Introduction
Cardiac ischemia/reperfusion (I/R) injury is associated with various etiological factors (1-3), including primary heart conditions, and neuropathic, vascular or systemic disorders. However, the pathological origin of I/R-induced cardiomyocyte death remains poorly understood. The neuropathic accumulation associated with myocardial I/R is thought to directly or indirectly activate sensory or sympathetic fibers that innervate the heart. However, as the underlying heart-specific neuronal pathway and mechanism are unknown, neurogenic therapeutic interventions often only have limited success.
It is well established that the autonomic nervous system serves a crucial physiological role in regulating cardiac function (4-9). Previous studies have demonstrated that some myocardial ischemic events are triggered by the autonomic nervous system, and a sympathetic-parasympathetic imbalance may lead to the pathophysiological development of myocardial ischemia (10,11). Our previous study revealed that an injection of retrograde tracer pseudorabies virus (PRV)-614 into the left ventricle wall in mice resulted in the retrograde infection of neurons in the intermediolateral nucleus of the spinal cord and the rostral ventrolateral medulla of the brainstem via the sympathetic pathway (12). The spinal cord has been implicated in the pathogenesis of cardiac injury caused by I/R (4,13-16). Furthermore, there is convincing evidence that the heart receives dense innervation from sensory afferent fibers, which peripherally release a variety of vasodilator neuropeptides, such as calcitonin gene-related peptide (CGRP) (17) and substance P (SP), in response to local stimuli (18). Despite extensive research in this area, the mechanisms underlying cardiac I/R injury are largely unknown. Therefore, there is an urgent requirement for more information and a genomic approach may prove helpful.
High-throughput RNA sequencing (RNA-seq) is a powerful tool that has been used to identify novel protein-coding and non-coding RNA transcripts involved in the regulation of gene expression (19-24). Recent research has focused on the cardiac long non-coding RNAs (lncRNAs) implicated in cardiac I/R injury (25-29); however, few studies have explored the important role of the spinal cord during focal cardiac I/R. Therefore, the present study was designed to identify the expression patterns of differentially expressed lncRNAs and mRNAs in the spinal cord under normal and cardiac I/R conditions, with the aim to gain a better understanding of the genetic mechanisms underlying the pathogenesis of cardiac I/R injury. The present study also determined the expression levels of various genes in the spinal cord at different time-points during cardiac I/R injury.
Materials and methods
Animals
A total of 24 male Sprague Dawley (SD) rats aged 8-10 weeks (200-240 g; specific pathogen-free grade; no. 42000600010250) were supplied by the Experimental Animal Research Center of Hubei Province (Hubei, China). The present experiment protocol was approved by the Institutional Ethical Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Hubei, China; no. TJ-A20150804). All animals were humanely treated according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (revised 2011) and the Guide for the Care and Use of Laboratory Animals (National Academic Press, USA; revised 2011) (30). Animals were housed in compliance with the Specific Pathogen-Free Animal Criteria, and maintained at a standard temperature of 21-23°C and 65±5% humidity under a 12-h light/dark cycle condition, with ad libitum access to artificial feed (food and water).
Myocardial I/R injury model
Surgical procedures to establish the myocardial I/R injury model were performed according to previously described methods (31-33).
Experimental protocol
Experiment A
A total of 6 SD rats were randomly allocated into 2 groups. The model group (n=3) received 2 h reperfusion following 30-min occlusion of the left anterior descending coronary artery by pulling the reversible trap (I/R group). The control group (n=3) received the same surgical procedure, without any occlusion of the coronary artery and reperfusion (sham group). T1-T4 spinal cord samples were collected for RNA-seq and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis.
Experiment B
A total of 18 SD rats were randomly assigned into 3 groups: i) A control group (n=6); ii) a 0.5 h group (0.5 h reperfusion following 30 min ischemia; n=6); and iii) a 2 h group (2 h reperfusion following 30 min ischemia; n=6). T1-T4 spinal cord segments were obtained for RT-qPCR analysis.
Tissue preparation and microarray gene expression analysis
The rats were sacrificed following the completion of the aforementioned experiments. Briefly, upon completion of the experiments, the rats were anesthetized by intraperitoneal injection with 100 mg/kg body weight ketamine and 10 mg/kg body weight xylazine (34). Then the rats were quickly decapitated to limit animal suffering and minimize the effects on the experimental results, and the T1-T4 spinal cord segments were immediately removed and frozen with liquid nitrogen for 1 min, then stored at −80°C until required. Total RNA from each animal was quantified using a mirVana miRNA Isolation kit (Ambion; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and RNA integrity was assessed according to the manufacturer's protocol, which included standard denaturing agarose gel electrophoresis (35-37). For RNA-seq, the micro-array work was performed by CapitalBio Technology Co. Ltd. (Beijing, China), whereby 6 tissue samples (3 model group samples and 3 control group samples) were used for mRNA and lncRNA microarray analysis (38).
The present study used an Agilent Array platform for microarray analysis. Tissue preparation and microarray hybridization were performed based on the manufacturer's standard protocols (Agilent Technologies, Inc., Santa Clara, CA, USA) with minor modifications. Briefly, the mRNA was purified from the total RNA once the ribosomal RNA was removed using an mRNA-ONLY Eukaryotic mRNA Isolation kit (Epicentre; Illumina, Inc., San Diego, CA, USA). Each sample was then amplified and transcribed into fluorescent complementary RNA (cRNA) along the entire length of the transcripts using a random priming method (39). The labeled cRNAs were hybridized onto the mouse lncRNA Array v2.0 (8×60 K; Arraystar, Inc., Rockville, MD, USA). Then the arrays were scanned using a G2505C Scanner (Agilent Technologies, Inc.).
Bioinformatics analysis
Gene Ontology (GO) annotations were employed to investigate the differentially expressed mRNAs and lncRNAs in the T1-T4 spinal cord segments, according to the GO database (www.geneontology.org/). For the functions of genes and their products, the GO database describes 3 biological functional groups: Biological process, Cellular compartment, and Molecular function. The present study conducted GO functional enrichment analysis on the differentially expressed mRNAs involved in protein-protein interaction (PPI) networks. In addition, the key regulatory pathways in the spinal cord that respond to I/R-induced cardiac injury were also investigated using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (www.genome.jp/kegg) and STRING database (string-db.org).
RT-qPCR analysis
The present study extracted total RNA from the upper thoracic spinal cord segments (T1-T4) (40) using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to our previous research (35-37). The primers for RT-qPCR were designed based on the lncRNA sequences (Table I), and were synthesized and purified at Invitrogen (Thermo Fisher Scientific, Inc.). The RT reactions were performed using a iScript™ cDNA Synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). RT-qPCR was performed using a ABI StepOnePlusä Multicolor system with the SsoAdvanced™ Universal SYBR®-Green Supermix (Bio-Rad Laboratories, Hercules, Inc.). The PCR thermocy-cling conditions were as follows: Initial denaturation at 95°C for 1 min followed by 40 cycles of 95°C for 15 sec, 60°C for 15 sec and 72°C for 45 sec. Compared with the averages for the housekeeping gene (GAPDH), the data were quantified using the 2−∆∆Cq method for relative fold-change, as described previously (41).
Statistical analysis
Data are expressed as the mean ± standard error of the mean. The data and statistical graphs were analyzed using the GraphPad Prism v6.02 package (GraphPad Software, Inc., La Jolla, CA, USA). Between-group counts were compared using a Student's t-test (Mann-Whitney U), and the data from three groups were analyzed by one-way analysis of variance followed by Dunn's post hoc test. P<0.05 was considered to indicate a statistically significant difference.
Results
Characteristics of ischemic myocardial tissues
With regard to the animals in the model group, there were evident cyanotic changes in the myocardium of the occluded area 30 min post-ischemia, and a reactive hyperemic response after refilling of the left anterior descending coronary artery. With regard to the samples in the model group, examination under a dissecting microscope revealed discoloration of the occluded distal myocardium at the early stage of reperfusion in the infarct region (data not shown).
Expression profiling of lncRNAs in the spinal cord 2 h post-cardiac I/R
To select possible targets of lncRNAs in the model and control groups, the present study detected up to 16,987 coding transcripts in the T1-T4 spinal cords 2 h post-reperfusion. A total of 3,621 upregulated and 13,366 downregulated lncRNAs were identified in the spinal cords. On average, 234 lncRNAs were upregulated in the spinal cords of the model group, compared with those in the control group, whereas an average of 7,746 lncRNAs were downregulated (>2.0-fold-change; P<0.05). The distributions of the log2 ratios of the lncRNAs in the model and control samples were nearly identical; Fig. 1 presents the heatmaps of the expression ratios (log2 scale) of the lncRNAs in the spinal cords. The top 20 up- and downregulated mRNAs are listed in Tables II and III.
Expression profiling of mRNAs in the spinal cord 2 h post-cardiac I/R
To explore the potential role of mRNAs in the T1-T4 spinal cords 2 h post-cardiac I/R, the present study determined the expression profiles of mRNAs by high-throughput RNA-seq. Of the 26,466 mRNAs that were quantified using reads per kilobase per million mapped reads (RPKM) values, 3,428 mRNAs were deregulated by 2-fold following I/R-induced cardiac injury, of which 767 mRNAs were upregulated and 2,661 mRNAs were downregulated; Fig. 2 presents the heatmaps of the expression ratios (log2 scale) of the mRNAs in the spinal cords. The top 20 up- and downregulated mRNAs are listed in Tables IV and V.
Hierarchical clustering analysis of the differentially expressed mRNAs and lncRNAs
Following the determination of the expression values of the differentially expressed genes (DEGs), the present study carried out hierarchical clustering analysis on the DEGs. As shown in Figs. 1 and 2, the differentially expressed mRNAs and lncRNAs clearly distinguished the cardiac I/R tissues from the control samples. In the cardiac I/R tissues, there were more downregulated genes than upregulated genes (Figs. 1 and 2).
Functional and pathway enrichment analyses
The significantly enriched GO terms (top 30) were comprised of 16 biological processes, 6 cellular compartments, and 8 molecular functions (Fig. 3). It was revealed that the differentially expressed biological processes in the spinal cords were primarily involved in the serotonin receptor signaling pathway, regulation of protein kinase B signaling, regulation of keratinocyte migration, and skeletal muscle satellite cell differentiation. The significantly enriched pathway terms (top 30) primarily involved KEGG pathways including 'olfactory transduction', 'arachidonic acid metabolism', the 'phosphoinositide 3-kinase-protein kinase B signaling pathway', 'extracellular matrix-receptor interaction', 'cytokine-cytokine receptor interaction' and 'neuroactive ligand-receptor interaction' (Fig. 4). The DEGs were analyzed with GO background significant enrichment, which demonstrated the number of genes associated with biological processes, cellular compartments and molecular functions (Fig. 5).
The results of the biological process analysis revealed that the DEGs involved in PPI networks were mainly enriched in 'neurological system processes' (P=1.92×10−11), 'sensory perception' (P=1.92×10−11), the 'detection of chemical stimuli' (P=5.10×10−10) and 'cell surface receptor signaling pathways' (P=4.23×10−10; Fig. 6). A total of 7 cellular components from GO terms were significantly enriched for the DEGs involved in PPI networks: 'Extracellular regions' (P=0.02), 'membrane parts' (P=1.54×10−5), 'extracellular spaces' (P=1.52×10−6), 'intrinsic components of the membrane' (P=1.29×10−8), the 'cell periphery' (P=1.76×10−7), 'integral components of the membrane' (P=1.61×10−8) and the 'plasma membrane' (P=3.22×10−7; Fig. 7). A total of 10 molecular functions from GO terms were significantly enriched for the DEGs involved in PPI networks: 'Molecular transducer activity' (P=6.13×10−14), 'signal transducer activity' (P=9.42×10−14), 'receptor activity' (P= 4.77×10−14), 'signal receptor activity' (P=4.77×10−15), 'transmembrane signal receptor activity' (P=1.31×10−15), 'serine-type peptidase activity' (P=9.57×10−5), 'endopeptidase activity' (P=0.02), 'olfactory receptor activity' (P=5.07×10−9), 'G-protein-coupled receptor activity' (P=3.03×10−14) and 'serine-type endopeptidase activity' (P=1.08×10−5) (Fig. 8).
RT-qPCR validation of lncRNA expression in the spinal cords 2 h post-cardiac I/R injury
To validate the reliability of the RNA sequencing results in the rats, the present study analyzed the differentially expressed lncRNAs, including 5 upregulated lncRNAs and 4 downregulated lncRNAs, by RT-qPCR analysis. T1-T4 spinal cord tissues were collected from the control and I/R groups 2 h post-reperfusion. The expression levels of 4 upregulated lncRNAs (NONRATT025386, NONRATT016113, NONRATT018298 and NONRATT018300) increased significantly in the I/R group when compared with those in the control group, whereas the expression level of one downregulated lncRNA (NONRATT002188) decreased significantly (Fig. 9). The RT-qPCR results for 3 lncRNAs (XR_589980.1, XR_598701.1 and XR_590197.1) were not consistent with the data from the RNA sequencing (XR_589980.1 decreased, and XR_598701.1 and XR_590197.1 increased post-reperfusion when compared with the control; Fig. 10).
Expression levels of 4 lncRNAs in the spinal cord 0.5 h post-cardiac I/R injury
The present results indicated that the expression levels of the lncRNA NONRATT025386 were significantly upregulated in the 0.5 h group when compared with the control group, whereas the expression levels of the lncRNAs NONRATT002188 and XR_590197.1 were signifi-cantly downregulated in the 0.5 h group compared with the control group. Furthermore, the lncRNA NONRATT016113 showed no significant difference between the two groups (Figs. 10 and 11).
Expression levels of 9 lncRNAs in the spinal cord at different time-points (0.5 and 2 h) following cardiac I/R injury
The present study collected samples from spinal cord tissues at 0.5 and 2 h post-cardiac I/R injury for RT-qPCR validation. The results indicated that the expression levels of the lncRNA NONRATT025386 were significantly upregulated in the 0.5 and 2 h groups compared with the control group. In addition, the expression levels of NONRATT016113, NONRATT018298 and NONRATT018300 were also significantly increased in the 2 h group, but not at 0.5 h, compared with the control group (Figs. 9–11). By contrast, the expression levels of the lncRNA NONRATT002188 were significantly downregulated in the 0.5 and 2 h groups when compared with the control group (Figs. 9–11).
Discussion
With regard to ischemic cardiac tissues, previous research has focused on multiple signaling pathways that regulate the critical balance between cell death and survival during cardiac I/R injury (42-47). The present study, to the best of our knowledge, for the first time provides evidence that suggests that many DEGs, pathways and biological processes of the T1-T4 spinal cord are implicated in myocardial I/R. Based on high-throughput RNA seq, 16,987 lncRNAs in the T1-T4 spinal cord tissues were identified, of which 3,621 were upregulated and 13,366 were downregulated (>2.0-fold-change; P<0.05). Among the 26,466 mRNAs that were quantified using RPKM values, 3,428 were deregulated by 2-fold following I/R-induced cardiac injury, of which 767 were upregulated and 2,661 were downregulated. According to these results, some differentially expressed lncRNAs were verified by RT-qPCR analyses.
Previous studies have shown that the spinal cord serves an important role in the pathogenesis of cardiac disease (48-51). The present study used a virally mediated trans-synaptic tracing method, by injecting the PRV virus into the rat heart and kidney, and these viruses were subsequently found in the lateral medial column of the spinal cord in the corresponding segment (12,52). This revealed the characteristics of the transcriptome in the T1-T4 spinal cord following cardiac I/R injury and the specific spinal segment that innervates the heart serves a significant role in cardiac I/R injury. Cheng et al (53) reported that melatonin regulation of the transcriptome was associated with the reversal of morphine tolerance by transcriptomic analysis of the L5-S3 segmental spinal cord. Mohrman et al (54) revealed the spinal cord transcriptomic and metabolic patterns in a excitotoxic injection injury model of syringomyelia. Niu et al (55) demonstrated the upregulation of tumor necrosis factor (TNF)-α in spinal cord neurons during coronary artery occlusion in rats, suggesting that TNF-α in the spinal cord may be associated with the nociception initiated by acute myocardial ischemia/infarction. Schulz et al (56) reported that connexin 43 in the spinal cord serves an important role in providing protection from cardiac I/R injuries. It is well known that myocardial ischemia creates an autonomic nervous system imbalance, and can trigger cardiac arrhythmias (57,58). Howard-Quijano et al (59) indicated that neuromodulation by spinal cord stimulation (SCS), in which a 4-pole lead was placed percutaneously in the T1-T4 epidural space, attenuated local cardiac sympathoexcitation from ischemia-induced increases in afferent signaling, reduced ventricular arrhythmias and improved myocardial function during acute ischemia. Liao et al (60) reported that chronic thoracic SCS at the T1-T3 level induced significant remodeling of cardiac sympathetic innervation over the peri-infarct and infarct regions, and was associated with improved left ventricular function and reduced myocardial norepinephrine spillover. The present study revealed that the differential expression of certain mRNAs and lncRNAs in the spinal cord affects the myocardial ischemic regions, suggesting that in the spinal T1-T4 segment these genes are involved in the response to cardiac injury. Although the functions of mRNAs and lncRNAs in the spinal cord are unclear, the present findings provide a novel paradigm for cardioprotection against I/R-induced myocardial injury.
The present results also revealed that some mRNAs in the spinal cord, including chemokine C-X-C motif ligand 1 (CXCL1), regulatory factor X4 (RFX4), WNT10a and interleukin (IL)-6, were differentially expressed following cardiac I/R. Haider et al (61) reported that the angiogenic potential of the mononuclear cell (MNC) secretome is regulated by CXCL-1 upregulation in spinal cord tissue, and factors in the MNC secretome may mitigate the pathophysiological processes of secondary damage following spinal cord injury, and may also improve functional outcomes in rats. Ashique et al (62) reported that the spinal cords of RFX4 mutants were correlated with defects in patterning and cilia formation, suggesting that RFX4 is a regionally specific transcriptional regulator of Sonic hedgehog signaling during the development of the central nervous system. Zhao et al (63) demonstrated that hyperbaric oxygen (HBO) reverses Wnt-10a upregulation induced by chronic constriction (CCI) injury in the dorsal root ganglion (DRG), spinal cord and hippocampus, suggesting that HBO attenuates CCI-induced rat neuropathic pain and inflammatory responses, potentially through regulation of the Kindlin-1/Wnt-10a signaling pathway. Although the detailed functions of many mRNAs from spinal cords are not fully understood, the present results provide novel insight into the molecular mechanisms underlying cardiac I/R injury.
It is well known that regulations of gene expression are varied over the time course (64-66). Our previous research associated with Itchy E3 ubiquitin protein ligase demonstrated that gene expression was significantly different in the C5-C8 spinal cord at 0.5 and 2 h following compound 48/80 injection when compared with the control group (36). Similar to the above method, we also screened key genes in myocardial tissues under 30 min cardiac ischemia following 2 h reperfusion compared with the sham group (67). Li et al (68) also indicated that impairment of sensory nerves with significant reductions in CGRP and SP in the DRG, ventricular myocardium and serum may be associated with an increase in myocardial vulnerability in acute cardiac I/R injury in diabetic rats. It was revealed that the injury was relatively evident at 2 h post-myocardial reperfusion, which may be considered as an acute cardiac I/R injury. Notably, this time point could be equivalent to the patients who received early percutaneous coronary intervention following myocardial infarction (69,70). Therefore, the present study chose 0.5 and 2 h post-reperfusion as the different time points of cardiac reperfusion injury. The present study revealed that there are significant differences in lncRNA and mRNA expression patterns at different time points of myocardial I/R, suggesting that the neural modulation of cardiac I/R injury may be temporal- and spatial-dependent.
In particular, our previous study also demonstrated that proton magnetic resonance spectroscopy was able to simultaneously detect and quantify the absolute concentrations of multiple metabolites within the spinal cord underlying α-Me-5-HT-evoked pruritus (71). Using the above method, we can also detect the changes of various metabolites in the spinal cord following cardiac I/R injury, which may deepen our understandings of the pathophysiology and pharmacological therapies for acute myocardial infarction.
The present study screened several differentially expressed mRNAs under cardiac I/R injury. In the future, whether the proteins encoded by the mRNAs are consistent with these mRNAs will be verified by immunoblotting. If so, the effects on cardiac I/R injury may be observed by activating or silencing the expression of one specific mRNA. Li et al (68) revealed that the regulation of the spinal cord served a significant role under cardiac I/R injury in diabetic neuropathic rats. Thus, it can be hypothesized that intervention on the spinal cord may have an important influence on cardioprotection in the future.
The present study used high-throughput RNA seq, coupled with RT-qPCR analysis, to demonstrate that the expression profiles of lncRNAs and mRNAs in spinal cords differed markedly between the control and 2 h groups, and ultimately identified 7,980 differentially expressed (>2-fold) lncRNAs (234 upregulated and 7,746 downregulated) and 3,428 mRNAs (767 upregulated and 2,661 downregulated). The expression patterns of several lncRNAs were confirmed by RT-qPCR. The results also indicated that the expression levels of the lncRNA NONRATT025386 were significantly upregulated in the 0.5 and 2 h groups compared with the control group, whereas the expression levels of NONRATT016113, NONRATT018298 and NONRATT018300 were significantly increased in the 2 h group when compared with the control group, although there was no statistically significant difference between the expression levels in the 0.5 h and control groups. Furthermore, the expression levels of the lncRNA NONRATT002188 were significantly downregulated in the 0.5 and 2 h groups compared with the control group.
In conclusion, this study revealed that high-throughput RNA seq can facilitate the systematic exploration of gene expression on a genome-wide scale, and can be used to investigate DEGs and lncRNA expression patterns in the spinal cords of rats during I/R-induced cardiac injury. In the search for better treatments for cardiac I/R injury, expanded sets of differentially expressed mRNAs and lncRNAs may prove very useful for identifying novel therapeutic targets.