Abstract

   Ischemia reperfusion (I/R) injury is an unavoidable event occurring
   during heart transplantation, leading to graft failures and lower
   long-term survival rate of the recipient. Several studies have
   demonstrated that microRNAs (miRNAs) are vital regulators of signalling
   pathways involved in I/R injury. The present study aims to quantify the
   altered expression levels of miRNA and mRNA upon I/R injury in a mouse
   heart transplantation model, and to investigate whether these miRNA can
   regulate genes involved in I/R injury. We performed heterotopic heart
   transplantation on mouse models to generate heart tissue samples with
   I/R and non-I/R (control). The expression levels of miRNAs as well as
   genes were measured in heart grafts by microarray and real time RT-PCR.
   miRNA alteration in cardiomyocytes exposed to hypoxia was also detected
   by qRT-PCR. We observed significant alterations in miRNA and gene
   expression profile after I/R injury. There were 39 miRNAs significantly
   downregulated and 20 upregulated up to 1.5 fold in heart grafts with
   I/R injury compared with the grafts without I/R. 48 genes were observed
   with 3 fold change and p<0.05 and 18 signalling pathways were enriched
   using Keggs pathway library. Additionally, hypoxia/reperfusion induced
   primary cardiomyocyte apoptosis and altered miRNA expression profiles.
   In conclusion, this is the first report on miRNA expression profile for
   heart transplantation associated with I/R injury. These findings
   provide us with an insight into the role of miRNA in I/R injury in
   heart transplantation.

Introduction

   Since the 1970s, heart failure (HF) prevalence has been increasing in
   the world as a consequence of a decline in coronary artery and
   cerebrovascular disease mortality [44][1]. Although there are many
   treatments available for HF patients, heart transplantation remains the
   best option for long-term survival for end-stage HF patients [45][2].
   However, this effective treatment for heart failure is severely
   affected by ischemia reperfusion (I/R) injury occurring during
   transplantation.

   Despite major achievements in heart transplantation, I/R injury is a
   major contributing factor in graft failure and longer ischemia time has
   shown to lower the long-term survival rate, especially for older
   patients [46][3]. In addition, due to a shortage of donors, physicians
   are forced to enlarge the donor pool by accepting marginal organs,
   which include organs from elderly or ill patients, and thus they are
   more susceptible to I/R injury [47][3]. Currently, there are no
   effective treatments against ischemia reperfusion injury. It is
   important to explore new alternative mechanisms involved in I/R injury
   during heart transplantation.

   miRNAs are endogenous, short, non-coding single-stranded RNAs that are
   approximately 20 nucleotides in length. miRNAs have emerged as a key
   player in physiology as well as pathophysiology attributable to its
   ability to downregulate gene expression through mRNA
   destabilization/degradation and translation repression by binding onto
   either 3′ UTR or 5′UTR of the mRNA [48][4]. Several studies have shown
   that miRNAs have the ability to regulate the expression profiles of
   genes in signalling pathways associated with heart diseases, including
   heart failure, hypertrophy, and ischemia reperfusion injury [49][5].
   Therefore, it is crucial to examine the role of miRNA in heart
   transplantation and its implications for I/R signalling pathways. In
   this study, for the first time, we report miRNA expression profiles in
   I/R injured heart grafts and also investigated mRNA expression profiles
   that may be affected by miRNAs.

Materials and Methods

Animals

   Eight weeks old C57BL/6 mice were purchased from Charles River
   Laboratory (Canada). All procedures involving mouse breeding and
   surgery were performed according to the guidelines of the Canadian
   Council of Animal Care and were approved by the Animal Use Subcommittee
   at the University of Western Ontario, Canada.

Induction of Cold Ischemia Reperfusion Injury and Heart Transplantation

   C57BL/6 mice were anesthetized with ketamine/protophin and injected
   with 1 ml heparin. Donor hearts were excised from mice and
   heterotopically implanted into the peritoneal cavity with the donor
   aorta anastomosed to the recipient abdominal aorta and the pulmonary
   artery connected to the inferior vena cava. For induction of cold I/R
   injury, donor hearts were preserved with University of Wisconsin (UW)
   solution at 4°C for 18 hours before implantation. Meanwhile, the rest
   of the excised heart was immediately implanted into the recipient to
   generate non cold ischemia injury heart (non-I/R) as controls. At the
   endpoint of experiments, mice were sacrificed by injection over dose of
   ketamine/protophin and heart grafts were harvested for future studies.

Histological Analysis

   At 24 hours post-transplantation, heart grafts were collected from mice
   and tissue slices were fixed in 10% formalin and processed for
   histology examination using standard techniques. Formalin tissue was
   embedded in paraffin and 5 µm sections were stained with hematoxylin
   and eosin stain (H&E).

Myeloperoxidase (MPO) Activity

   To detect neutrophil infiltration, MPO activity was detected in heart
   tissues. Paraffin tissue sections were stained with MPO antibody (Santa
   Cruz, San Diego, CA) following the manufacturer’s instruction. Each
   slide was examined by light microscopy at 200× magnification.

Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labelling
(TUNEL) Assay

   To detect cell apoptosis in heart grafts, TUNEL assay was performed on
   paraffin tissue sections using an in situ cell death detection kit
   according to the manufacturer’s instruction (Roche, Mississauga, ON,
   Canada). Sections were counter-stained with hematoxylin. Each slide was
   examined by light microscopy at 200× magnification.

microRNA/RNA Extraction

   Fresh heart tissues or primary cardiomyocytes were collected and
   subjected to the extraction of miRNA/RNA. miRNAs were extracted using a
   miRNeasy mini Kit (Qiagen, Ontario, Canada). RNA quality was assessed
   using the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Palo
   Alto, CA) and the RNA 6000 Nano kit (Caliper Life Sciences, Mountain
   View, CA). One microgram of total RNA was used to synthesize cDNA for
   measuring miRNA expression using miRScript II kit (Qiagen, Ontario,
   Canada) according to the manufacturer’s manual.

miRNA Microarray

   All sample labeling and GeneChip processing were performed at the
   London Regional Genomics Centre (Robarts Research Institute, London,
   Ontario, Canada; [50]http://www.lrgc.ca). One microgram of total RNA
   was labeled using the Flash Tag Biotin HSR kit from Genisphere
   ([51]http://www.genisphere.com/array_detection_flashtag_biotin.html).
   Samples were then hybridized to Affymetrix miRNA 3.0 arrays for 16
   hours at 48°C. All washing steps were performed by a GeneChip Fluidics
   Station 450 and GeneChips were scanned with the GeneChip Scanner 3000 7
   G (Affymetrix, Santa Clara, CA) using Command Console v3.2.4. Partek
   was used to determine ANOVA p-values and fold changes for miRNAs.
   Species annotations were added and used to filter only those miRNA
   found in Mus musculus. miRNA expression data were submitted to the
   National Center for Biotechnology Information Gene Expression Omnibus
   ([52]http://www.ncbi.nlm.nih.gov/geo/) under accession number
   [53]GSE50885.

Gene Expression Microarray

   Single stranded complementary DNA (sscDNA) was prepared from 200 ng of
   total RNA as per the Ambion WT Expression Kit for Affymetrix GeneChip
   Whole Transcript WT Expression Arrays
   ([54]http://www.ambion.com/techlib/prot/fm_4411973.pdf, Applied
   Biosystems, Carlsbad, CA). Total RNA was first converted to cDNA,
   followed by in vitro transcription to make cRNA. 5.5 µg of single
   stranded cDNA was synthesized; end labeled and hybridized, for 16 hours
   at 45°C, to Mouse Gene 1.0 ST arrays. All washing steps were performed
   by a GeneChip Fluidics Station 450 and GeneChips were scanned with the
   GeneChip Scanner 3000 7 G (Affymetrix, Santa Clara, CA) using Command
   Console v3.2.4. Partek was used to determine ANOVA p-values and fold
   changes for genes. Gene expression data were submitted to the National
   Center for Biotechnology Information Gene Expression Omnibus
   ([55]http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE
   50884.

Quantitative Reverse Transcriptase-polymerase Reaction (q-PCR) for miRNA
Expression

   miRNAs were extracted from I/R and non-I/R heart tissues using miRNeasy
   mini Kit (Qiagen, Ontario,Canada), then cDNA were synthesized using
   miRScript II RT Kit (Qiagen, Ontario). Synthesized cDNA samples were
   then subjected to real time RT-PCR (Stratagene mx3005P) using miRScript
   SYBR Green PCR Kit (Qiagen, Ontario) according to instruction of the
   kit supplier. Thermal profiling for the real time PCR was an initial
   activation step at 95°C for 10 mins, followed by 40 cycles of: 95°C for
   15 s, 55°C for 30 s, 70°C for 30 s. Expression levels between I/R and
   non-I/R were quantitatively compared using the ΔΔCt method with SNorD6
   as the endogenous control for miRNA expression.

Primary Neonatal Cardiomyocytes Culture

   Neonatal ventricular cardiomyocytes were cultured as described by the
   Feng group [56][6]. Briefly, ventricular tissues from C57BL/6 mice were
   isolated and minced within 24 h after birth. Subsequently,
   cardiomyocytes were dispersed by incubation in a D-Hank’s buffer
   supplemented with 0.5 mg/ml Liberase (Roche, Worthington Biochemical,
   Lakewood, NJ) and the cellular suspension was filtered through a
   polypropylene macro porous filter (mesh opening 105 µm, Spectra Mesh;
   Spectrum Medical Industries). The suspension was then centrifuged at
   200 g for 5 min, and the cellular pellet was suspended in medium 199
   (M199) with 10% FBS and penicillin-streptomycin (50 µg/ml; GIBCO-BRL).
   The cellular suspension was pre-plated for 1.5 h at 37°C in 5% CO[2] to
   remove any non-cardiomyocytes. Cell density was adjusted to 10^6
   cells/ml using M199 supplemented with 10% FBS, and cells were seeded (5
   × 10^5) in polystyrene, nonpyrogenic24-well culture plates (Becton
   Dickinson, Franklin Lakes, NJ) precoated with 1% gelatin. Cells were
   incubated in 5% CO[2] at 37°C.

Hypoxia/Reperfusion Model for Cell Culture

   Isolated primary cardiomyocytes were cultured in a 24 well plate
   pre-coated with 1% gelatine for overnight. Culture medium were replaced
   by 250 µl deoxygenated DMEM medium without FBS and antibiotics and then
   placed in a chamber with 2% O[2] at 37°C for 45 min. After hypoxia,
   cells were reoxygenated by adding 250 µl of complete medium
   supplemented with 10% FBS and cultured at 5% CO[2,] 37°C, for 24 h.

Annexin-V Staining and Flow Cytometry

   Cardiomyocytes were harvested using Trypsin and suspended in PBS
   solution with 2% FBS. Cells were double stained with Annexin-V and PI
   using Annexin-V kit (ebioscience, San Diego, CA) according to the
   manufacturer’s instruction. The fluorescence from the stained cells was
   measured by flow cytometry (BD bioscience, San Jose, CA).

Potential miRNA Targets

   For selected miRNA that were significantly altered by I/R injury in
   heart transplantation, online computational algorithm analyses
   (Targetscan and FINDTAR3) were used to predict potential target genes.
   Based on scientific literature, target genes that are known to be
   involved in I/R injury pathways were selected for further analysis.

Gene Expression

   Total RNA isolations were performed using the TRIzol reagent
   (Invitrogen), and cDNA was synthesized using oligo-(dT) primer and
   reverse transcriptase (Invitrogen) according to the manufacturer’s
   protocol. Synthesized cDNA samples were then subjected to real time
   RT-PCR (Stratagene mx3005P and Bio-Rad CFX) with SYBR Green and a final
   primer concentration of 80 nM. Thermal profiling for the real time PCR
   was the initial activation step at 95°C for 10 mins, followed by 40
   cycles of: 95°C for 30 s, 58°C for 30 s, 72°C for 30 s. Expression
   levels between I/R and non-I/R were quantitatively compared using the
   ΔCt method with mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
   as the endogenous control for target gene expression.

Western Blotting

   Heart tissues were homogenized with RIPA buffer containing 5 µg/ml PMSF
   as the manufacturer’s instruction (Invitrogen, Life Tech., Ontario,
   Canada) and centrifuged at 10,000 g for 10 mins. Protein concentration
   was measured using a Bradford assay (Bio-Rad, Mississauga, Ontario,
   Canada). 40 µg of total proteins were separated by 12% SDS-PAGE and
   transferred onto nitrocellulose membranes. After blocking for 1 h in
   TBS supplemented with 5% milk and 0.1% Tween20, membranes were blotted
   with rabbit anti mouse polyclone angiopoietin 1 Ab (used at 1∶500
   dilution, Abcam. Toronto, ON, Canada) and developed using
   HRP-conjugated anti-rabbit IgG (1∶5,000 dilution) (Santa Cruz, Dallas,
   Texas) and enhanced chemiluminescence (Bio-Rad).

Statistical Analysis

   All data are presented as means ± SEM. Statistical comparisons between
   two groups were performed using student’s t-test and ANOVA was applied
   for the microarray data. Statistical significance was determined as
   P<0.05.

Results

I/R Induced Cardio Graft Damage

   Currently during organ transplantations, donor organs are preserved
   using a hypothermal static method to reduce ischemia reperfusion
   injury. Thus, I/R injury in organ transplantation is usually referred
   to as cold I/R, differentiating it from that in non-transplanted.
   Accordingly, we used this hypothermal static method to preserve donor
   organs for a variety of periods in order to induce cold
   ischemia-reperfusion injury. Histopathological staining (H&E, apoptosis
   and neutrophil infiltration) were applied to assess the severity of I/R
   injury in grafts. We found that 18 h cold ischemia induced I/R injury -
   supported by results from H&E, TUNEL assay, and MPO assay. H&E staining
   ([57]Figure 1A) shows disrupted architecture, necrosis, and
   degeneration in I/R groups compared to the control (non-I/R) group.
   TUNEL assay ([58]Figure 1B) shows an increased number of apoptotic
   cells in I/R groups. As shown by MPO assay ([59]Figure 1C), increased
   myeloperoxidase activity was observed in the I/R group, indicating
   there are neutrophils infiltrating into grafts.

Figure 1. 18 h cold ischemia-reperfusion caused I/R injury in heart grafts.

   [60]Figure 1
   [61]Open in a new tab

   Donor hearts were isolated from C57BL/6 mice, infused with UW solution
   and preserved in UW solution at 4°C for 18 h. After 18 h preservation,
   donor hearts were implanted into syngeneic recipient C57BL/6 mice. At
   day 2 after transplantation, the heart grafts were harvested and fixed
   in 10% formalin. The paraffin heart sections were subjected to HE
   staining, TUNEL assay, and MPO assay. (A) HE staining. (B) TUNEL assay
   for apoptosis. (C) MPO assay. Representative images were from
   experiments.

I/R Injury Altered miRNA Expression Profiles in Heart Grafts

   In order to characterize the miRNA expression profile that regulates
   genes involved in I/R injury in heart transplantation, we performed a
   microarray assay using Affymatrix: GeneChip miRNA 3.0 Array that
   contains 1111 mouse probe sequences. Heart grafts (n = 3/group) with
   I/R or with non-I/R (control) were collected to extract miRNA at day 2
   post transplantation. Microarray assays showed that miRNA were
   expressed differentially in heart grafts. A total of 59 miRNA were
   significantly altered with the criteria of 1.5 fold change with P<0.05
   ([62]Table 1). Out of the 59 altered miRNAs, 39 were downregulated in
   heart grafts with I/R injury compared with the grafts without I/R,
   while 20 miRNA were upregulated ([63]Table 1). As shown in a pie graph
   of miRNA distribution based on their fold changes in expression
   ([64]Figure 2A), the majority of altered miRNA (49 out of 59) fell into
   the range of 1.5 to 3 fold up or downregulation. Only ten miRNAs (five
   up-regulated and another five down-regulated) displayed over three fold
   changes between two groups. Each individual altered miRNA with >3 fold
   change is shown in [65]Figure 2B, indicating that the miRNA changes fit
   a Poisson distribution. Additionally, signalling pathway enrichment
   analysis was conducted based on miRNAs with 1.5 fold change and p<0.05,
   71 signalling pathways were selected (enrichment P<0.05) using the Kegg
   pathway library.

Table 1. miRNA altered in heart transplantation.

   miRNA Fold-Change(Exp vs. Control) SS(Error) Fold-Change(Exp vs
   Control) p-value(Exp vs. ontrol)
   mmu-miR-490-5p_st −6.24593 1.23515 Down 0.0203434
   mmu-miR-326_st −5.24883 0.284485 Down 0.00340912
   mmu-miR-490-3p_st −3.65425 1.19612 Down 0.0477261
   mmu-miR-1843b-5p_st −3.2084 0.636683 Down 0.0280223
   mmu-miR-345-3p_st −3.1504 0.691868 Down 0.0325233
   mmu-miR-671-3p_st −2.92478 0.667747 Down 0.0368904
   mmu-miR-24-1-star_st −2.85064 0.446847 Down 0.0232897
   mmu-miR-181a-2-star_st −2.7874 0.358902 Down 0.0183911
   mmu-miR-1943_st −2.62098 0.223826 Down 0.0113917
   mmu-miR-425-star_st −2.37163 0.416558 Down 0.0351811
   mmu-miR-181c-star_st −2.3374 0.0854605 Down 0.00415107
   mmu-miR-185-star_st −2.32242 0.115686 Down 0.00655456
   mmu-miR-491_st −2.2317 0.456292 Down 0.0473795
   mmu-miR-328_st −2.20344 0.412421 Down 0.0434945
   mmu-miR-2183_st −2.11913 0.0630796 Down 0.00381541
   mmu-miR-3058_st −2.01364 0.183612 Down 0.0208451
   mmu-miR-128_st −1.98828 0.0715253 Down 0.00590299
   mmu-miR-874-star_st −1.977 0.110185 Down 0.0111359
   hp_mmu-mir-181a-2_st −1.96297 0.169814 Down 0.0207353
   mmu-miR-423-3p_st −1.93968 0.261325 Down 0.0381555
   mmu-miR-324-3p_st −1.91486 0.201859 Down 0.0287916
   mmu-miR-532-3p_st −1.89947 0.18219 Down 0.0260086
   mmu-miR-181a-1-star_st −1.86983 0.121223 Down 0.0160835
   mmu-miR-467a-star_st −1.822 0.238043 Down 0.0435423
   mmu-miR-433_st −1.78743 0.166247 Down 0.0299398
   mmu-miR-351-star_st −1.75 0.0465557 Down 0.00574918
   mmu-miR-5123_st −1.74629 0.132341 Down 0.024712
   hp_mmu-mir-181a-2_x_st −1.74163 0.175927 Down 0.0362254
   mmu-miR-669a-3p_st −1.69692 0.198551 Down 0.0475402
   mmu-miR-295-star_st −1.68704 0.195156 Down 0.047832
   mmu-miR-470_st −1.64899 0.159001 Down 0.0414325
   mmu-miR-139-3p_st −1.64413 0.0621587 Down 0.012078
   hp_mmu-mir-128-2_x_st −1.61738 0.0527945 Down 0.0105619
   mmu-miR-362-5p_st −1.60748 0.155159 Down 0.0457828
   hp_mmu-mir-133a-1_x_st −1.59055 0.125522 Down 0.0371386
   mmu-miR-190b-star_st −1.55534 0.030848 Down 0.00627936
   mmu-miR-324-5p_st −1.53951 0.0934193 Down 0.0306476
   mmu-miR-210_st −1.52691 0.0256112 Down 0.00543635
   mmu-let-7i-star_st −1.51651 0.0849839 Down 0.0297218
   hp_mmu-mir-128-2_st 1.5022 0.112589 up 0.045064
   hp_mmu-mir-5112_st 1.50223 0.0196402 up 0.00415309
   hp_mmu-mir-466c-1_x_st 1.62092 0.162286 up 0.0463585
   hp_mmu-mir-101a_x_st 1.66217 0.129606 up 0.0306606
   hp_mmu-mir-466b-4_x_st 1.7585 0.158651 up 0.0303406
   mmu-miR-705_st 1.89157 0.269444 up 0.0436911
   hp_mmu-mir-5130_st 1.96821 0.127422 up 0.013874
   mmu-miR-1949_st 2.02064 0.0914098 up 0.00783574
   mmu-miR-5099_st 2.15647 0.30996 up 0.0324799
   mmu-miR-328-star_st 2.46223 0.335471 up 0.0237448
   mmu-miR-5130_st 2.57367 0.653544 up 0.0492977
   mmu-miR-744_st 2.59709 0.0620819 up 0.00185233
   mmu-miR-714_st 2.65014 0.567341 up 0.0383118
   mmu-miR-346-star_st 2.87902 0.78242 up 0.0466927
   mmu-miR-1896_st 2.91753 0.789354 up 0.0457703
   mmu-miR-1982-star_st 3.12965 0.771611 up 0.0379458
   mmu-miR-2137_st 3.14534 0.670176 up 0.0313255
   mmu-miR-3104-5p_st 3.44502 1.09737 up 0.048136
   mmu-miR-1893_st 4.59124 0.681606 up 0.01495
   mmu-miR-711_st 9.14388 3.24 up 0.0435551
   [66]Open in a new tab

   Note: Exp: grafts with 18 h cold-ischemia and reperfusion; Control:
   grafts without 18 h cold ischemia.

Figure 2. miRNA expression in heart grafts.

   [67]Figure 2
   [68]Open in a new tab

   Donor hearts were treated and transplanted into syngeneic recipient
   C57BL/6 mice as described in [69]Figure 1. miRNAwere extracted and
   detected by miRNA array or qPCR at day 2 after transplantation. (A) A
   heat map of miRNA microarrays. (B)A pie graph of miRNA distribution.
   (C). Altered miRNA with its fold change.

   We further confirmed the expression of miRNA selected from miRNA
   microarray results by quantitative PCR ([70]Figure S1). The expressions
   of miR-711, miR-714, miR-744, miR-2137, miR-5130, miR-1892, miR-328,
   miR-346, miR-5099, and miR-705 were significantly upregulated in I/R
   injured heart grafts, while miR-490, miR-491, miR-210, miR-362, miR-24,
   miR-423, miR-128, miR-328, miR -181, and miR-532 were downregulated.
   According to intensity of fluorescence detected on miRNA microarray and
   Ct values of qPCR, the expression levels of these miRNA in heart
   tissues varied to a great extent between each other. For example,
   miR-2137, miR-5130 and miR-5112 were highly expressed in heart tissues;
   miR-490, miR-491, miR-181, miR-362, miR-425, and miR-3104 were
   expressed at quite a low level (Ct value ∼ over 30), whereas 32 out of
   those 58 altered miRNA were expressed at an extremely low level in
   hearts and there were almost no Ct value detected by qPCR.

miRNA Expression Profile was Dynamically Changed in Heart Grafts

   Literature has reported that miRNA expression changes dynamically
   [71][7]. Accordingly, we investigated whether miRNA expression changes
   over time in hearts post transplantation. We extracted grafts with I/R
   and with non-I/R at day 7 after transplantation and detected the
   expression level of miRNAs (miR-711, miR -714, miR-744, miR -2137, miR
   -5130, miR -346, miR -490, miR -491, miR -24, and miR -328). As shown
   in [72]Figure 3, the expressions of miR-711, miR-714, miR-744, miR
   -2137, miR -5130, miR -346, and miR -328 was still upregulated in the
   I/R group, but the expression of miR-24 and miR-490 were downregulated,
   compared to the control group grafts. The expression of miR-491 was
   slightly but not significantly upregulated in I/R injured grafts.
   Compared with grafts taken out on day 2 post-transplantation, the
   expression of miR-2137, miR-714, miR-744, miR -2137, miR -5130, miR
   -346, and miR -328 were slightly decreased, whilst the expression of
   miR-711 continued its upregulation in the I/R injured grafts.

Figure 3. miRNA expression in heart grafts on day 7 post transplantation.

   [73]Figure 3
   [74]Open in a new tab

   Donor hearts were treated and transplanted into syngeneic recipient
   C57BL/6 mice as described in [75]Figure 1. miRNA were extracted and
   detected by qPCR on day 7 after transplantation.

Hypoxia/Reperfusion Incurred Cardiomyocyte Apoptosis and miRNA Expression
Change In Vitro

   Cardiomyocyte is a main component of heart tissue. Given that prolonged
   cold ischemia changed miRNA expression profiles in heart grafts, we
   proposed to determine what the miRNA profile would be in cardiomyocytes
   in response to hypoxia/reperfusion stress in vitro and whether it was
   in line with that from heart tissues in vivo. We isolated and cultured
   primary cardiomyocytes from neonatal mice and subjected them to
   hypoxia/reperfusion stress by exposing them to a 2% O[2] chamber for 45
   mins and then reoxygenation for 24 h by incubation of cells in a 5%
   CO[2] and 95% O[2] incubator. We first measured cell apoptosis/death by
   double staining with Annexin-V and PI to confirm cell injury induced by
   hypoxia/reperfusion. Results showed that 45 mins hypoxia/reperfusion
   induced cardiomyocyte apoptosis evidenced by the increased percentage
   of apoptotic cells ([76]Figure 4A) in hypoxia/reperfusion treated cells
   as compared with cells without hypoxia stress.

Figure 4. Hypoxia induced apoptosis of primary cardiomyocytes and altered
miRNA expression in primary cardiomyocytes.

   [77]Figure 4
   [78]Open in a new tab

   Primary cardiomyocytes were isolated from neonatal C57BL/6 mice
   cultured in vitro and then subjected to a hypoxia environment. Cell
   apoptosis was detected by staining with Annexin–V and followed by flow
   cytometry analysis 24 h after hypoxia. miRNAs were extract from above
   cells and the expression of miRNA were detected by qPCR.

   Next, we measured the expression of miRNA in hypoxia/reperfusion
   -treated cardiomyocytes by qPCR. As compared with cells under normxia,
   miR-711, miR-714, miR-328, miR-346, miR-210, miR-744, miR-5130,
   miR-181a and miR-2137 were significantly over-expressed in
   hypoxia/reperfusion treated cardiomyocytes, while the expression of
   miR-491, miR-211, miR-532, miR-185, miR-425, miR-128, miR-24 was
   down-regulated ([79]Figure 4B). There was no significant difference in
   the expression of miR-490 between the two groups ([80]Figure 4B). As
   expected, miR-2137, miR-210, miR-5130, and miR-328 were highly
   expressed in cardiomyocytes, while miR-490, miR-491, and miR-211 were
   expressed at a low level.

Prolonged Cold I/R Changed Gene Expression Profile and Signalling Pathways in
Heart Grafts

   miRNA functions as a negative regulator of gene expression. Similarly,
   using microarray assay, we investigated the global gene expression
   changes in I/R injured heart grafts. Heart grafts with or without
   prolonged ischemia were harvested at day 2 after implantation and
   subjected to gene expression microarray assay ([81]Table S1). I/R
   greatly altered gene expression profiles in heart grafts as shown in a
   heat map. The expression for the majority of significant altered genes
   (P<0.05) was increased or decreased 1.5–2 fold between two groups and
   also fits in a Poisson distribution ([82]Figure 5A and 5B). Most of the
   altered genes were expressed in heart tissue at a moderate level with
   fluorescence intensities of around 1000–2000 units in microarray
   ([83]Figure 5C). Given a three-fold change and P<0.05 (up and down) in
   differential expression as a cut-off, the number of altered genes was
   reduced to 48; 36 of them were downregulated, and 12 genes were up
   regulated ([84]Table 2). Among them, Angiopoietin 1(ANG1) decreased to
   the greatest degree, which was confirmed by the Western blot
   ([85]Figure 5D).

Figure 5. Gene expression in heart grafts detected by microarray assays.

   [86]Figure 5
   [87]Open in a new tab

   Donor hearts were treated and transplanted into syngeneic C57BL/6
   recipient mice. At day 2 post transplantation, total RNAs were
   extracted from grafts and gene expression in grafts were detected by
   microarray assays. (A) A Poisson distribution of gene expression. (B)
   Fluorescence intensities of altered genes detected by microarray
   assays. (C) Altered genes with two fold changes. (D) Angiopoietin 1
   expression by Western Blotting.

Table 2. Annotated genes regulated in mouse heart transplants.

   Gene p-value Fold-Change (Exp vs. Control) Function annotation
   Angpt1 0.008529 −19.3306 receptor binding
   Asb15 0.0182505 −7.53081
   Gpr22 0.0024004 −6.50553 signal transducer activity
   Lrrc10 0.0475174 −5.97936
   Klhl38 0.0359302 −5.9631
   Grm1 0.0210762 −5.86236 PLC activating G-protein coupled glutamate
   receptor activity
   Il15 0.0122715 −5.71981 cytokine activity
   Iigp1 0.0378235 −5.64458 nucleotide binding
   Art1 0.0219665 −5.48431 NAD+ ADP-ribosyltransferase activity
   Ucp3 0.0087787 −4.93265 oxidative phosphorylation uncoupler activity
   Adh1 0.0416643 −4.91006 nucleotide binding
   Mylk3 0.00744 −4.62612 nucleotide binding
   Fam40b 0.0144888 −4.56915
   Csdc2 0.0223918 −4.44072 nucleic acid binding
   Tmtc1 0.0141671 −4.27416
   Kcnj3 0.0285984 −4.2726 ion channel activity
   Car14 0.0368218 −4.11809 carbonate dehydratase activity
   Asb2 0.0086771 −4.10112
   Tmem100 0.0122261 −4.00128
   Slit2 0.0351409 −3.99089 GTPase inhibitor activity
   Tnni3k 0.01648 −3.90863 nucleotide binding
   Gm4951 0.0269588 −3.81868 no
   Lpin1 0.0219087 −3.66845 RNA polymerase II transcription factor binding
   Filip1l 0.0181653 −3.62773
   Ppp1r3a 0.0463544 −3.49446 protein serine/threonine phosphatase
   activity
   Hfe2 0.0235755 −3.47549 protein binding
   Slc2a4 0.0300101 −3.47473 transporter activity
   Ppargc1a 0.0280838 −3.30582 nucleotide binding
   Mapk10 0.0245692 −3.29025 nucleotide binding
   Slc38a3 0.0377629 −3.24102 L-histidine transmembrane transporter
   activity
   Adck3 0.0251863 −3.2227 nucleotide binding
   Pnmt 0.0497843 −3.13239 phenylethanolamine N-methyltransferase activity
   Fuca2 0.0231706 −3.10097 catalytic activity
   Asb11 0.0384006 −3.07244
   Ube2ql1 0.0294376 −3.04395 nucleotide binding
   Hfe 0.0433132 −3.00196 protein binding
   Trim16 0.0428384 3.10174 DNA binding
   Sfn 0.0394142 3.14658 protein binding
   Tnfrsf23 0.0115847 3.26603 receptor activity
   Apln 0.0152681 3.36778 receptor binding
   Cyr61 0.0336177 3.38045 integrin binding
   Pgf 0.0303102 3.53196 growth factor activity
   Loxl4 0.0059025 3.71021 protein-lysine 6-oxidase activity
   Prnd 0.0159526 4.75449 copper ion binding
   Gdf15 0.0093257 4.8554 growth factor activity
   Fosl1 0.0168812 5.48284 DNA binding
   Plk2 0.0104977 5.50407 nucleotide binding
   Crct1 0.01037 7.60055 protein binding
   [88]Open in a new tab

   Note: Exp: grafts with 18h cold-ischemia and reperfusion;Control:
   grafts without 18 h cold ischemia.

   To clarify which signalling pathways were affected by I/R in heart
   transplantation, we applied the KEGG library and performed enrichment
   analysis for microarray data. Eighteen signalling pathways were
   enriched with the criteria of 2 fold changes and p<0.05 ([89]Table 3),
   which include the Insulin, Tyrosine metabolism, cell cycle, gap
   junction, calcium, energy metabolism, p53, cardio function associated
   signalling pathways and some cancer signalling pathways. Less than 10%
   genes listed on those pathways significantly changed with more than 2
   folds. [90]Figure 6 shows the information of these genes from the 18
   altered signalling pathways listed on [91]Table 3. Most genes from
   those pathways were downregulated, except genes in the p53 and one
   carbon pool by folate, bladder cancer, acute myeloid leukemia and
   transcriptional misregulation in cancer pathways were upregulated. The
   Gap junction pathway evenly consisted of both up and downregulated
   genes.

Table 3. Enriched signalling pathways regulated in heart transplantation.

   Pathway Name EnrichmentScore p-value Enrichment % genes^a # genes[92]^b
   # genes[93]^c # genes[94]^d Database
   Arrhythmogenic right ventricular cardiomyopathy (ARVC) 9.7642 5.75E-05
   9.58904 7 89 6829 kegg
   Calcium signaling pathway 8.48247 0.000207 5.40541 10 86 6720 kegg
   Gap junction 6.56392 0.00141 6.66667 6 90 6811 kegg
   Adipocytokine signaling pathway 5.81675 0.002977 6.94444 5 91 6828 kegg
   Dilated cardiomyopathy 4.86527 0.00771 5.55556 5 91 6810 kegg
   Energy Metabolism 4.60156 0.010036 4 7 89 6727 kegg
   p53 signaling pathway 4.22517 0.014623 5.7971 4 92 6830 kegg
   Insulin signaling pathway 4.17598 0.01536 4.08163 6 90 6754 kegg
   Tyrosine metabolism 3.99802 0.018352 7.31707 3 93 6857 kegg
   Bladder cancer 3.81036 0.02214 6.81818 3 93 6854 kegg
   Hypertrophic cardiomyopathy(HCM) 3.6154 0.026906 4.81928 4 92 6816 kegg
   One carbon pool by folate 3.59657 0.027418 10.5263 2 94 6878 kegg
   Mineral absorption 3.52912 0.029331 6.12245 3 93 6849 kegg
   Fatty acid metabolism 3.477 0.0309 6 3 93 6848 kegg
   Drug metabolism - cytochromeP450 3.46478 0.03128 4.5977 4 92 6812 kegg
   Type II diabetes mellitus 3.28047 0.037611 5.55556 3 93 6844 kegg
   Transcriptional misregulationin cancer 3.27588 0.037784 3.31492 6 90
   6720 kegg
   Acute myeloid leukemia 3.01689 0.048953 5 3 93 6838 kegg
   [95]Open in a new tab

   note: ^a% genes in pathway that are present;
   ^b

   numbers of genes in list and in the pathway;
   ^c

   numbers of genes in list, not in pathway;
   ^d

   numbers of genes not in list, not in pathway.

Figure 6. Genes in 18 altered signalling pathways.

   [96]Figure 6
   [97]Open in a new tab

   Gene expression in the heart grafts was detected by microarrays as
   describe in [98]Figure 5. Signalling pathways were enriched using the
   Kegg library with 2 fold change and p<0.05.

Discussion

   I/R injury is recognized as a primary factor leading to graft
   dysfunction [99][8] and graft failure [100][9], [101][10]. It is
   important to understand molecular mechanisms of I/R injury for the
   development of therapies against I/R injury. The current study, for the
   first time, reported on the expression profile of miRNA in I/R injured
   heart grafts in heart transplantation. The findings of our study
   demonstrate that miR-711, miR-2137 miR-705, miR-5130, miR-346, miR-714,
   and miR-744 were significantly upregulated (>2 fold change) in I/R
   injured hearts, while miR-210, miR-490, miR-491, miR-425, miR-423-3p,
   and miR-532-3p were downregulated. The study also demonstrates that 250
   genes and 18 signalling pathways were significantly altered with more
   than 2 fold changes by I/R injury by cDNA microarray assay.

   microRNAs have emerged as a vital regulator in many physiological and
   pathological pathways. Deregulation of miRNAs associated with different
   forms of ischemia reperfusion injury [102][11]. A substantial number of
   miRNA including miR-1 [103][12], miR-15 [104][13], miR-21 [105][14],
   [106][15], miR-24 [107][16], [108][17], [109][18], miR-499 [110][19],
   and the miR-17-92 family [111][20], miR-124 [112][21], miR-15a/b
   [113][22] (2012), miR-93 [114][23], miR-29 family [115][24], [116][25],
   miR-146a [117][26], [118][27], miR-145/451 [119][28], miR-384-5p
   [120][29], miR-424 [121][30], and miR-494 [122][31] have been
   identified in I/R injury. It has been demonstrated that ischemia
   precondition (IPC) increases cardiac expression of miRNA-1, miRNA-21
   and miRNA-24. IPC-regulated miRNA-21 reduces cell apoptosis by
   repressing the programmed cell death 4 (PDCD4) gene, which results in
   preventing the heart from I/R injury [123][32]. It has also been
   reported that inhibition of miR-15 prevents cardiac ischemia injury in
   a porcine coronary arterial ligation model [124][33]. However, in this
   study we did not observe the above miRNAs change in heart grafts. The
   reason for this might be that miRNA expression changes dynamically and
   miRNA expression varies between different models [125][34], [126][35].

   In this study, we observed that miR-711 was significantly up-regulated
   both in I/R injured heart grafts and hypoxia/reperfusion treated
   primary cardiomyocytes. Despite only a few studies of miR-711 have been
   reported, available data have shown that miR-711 is expressed in many
   types of cells [127][36], [128][37], [129][38] and is upregulated under
   different stresses [130][39], [131][40]. For example, a chemical
   palmitate used to induce insulin resistance increases the expression of
   miR-711 in mouse muscle C2C12 cells [132][41]. A study has also shown
   that miR-711 was significantly upregulated in the myocardium with acute
   myocardial infarction on day 14 post ischemia [133][42]. Tranter et al
   reported shows that cardiac ischemic preconditioning (IPC) of the in
   vivo mouse heart results in decreased levels of miR-711 which was
   dependent on NF-kB, and that miR-711 post-transcriptionally suppresses
   Hsp70.3 [134][43]. A more recent study reported that Pioglitazone (an
   insulin sensitizing drug with cardio protective effect, it attenuates
   cardiac fibrosis) increased miR-711 levels in myocardial infarction
   rats and miR-711 directly targeted and downregulated SP1, leading to
   reduced collagen-I levels [135][44]. However, the expression level of
   SP1 and HSP70 was not altered in I/R injured heart tissues in this
   study (data not shown). It may be attributed to differences in animal
   model and injury. Predicted by TargetScan and FINDTAR3, Angiopoietin 1
   (ANG1) is a putative target of miR-711. Our data also showed that ANG1
   was significantly downregulated in I/R injured hearts and
   hypoxia-treated cardiomyocytes, suggesting ANG1 might be a target of
   miR-711. Supportively, Lee et al [136][45] demonstrated that ANG1 can
   exert cardio protective effects by preventing vascular leakage and
   cardiomyocyte death by inhibiting activities of Caspase 3 and Caspase
   9. Further study on miR-711 function will help us understand the
   regulatory roles of miR-711.

   Additionally, miR-2137, miR-1893, miR-744, miR-705 and miR-714 are
   highly expressed in heart tissue and cardiomyocytes as well, suggesting
   that these miRNA are important for cardiomyocytes survival and growth.
   However, there are no reports available on miR-2137 and 1893 regarding
   their functions. Performing computational analysis using TargetScan,
   two conserved genes, retrograde golgi transport homolog (RGP1) and
   pleckstrin and Sec7 domain containing (Psd), were predicted as targets
   of miR-2137, while another 144 genes including calmodulin binding
   transcription activator 1(Camta1) and furry homolog-like (Fryl) were
   predicted irrespective of site conservation. The data from the gene
   expression microarray assay showed that the Camta1 gene was
   significantly downregulated in I/R injured grafts. It has been shown
   that CAMTA 1 can activate the expression of the anti-proliferative
   cardiac hormone natriuretic peptide A (NPPA) in the heart and is
   recognised as a tumor suppressor [137][46]. Another family member of
   CAMTA, the transcriptional coactivator CAMTA2 stimulates cardiac growth
   by opposing class II histone deacetylases, while the loss of CAMTA2
   promotes cardiomyocyte hypertrophy [138][47]. Taken together, the
   increased expression of miR-2137 may play a role in I/R injury in heart
   transplantation through regulating CAMTA 1.

   miR-705, miR-714 and miR-744 have not been extensively investigated
   yet. It has been reported that the expression of miR-714 and miR-744
   are significantly higher in mice aorta with vascular calcification
   [139][48]. miR-744 is known to be expressed in cardiac valves [140][49]
   and involved in cancer cell growth and proliferation [141][50],
   [142][51]. Recent studies have shown that miR-744 targets TGF-β and
   eukaryotic translation elongation factor 1 alpha 2(eEF1A2) which is
   able to promote cell growth and inhibit apoptosis [143][52], [144][53],
   [145][54]. Our data showed that eEF1A2 was decreased in I/R injured
   hearts, implying that there may be a causative relationship between
   miR-744 and eEF1A2. Predicted by TargetScan, miR-705 has 89 putative
   targets including Transmembrane BAX inhibitor motif containing 1
   (TMBIM1) with conservative sites. TMBIM1 has been well documented as a
   part of the Bax Inhibitor-1(BI-1) family that has a similar
   anti-apoptotic function as Bcl-2 and miR-705 induced inactivation of
   TMBIM1 may be responsible for the increase rate of apoptosis after I/R
   injury [146][55]. Although our cDNA microarray data did not show
   significant changes of TMBIM1 expression at the mRNA level, we can not
   rule out TMBIM1 as a putative target of miR-705 because that miRNA
   functions via translation repression as well. Our data also showed that
   miR-346 was highly expressed in normal heart tissues and upregulated by
   I/R. There are 90 putative targets of miR-346 predicted by the
   TargetScan. Most studies on miR-346 have been investigated in a
   rheumatoid arthritis model [147][56], [148][57]. The expression of
   miR-346 was positively correlated with the severity of ischemic injury
   in a mouse hepatic ischemia/reperfusion injury model [149][58]. It has
   been demonstrated that miR-346 can target receptor-interacting protein
   140 (RIP140), TNFα, Leukemia inhibitory factor (LIF), IL18 and antigen
   peptide transporter 1 (TAP1) [150][56], [151][59], [152][60]. The
   expression of LIF was decreased in the I/R injured heart grafts (data
   not shown), indicating miR-346 may negatively regulate LIF.

   miR-24 has previously shown a protective effect on I/R injury
   [153][16], [154][17], [155][18]. We observed a decrease in the
   expression of miR-24 in the I/R injured heart grafts but it was not
   significant. However, we did see a significant reduction of miR-24 in
   hypoxia-treated primary cardiomyocytes, which is consistent with
   reported literature [156][16], [157][17], [158][18]. Since an entire
   heart tissue consists of multiple lineages of cells such as
   cardiomyocyte, endothelial cell, and fibroblast cell, the expression of
   miR-24 in heart is an accumulation from all cells in the heart, and
   other cells may buffer the reduction of miR-24 in cardiomyocytes.

   The data from gene expression microarray showed that 48 genes (36
   downregulated and 12 up-regulated) were significantly changed with more
   than three fold in prolonged cold I/R. Among them, some genes have been
   demonstrated to be involved in the growth and function of cardiac
   cells. For examples, growth of Ankyrin repeat and suppressor of
   cytokine signaling box-containing protein (ASB) 15 regulates myoblast
   differentiation [159][61]; G protein-coupled receptors 22 (GPCRs 22)
   which are highly expressed in cardiac myocytes and coronary arteries
   plays an essential role in the regulation of cardiac contractile
   function and cardiomyocyte apoptosis [160][27] and Leucine-rich repeat
   containing 10 (Lrrc10), a cardiac-specific factor, is crucial for
   proper cardiac development. Deletion of Lrrc10 in mice results in
   dilated cardiomyopathy [161][62]. Furthermore, uncoupling protein
   3(UCP3) [162][63], angiopoietin 1 [163][45] and growth differentiation
   factor-15 (GDF-15) [164][64] are anti-apoptotic genes and show
   protective effects on I/R injury in non-transplantation settings.
   Therefore, those altered genes might be good targets for the prevention
   of I/R injury in heart transplantation.

   In conclusion, our study demonstrates that ischemia reperfusion injury
   in heart tissue after transplantation is associated with an altered
   miRNA profile, which will help us to understand roles of miRNA in I/R
   injury. In addition, this study provides insights into the mechanisms
   involved in I/R injury and investigate specific miRNA that regulate
   genes associated with signalling pathways involved in I/R injury. As
   miRNAs hold great potential as therapeutic targets, expression levels
   can be modified to prevent, or revert ischemia reperfusion injury that
   occurs during heart transplantation - minimizing graft failure and
   increasing graft long-term survival rate.

Supporting Information

   Figure S1

   miRNA expression detected by qPCR. miRNA was extracted from heart
   grafts at day 2 post transplantation as described in [165]Figure 2.
   cDNA was synthesized using miRScript II RT Kit. The expression of miRNA
   was detected by qPCR using SYBRGreen systems.

   (TIF)
   [166]Click here for additional data file.^ (649.2KB, tif)
   Table S1

   Gene expression with two fold changes and p<0.05 in the heart grafts of
   I/R injury vs.non-IR, detected by gene expression microarray assays.

   (XLS)
   [167]Click here for additional data file.^ (72.5KB, xls)

Acknowledgments