Molecular evidence of stress-induced acute heart injury in a mouse model simulating posttraumatic stress disorder Ji-Hoon Choa,1, Inyoul Leea,1, Rasha Hammamiehb,1, Kai Wanga,1, David Baxtera, Kelsey Scherlera, Alton Etheridgea, Alena Kulchenkoa, Aarti Gautamb, Seid Muhieb, Nabarun Chakrabortyb, David J. Galasc, Marti Jettb, and Leroy Hooda,2 aInstitute for Systems Biology, Seattle, WA 98109; bIntegrative Systems Biology, US Army Center for Environmental Health Research, Fort Detrick, MD 21702; and cPacific Northwest Diabetes Research Institute, Seattle, WA 98122 Contributed by Leroy Hood, January 7, 2014 (sent for review December 2, 2013) Posttraumatic stress disorder (PTSD) is a common condition induced and subsequently, relate these disease-perturbed dynamical net- by life-threatening stress, such as that experienced by soldiers under works to the pathophysiology of the disease (10, 11). This ap- battlefield conditions. Other than the commonly recognized behav- proach may lead to more informative diagnostic markers for ioral and psychological dysfunction, epidemiological studies have identifying the disease early, provide information as to which also revealed that PTSD patients have a higher risk of other diseases, organs are disease-involved, and provide insights into therapeutic such as cardiovascular disorders. Using a PTSD mouse model, we approaches for reversing the progression of the disease. investigated the longitudinal transcriptomic changes in heart tissues Individuals with PTSD also have a higher risk of cardiovas- after the exposure to stress through intimidation. Our results revealed cular conditions, with an increased basal heart rate and blood acute heart injury associated with the traumatic experience, reflecting pressure, higher risk for hypertension and stroke, altered platelet the underlying biological injury processes of the immune response, activity, and elevated blood cholesterol and triglyceride levels extracellular matrix remodeling, epithelial-to-mesenchymal cell tran- (12–15). An 1871 report noted that serious cardiac disorders (car- sitions, and cell proliferation. Whether this type of injury has any long- diomyopathies, heart failure, heart pain, etc.) were a consequence term effects on heart function is yet to be determined. The of extended stress exposures in soldiers from the US Civil War (16). differing responses to stress leading to acute heart injury in different This report led to our interest in examining the effects of short-term inbred strains of mice also suggest that this response has a genetic stress exposure on heart tissues in the PTSD mouse model. as well as an environmental component. Accordingly, the results We adapted an aggressor-exposed social stress mouse model from this study suggest a molecular basis for the observed higher risk for PTSD by pairing aggressor mice (SJL strain) with different of cardiovascular disorders in PTSD patients, which raises the strains of subservient mice. This model generated a simple and likelihood of cardiac dysfunction induced by long-term stress reproducible set of PTSD-associated conditions in the sub- exposures. servient mice, such as avoidance behaviors (17). The reporting of myocarditis in short-term stressed subservient mice suggests that systems biology | transcriptome | microRNA stress-induced PTSD may also cause transitory heart injury. Through a detailed longitudinal transcriptomic study [mRNAs and microRNAs (miRNAs)] of the heart tissues in the PTSD n a modern society, the overall lifetime probability of an in- dividual being exposed to a traumatic event, such as a traffic I Significance accident, physical assault, or combat situation, is 40–90% in the general population (1). However, the overall lifetime chance of an individual suffering posttraumatic stress disorder (PTSD) is Exposure to extremely stressful conditions is common, and the lower, about 7–12% (1, 2). At any given time, about 7% of the effect of such exposure on neuropsychiatric function is well- population in the United States suffers from some form of PTSD documented with posttraumatic stress disorder (PTSD). Epi- (3). Hence, PTSD is perhaps the most common psychological demiological studies reveal a higher risk for cardiovascular disorder. The fact that not all individuals develop PTSD after conditions among individuals exposed to traumatic events. experiencing one or more traumatic events suggests there may However, the underlying molecular mechanism for ailments also be a genetic contribution to the development of the disease. associated with stress exposure is yet to be fully understood. Patients with PTSD frequently show sociobehavioral problems Our study with animal models revealed genetically associated and have a higher risk of drug or alcohol abuse. In addition, stress-induced tissue injuries on peripheral organs, including patients with PTSD often display structural changes in the pre- the heart. Longitudinal transcriptomics studies uncovered de- frontal cortex, the amygdala, and the hippocampus and bio- tailed molecular events involved in stress-related heart damage chemical changes, such as a higher ratio of norepinephrine/ followed immediately by tissue-repairing processes; whether cortisol in urine, higher total cholesterol levels in the blood, and this injury and repairing process causes long-term effects is elevated norepinephrine levels in the cerebrospinal fluid (4–8). uncertain. Our findings on heart injury in a PTSD mouse model These findings indicate a disruption of the normal hypotha- clearly indicate physiological changes arising from stress. lamic–pituitary–adrenal function and the autonomic nervous system in PTSD patients (9). A systems approach to the study of Author contributions: I.L., R.H., K.W., D.J.G., M.J., and L.H. designed research; J.-H.C., I.L., the molecular changes associated with a stress-induced, PTSD- R.H., K.W., D.B., K.S., A.E., A.K., A.G., S.M., and N.C. performed research; J.-H.C. contrib- like mouse model will allow us to gain a better understanding of uted new reagents/analytic tools; J.-H.C., I.L., R.H., K.W., A.E., and L.H. analyzed data; and the disease. It also allows us to evaluate the effects of PTSD-like J.-H.C., I.L., K.W., A.E., D.J.G., and L.H. wrote the paper. stress exposure on organs other than the brain. This systems The authors declare no conflict of interest. approach is embedded in the concept that disease arises as Data deposition: The data reported in this paper have been deposited in the Gene Ex- a consequence of disease-perturbed networks in relevant tissues pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE52875). initiated by either genetic and/or environmental stimuli—and the 1J.-H.C., I.L., R.H., and K.W. contributed equally to this work. fact that we can study dynamically, from onset to the final stage 2To whom correspondence should be addressed. E-mail: [email protected]. of the disease, the behavior of these disease-perturbed networks This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. through -omic analyses in relevant tissues from animal models 1073/pnas.1400113111/-/DCSupplemental. 3188–3193 | PNAS | February 25, 2014 | vol. 111 | no. 8 www.pnas.org/cgi/doi/10.1073/pnas.1400113111 Downloaded by guest on September 24, 2021 mouse, we observed the involvement of key molecular processes, identified, which suggests an active ongoing tissue repair process including an inflammatory response, extracellular remodeling and, in the heart that is complete by 10 d. The T10R1 group showed epithelial-to-mesenchymal transitions (EMTs), in PTSD-induced fewer changes between the control and stressed mice compared heart injury. These findings may provide possible molecular with the T5R1 group, which indicates that a similar repair pro- explanations for the higher risk of cardiovascular diseases in cess seems to be initiated early and is functioning even under the patients with PTSD. stress environment (from 455 DEGs to 40 DEGs). This con- tention is supported by the fact that the changing expression Results patterns of the DEGs identified in the T5R1 group are similar to We designed our initial study (study I) with four experimental those patterns seen in the T10R1 group, despite the observation conditions, in which we varied the length of time that subservient that most of them did not show greater than ±1.5-fold changes mice were exposed to aggressor mice as well as the length of rest and had an FDR < 0.1 (hence, missing our selection criteria for time after the exposure [number of days of trauma exposure (T); DEGs; only 31 of 455 genes were classified as DEGs in T10R1). number of days of rest after exposure (R)]. The conditions in- Enrichment analyses of Kyoto Encyclopedia of Genes and cluded were short exposure–short rest (T5R1), short exposure– Genomes (KEGG) pathways and gene ontology (GO) terms with long rest (T5R10), long exposure–short rest (T10R1), and long 377 up-regulated genes identified between the two groups (of exposure–long rest (T10R28) (Table 1). SJL mice were used as 464 DEGs total) revealed a strong association of cell cycle and the aggressor strain, and C57BL/6j, DBA/2j, and BALB/cj mice ECM remodeling activities—consistent with active tissue re- were used as subservient strains. Multiple subservient strains pair processes. The full list of enriched GO terms is given in were used to identify the shared features of this stress exposure Tables S1 and S2. No significantly enriched pathways or GO and be able to subtract away the strain-specific (biological or ge- terms were associated with 87 down-regulated genes identified netic) features—an important aspect of using a systems approach from either the T5R1 or T10R1 group. The 31 shared DEGs to reduce biological noise associated with the strain-specific fea- between T5R1 and T10R1 groups showed enrichment in ECM- tures of the response. We were also interested in whether the related functions, including cell adhesion and collagen fibril different strains exhibited different responses to stress under organization.