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. Author manuscript; available in PMC: 2021 Mar 1.
Published in final edited form as: Stress. 2019 Jul 26;23(2):125–135. doi: 10.1080/10253890.2019.1641081

Myocardial Fibrosis, Inflammation, and Altered Cardiac Gene Expression Profiles in Rats Exposed to a Predator-Based Model of Posttraumatic Stress Disorder

Boyd R Rorabaugh a,*, Nathaniel W Mabe b, Sarah L Seeley a, Thorne S Stoops a, Kasey E Mucher c, Connor P Ney c, Cassandra S Goodman c, Brooke J Hertenstein c, Austen E Rush c, Charis D Kasler c, Aaron M Sargeant d, Phillip R Zoladz c
PMCID: PMC6982550  NIHMSID: NIHMS1536593  PMID: 31347429

Abstract

People who are exposed to life-threatening trauma are at risk of developing posttraumatic stress disorder (PTSD). In addition to psychological manifestations, PTSD is associated with an increased risk of myocardial infarction, arrhythmias, hypertension, and other cardiovascular problems. We previously reported that rats exposed to a predator-based model of PTSD develop myocardial hypersensitivity to ischemic injury. The present study characterized cardiac changes in histology and gene expression in rats exposed this model. Male rats were subjected to two cat exposures (separated by a period of 10 days) and daily cage-mate changes for 31 days. Control rats were not exposed to the cat or cage-mate changes. Ventricular tissue was analyzed by RNA sequencing, western blotting, histology, and immunohistochemistry. Multifocal lesions characterized by necrosis, mononuclear cell infiltration, and collagen deposition were observed in hearts from all stressed rats but none of the control rats. Gene expression analysis identified clusters of upregulated genes associated with endothelial to mesenchymal transition, endothelial migration, mesenchyme differentiation, and extracellular matrix remodeling in hearts from stressed rats. Consistent with endothelial to mesenchymal transition, rats from stressed hearts exhibited increased expression of α-smooth muscle actin (a myofibroblast marker) and a decrease in the number of CD31 positive endothelial cells. These data provide evidence that predator-based stress induces myocardial lesions and reprogramming of cardiac gene expression. These changes may underlie the myocardial hypersensitivity to ischemia observed in these animals. This rat model may provide a useful tool for investigating the cardiac impact of PTSD and other forms of chronic psychological stress.

Keywords: Posttraumatic stress disorder, stress, heart, predator stress, cardiac remodeling, cardiovascular

Lay Summary

Chronic predator stress induces the formation of myocardial lesions characterized by necrosis, collagen deposition, and mononuclear cell infiltration. This is accompanied by changes in gene expression and histology that are indicative of cardiac remodeling. These changes may underlie the increased risk of arrhythmias, myocardial infarction, and other cardiac pathologies in people who have post-traumatic stress disorders or other forms of chronic stress.

Introduction

Posttraumatic stress disorder (PTSD) is a disabling mental health disorder that develops following wartime combat, assault, rape, motor vehicle accidents, terrorist attacks, and other traumatic experiences. People with PTSD experience psychological distress by repeatedly reliving their trauma through intrusive flashback memories (Ehlers et al., 2002; Ehlers et al., 2004; Reynolds and Brewin, 1999). This is accompanied by debilitating symptoms such as nightmares, emotional numbing, avoidance of stimuli associated with the trauma, and persistent arousal and hypervigilance (Nemeroff et al., 2006; Pitman et al., 2012; Zoladz and Diamond, 2013).

In addition to psychological manifestations, PTSD is also associated with cardiovascular abnormalities including hypertension, increased risk of cardiac arrhythmias, atherosclerosis, and increased risk of myocardial infarction (Ahmadi et al., 2011; Beristianos et al., 2016; Khazaie et al., 2013; Vaccarino et al., 2013). However, little is known about the mechanisms that underlie the increased risk of cardiovascular disease in PTSD patients. The direct impact of PTSD on the heart is difficult to study in humans because PTSD is associated with increased rates of smoking, sedentary lifestyle, diabetes, depression, and other factors that influence cardiovascular function and potentially confound efforts to assess the direct impact of posttraumatic stress on the heart (Dedert et al., 2010; Zen et al., 2012). Animal models provide a tool to examine the impact of posttraumatic stress on the heart in the absence of these confounding variables.

Cat exposure is a well described and ethologically relevant stressor that produces an intense fear response in rats (Blanchard et al., 2003; Hubbard et al., 2004; Wilson et al., 2013; Wilson et al., 2014a; Wilson et al., 2014b). Our laboratory uses a cat-based model of psychosocial stress that incorporates features that are known to promote the development of PTSD including acute intense stress (predator exposure), lack of control over the stress (immobilization during predator exposure), a lack of social support (daily cage mate changes), and a re-experiencing of the stressful event (second predator exposure). Exposure to this animal model results in physiological and behavioral abnormalities that are remarkably similar to those observed in people with PTSD including a robust fear-conditioned memory of the trauma exposure, increased anxiety, exaggerated startle response, impaired memory for new information, enhanced negative feedback of the hypothalamic-pituitary-adrenal axis, decreased basal corticosterone, and increased hormonal and cardiovascular reactivity to an acute stressor (Zoladz et al., 2008; Zoladz et al., 2012; Zoladz et al., 2013; Zoladz et al., 2015). Some of these changes are reversed by drugs such as sertraline, clonidine, and tianeptine that have efficacy in the treatment of patients who have PTSD (Wilson et al., 2014a; Zoladz et al., 2013).

Multiple studies have demonstrated an increased risk of myocardial infarction and other cardiovascular disorders in people with PTSD (Ahmadi et al., 2011; Beristianos et al., 2016; Khazaie et al., 2013; Vaccarino et al., 2013). We previously reported that rats exposed to predator stress develop myocardial hypersensitivity to ischemic injury. (Rorabaugh et al., 2015; Rorabaugh et al., 2018) The goal of the present study was to use this stress model to characterize changes in myocardial histology and gene expression that may make the heart more vulnerable to myocardial infarction and other cardiac disorders. Our data provide evidence that predator-based psychosocial stress induces the formation of cardiac lesions characterized by necrosis, collagen deposition, infiltration of immune cells, and changes in gene expression that are indicative of cardiac tissue remodeling. These stress-induced changes may underlie the increased risk of cardiac disorders associated with PTSD and other chronic stress disorders.

Methods

Animals.

Male Sprague-Dawley rats (6-7 weeks of age at the beginning of the study) from an established breeding colony at Ohio Northern University were used for all experiments. The rat colony originated from Sprague-Dawley rats obtained from Charles River Laboratories (Boston, MA; Strain Code 001). The rats were housed on a 12-h light/dark cycle (lights on at 0700) in standard Plexiglas cages (two per cage) with free access to food and water. The animals originated from 4 different litters. Siblings were divided between stressed and nonstressed experimental groups to avoid the potential for litter bias. All procedures were approved by the Institutional Animal Care and Use Committee and were performed in compliance with the recommendations published in the 8th edition of The Guide for the Care and Use of Laboratory Animals.

Psychosocial Stress Procedure.

Rats were randomly assigned to “psychosocial stress” or “no stress” experimental groups. The stress regimen has been previously described and validated by multiple laboratories (Rorabaugh et al., 2015; Zoladz et al., 2008; Zoladz et al., 2013; Zoladz et al., 2015). Rats in the psychosocial stress group were given two acute stress sessions (exposure to a cat), which were separated by 10 days. The first stress session took place during the light cycle (between 0800 and 1300), and the second stress session took place during the dark cycle (between 1900 and 2100) so that the animals could not predict when they might re-experience the predator exposure.

On Day 1, rats in the psychosocial stress groups were immobilized in plastic DecapiCones (Braintree Scientific; Braintree, MA) and placed in a perforated wedge-shaped Plexiglas enclosure (Braintree Scientific; Braintree, MA; 20 × 20 × 8 cm). This enclosure was then taken to a cat housing room and placed in a metal cage (61 × 53 × 51 cm) with an adult female cat for 1 h. The Plexiglas enclosure prevented any contact between the cat and rats, but the rats were still exposed to all non-tactile sensory stimuli associated with the cat. Canned cat food was smeared on top of the enclosure to direct the cat’s attention toward the rats. An hour later, the rats were returned to their home cages. Rats in the no stress groups remained in their home cages during the 1-h stress period.

Social Stress.

Rats in the psychosocial stress groups were exposed to unstable housing conditions starting on the day of the first cat exposure (Day 1) and continuing throughout the paradigm until behavioral testing (Day 32). Rats in the psychosocial stress group were housed two animals per cage, but every day their cage mates were changed. Therefore, no rats in the psychosocial stress groups had the same cage mate on two consecutive days during the 31-day stress period. Rats in the no stress groups were also housed two animals per cage. However, nonstressed animals were cohoused with the same cage mate throughout the experiment. Nonstressed animals were handled daily to control for handling effects on stressed animals.

Behavioral Testing.

Three weeks following the second stress session (Day 32), rats were tested on the elevated plus maze (EPM) to assess anxiety-like behavior. Rats were placed on the EPM for 5 min, and their behavior was videotaped by a JVC hard disk camera hanging above the EPM and scored offline by two separate investigators who were blind to the experimental conditions of the animals. Time spent in the open arms of the maze was used as a measure of anxiety, and the total number of closed arm entries was used to assess locomotor activity.

RNA-sequencing.

Stressed and nonstressed rats (5 animals / group) were anesthetized with sodium pentobarbital (100 mg/kg ip), and hearts were quickly isolated and perfused for 5 minutes on a Langendorff isolated heart system to flush blood from the tissue. The left ventricle was immediately flash frozen in liquid nitrogen and stored at −80°C until it was used for RNA isolation. Total RNA was isolated using TRIzol reagent (Fisher Scientific Catalog # 15596026) according to the manufacturer’s instructions. RNA was DNase I digested with a Turbo DNA-Free kit from Fisher Scientific (Catalog # AM1907) according to the manufacturer’s protocol. The RNA was shipped on dry ice to the Michigan State University Research Technology Support Facility Genomics Core (East Lansing, MI). The RNA from each rat was quantified using a Quibit dsDNA High Sensitivity Assay (Thermo Fisher Cat #Q32851) and analyzed on an Agilent 2100 Bioanalyzer RNA Pico Chip. RNA sequencing libraries were made from each rat heart using the Illumina TruSeq Stranded mRNA library preparation kit. Equimolar amounts of libraries were pooled for multiplexed sequencing on a single Illumina NextSeq 500 lane in a 75 base pair single end format. Sequencing data were demultiplexed and converted to FastQ format.

Raw Fastq files underwent quality control processing with FastQC (Andrews, 2010) software and were further processed with TrimGalore (Andrews, 2012) to remove low quality bases and to trim adapter sequences. Reads passing quality controls were aligned to the Rn6 version of the rat genome using STAR aligner software.(Dobin et al., 2013) Gene count files on the reverse strand were generated from alignment files with FeatureCounts (Liao et al., 2014) software using the RNor_6.0 ENSEMBL genome assembly. Differential gene expression analysis was produced in R (v3.3.3) software with the DESeq2 package (Love et al., 2014). Adjusted p-values (false discovery rate) were calculated in DESeq2 using the Benjamini-Hochberg equation (Benjamini Y and Y, 1995). Log2-transformed, median-centered gene expression and genes within the defined gene ontology families were selected for heatmap generation using ‘pheatmap’ package in R. Volcano plots to visualize transcriptional changes were produced with GraphPad 7 software. Genes considered significant (adjusted p value < 0.05) were analyzed by gene ontology in R with ‘ClusterProfiler’ software (Yu et al., 2012).

Data availability statement.

Raw data from next-generation sequencing experiments were deposited in the National Center for Biotechnology Information Sequence Read Archive and are available under BioProject ID number PRJNA498684. Differential gene expression analysis of these data is shown at https://figshare.com/s/5f32d3c03635c9bf46f5.

Western Blots.

Frozen ventricular tissue (from the same rats described above for RNA sequencing) was used for western blotting with antibodies for alpha smooth muscle actin (Cell Signaling Technology, Danvers, MA; Catalog #19245), T-cadherin (Abcam, Cambridge, MA; Cat #ab167407), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology, Danvers, MA; Catalog #2118). Alpha smooth muscle actin and T-cadherin bands were normalized to those of GAPDH.

Histology.

Stressed and nonstressed rats (6 animals / group) were euthanized with sodium pentobarbital (100 mg / kg, i.p.) followed by opening of the chest cavity. Hearts were removed, immersed in 10% neutral buffered formalin, and submitted to the Comparative Pathology and Mouse Phenotyping Shared Resource at The Ohio State University for tissue trimming, processing, and staining. Hearts were trimmed by longitudinal bisection perpendicular to the plane of the interventricular septum (Berridge et al., 2016). Two 5-µm thick sections of paraffin-embedded heart tissue were stained with hematoxylin and eosin (H&E) or Masson’s trichrome stains and evaluated microscopically by a board certified veterinary pathologist who was blinded to treatment groups. Histology slides were digitally scanned at Charles River Laboratories in Spencerville, Ohio using a Leica ScanScope XT scanner at 20x magnification and Aperio ImageScope [v12.3.0.5056] viewing software. Microscopic evaluation included scoring of the following criteria on a scale of 1 to 10 where 1 represented no visible lesions and 2 through 10 represented ascending levels of severity of myofiber necrosis (1-10), mononuclear cell infiltrate (1-10), or fibrosis (1-10).

Hearts from stressed and nonstressed rats (6 animals / group) were analyzed by immunohistochemistry using a CD31-specific antibody (Abcam, Cambridge, MA; Catalog # ab-28364). Five randomly chosen regions of the stained ventricular myocardium were photographed at 400 X magnification and the number of CD31 positive cells in each field was averaged to determine the number of CD31 cells for each heart. This process was repeated for each heart by 3 independent investigators who were blinded to treatment groups. The mean value of the three investigators was used to represent the number of CD31 positive cells for each heart.

Statistical Analyses.

Data are presented as the mean ± S.E.M. The unpaired Student’s t test was used to compare data from stressed and nonstressed experimental groups in the elevated plus maze, western blots, histology, and immunohistochemistry. These analyses were performed using Graphpad Prism 7 software. P values < 0.05 were regarded as statistically significant. P values for normalized counts from RNA sequencing were adjusted using R (v3.3.3) software with DESeq2 as previously described (Love et al., 2014) (Benjamini Y and Y, 1995) to minimize the frequency of false discovery. Adjusted P values < 0.05 were regarded as statistically significant.

Results

Anxiety-related behavior in the elevated plus maze.

Previous work demonstrated that rats subjected to this stress model display anxiety-related behavior in the elevated plus maze (Rorabaugh et al., 2015; Zoladz et al., 2008; Zoladz et al., 2013). Consistent with previous studies, rats exposed to 31 days of psychosocial stress (n = 11) spent significantly (t(21) = 9.13, p < 0.0001) less time than nonstressed rats (n = 12) in the open arms of the maze (Fig. 1A). The number of closed arm entries was similar (p = 0.47) for stressed and nonstressed rats (Fig. 1B), indicating that stress had no significant effect on overall locomotor activity. These data provide behavioral validation of the efficacy of the stress regimen in rats that were subsequently used for cardiac analyses. Data from one stressed rat was excluded because the amount of time that it spent in the open arms of the elevated plus maze was more than 5 standard deviations from the mean.

Fig. 1. Stressed rats exhibit anxiety-related behavior in the elevated plus maze.

Fig. 1.

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Rats exposed to 31 days of psychosocial stress spent significantly less time than nonstressed rats in the open arms of the elevated plus maze (A). Stressed and nonstressed rats had similar numbers of entries into the closed arms, indicating that stress had no significant effect on locomotor activity (B). Data represent the mean ± S.E.M. of 11-12 animals per group.

Body weight.

Body weights of control and stressed rats were similar on day 1 of the stress protocol (278 ± 19 g and 290 ± 14 g for control and stressed animals, respectively; p = 0.61), and both experimental groups gained similar amounts of weight over the course of the 31-day stress regimen (187 ± 12 g and 185 ± 9 g; p = 0.92).

Stress-induced changes in cardiac gene expression.

RNA sequencing analysis was performed using RNA isolated from the left ventricular tissue from 5 stressed and 5 nonstressed rats. Principal component analysis (PCA) indicated that ventricles from nonstressed rats had variable baseline gene expression patterns that separated on principal component 1 (PC1), while ventricular tissue from all 5 stressed rats clustered closely and were separated by principal component 2 (PC2). (Fig. 2A). These data indicate that stress induced distinct and reproducible transcriptional changes in rat ventricles.

Fig. 2. RNA-sequencing of ventricular tissue from stressed and nonstressed rats.

Fig. 2.

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Principal component analysis of gene expression profiles indicated that hearts from nonstressed rats have variable baseline gene expression that separated on principal component 1 (PC1), while hearts from all 5 stressed rats clustered closely and were separated by principal component 2 (PC2) (A). A volcano plot illustrating gene expression alterations and P values for 12,218 genes. Genes of interest are annotated in red (B). Gene ontology analysis identified significantly enriched gene family clusters for upregulated (red) or downregulated (black) genes (C). Genes were ranked in order of most to least enriched in stressed hearts and analyzed by Geneset Enrichment Analysis (GSEA). The enrichment plot shows the normalized enrichment score (NES) for genes associated with endothelial-to-mesenchymal transition (D). Heatmap showing median-centered, log2-fold changes for select upregulated genes identified from Gene Ontology families in panel C (E).

In light of our finding that stressed hearts undergo transcriptional alterations, we next sought to define these transcriptional programs. Out of 12,218 detected genes, 481 genes were significantly (adjusted P-value < 0.05) upregulated and 660 genes were significantly downregulated (Fig. 2B; the differential gene expression analysis for the entire data set is available at https://figshare.com/s/5f32d3c03635c9bf46f5). Gene ontology analysis indicated that upregulated genes were primarily associated with angiogenesis (GO:0001525) and endothelial cell migration (GO:0043542) (Fig. 2C; Table 1), mesenchyme development (GO:0060485) and mesenchymal differentiation (GO:0048762) (Fig. 2C; Table 2), and extracellular matrix organization (GO:0030198) (Fig. 2C; Table 3). Interestingly, downregulated genes were only associated with one gene cluster (protein polyubiquitination; GO:0000209) (Fig. 2C), suggesting that only upregulated genes fall within defined biologically relevant processes. Additionally, we ranked genes in order from increasing to decreasing log2-fold expression changes in stressed hearts and performed gene set enrichment analysis (GSEA) to identify pathways associated with stress. We found that ‘Endothelial to Mesenchymal Transition’ was among the top enriched gene sets (Normalized Enrichment Score=1.26, P=0.03) (Fig 2D). Thus both analyses (gene ontology and gene set enrichment analysis) are consistent with increased formation of mesenchymal tissue. These data suggest that stressed hearts may have an increase in fibrotic tissue. Notably, many genes that were significantly upregulated have been associated with cardiac fibrosis, including PDGFB,(Gallini et al., 2016) TBX2,(Shirai et al., 2009) SPARC,(Vaughan et al., 2018) and ACTA2.(Teekakirikul et al., 2010; Zeisberg et al., 2007) (Figure 2E). Differential gene expression analysis data for the entire data set is available at https://figshare.com/s/5f32d3c03635c9bf46f5.

Table 1.

Selected changes in the number of mRNA transcripts (normalized counts) for genes in the angiogenesis (GO:0001525) and endothelial cell migration (GO:0043542) gene ontology clusters.

Gene Symbol NCBI Reference # Normalized Counts Adjusted P value
Nonstressed Stressed
RAPGEF3 NM_021690 1,597 ± 67 1,869 ± 93 1.64 E-06
PLXND1 NM_001107881 5,576 ± 340 8,524 ± 113 4.79 E-07
CDH13 NM_138889 1,504 ± 47 2,270 ± 129 6.34 E-06
TIE1 NM_053545 3,639 ± 192 4,347 ± 146 5.90E-05
FLT4 NM_053652 396 ± 38 605 ± 22 0.004
FLNA NM_001134599 6,078 ± 176 7,931 ± 250 0.0001
NOS3 NM_021838 2,291 ± 122 3,078 ± 125 0.003
EPHA2 NM_001108977 198 ± 7 298 ± 12 0.0002
NOTCH1 NM_001105721 3,126 ± 194 4,405 ± 107 0.0002
NOTCH3 NM_020087 1,683 ± 102 2,104 ± 43 0.03
NOTCH4 NM_001002827 1,387 ± 94 1,869 ± 93 0.01
VEGFB NM_053549 1,976 ± 245 3,329 ± 336 0.007
NRP2 NM_030869 2,305 ± 100 3,677 ± 261 0.0001
SPARC NM_012656 44,383 ± 1,024 51,715 ± 11,878 0.0006
FGFR1 NM_024146 1,550 ± 130 2,121 ± 39 0.008
RASIP1 NM_001106261 1,113 ± 47 1,433 ± 42 0.004
PDGFB NM_031524 693 ± 54 1,058 ± 40 0.0003
EFNA1 NM_053599 538 ± 12 657 ± 28 0.04
EFNB2 NM_001107328 1,351 ± 28 1,565 ± 28 0.03
PXN NM_001012147 2,030 ± 50 2,364 ± 48 0.02
PTPRM NM_001168632 2,625 ± 154 3,277 ± 33 0.02
ANGPT2 NM_134454 200 ± 21 284 ± 18 0.048
BMP4 NM_012827 164 ± 7 223 ± 14 0.03
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Data represent the mean ± SEM of 5 hearts for each group.

Table 2.

Selected changes in the number of mRNA transcripts (normalized counts) for genes in the mesenchyme development (GO:0060485) and mesenchyme differentiation (GO:0048762) gene ontology clusters.

Gene Symbol NCBI Reference # Normalized Counts Adjusted P value
Nonstressed Stressed
ACTA2 NM_031004 1,285 ± 105 1,813 ± 47 0.004
FLNA NM_001134599 6,079 ± 176 7,931 ± 250 0.0001
NRP2 NM_030869 2,305 ± 100 3,318 ± 180 0.0001
ERG NM_133397 894 ± 41 1,284 ± 87 0.003
CORO1C NM_001109327 1,449 ± 168 2,248 ± 89 0.005
DAB2IP NM_138710 2,105 ± 101 2,714 ± 93 0.005
BMP4 NM_012827 164 ± 7 223 ± 14 0.03
SEMA7A NM_001108153 1,631 ± 42 1,961 ± 45 0.009
SEMA5B NM_001107091 66 ± 12 123 ± 8 0.02
SEMA3F NM_001108185 432 ± 73 709 ± 29 0.02
TBX2 NM_001107033 147 ± 22 242 ± 12 0.02
NOTCH1 NM_001105721 3,126 ± 194 4,405 ± 107 0.0002
LAMA5 NM_001191609 2,083 ± 63 2,573 ± 140 0.03
PHLDB1 NM_001191578 7,833 ± 455 9,989 ± 504 0.03
FGFR1 NM_024146 1,550 ± 130 2,121 ± 39 0.008
EFNA1 NM_053599 538 ± 12 657 ± 28 0.04
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Data represent the mean ± SEM of 5 hearts for each group.

Table 3.

Selected changes in the number of mRNA transcripts (normalized counts) for genes in the extracellular matrix assembly (GO:0085029) and extracellular matrix organization (GO:0030198) gene ontology clusters.

Gene Symbol NCBI Reference # Normalized Counts Adjusted P value
Nonstressed Stressed
MYH11 NM_001170600 2,117 ± 227 3,019 ± 103 0.02
ELN NM_012722 1,799 ± 93 2,416 ± 199 0.03
PHLDB1 NM_001191578 7,833 ± 455 9,989 ± 504 0.03
PXDN NM_001271261 3,835 ± 108 5,615 ± 134 3.22E-11
VTN NM_019156 1,322 ± 87 1,746 ± 79 0.01
SERPINH1 NM_017173 7,703 ± 364 9,430 ± 234 0.02
FLOT1 NM_022701 2,132 ± 190 2,792 ± 60 0.03
LAMB2 NM_012974 19,323 ± 652 2,3729 ± 1440 0.04
THBS2 NM_001169138 1,253 ± 40 1,557 ± 75 0.02
ADAMTS9 NM_001107877 886 ± 41 1,174 ± 41 0.003
NOTCH1 NM_001105721 3,126 ± 194 4,405 ± 107 0.0002
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Data represent the mean ± SEM of 5 hearts for each group.

Stress-induced cardiac fibrosis and immune cell infiltration.

RNA sequencing indicated significant upregulation of genes involved in angiogenesis, cell migration, differentiation into mesenchymal tissue, and assembly /organization of extracellular matrix (Fig. 2; Tables 1 – 3). Thus, H&E and Masson’s trichrome staining were used to determine whether these changes in gene expression reflected histological changes in the myocardium. Multifocal lesions characterized by myofiber necrosis (Fig. 3A), infiltration of mononuclear immune cells (Fig. 3B), and collagen deposition (Fig. 3C) were identified in the ventricular walls of all (n = 6) stressed rats. These lesions were absent from the hearts of all (n = 6) nonstressed rats with the exception of 1 animal that exhibited minor fibrosis (score = 2) without necrosis or mononuclear cell infiltration.

Fig. 3. Stress induces myocardial lesions characterized by fibrosis, necrosis, and mononuclear cell infiltration.

Fig. 3.

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Hearts from stressed and nonstressed rats were evaluated by H&E and Masson’s trichrome staining. Hearts from stressed rats developed multifocal lesions characterized by necrosis (A,D), infiltration by mononuclear immune cells (B,D), and collagen deposition (C,D). Data represent the mean ± S.E.M. of 6 animals. Representative photographs of heart sections are shown in panel D. Blue circles in photographs of subgross heart images indicate heart regions that were photographed at higher (15.8 X) magnification. Scale bars in panel D represent 200 µm.

RNA sequencing and western blots indicated that α-smooth muscle actin was upregulated at both the mRNA transcript (t(8) = 4.60, p < 0.005) (Fig. 4A) and protein (t(8) = 2.69, p < 0.05) (Fig. 4B) levels in stressed heart. Increased expression of this myofibroblast marker is consistent with the observed deposition of collagen in stressed hearts (Fig. 3C) (van Putten et al., 2016). We also observed that T-cadherin (CDH13) was significantly upregulated at the mRNA transcript (t(8) = 5.56, p < 0.0005) (Fig. 4C) and protein (t(8) = 4.80, p = 0.001) (Fig. 4D) levels in hearts from stressed rats. These stress-induced changes are consistent with fibrosis and extracellular matrix remodeling (Asada et al., 2007; Frismantiene et al., 2014).

Fig. 4. Stress upregulates myocardial expression of α-smooth muscle actin and T-cadherin.

Fig. 4

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RNA sequencing identified upregulation of mRNA transcripts encoding α-smooth muscle actin (encoded by ACTA2) (A) and T-cadherin (encoded by CDH13) (C) in hearts from rats exposed to 31 days of psychosocial stress compared to hearts from nonstressed rats. Western blots confirmed significant increases in the expression of α-smooth muscle actin (B) and T-cadherin (D) at the protein level. Data in panels A and C represent the mean ± S.E.M. of 6 hearts. Data in panels B and D represent the mean ± S.E.M. of 5 hearts.

Impact of stress on CD31 Positive cells.

RNA sequencing indicated that hearts from stressed rats exhibited upregulation of genes involved in angiogenesis, blood vessel morphogenesis, and endothelial cell migration (Fig. 2). CD31 is commonly used as an endothelial marker (Lv et al., 2018; Tian et al., 2018; Wang et al., 2018). Thus, we compared the number of CD31 positive cells in high magnification (400 X) images of ventricles from stressed and nonstressed hearts. Immunohistochemistry indicated a significant (t(10) = 2.43, p < 0.05) decrease in the number of CD31 positive cells in ventricles from stressed rats compared to ventricles from nonstressed rats (Fig. 5).

Fig. 5. Stressed hearts exhibit a decrease in the number of CD31 positive cells.

Fig. 5.

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Predator-based psychosocial stress decreased the number of CD31-positive cells in the ventricular myocardium. Ventricular tissue was stained with a CD31-specific antibody, and the number of CD31 positive cells were counted by in 5 high (400 X) magnification fields for each heart by three independent investigators that were blinded to animal treatment. Data represent the mean ± S.E.M. of 6 hearts for each group.

Discussion

PTSD and other forms of chronic psychosocial stress are associated with an increased risk of cardiovascular abnormalities including hypertension, endothelial dysfunction, atherosclerosis, cardiac arrhythmias, and myocardial infarction (Ahmadi et al., 2011; Beristianos et al., 2016; Grenon et al., 2016; Khazaie et al., 2013; Vaccarino et al., 2013). We previously reported that rats exposed to the predator- stress model used in this study develop myocardial hypersensitivity to ischemic injury (Rorabaugh et al., 2015; Rorabaugh et al., 2018). The present work demonstrated that these animals also develop multifocal cardiac lesions characterized by myofiber necrosis, fibrosis, and infiltration by mononucleated immune cells (Fig. 3). This was accompanied by changes in gene expression associated with endothelial cell migration, mesenchymal development, and extracellular matrix organization (Fig. 2; Table 1, Table 2, Table 3). In addition, changes in the expression of markers for endothelial to mesenchymal transition were observed at both the mRNA (Fig. 2) and protein levels (Fig. 4, Fig. 5). These data provide evidence of cardiac stress and tissue remodeling in rats exposed to predator stress . These changes in histology and gene expression may underlie our previous observation that rats exposed to this model develop myocardial hypersensitivity to ischemic injury.(Rorabaugh et al., 2015; Rorabaugh et al., 2018)

Cho et al. previously used DNA microarray analysis to assess changes in gene expression in the mouse heart following 1-10 days of exposure to a social defeat model (Cho et al., 2014). Similar to the current study, these investigators identified changes in gene expression that were indicative of extracellular matrix remodeling, angiogenesis, and the formation of mesenchymal tissue. This prior work differed from the present study in fundamental ways including the species (mouse vs rat), stress model (stress induced by an aggressor mouse vs a predator), the duration of stress (1-10 days vs 31 days), and the method of assessing gene expression (DNA microarray vs RNA sequencing). Despite these differences, similar stress-induced changes in gene expression were observed, suggesting that increased expression of genes related to these ontologies represent a fundamental cardiac response to psychosocial stress.

Rats exposed to the psychosocial stress model used in the current study exhibit significant increases in superoxide, peroxynitrite, and total reactive oxygen species in circulating blood, brain, and adrenal tissue (Ebenezer et al., 2016; Wilson et al., 2013). These animals also exhibit increased expression of pro-inflammatory cytokines (interleukin-1β and interleukin-18), decreased expression of anti-inflammatory cytokines (interleukin-10 and interleukin-4), and increased expression of the NALP3 inflammasome in the brain(Wilson et al., 2013). However, these proinflammatory markers have not previously been assessed in hearts of animals exposed to this model. RNA sequencing did not identify significant changes in the presence of mRNA transcripts encoding pro-inflammatory cytokines in the heart. However, the fact that hearts from stressed rats were infiltrated with mononucleated immune cells provides evidence that this form of stress produces an inflammatory response in the heart.

The gene expression profile, increased secretion of collagen, increased expression of α-smooth muscle actin, and the decrease in the number of CD31 positive cell are consistent with endothelial to mesenchymal transition in hearts from stressed rats. In addition to these phenotypic markers, stressed hearts also exhibited upregulation of genes (Zeb1, bone morphogenetic protein-4, notch 1, β-catenin) (Cho et al., 2018) that are known to mediate endothelial to mesenchymal transition (Table 1). However, we did not trace fibroblast lineage to determine whether stressed hearts contain an increase in the number of fibroblasts of endothelial origin. Thus, we cannot exclude the possibility that the decrease in endothelial cells and the increase in mesenchymal markers (collagen and α-smooth muscle actin) occurred independent of each other rather than through the process of endothelial to mesenchymal transition.

T-cadherin was upregulated in stressed hearts at both the mRNA transcript and protein levels. Previous work demonstrated that T-cadherin is involved in the organization of collagen fibrils in vitro (Frismantiene et al., 2014) and that it promotes remodeling of the vascular wall following vascular injury (Kudrjashova et al., 2002). Asada et al. (Asada et al., 2007) reported that T-cadherin was upregulated in a rat model of liver fibrosis. Thus, it is not surprising that T-cadherin was upregulated in the heart under conditions that promote collagen deposition and fibrosis. However, we are unaware of any previous studies in which T-cadherin has been associated with extracellular matrix remodeling or fibrosis in the heart. Further work is needed to better understand the role of T-cadherin in myocardial fibrosis.

The observed decrease in the number of CD31 positive cells in stressed hearts (Fig. 5) seemed contradictory to the outcome of gene ontology analysis (Fig. 2C) which suggested that stressed hearts exhibit a gene expression profile indicative of increased angiogenesis. However, it should be noted that there is overlap in the content of these gene clusters. Several genes that were significantly upregulated (FLNA, NRP2, BMP4, FGFR1, EFNA1) at the mRNA level are part of both the angiogenesis (Table 1) and mesenchyme development gene clusters (Table 2). We have performed western blots on ventricular homogenates from both stressed and nonstressed rats for a number of proteins that are associated with angiogenesis including endothelial nitric oxide synthase, vascular endothelial growth factor receptor 3, and vascular cell adhesion molecule 1 (data not shown) and found no stress-induced effect on the expression of these angiogenesis markers at the protein level. These data suggest that overlap between gene clusters may have caused gene ontology analysis to falsely identify myocardial angiogenesis as a stress-induced response in these animals.

PTSD is associated with an increased risk of developing cardiac disorders (Ahmadi et al., 2011; Beristianos et al., 2016; Khazaie et al., 2013; Vaccarino et al., 2013). The identification of pharmacotherapies that can prevent the development of cardiac disorders in patients with PTSD or other forms of chronic psychological stress would be of great clinical value. Increased sympathetic signaling is a hallmark feature of PTSD, and sympatholytics such as clonidine and propranolol have demonstrated efficacy in treating trauma-related nightmares, intrusive thoughts, and hyperarousal in some patients with PTSD. (Alao et al., 2012; Belkin and Schwartz, 2015; Boehnlein and Kinzie, 2007; Kinzie and Leung, 1989; Lindgren et al., 2013; Onder et al., 2006) Clonidine prevents the development of some of the PTSD-like physiological (increased systolic and diastolic blood pressure, tachycardia, and adrenal hypertrophy) and behavioral (anxiety in elevated plus maze and increased startle response) effects induced by this animal model.(Zoladz et al., 2013) However, neither clonidine or propranolol prevent the development of myocardial hypersensitivity to ischemia in this rat model,(Rorabaugh et al., 2018) indicating that this stress-induced effect is not the result of excessive sympathetic signaling. The current data suggest that anti-inflammatory agents or pharmacotherapies that inhibit endothelial to mesenchymal transition and the deposition of collagen may be more effective approaches to protecting the heart from the adverse effects of psychosocial stress.

In conclusion, our data indicate that predator stress promotes myocardial necrosis, collagen deposition, infiltration of immune cells and reprogramming of myocardial gene expression. These effects are consistent with the observation that animals exposed to this model develop myocardial hypersensitivity to ischemic injury (Rorabaugh et al., 2015; Rorabaugh et al., 2018). They are also consistent with previous work demonstrating that people who develop PTSD and other forms of chronic stress are at increased risk of developing cardiac disorders such as myocardial infarction and arrhythmias (Ahmadi et al., 2011; Beristianos et al., 2016; Khazaie et al., 2013; Vaccarino et al., 2013). This predator-based model of psychosocial stress may provide a useful tool to understand the mechanism by which PTSD impacts the heart and to identify therapeutic strategies to prevent the development of heart diseases in people who suffer from PTSD and other chronic stress disorders.

Acknowledgements

The authors thank Dr. Kevin Childs (Michigan State University Research Technology Support Facility Genomics Core) for help with the RNA sequencing experiments and Dr. Krista LePerle (The Ohio State University Comparative Pathology and Mouse Phenotyping core facility) for preparing hearts for histological analysis.

Funding: This work was supported by a grant from the National Heart Lung Blood Institute (R15HL132322) to BRR and a core grant from the National Cancer Institute (P30CA016058) to The Ohio State University Comparative Pathology and Mouse Phenotyping core facility.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Raw data from next-generation sequencing experiments were deposited in the National Center for Biotechnology Information Sequence Read Archive and are available under BioProject ID number PRJNA498684. Differential gene expression analysis of these data is shown at https://figshare.com/s/5f32d3c03635c9bf46f5.

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