Ischaemic post‐conditioning in rats: Responder and non‐responder differ in transcriptome of mitochondrial proteins

Abstract Ischaemic post‐conditioning (IPoC) is a clinical applicable procedure to reduce reperfusion injury. Non‐responsiveness to IPoC possibly caused by co‐morbidities limits its clinical attractiveness. We analysed differences in the expression of mitochondrial proteins between IPoC responder (IPoC‐R) and non‐responder (IPoC‐NR). Eighty rats were randomly grouped to sham, ischaemia/reperfusion (I/R), IPoC or ischaemic pre‐conditioning (IPC, as positive cardioprotective intervention) in vivo. Infarct sizes were quantified by plasma troponin I levels 60 minutes after reperfusion. After 7 days, rats were sacrificed and left ventricular tissue was taken for post hoc analysis. The transcriptome was analysed by qRT‐PCR and small RNA sequencing. Key findings were verified by immunoblots. I/R increased plasma troponin I levels compared to Sham. IPC reduced troponin I compared to I/R, whereas IPoC produced either excellent protection (IPoC‐R) or no protection (IPoC‐NR). Twenty‐one miRs were up‐regulated by I/R and modified by IPoC. qRT‐PCR analysis revealed that IPoC‐R differed from other groups by reduced expression of arginase‐2 and bax, whereas the mitochondrial uncoupling protein (UCP)‐2 was induced in IPC and IPoC‐R. IPoC‐R and IPoC‐NR synergistically increased the expression of non‐mitochondrial proteins like VEGF and SERCA2a independent of the infarct size. Cardiac function was more closely linked to differences in mitochondrial proteins than on regulation of calcium‐handling proteins. In conclusion, healthy rats could not always be protected by IPoC. IPoC‐NR displayed an incomplete responsiveness which is reflected by different changes in the mitochondrial transcriptome compared to IPoC‐R. This study underlines the importance of mitochondrial proteins for successful long‐term outcome.


| INTRODUC TI ON
Today, early reperfusion is the key strategy to overcome acute ischaemia/reperfusion (I/R) injury. However, although successful reperfusion greatly improves the acute outcome, the long-term prognosis of patients depends on subsequent cardiac remodelling. 1 Procedures such as ischaemic pre-conditioning (IPC) or ischaemic post-conditioning (IPoC) have been shown to reduce reperfusion injury and should improve also the long-term outcome. In clinical routine, IPoC is more relevant than IPC, but although studied for years rather extensively it is not yet established in clinical routine. One of the great disadvantages is the relative high number of non-responders. 2 Although this is often associated to co-morbidities such as advanced age, dyslipidemia, diabetes or hypertension as well as to patient-specific medications neither clinical nor pre-clinical studies show a unique relationship between IPoC and protection. [3][4][5] Specifically, in rats, different reports found either no or small protection [6][7][8][9] or effective protection [10][11][12][13][14] with and without co-morbidities. [15][16][17][18] In order to explain the large outcome differences in pre-clinical studies, it may be argued that differences between protocols, source of animals, sex and age of animals, animal handling and other variables are responsible for inconsistent results. To overcome these limitations, we started a study in which experimental variables were minimized. Nevertheless, rats undergoing IPoC showed heterogeneous responsiveness separating them in true responders and non-responders. Transcriptome analysis was used as an unbiased evaluation according to the ESC guidelines to identify differences between both groups. 19 The analysis revealed that IPoC similarly affected transcriptome response in non-mitochondrial proteins but displayed significant differences in the transcriptome of mitochondrial proteins. As a potential source of regulation, differences in mitochondrial transcriptome were ale found for microRNA (miR) related to mitochondria.

| MATERIAL S AND ME THODS
The investigation confirms the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). The study was approved by the local ethics committee of the University of Szeged. GmbH) was given sc. to alleviate post-operative pain. One hour after the onset of reperfusion, venous blood was sampled from the right femoral vein for troponin I determination. Rats were allowed to recover for 7 days after coronary occlusion.

| Experimental groups
Animals were randomly assigned to four experimental groups: (a) sham operated, (b) ischaemic control, (c) ischaemic pre-conditioning (IPC) and (d) ischaemic post-conditioning (IPoC). Sham operated rats underwent the same procedure detailed above without occluding the LAD. IPC was performed by occluding the LAD in three cycles for 3 minutes interrupted with 5 minutes perfusions prior to 30-minute coronary occlusion. IPoC was performed by three cycles of 10-second reperfusion/10-second re-occlusion at the end of 30-minute coronary occlusion. Figure 1 gives an overview about the experimental design. The study protocol confirm to the recommendation given previously. 20

| Echocardiography
Cardiac morphology and function were also assessed by transthoracic echocardiography in the post-ischaemic heart 7 days after 30-minute occlusion and reperfusion of the LAD. Echocardiography was performed as described previously. 21,22 The observer was blinded to the animal groups. Briefly, rats were anaesthetized with sodium pentobarbital (Euthasol, 40 mg/kg bodyweight, i.p.), the chest was shaved and the animal was placed in supine position onto a heating pad. Two-dimensional and M-mode echocardiographic examinations were performed in accordance with the criteria of the American Society of Echocardiography with a Vivid 7 Dimension ultrasound system (General Electric Medical Systems) using a phased array 5.5-12 MHz transducer (10S probe). Data of three consecutive heart cycles were analysed (EchoPac Dimension software, General Electric Medical Systems) and then the mean values of the three measurements were calculated and used for statistical evaluation. Systolic and diastolic wall thicknesses were obtained from parasternal short-axis view at the level of the papillary muscles.
The left ventricle diameters were measured by means of M-mode echocardiography from short-axis views between the endocardial borders. Functional parameters including fractional shortening and ejection fraction were calculated on short-axis view images.

| Measurement of cardiac troponin I release in plasma
Plasma samples were collected into heparinized tubes (Sarstedt) from the femoral vein at the 60th minute of reperfusion, and plasma was separated to determine cardiac troponin I (TnI) release after acute myocardial infarction. Plasma TnI concentration was determined by a conventional ELISA kit (Life Diagnostics, Inc) according to the recommendations of the manufacturer. Briefly, plasma samples were diluted 4 to 40 times according to the treatment protocol (ie, sham or ischaemic) and to previous pre-analyses to get absorbances in the range of standard absorbances. Diluted samples were allowed to react simultaneously with 2 antibodies against rat TnI (1 is immobilized on the microtitre wells, and the other is conjugated to horseradish peroxidase [HRP] in soluble phase), resulting in TnI being sandwiched between the solid phase and HRP-conjugated antibodies. After 1 hour of incubation at room temperature on a plate shaker, the wells were washed with wash solution to remove unbound HRPconjugated antibodies. A solution of tetramethylbenzidine, a HRP substrate, was then added and incubated for 20 minutes, resulting in the development of a blue colour. The colour development was stopped by addition of 1 N HCl, which changed the colour to yellow. The concentration of TnI was proportional to the absorbance at 450 nm.

| RNA isolation and real-time RT-PCR
Total RNA was isolated from the LV using peqGold TriFast (peqlab, Biotechnologie GmbH) according to the manufacturer's protocol. To remove genomic DNA contamination, RNA samples were treated with 1 U DNase/µg RNA (Invitrogen) for 15 minutes at 37°C. One microgram of total RNA was used in a 10 µL reaction to synthesize cDNA   Table S1.
RT-PCR of cardiomyocytes was performed as described before.
The amplification of the PCR products was performed under the fol-

| Prediction of the microRNA-target interaction network
Expected expression changes of target genes due to miR mediated post-transcriptional regulation was predicted by the miRNAtarget™ software (https://mirna target.com; Pharmahungary), which was validated in previous studies. 28  were used to compare our predicted miR-target list and the IMPI list. Mitochondria related miR-target subnetwork was constructed from target genes with a Gene Ontology mitochondrial annotation or with an IMPI score above 0.7 and from miRs interacting with these mitochondrial targets.

| Isolation and cultivation of cardiomyocytes
Ventricular heart muscle cells were isolated from male rats as described in greater detail previously. 38

| Determination of cell contraction
Cell shortening was measured as initially described in greater detail. 39 Briefly, isolated cardiomyocytes were allowed to contract at room temperature and analysed using a cell-edge detection system.
Cells were stimulated via two AgCl electrodes with biphasic electrical stimuli composed of two equal but opposite rectangular 50-V stimuli of 0.5 ms duration. Each cell was stimulated for 1 minutes at a frequency of 2.0 Hz. Every 15 seconds contractions were recorded.
The mean of these four measurements was used to define the cell shortening of a given cell. Cell lengths were measured via a line camera (data recording at 500 Hz). Data are expressed as cell shortening normalized to diastolic cell length (dL/L (%)).

| Immunoblotting
Cells or tissue was lysed in lysis buffer [composition: Tris·Cl

| Statistics
Data are expressed as indicated in the legends. ANOVA and the

| Effect of IPC and IPoC on infarct size, function and hypertrophy
In total 80 rats, subdivided in four different treatment groups (Sham, I/R, IPC, IPoC), underwent surgical procedure and plasma troponin I values, as a surrogate parameter for infarct size, was quantified sixty minutes later (Figure 2A). The data revealed the expected increase of plasma troponin I (TnI) after I/R in comparison to sham and reduced plasma levels after IPC in comparison to I/R. However, with regard to IPoC results were heterogeneous. We determined two groups of rats. One had extremely low TnI levels, subsequently termed the IPoC responders (IPoC-R) and the others had TnI levels indistinguishable from I/R, subsequently termed IPoC non-responders (IPoC-NR). During the following week three rats from the sham group and one rat from the IPC group died and could not be considered further. The subsequent analysis was therefore performed with five groups: Sham (n = 17), I/R (n = 20), IPC (n = 19), IPoC-R (n = 8) and IPoC-NR (12). Seven days after induction of I/R, the subsequent functional analysis showed increased left ventricular end-systolic dimension (LVESD) and fractional shortening (FS) after I/R compared to sham (Table 1). In the three protection groups, FS of the IPC group was better than IPoC-R and both were better than IPoC-NR, with IPoC-NR remain indistinguishable from I/R (Table 1). Similarly, I/R and IPoC-NR had an increased hypertrophy index (heart weight to bodyweight; HW/BW; Figure 2B). However, HW/BW of IPoC-R but not IPC was indistinguishable from sham. These data suggest that rats of the IPoC-NR group did not respond to the protocol but also suggests differences between the long-term adaptation between IPC (better preserved function) and IPoC-R (less hypertrophy but less effective in terms of function compared to IPC). To analyse the response to post-infarct remodelling, transcriptional changes in the different tissues were analysed. Therefore, we randomly selected five samples from each group for further analysis. This was done to have equal groups sizes for the subsequent analysis and keeping at least three samples from the smallest group for Western blotting.

| Effect of IPoC on transcriptome adaptation
Differences in the mRNA expression of different genes between groups are sensitive molecular fingerprints to analyse the effect of protection protocols on the subsequent remodelling. 19 Our focus was on differences between the IPoC-R and IPoC-NR group. As shown in Table 2  the expression of mRNA expression of SERCA2a ( Figure 3A) and vascular endothelial growth factor (VEGF)-A ( Figure 3B). This finding, the strongest signal in IPoC-R, was further validated on the protein level ( Figure 3C). We verified that adult rat ventricular cardiomyocytes constitutively express VEGF and VEGF receptors ( Figure 3D) and found that VEGF induced the expression of SERCA2a ( Figure 3E) and increased load-free cell shortening ( Figure 3F) when administered for 24 hours. Load-free cell shortening was increased by 8.3% from 10.30 ± 0.25% of diastolic cell length to 11.16 ± 0.19%.
Furthermore, we identified JDP2, an inhibitor of activator protein-1 (AP-1), to be specifically down-regulated in IPoC-R. JDP2 showed the strongest difference between IPoC-R and IPoC-NR.
This suggests that down-regulation of JDP2 and thereby activation of AP-1 may be involved in the process of adaptation in IPoC-R.

| Effect of IPoC on mitochondrial proteins
Next, we looked on mitochondrial proteins in more depth as mitochondria are key regulators of cardiac protection. 40  showed the strongest down-regulation in IPoC-NR ( Figure 4).

| Effect of IPoC on microRNA transcriptome
The regulation of mRNA expression depends on the activation of transcription factors that modify the de novo synthesis of mRNA.
Furthermore, the mRNA expression also depends on mRNA stability that can be targeted by miRs. We, therefore, extended our analysis and investigated the effect of IPoC on miRs. In total, 627 miRs were read and 286 miRs showed differential expression if only P-values were considered, from which 28.7% (n = 82) were up-regulated by I/R ( Figure 5). Among them were miR-34b-3p, miR-34c-3p, miR-195-3p, miR-130b-5p and miR-155-5p that were previously also found in pigs therefore are evolutionary conserved. From these miRs 28% (n = 23) were differentially regulated in the IPoC-R group. The strongest effects were on miR-199a-3p and miR-148a-5p (P < .01). Of note, both TA B L E 2 Differentially expressed mRNAs (vs Sham, x-fold, means ± SD) and effect of protection

TA B L E 3
Genes that are differentially expressed by IPoC-R and IPoC-NR and its comparison to IPC were not regulated by IPC. Only one of IPoC-dependent down-regulated miRs that are normally increased by I/R was down-regulated in all three conditioning groups (miR-122-5p). In only one case, the down-regulation by IPoC-R was found for IPC but not IPoC-NR (rno-miR-34c-5p). In four cases, the expression was even further enhanced in IPoC-NRs (miR-455-5p, miR-195-3p, miR-271a-5p and miR-148a-3p).
I/R caused also a down-regulation of 26.9% (n = 77) miRs. Nearly all of them were affected by any conditioning method (94.8%). This effect was influenced in IPoC-R in 26.0% (n = 20). In six cases, this was also seen in IPC-treated rats, therefore linked to smaller infarcts rather than to specific effects of IPoC. In one case, down-regulation was enhanced in IPoC-NRs (miR-133a-3p). At last, 18 miRs that were not differentially expressed by I/R but either reduced (n = 10) or increased (n = 8) in the IPoC-R. The strongest down-regulated miR induced one was miR-532-5p. Neither IPC nor IPoC-NR influenced the expression of these miRs.

| Effect of IPoC on mitochondrial related microRNA-target subnetwork
As mentioned before, mitochondrial proteins are significantly affected by IPoC. To understand the contribution between miR regulation and mitochondrial transcriptome in more depth, mitochondrial related miR-target subnetworks were analysed. In total, we found 21 miRs that are up-regulated by I/R and affected by IPoC.

| D ISCUSS I ON
The most important finding of our study is that IPoC in healthy rats leads to a heterogeneous responsiveness as monitored by an infarct size surrogate parameter. Even in those cases in which IPoC was associated with small infarcts, IPoC differed from IPC, which was always cardioprotective in the same animal model. The main difference was less hypertrophy (as measured by heart weight to bodyweight). However, similarities between IPoC-R and IPoC-NR in the subsequent remodelling process as seen by equal regulation of some mRNAs indicate that the responsiveness to IPoC in IPoC-NRs is rather incomplete than completely missing. Previous studies reported about differences in the anti-arrhythmic property of IPoC and IPC. 41 Thus, differences between both types of In this study, we grouped IPoC-treated rats into responders and non-responders based on TnI plasma levels. In terms of cardioprotection by conditioning, this is not the gold standard. However, we previously showed that plasma TnI correlates with infarct size as quantified by TTC staining that served as gold standard. 20,44 Furthermore, IPC successfully protected the hearts but a heteroge-

2.0
Our study standardized these variables as best as possible. However, although in principle the IPoC protocol was sufficient to protect rats, IPoC reduced infarct size not in all rats, indicating an endogenous heterogeneity. Our study does not identify a master molecule that is responsible for this difference. However, strong differences were found between IPoC-R and IPoC-NR for the expression of JDP2 mRNA. JDP2 is an intrinsic inhibitor of the activator protein-1 (AP-1). Transgenic over-expression of JDP2 reduces the expression of SERCA2a thereby impairing the responsiveness to β-adrenoceptor stimulation. 45 In the current study we found a down-regulation of JDP2 in IPoC-R and vice versa a strong up-regulation of SERCA2a (mRNA and protein). Another mRNA similarly strongly regulated by IPoC was VEGF. At least in endothelial cells, VEGF induction is AP-1-dependent. 46  is that electron transport chain of mitochondria is bypassed. As a result, it is expected that UCPs reduce oxidative stress in mitochondria but decrease ATP production. 40 However, alternative functions of UCP-2 have been proposed due to its low uncoupling efficiency. 47 Among them, UCP-2 may attenuate glucose uptake. Interestingly, UCP-2 was up-regulated in post-infarct hearts only in IPC and (more significant) in IPoC-R. Both groups had the best functional recovery suggesting that at least in the post-infarct hearts UCP-2 expression is required for improvement of function. One may speculate that increasing the expression of UCP-2 in these hearts stabilizes metabolic flexibility in rat hearts.
Changes in the mRNA expression of molecules that participate in processes such as fibrosis, inflammation and electrophysiology are well described in post-infarct hearts and therefore it is not surprising that we found multiple changes in these rats after seven days. However, the underlying level of regulation is less known.
In principle, this can be performed by activation of transcription factors or modulation of miRs that target specific mRNAs, thereby shorten their halflife and expression level. IPC and IPoC affect the expression of miRs within 2 hours of reperfusion but whether this is relevant for the subsequent remodelling process remained unclear. 48 In response to I/R we found 82 miRs up-regulated among them were five previously also found in a pig model. 43 This indicates a strong evolutionary regulation and a common cluster of miRs and subsequent mRNAs affected by I/R. However, in the current study changes between I/R and IPoC-R were in the focus. Here

F I G U R E 5
Overview of miRs analysis and comparison of overall changes on the basis of differences producing a P < .05

Influenced by IPoC-R
199a-3p and miR-148a-5p showed the greatest effects. Both miRs were induced by I/R and this induction was attenuated by IPoC-R.
Transgenic mice with disruption of miR-199 showed an adaptive type of hypertrophy and increased expression of PGC-1α. 49 Here, we found a down-regulation of miR-199 in the IPoC-R group compared to I/R and an increase in PGC-1α (from 0.48 ± 0.19 to 0.70 ± 0.14). Furthermore, a down-regulation of miR-199 was linked to an increased activity of the proteasome systems in end-stage dilative cardiomyopathy. 50 In our study, IPoC-R group were unable to increase their heart weights but had the largest left ventricular end-diastolic dimensions among the protection groups. The lack of hypertrophic responsiveness to IPoC in responders may be the consequence of increased activity of the proteasome linked to miR-199 down-regulation as well. Finally, miR-199 was increased in plasma samples from patients with acute myocardial infarction and experiments with H9c2 cells further suggesting a protective role for miR-199 down-regulation against hypoxia-dependent cytotoxicity. 51 In hepatocellular carcinoma cells, miR-199a-3p suppresses VEGF release and VEGF receptor expression 52 . This suggests that miR-199 down-regulation in the IPoC-R participates in the F I G U R E 6 Mitochondrial-associated miRNAs and linkage to potential targets found in post-ischaemic hearts subsequent protective remodelling including VEGF-dependent pathways. These suggestions make miR-199 an interesting candidate to be targeted by IPoC or alternatively by other procedures.
MiR-148a-5p, however, has not yet been associated with cardiovascular diseases but may require more attention in future studies.
Detailed analysis of mitochondrial network pathways identified solute carrier family 25, member 44 (Slc25a44) as the main target mRNA that by prediction should be down-regulated. Interestingly, Slc25a44 is linked to miR-222-3p. This miR is induced by I/R and in IPoC-NRs but not in IPC and IPoC-R. Collectively, these data suggest an important role for miRs for the regulation of mitochondrial-specific genes in I/R.
In conclusion, this study shows that IPoC-NR (as classified by infarct size) still react to IPoC by molecular adaptations distinct from IPC and I/R but comparable to IPoC-R. This adaptation, however, is incomplete. We found inter-individual variability in response to IPoC in healthy rats specifically in mitochondrial-specific adaptations suggesting that non-responsiveness is not simply linked to co-morbidities. A high inter-individual variability may limit the availability of IPoC in other species as well. However, as certain types of co-morbidities affect mitochondrial function this may be a link to reduced responsiveness.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data needed to evaluate the conclusions in the paper are present in the paper and/or supplements. Additional data related to this paper may be requested by the author.