Abstract Background Ischemic heart disease is a leading cause of mortality. To study this disease, ischemia/reperfusion (I/R) models are widely used to mimic the process of transient blockage and subsequent recovery of cardiac coronary blood supply. We aimed to determine whether the presence of the growth hormone secretagogues, ghrelin and hexarelin, would protect/improve the function of heart from I/R injury and to examine the underlying mechanisms. Methodology/Principal Findings Isolated hearts from adult male mice underwent 20 min global ischemia and 30 min reperfusion using a Langendorff apparatus. Ghrelin (10 nM) or hexarelin (1 nM) was introduced into the perfusion system either 10 min before or after ischemia, termed pre- and post-treatments. In freshly isolated cardiomyocytes from these hearts, single cell shortening, intracellular calcium ([Ca2+] i ) transients and caffeine-releasable sarcoplasmic reticulum (SR) Ca2+ were measured. In addition, RT-PCR and Western blots were used to examine the expression level of GHS receptor type 1a (GHS-R1a), and phosphorylated phospholamban (p-PLB), respectively. Ghrelin and hexarelin pre- or post-treatments prevented the significant reduction in the cell shortening, [Ca2+] i transient amplitude and caffeine-releasable SR Ca2+ content after I/R through recovery of p-PLB. GHS-R1a antagonists, [D-Lys3]-GHRP-6 (200 nM) and BIM28163 (100 nM), completely blocked the effects of GHS on both cell shortening and [Ca2+] i transients. Conclusion/Significance Through activation of GHS-R1a, ghrelin and hexarelin produced a positive inotropic effect on ischemic cardiomyocytes and protected them from I/R injury probably by protecting or recovering p-PLB (and therefore SR Ca2+ content) to allow the maintenance or recovery of normal cardiac contractility. These observations provide supporting evidence for the potential therapeutic application of ghrelin and hexarelin in patients with cardiac I/R injury.

Citation: Ma Y, Zhang L, Edwards JN, Launikonis BS, Chen C (2012) Growth Hormone Secretagogues Protect Mouse Cardiomyocytes from in vitro Ischemia/Reperfusion Injury through Regulation of Intracellular Calcium. PLoS ONE 7(4): e35265. https://doi.org/10.1371/journal.pone.0035265 Editor: Alfred Lewin, University of Florida, United States of America Received: September 13, 2011; Accepted: March 14, 2012; Published: April 6, 2012 Copyright: © 2012 Ma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by funding from the Australian National Health and Medical Research Council and the University of Queensland. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Cardiac ischemia is one of the leading causes of mortality in the world. It is caused by a temporary interruption of blood flow in the arteries of the heart [1]. The primary clinical therapeutic strategy for treatment of cardiac ischemia is reperfusion. However, reperfusion can cause additional injury to the heart [1], [2]. Recovery of cardiac function following ischemia is critically dependent on the time spent under ischemic conditions and reperfusion [3]. In vitro global and in vivo regional ischemia/reperfusion (I/R) models have been developed to examine experimentally cardiac ischemia and subsequent reperfusion of the ischemic heart. The in vivo regional I/R model mimics atherosclerosis by ligating the left anterior descending coronary artery. The global I/R model for in vitro study blocks all perfusion of the heart for a given period. The latter can be easily implemented and affects a larger area with a less variability among different regions [4]. It is used to mimic the process of cardiac arrest and cardiac surgery [5]. This model is also more appropriate for obtaining isolated cells which have been through similar ischemia conditions without the regional differences often observed in regional I/R models. In the past 50 years, great progress has been made to clarify the metabolic changes that occur following I/R [1], [2], [6], [7]. During ischemia, depletion of oxygen and ATP inhibits SR Ca2+ ATPase (SERCA2a) and Na+-K+ ATPase activities. This results in an accumulation of intracellular Ca2+ ([Ca2+] i ) and Na+ ([Na+] i ) [1], [2], [6], [7]. The subsequent reintroduction of oxygen during reperfusion leads to the generation of large amounts of reactive oxygen species (ROS), causing increased oxidative stress and subsequent damage to the plasma and SR membranes resulting in further increases in [Ca2+] i . The combined effects of ROS and [Ca2+] i overload also favor the opening of the mitochondrial permeability transition pore (mPTP), which induces cardiomyocyte apoptosis and necrosis [1], [2], [6], [7]. Ghrelin is a 28 amino acid peptide produced in the stomach and is an endogenous ligand of the growth hormone secretagogue (GHS) receptor type 1a (GHS-R1a) [8]. A synthetic analogue of ghrelin, hexarelin, also binds and activates the GHS-R1a [9], [10], [11]. Ghrelin mainly exists in the pituitary and gastrointestinal system [8], [12], while the distribution of its receptor GHS-R1a is ubiquitous and has been confirmed in the myocardium [12], [13]. Although ghrelin may bind to receptors other than GHS-R1a [14], [15], [16], its main target is GHS-R1a. Previous studies have confirmed the protective effects of GHS on whole heart function after I/R. Administration of ghrelin in vitro to I/R rat hearts was shown to reduce the infarct size [17], and enhance cardiac function [18] through the activation of PKC [17]. These effects are likely initiated by the binding of ghrelin to its receptor, GHS-R1a [18]. Further studies in rats pre-treated with GHS for 7 days in vivo prior to in vitro I/R injury showed an improvement in cardiac function [10] and attenuation of myocardial injury and apoptosis through the inhibition of endoplasmic reticulum (ER) stress [19]. Similarly, hexarelin has also been shown to play a cardioprotective role in I/R hearts from rodents [10], [17], [20]. As discussed above, whole heart functional studies employing the in vivo and in vitro I/R models have revealed some potential mechanisms of the cardioprotective effects of GHS. Detailed cellular and molecular pathways employed by GHS through activation of GHS-R1a after cardiac I/R remain elusive. Since [Ca2+] i plays a critical role in cardiomyocyte contraction and I/R injury, in this study we investigated the alterations in and regulation of [Ca2+] i homeostasis in isolated mouse cardiomyocytes with or without I/R and GHS treatment.

Methods Animals and Chemicals All experiments conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No.85–23, revised 1996), and the protocol was approved by the Animal Ethics Committee of the University of Queensland (AEC # SBMS/814/07/NHMRC). All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Human ghrelin was obtained from Auspep (Parkville, Australia). Hexarelin was obtained from GL Biochem (Shanghai, China). Pentobarbital sodium was purchased from Virbac Pty Ltd (Australia). Heparin sodium salt was purchased from Sigma Aldrich (St. Louis, MO, USA). Fura 2-AM was purchased from Invitrogen (Eugene, Oregon, USA). [D-Lys3]GHRP-6 was purchased from Anaspec Inc. (San Jose, CA). BIM28163 was kindly provided by Michael D. Culler (Ipsen Pty Ltd, Australia). Other chemicals for recording solutions were purchased from Sigma (St. Louis, MO, USA). In vitro Ischemia/Reperfusion Model and Preparation of Ventricular Myocytes Adult male C57/B1 mice (7 to 9 weeks old) weighing between 34 g and 36 g were anesthetized with sodium pentobarbitone (40 mg/kg, ip) containing heparin (500 Units, ip). The heart was rapidly excised, cannulated and perfused retrogradely via the aorta with Tyrode solution at 3 ml/min on a Langendorff perfusion apparatus (composition in mM: 10 HEPES, 143 NaCl, 5.4 KCl, 0.5 MgCl 2 , 10 Glucose, 20 Taurine, 1.5 CaCl 2 ; pH 7.4; bubbled with 100% O 2 at 37°C). The times for stabilization, ischemia and reperfusion were similar to previous studies [3], and are generally considered the most appropriate for functional studies using an in vitro I/R model. After 20 min of stabilization, the heart was subjected to 20 min of no-flow global ischemia followed by 30 min of reperfusion. Control hearts were continuously perfused for 70 min. Ghrelin (10 nM) or hexarelin (1 nM) was administered in the perfusion solution before or after ischemia for 10 min [13], termed GHS pre-treatment and post-treatment respectively. In some experiments, the GHS-R1a antagonist [D-Lys3]-GHRP-6 (200 nM) or BIM28163 (100 nM) was introduced into the perfusion system 5 min before the onset of ischemia and remained present throughout (15 min in total). Following perfusion, cardiomyocytes were isolated from the left ventricle of each heart with Tyrode solution containing 100 µM CaCl 2 , 0.6 mg/ml collagenase Type II (Worthington, NJ, USA) and 0.1 mg/ml proteinase type XXIV (Sigma, MO, USA). The Ca2+ level was gradually increased to 1.5 mM over 30 min. The yield of this isolation was usually around 60 – 70%. Only cardiomyocytes that were quiescent with a rod shape, sharp edges and clear striations were used in this investigation. At least 3 hearts were used in each group. Measurement of Sarcomere Shortening Sarcomere shortening was measured as previously described [21]. In brief, cardiomyocytes were electrically stimulated at 0.5 Hz until contractions became uniform. Following this, 10 – 20 consecutive contractions were recorded. The percentage of sarcomere shortening, time-to-peak shortening and time-to-90% relaxation were determined by IonWizard software (IonOptix Corporation, MA). Measurement of Intracellular Ca2+ Transients and SR Ca2+ Content The isolated and Ca2+-tolerant cardiomyocytes were loaded with 5 µM Fura-2 AM (Invitrogen, CA, USA) for 10 min at room temperature. Cardiomyocytes were observed through a Nikon fluor ×40 oil immersion objective and positioned for recording of Fura-2 fluorescence signals. During field stimulation at 0.5 Hz, cytoplasmic Fura-2 was excited by an IonOptix Hyperswitch dual-excitation light source (IonOptix Corporation, MA) at 340 and 380 nm and emitted light collected in a photomultiplier tube. [Ca2+] i concentration was inferred from the ratio (R) of the intensity of the emitted fluorescence signals. Amplitude, time-to-peak, time-to-90% decay, and rate of rise (dR/dt) of the derived [Ca2+] i transients were determined by IonWizard software. For estimation of SR Ca2+ content, cardiomyocytes with cytoplasmic Fura-2 were paced at least 15 times at 0.5 Hz and then stopped. About 30s later, 10 mM caffeine was added to induce SR Ca2+ release. The area under the caffeine-induced [Ca2+] i transient (area under curve, AUC) and its amplitude were used as a reflection of the SR Ca2+ content [22]. Time-to-90% decay of caffeine-induced increase in [Ca2+] i was also measured to estimate the Ca2+ clearance ability of Na+-Ca2+ exchanger (NCX). RT–PCR Total cellular RNA was extracted from left ventricle, septum and right ventricle of mouse hearts using a TRIzol Plus RNA Purification kit (Invitrogen, CA, USA). Single-stranded cDNA was synthesized from 2 µg total RNA with an iScript cDNA Synthesis kit (Bio-Rad Laboratories, CA, USA) following the manufacturer’s instructions. PCR was performed using JumpStart Taq DNA polymerase (Sigma, MO, USA), the cDNA generated above and the corresponding primers for GHS-R1a [23] (Forward: TCATCGATCACAGCCATGT; Reverse: AAGCCAAACTGACCATGT; Tm = 64°C, 40 cycles). Mouse 18s rRNA was amplified as a control. Following our previous report [13], liver and pituitary were chosen as negative and positive controls respectively for GHS-R1a. PCR products were separated by agarose gel electrophoresis (2%), stained with ethidium bromide and visualized under UV light. Western Blotting Protein expression of the GHS receptor GHS-R1a and the phosphorylated phospholamban (p-PLB)/phospholamban (PLB) that are essential for SERCA2a activity were examined by Western blot analysis according to previous reports [24], [25]. In brief, proteins were extracted from isolated cardiomyocytes (GHS-R1a) or minced mouse left ventricles (p-PLB and PLB) in lysis buffer. Extracted proteins (100 µg) were denatured at 37°C for 30 min (p-PLB and PLB) or 70°C for 10 min (GHS-R1a) in 2× sample buffer, separated on 10–15% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. After blocking, the membrane was incubated with polyclonal rabbit anti- mouse phospholamban (phospho S16,1∶1000; abcam, Cambridge, MA, USA), polyclonal rabbit anti- mouse phospholamban (1∶1000; abcam) or polyclonal goat anti-human GHS-R1a (1∶1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) primary antibodies overnight at 4°C before incubation with the corresponding secondary antibodies (1∶5000) and detection with enhanced chemiluminescence (Pierce) according to the manufacturer’s instructions. Mouse GAPDH (1∶2000; Milipore, Billerica, MA, USA) was used as the internal control to allow semi-quantitative densitometry analysis on scanned films using ImageJ software [24]. Statistical Analysis All data were expressed as mean ± S.E.M. One-way ANOVA with Tukey post hoc test was carried out for multiple comparisons as appropriate. In all comparisons, the differences were considered to be statistically significant at a value of P < 0.05.

Acknowledgments We thank the careful editing of Dr Tamara Paravicini at the School of Biomedical Sciences, University of Queensland.

Author Contributions Conceived and designed the experiments: LZ JNE BSL CC. Performed the experiments: YM. Analyzed the data: YM. Contributed reagents/materials/analysis tools: JNE BSL CC. Wrote the paper: YM LZ JNE BSL CC.