Sequential activation of different pathway networks in ischemia-affected and non-affected myocardium, inducing intrinsic remote conditioning to prevent left ventricular remodeling

We have analyzed the pathway networks of ischemia-affected and remote myocardial areas after repetitive ischemia/reperfusion (r-I/R) injury without ensuing myocardial infarction (MI) to elaborate a spatial- and chronologic model of cardioprotective gene networks to prevent left ventricular (LV) adverse remodeling. Domestic pigs underwent three cycles of 10/10 min r-I/R by percutaneous intracoronary balloon inflation/deflation in the mid left anterior descending artery, without consecutive MI. Sham interventions (n = 8) served as controls. Hearts were explanted at 5 h (n = 6) and 24 h (n = 6), and transcriptomic profiling of the distal (ischemia-affected) and proximal (non-affected) anterior myocardial regions were analyzed by next generation sequencing (NGS) and post-processing with signaling pathway impact and pathway network analyses. In ischemic region, r-I/R induced early activation of Ca-, adipocytokine and insulin signaling pathways with key regulator STAT3, which was also upregulated in the remote areas together with clusterin (CLU) and TNF-alpha. During the late phase of cardioprotection, antigen immunomodulatory pathways were activated with upregulation of STAT1 and CASP3 and downregulation of neprilysin in both zones, suggesting r-I/R induced intrinsic remote conditioning. The temporo-spatially differently activated pathways revealed a global myocardial response, and neprilysin and the STAT family as key regulators of intrinsic remote conditioning for prevention of adverse remodeling.


Porcine Model of Ischemic Preconditioning
The pigs received an intramuscular injection of 12 mg/kg ketamine hydrochloride, 1 mg/kg xylazine and 0.04 mg/kg atropine, with inhalation anesthesia with isoflurane and O 2 . After reaching deep anesthesia, pigs were intubated and the anaesthesia was continued with an anesthetic gas mixture of 1.5-2.5 vol% isoflurane, 1.6-1.8 vol% O 2 and 0.5 vol% N 2 O. After surgical preparation of the right femoral artery, a 6F introduction sheath (Medtronic Inc, Minneapolis, MN) was placed followed by intra-arterial administration of 200 IU/kg of unfractionated heparin. A 6F coronary catheter (Medtronic Inc, Minneapolis, MN) was placed into the abdominal aorta and selective angiography of the left coronary arteries was performed. After placement of a guidewire (Medtronic Inc, Minneapolis, MN), a coronary balloon dilation catheter (2.75 mm of diameter and 12 mm of length, Medtronic Inc, Minneapolis, MN), was placed into the left anterior descending coronary artery, below the origin of the second diagonal branch. Coronary occlusion was performed using 6 atm inflation pressure, in order to avoid injury of the endothelium of the vessel. Coronary angiography was performed injecting non-ionic contrast media (Takeda, Zürich, Switzerland) to prove the occlusion or the reperfusion of the coronary artery. Electrocardiogram, blood gas and hemodynamics were monitored continuously during the procedure. The study corresponds to the ARRIVE guidelines [S1].

Effect of IPC on 30 days cardiac function after acute myocardial infarction (AMI) in SWOP.
At 26h, during SWOP, the animals underwent myocardial infarction by induction of 90 min sustained ischemia by occlusive inflation of a percutaneous coronary balloon in the mid LAD followed by deflation of the balloon (reperfusion). After 30 days cardiac magnetic resonance imaging (cMRI) was performed to compare the impact of ischemic injury between both groups.

Invasive LV hemodynamics
The following parameters were recorded via a pigtail catheter directly before and after the r-I/R stimulus as well as at follow-up in the r-I/R [5h], r-I/R [24h] and control groups: systolic and diastolic aortic pressure, LV systolic and diastolic pressures (LV SP and LV DP), LV end-diastolic pressures (LV EDP), and a parameter of LV contractility expressed as dP/dt.

Echocardiography
To assess the systolic and diastolic movement of the LV, transthoracic echocardiography was performed at baseline, immediately after the r-I/R stimulus and at 5h and 24h follow-up in the r-I/R [5h], r-I/R [24h] and Control groups. LV diastolic and systolic diameter and fractional shortening were measured, the ejection fraction was determined using Simpson's method and regional wall motion abnormalities were evaluated visually by blinded investigators. Diastolic parameters such as isovolumetric relaxation time (IVRT), velocity of the E and A waves and their ratio (E/A) were also determined. cMRI cMRI was performed at day 30 after reperfused myocardial infarction in the IPC-AMI and AMI groups using a 1.5-T clinical scanner (Avanto, Siemens, Erlangen, Germany) with a phased-array coil and a vector electrocardiogram system (Supplementary Material). The enddiastolic and end-systolic volumes of the LV (LV EDV and LV ESV), LV ejection fraction (LV EF), RV ejection fraction (RV EF), cardiac output (CO), left ventricular mass (LVM), myocardial scar tissue area of the LV and area of transmural myocardial infarction of the LV were measured. Using the 17-segment model, the LV segmental wall contraction velocities of the ischemia-affected segments (apical-distal anterior and septal anterior) and remote area (proximal anterior) were calculated at the time point of peak ejection (cm/s).

Histology, immunofluorescence analyses, and blood marker analyses
Representative tissue samples from proximal (non-ischemic), mid and distal (ischemiaaffected) anterior wall regions were stored in 7.5% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (HE).
Fluorescence immunohistochemistry was performed using alpha actin antibody (Abcam, Cambridge, UK) and counterstaining with DAPI (Abcam). Circulating troponin I (normal value in pigs <0.15 ng/mL), myoglobin (normal range 10-95 ng/mL) and creatine kinase (CK, normal range <100 U/L) were determined at baseline, immediately after the IPC stimulus and at the 5h and 24h follow-up using porcine Cardiac Troponin I Type 3 ELISA (MyBioSource, San Diego, CA, USA), porcine myoglobin ELISA (West Chester, PA) and porcine creatine kinase ELISA (Abcam, Cambridge, UK). Data were compared to that of the sham-operated controls.

Gene Expression Analysis Using Next Generation Sequencing and Real Time PCR (RT-PCR)
Tissue samples from the entire LV were stored in RNAlater (Qiagen, Germany) at -20°C. Total RNA was isolated from the tissue samples using the RNeasy Microarray Tissue Mini Kit (Qiagen, Germany). RNA quality was checked on RNA Nano chips using the Agilent 2100 Bioanalyzer platform (Agilent Technologies). a) Next generation sequencing (NGS). mRNA of myocardial probes from the basal, mid and apical anterior wall segments of preconditioned and control animals were analyzed. Total RNA (500 ng) were poly-A enriched (NEBNext Poly(A) mRNA Magnetic Isolation Module) and used for library preparation with the NEBNext Ultra Directional RNA Library Prep Kit for Illumina and final libraries were quality controlled on a Fragment Analyzer (Advanced Analytical Technologies) and quantified by digital droplet PCR (QX100™ Droplet Digital™ PCR System, Bio-Rad) and the ddPCR Library Quantification Kit for Illumina (Bio-Rad). Equimolarly pooled samples were sequenced on a HiSeq 2500 to a mean depth of 20.1 million (SD ±5.8 million) paired end reads. After quality check of demultiplexed raw reads by FastQC, reads were mapped to the Sus scrofa genome (Sscrofa10.2) by RNA-Seq Unified Mapper (RUM v2.0.4). Mapped reads were in a range of 91.3% to 93.6% and these mapped reads were counted into the Sus scrofa Ensembl gene model Sscrofa10.2 release 73 (using htseq-count from the HTSeq Python framework to work with high-throughput sequencing data, PMID: 25260700, with "-m union -s reverse"). All further analyses were done in R v3.1.0 (R Core Team, R Foundation for Statistical Computing, "R: A Language and Environment for Statistical Computing", 2014, Vienna, Austria, http://www.R-project.org) with R-packages limma v33. Significantly differentially expressed genes were determined with linear models in limma after voom transformation and gene weights calculation between different contrasts with region and individual pigs as confounding factors. Linear models for each gene were fitted and the estimated coefficients and standard errors for all contrasts were computed. Significantly deregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were analyzed by Signaling Pathway Impact Analysis (SPIA) and illustrated by pathview (node colors represent log 2 fold changes). Biological interpretation of significant gene lists was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 [S7]. Principal Component Dimensionality reduction of normalized log 2 gene expression values was performed with the non-linear dimensionality reduction method Isomap [S8], and implemented in the R-package RDRToolbox 1.10.0. Three dimensions were calculated with the modified version of the original Isomap algorithm including the nearest and farthest n-1 neighbors. KEGG pathway enrichment analysis with STRING interaction network was performed to identify the significantly deregulated genes connecting the significantly activated pathways. b) RT-PCR of Target Genes. mRNA from myocardial probes of the entire LV of representative animals of each group was reverse transcribed to cDNA (Qiagen, Germany) and mRNA expression was quantified by RT-PCR (Applied Biosystems 7500 Real-Time PCR System, Life Technologies, USA). Genes of interest were selected based on suspected involvement in cardioprotection (i.e. HIF-1α, MEF2C, and caspase-3) and based on the results of the NGS analysis (i.e. HK2, ERCC4 and CLU). The primers for the target sequences were designed using Primer3 software (http://primer3.wi.mit.edu/primer3web_help.htm; Microsynth, Switzerland) (Supplemental Table 1). Target gene expression rates were normalized to the geometric means of three housekeeping genes that were selected as endogenous controls due to stable expression (i.e. GAPDH, HPRT1, and PPIA). The relative gene expression level was calculated using the ΔCt method. The expression changes were calculated relative to expression levels in the normal myocardium of control animals that underwent the sham procedure.

Statistics
Continuous data are reported as median and interquartile ranges (IQR). Differences between the groups were tested by the means of two-sided Kruskal-Wallis-Test and the Mann-Whitney-U non-parametric tests, at the alpha level of 0.05. A difference was considered statistically significant at p<0.05. Data analyses and interpretations were performed by an experienced observer who was blinded to the randomization and to all study results. Statistical analysis was performed using SPSS 18.0 software (SPSS Inc, USA).
For gene array analysis, to test for all differences between regions of interest (proximal, mid and distal anterior wall regions) and groups (IPC [5h] and IPC [24h] vs. Control), a linear model for each gene was fitted, and the coefficients with standard errors were computed. Moderated t-statistics, F-statistics, and the log-odds of differential expression were computed by empirical Bayes shrinkage of the standard errors towards a common value. A false discovery rate (FDR) below 5% was considered statistically significant. An FDR cut-off of 10% was considered for some contrast, for relevant gene lists for biomedical interpretation. Unsupervised hierarchical cluster analysis was performed using the Euclidean distance as the distance function and the Ward algorithm in R, using centered and scaled log2 expression values. Principal Component Analysis (PCA) was conducted for centered and scaled values.  Figure S1. SPIA two-way evidence plots for identification of significantly changed cellular pathways.

Supplementary References
Pathways with FDR <5 % are highlighted in red, and pathways with FDR between 5 and 10 % in blue.

Mid anterior
Proximal anterior

Supplementary Figure S2. Ca-signaling pathway
Genes involved in activated Ca-signaling pathway at 5h (group r-I/R [5h]; A) and 24h (group r-I/R [24h]; B) after repetitive ischemia/reperfusion (r-I/R) in the r-I/R-affected (distal anterior) region. In the ischemic distal anterior region, GPCR, ADCY, SERCA, CALM and IP3R were up-and CAV3, PMCA, PKC, MLCK and CAMK were downregulated at 5h, whereas similar regulation but in a less explicit manner was found at 24h. Relative expression of differentially expressed genes is color-coded (red overexpression, green downregulation).

Supplementary Table S2. Hemodynamic and echocardiographic parameters at baseline, immediately after and at 5h Group r-I/R [5h] and 24h Group r-I/R [24h] following the repetitive ischemia/reperfusion (r-I/R) stimulus and in sham-operated animals.
Variables are displayed as mean±sd.