Available online at www.jbr-pub.org Open Access at PubMed Central

The Journal of Biomedical Research, 2016, 30(5): 380–385

Original Article

Effects of epinephrine on -related expressions in cultured rat cardiomyocytes

Henry Liu1, ✉, Lisa Sangkum1, Geoffrey Liu1, Michael Green1, Marilyn Li2, Alan Kaye3 1 Department of Anesthesiology & Perioperative Medicine, Drexel University College of Medicine, Hahnemann University Hospital, Philadelphia, PA 19102, USA; 2 Division of Genomic Diagnostics, Dept. of Pathology & Laboratory Medicine, Children’s Hospital of Philadelphia, Perelman School of Medicine University of Pennsylvania, Philadelphia, PA 19104, USA; 3 Department of Anesthesiology, LSUHSC-New Orleans, New Orleans, LA 70112, USA

Abstract Epinephrine is often used for the treatment of patients with heart failure, low cardiac output and cardiac arrest. It can acutely improve hemodynamic parameters; however, it does not seem to improve longer term clinical outcomes. Therefore, we hypothesized that epinephrine may induce unfavorable changes in of cardiomyocyte. Thus, we investigated effects of epinephrine exposure on the mediation or modulation of gene expression of cultured cardiomyocytes at a genome-wide scale. Our investigation revealed that exposure of cardiomyocytes to epinephrine in an in vitro environment can up-regulate the expression of -2 gene (+ 2.1 times), and down-regulate the gene expression of 1 ( – 3.7 times), plasminogen activator inhibitor-1 ( – 2.4 times) and SPARC-related modular calcium-binding -2 ( – 4.5 times). These changes suggest that epinephrine exposure may induce inhibition of angiogenesis-related gene expressions in cultured rat cardiomyocytes. The precise clinical significance of these changes in gene expression, which was induced by epinephrine exposure, warrants further experimental and clinical investigations. Keywords: epinephrine, angiogenesis, gene expression, cardiomyocytes, angiopoietin-2, , plasminogen activator inhibitor-1, SPARC-related modular calcium-binding protein

Introduction Epinephrine, as an adrenergic receptor agonist, is not only a potent stimulator of myocardial contractility but Patients with heart failure, septic shock, low cardiac also, a strong stimulator of cardiac chronotropic effect. output syndrome and other clinical scenarios, will often Epinephrine is administered to improve hemodynamic [1] necessitate inotropic therapy . Among those com- parameters, such as cardiac output (CO), mean arterial monly used inotropic agents, epinephrine is one of the blood pressure (MAP), and systemic vascular resistance most potent and most frequently used inotropes in (SVR). The use of epinephrine seems to improve short clinical practice, especially in cases when inotropic term clinical outcomes. Donnino's group in Boston support is warranted perioperatively and in patients with analyzed the data from Get With the Guidelines- [2–3] cardiac arrest . Resuscitation database (formerly National Registry of

✉ Corresponding author: Henry Liu, MD, Department of Anesthe- March 2016, Epub 30 April 2016 siology, Drexel University College of Medicine Philadelphia, PA CLC number: R542.2, Document code: A 19102, [email protected]. The authors reported no conflict of interests Received 16 February 2016, Revised 18 March 2016, Accepted 29 © 2016 by the Journal of Biomedical Research. All rights reserved. doi:10.7555/JBR.30.20160024 Effects of epinephrine on angiogenesis-related gene expressions 381

Cardiopulmonary Resuscitation, NRCPR). The data- epinephrine was added to the culture medium to make a base is sponsored by the American Heart Association final concentration of epinephrine of 1μmol/L. The cells (AHA) and contains prospective data from 570 were then cultured for 48 hours without medium American hospitals collected from 1 January 2000 to change. Flasks with similar culture medium and 19 November 2009. They found that in patients with identical concentration of H9C2 cells without epinephr- non-shockable cardiac arrest in a hospital setting, earlier ine served as controls; this technique is modified from administration of epinephrine is associated with a higher the technique reported by Merten[10]. Each test was probability of return to spontaneous circulation, survi- done in triplicates, two serves as experimental, one val in hospital, and neurologically intact survival[4]. serves as control. At 48 hours, the cell culture was There have been other studies which also supported the stopped and total RNA was extracted from cultured concept that epinephrine improves survival for out of cardiomyocytes and purified by using Trizol (Therma hospital arrest patients and improves survival for in- Fisher Scientific, Waltham, MA, USA) and RNeasy hospital cardiac arrests[5], at least in selective groups of cleanup (Qiagen, Inc., Valencia, CA, USA) accord- patients[6–7]. However, Hiyashi et al. evaluated the ing to the manufacturer's protocol. The total RNA yield outcomes of 3,161 adult non-traumatic bystander- was quantified by NanoDrop 1,000 Spectrophotometer witnessed cardiac arrest patients. They found that the (Therma Fisher Scientific, Waltham, MA USA) and the only group which potentially benefited from epinephr- quality was verified by gel electrophoresis. ine administration was those patients with cardiac arrest from ventricular fibrillation. Otherwise, epinephrine Microarray groups had a significantly lower rate of neurologically The RNA was then used for whole genome gene intact 1-month survival than the non-epinephrine group expression evaluation. This array contains more than (4.1% vs. 6.1%, P = 0.028)[3]. Furthermore, data from 41,000 rat . The cDNA was synthesized from the the European ALARM-HF study indicates that cate- RNA samples and used to synthesize fluorescent cRNA. cholamine inotropes should be used cautiously, as they Labeled cRNA samples were hybridized to the Whole have been demonstrated to increase the risk of in- Rat Genome Oligo Microarray slides (Agilent Technol- hospital mortality[2,8]. Additional studies have revealed ogies, Santa Clara, CA, USA). After hybridization, that a combination of inotropes can cause increased arrays were washed and scanned. hospital mortality[9]. We, therefore, hypothesized that epinephrine admin- Data analysis istration may alter gene expression in cardiomyocytes, Data were imported into GeneSpring GX 11 software which may contribute to observed adverse clinical (Agilent Technologies, Santa Clara, CA) as 20 one- outcomes. Our present investigation was conducted to color arrays and normalized to the median per chip and investigate gene expression changes after exposure of the median value per gene across all arrays. Parameter cardiomyocytes to epinephrine. data were added in order that microarrays could be grouped by time and treatment. Guided workflow Methods and materials returned several gene lists. These were analyzed for fi Cell culture signi cant and pathway hits based on passing P value criterion (P < 0.05). Epinephrine- Rat cardiomyocyte cell line H9C2 (ATCC, Rockville, induced gene expressional changes related to angiogen- Maryland) was utilized and cardiomyocytes were esis were identified (P < 0.05). inoculated at 0.5 M/mL and cultured at 37°C, 5% fi CO2 in Dulbecco's modi ed Eagle's medium (DMEM) Results with 10% fetal calf serum (FCS) in 25 mL flasks, with penicillin (100 U/mL), and streptomycin (100 mg/mL). The results of the present investigation are listed in The cells were kept overnight and the next morning, Table 1 and Fig. 1. Up-regulated genes included

Table 1 Genes with altered expressions in cultured rat cardiomyocytes exposed to epinephrine. Increased gene expressions Decreased gene expressions ANGPT2 (2.12 times) NRG1( – 3.653 Times) SERPINE1 ( – 2.42 times) SMOC2 ( – 4.52 times)

Note: (ANGPT2 (Angiopoietin-2); NRG1 (Neuregulin1); SERPINE1 (plasminogen activator inhibitor-1; PAI-1 is a serine protease inhibitor); SMOC2 (SPARC-related modular calcium-binding protein-2). P < 0.05. 382 Liu H et al. J Biomed Res, 2016, 30(5)

Fig. 1 Gene expression changes related to angiogenesis after exposure of cardiomyocytes to epinephrine for 48 hours. Red-highlighted indicates increased expression while green-highlighted indicates decreased expression. The numbers show the times of the gene expression changes. Effects of epinephrine on angiogenesis-related gene expressions 383

ANGPT2 (Angiopoietin-2, a natural antagonist for pathway activates β1- and N-cadherin in LSK- ANGPT1 and TIE), which was increased by 2.12 TIE-2 + cells and promotes hematopoietic stem cell times. Down-regulated gene expression included NRG1 (HSC) interactions with extracellular matrix and its (Neuregulin1, a peptide essential for the development of cellular components. Although Ang2 and Ang3 behave heart), which was decreased by – 3.653 times; SER- as competitive antagonists, they function by binding to PINE1 (plasminogen activator inhibitor-1; PAI-1 is a their prospective physiologic receptors, Tie-1 and Tie-2. serine protease inhibitor), expression was decreased Though it is still controversial which of the Tie 2.42 times. SMOC2 (SPARC-related modular calcium- receptors mediate functional signals downstream of binding protein-2) expression was down-regulated by- Ang1 stimulation, it is clear that at least Tie-2 is capable 4.52 times. of physiologic activation as a result of binding the [15–16]. Up-regulation of Ang2 by 2.1 Discussion times may potentially inhibit the angiogenic effects of Ang1 stimulation and can be harmful to ischemic Epinephrine remains a widely used pharmacological myocardial survival. agent in clinical practice. The results of the present NRG1 encodes neuregulin1, which is a peptide investigation revealed significant changes in gene essential for cardiac development[17]. Neuregulin 1 is expression that may provide a link in understanding one of the four in the neuregulin family which the clinical effects of epinephrine administration in acts on the epidermal receptor (EGFR) different pathological settings. family of receptors[18]. Neuregulin 1 is produced in ANGPT2 was significantly up-regulated, increasing many isoforms by , which allows it by 2.1 times. NRG1 had significantly down-regulated to perform a wide variety of functions. These isoforms gene expression by 3.7 times, SERPINE1 gene expres- include heregulins (HRGs), glial growth factors sion was down-regulated by 2.4 times, and SMOC2 (GGFs), and sensory and motor neuron-derived factor gene expression was down-regulated by 4.5 times. In (SMDF). These isoforms are tissue-specific and sig- this regard, it remains controversial as to the precise nificantly differ in their structures. All HRG isoforms effects of epinephrine on short and long-term clinical contain immunoglobulin (Ig) and epidermal growth outcomes in patients with cardiac arrest, heart failure factor-like (EGF-like) domains. GGF and GGF2 iso- and/or other clinical scenarios which necessitate forms also contain a kringle-like sequence plus immu- – inotropic support[2 3]. noglobulin and EGF-like domains; and the SMDF Angiogenesis is a physiological process through isoform shares only the EGF-like domain with other which new vascular structures form from preexisting isoforms[19]. The receptors for all NRG1 isoforms are blood vessels[11]. Angiogenesis proceeds through the ERBB family of kinase transmembrane sprouting, endothelial cell migration, proliferation, and receptors [the ERBB family includes Her1 (EGFR, vessel destabilization and stabilization. This process is ErbB1), Her2 (Neu, ErbB2), Her3 (ErbB3), and Her4 different from vasculogenesis, the latter is the formation (ErbB4)][18]. Through they displayed interaction with of endothelial cells from mesoderm cell precursors[11]. ERBB receptors, NRG1 isoforms induce the growth and Angiogenesis is responsible for most blood vessel differentiation of epithelial, neuronal, glial, and poten- growth during development and in disease[12]. Angio- tially other types of cells. Neuregulin-1, as a cardioac- genesis is a critical process in wound healing and tive growth factor released from endothelial cells, is survival of ischemic myocardium, pro-angiogenic necessary for cardiac development, structural main- therapy has emerged as having promising potential in tenance, and functional integrity of the heart. NRG-1 cardiac repair[13]. Therapeutic angiogenesis, neovascu- and its receptor family ErbB can play a beneficial role in larization of ischemic myocardium, can hamper the the treatment of chronic heart failure (CHF) by progression of heart failure[14]. promoting survival of cardiomyocytes, improving ANGPT2 is a gene which encodes angiopoietin-2, a sarcomeric structure, balancing Ca2+ homeostasis, and natural antagonist for Ang1 (angiopoietin-1) and Tie-2 enhancing cardiac pumping function[18]. The subse- ( receptor-2), both are stimulants for quent effectors of NRG-1/ErbB can include cardiac- angiogenesis. The angiopoietins are protein growth fac- specific myosin light chain kinase (cMLCK), Protein tors which regulate the process of angiogenesis, the phosphatase type 1 (PP1), sarcoplasmic reticulum Ca2+- formation of new blood vessels. There are at present ATPase 2 (SERCA2), and focal adhesion kinase (FAK). four identified angiopoietin subtypes: Ang1, Ang2, The beneficial effects of neuregulin-1 make recombi- Ang3, and Ang4. Ang1 and Ang4 function as agonistic nant human neuregulin-1 (rhNRG-1) a potential treat- or activating ligands for Tie2. Tie-2/Ang1 signaling ment of CHF[20]. Decreased expression of neuregulin1 ( 384 Liu H et al. J Biomed Res, 2016, 30(5)

– 3.7 Times) may potentially hamper angiogenesis. that could benefit from the angiogenic process to revive SERPINE1 encodes plasminogen activator inhibitor- dysfunctional or dying myocardium. 1(PAI-1). PAI-1 is a serine protease inhibitor (serpin) Whether these epinephrine-induced gene expression that functions as the key inhibitor of tissue plasminogen changes lead to clinically observed long-term and/or activator (tPA) and urokinase (uPA), the two important short-term adverse outcomes in patients treated with activators of plasminogen. Besides PAI-1 which func- epinephrine is not clear yet. Further studies, especially tions as a serine protease inhibitor (serpin) protein in vivo and human studies, will be needed to clarify the (SERPINE1), protease nexin also acts as an inhibitor importance of these gene expression changes and their of tPA and urokinase. PAI-1 is mainly produced by the clinical implications in mediation or modulation of endothelium but also secreted by other tissue types important complex pathways that ultimately influence (such as adipose tissue). PAI-1 inhibits the serine morbidity or mortality. proteases tPA and uPA/urokinase, and thus it is an In conclusion, our present investigation utilized inhibitor of fibrinolysis, the physiological process that cultured cardiomyocytes to study the effects of epi- degrades blood clots[21]. PAI-1 also inhibits the activity nephrine on the angiogenesis-related gene expressions. of matrix metalloproteinases. Congenital deficiency of The results of our study demonstrated epinephrine- PAI-1 can lead to a hemorrhagic diathesis (a tendency to induced upregulation of ANGPT2 and downregulation hemorrhage)[22]. PAI-1 is present in increased levels in of angiopoietin-2, NRG1, and SMOC2. The results many disease states, as well as in obesity and the suggest that epinephrine exposure may inhibit the metabolic syndrome. It has been linked to the increased angiogenic process, potentially devastating to injured occurrence of thrombosis in patients with these condi- or ischemic myocardium. Whether these changes lead to tions. In inflammatory conditions in which fibrin is clinically observed adverse outcomes is not clear. deposited in tissues, PAI-1 appears to play a significant Further studies will be needed to illustrate the impor- role in the progression to fibrosis. Lower level of PAI-1 tance of these gene expression changes. would presumably lead to less suppression of fibrino- lysis and conversely a more rapid degradation of fibrin. Acknowledgements A recent study reported that inhibition of PAI-1 may This study was supported by internal funding from decrease angiogenesis[23]. 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