release of miR-144 after rIPC, we only performed a single rIPC protocol (4 9 5 min cycles of limb ischemia/reperfusion), with measurement of circulating levels at baseline and 15 min after the end of rIPC. Our subsequent studies of cardioprotection were performed in isolated hearts. While this study design allows the dissection of specific myocardial effects of rIPC and miR-144, it fails to study the possible additional effects of these interventions on other potential mediators of IR injury, such as platelet and neutrophil effects. Indeed, given the known effect of rIPC on neutrophil function, for example, it is possible that we have underestimated the overall benefit, and future studies will require in vivo assessment of cardioprotection. Finally, to establish whether a circulating miR-144 level may be a biomarker of preconditioning, future studies should establish the site of release and relationship of miR-144 levels to, for example, the number and duration of rIPC cycles, to the effect of known antagonists of rIPC, and the relationship between levels of miR-144 and the degree of cardioprotection. In summary, our findings describe a pivotal role of miR144 in rIPC-induced cardioprotection. As such, it may represent both a biomarker of rIPC as well as, at least one of, the humoral factor(s) previously associated with the cardioprotection induced by transient limb ischemia. Furthermore, beyond its role in rIPC, miR-144 represents a potential therapy to modify the consequences of ischemia– reperfusion injury in a variety of cardiovascular diseases.

Page 13 of 15 Acknowledgments This work was supported by grants from Foundation Leducq and The Canadian Institutes of Health Research. Conflict of interest ANR has applied for a patent in regard to the properties of miR-144.

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