- Email: [email protected]
Glycine prevents pressure overload induced cardiac hypertrophy mediated by glycine receptor
Biochemical Pharmacology 123 (2017) 40–51
Contents lists available at ScienceDirect
Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm
Glycine prevents pressure overload induced cardiac hypertrophy mediated by glycine receptor Yan Lu a,b,1, Xudong Zhu a,1, Jinjie Li a, Ru Fang a, Zhuoyun Wang a, Jing Zhang a, Kexue Li a, Xiaoyu Li a, Hui Bai a, Qing Yang a, Jingjing Ben a, Hanwen Zhang a, Qi Chen a,⇑ a b
Atherosclerosis Research Center, Key Laboratory of Cardiovascular Disease and Molecular Intervention, Nanjing Medical University, Nanjing 210029, People’s Republic of China Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 28 June 2016 Accepted 4 November 2016 Available online 9 November 2016 Chemical compounds studied in this article: Glycine (Pubchem CID: 750) Angiotensin II (Pubchem CID: 172198) Keywords: Cardiac hypertrophy Glycine Glycine receptor ERK1/2 signaling
a b s t r a c t As a major amino acid, glycine has multiple functions in metabolism, growth, immunity, cytoprotection, and survival. The aim of this study was to determine the effects of glycine on pathologic cardiac hypertrophy and the mechanism underlying it. Pre-treatment with glycine significantly attenuated murine cardiac hypertrophy induced by transverse aortic constriction or by administration of angiotensin II (Ang II). This action was associated with a suppressive extracellular signal-regulated kinase 1/2 phosphorylation in myocardium. The cardioprotective effect of glycine disappeared when endogenous glycine receptor a2 was knocked down by mRNA interference in rats. Co-culture experiments revealed that glycine could also antagonize Ang II stimulated release of transforming growth factor b and endothelin-1 by cardiomyocytes, which prevented an over-production of collagens in rat fibroblasts. These results, for the first time, demonstrate that glycine may be a novel cardioprotector against pressure overload induced cardiac hypertrophy. Thus, glycine would be useful in the prevention of cardiac hypertrophy and heart failure. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Despite great advances in the understanding and treatment of heart failure, the disease remains a leading cause of death worldwide [1,2]. Heart failure is structurally characterized by pathologic hypertrophy of the myocardium which temporarily preserves pump function and reduces ventricular wall stress. However, prolonged cardiac hypertrophy can cause arrhythmias, dilated cardiomyopathy and heart failure [3,4]. In contrast to physiological hypertrophy, pathological hypertrophy is characterized by accumulation of interstitial collagen and cell death, both of which contribute to increased risk for myocardial infarction, arrhythmia and sudden death. Therefore, it would be of great therapeutic interest to prevent pathological hypertrophy. Glycine is a major amino acid in mammals and other animals. It plays an important role in metabolism, growth, development, immunity, cytoprotection, and survival [5,6]. Recent studies have shown a few beneficial effects of glycine on cardiomyocytes under ischemia-reperfusion (I/R) conditions. For example, 3 mM glycine ⇑ Corresponding author at: Atherosclerosis Research Center, Key Laboratory of Cardiovascular Disease and Molecular Intervention, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, People’s Republic of China. E-mail address: [email protected] (Q. Chen). 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.bcp.2016.11.008 0006-2952/Ó 2016 Elsevier Inc. All rights reserved.
increases the cell viability of isolated rat hearts after I/R [7]. Infusion of glycine into animal donor hearts is good for right ventricular function after transplantation [8]. Glycine can inhibit the LPS induced increase in cytosolic Ca2+ concentration and tumor necrosis factor-a (TNF-a) production in cardiomyocytes by activating a glycine receptor (glyR) [9]. The antioxidant N-2mercaptopropionyl glycine has been reported to attenuate cardiac hypertrophy induced by TAC in mice [10]. However, whether glycine has an impact on cardiac hypertrophy is unknown. In the current study, we demonstrate that glycine significantly attenuates murine left ventricular (LV) hypertrophy and cardiac fibrosis induced by either transverse aortic constriction (TAC) or angiotensin II (Ang II) administration. Mechanistically, we show that the cardioprotective effect of glycine may be via glyR a2 coupling to inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and preventing production of transforming growth factor-b (TGF-b) and endothelin-1 (ET-1) by cardiomyocytes. 2. Materials and methods 2.1. Animals and treatments All aspects of the animal care and experimental protocols were in accordance with the Guide for the Care and Use of Laboratory
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
Animals (NIH publication, 8th edition, 2011) and approved by the Experimental Animal Care and Use Committee of Nanjing Medical University. Male 8-week-old C57BL/6J mice, male Sprague-Dawley rats each weighing 250 ± 30 g, and neonatal Sprague-Dawley rats (0–3 day old) were obtained from the Animal Center of Nanjing Medical University. All animal experiments performed in this study adhered to the protocols approved by the Institutional Animal Care and Use Committee of the Nanjing Medical University. Cardiac hypertrophy and heart failure were induced by TAC surgery or by Ang II administration by using an osmotic pump as described [11]. Glycine (Sigma-Aldrich, St Louis, USA) was suspended in saline for intraperitoneal injection. Mice were anaesthetized with an intraperitoneal injection of ketamine (100 mg/ kg) and xylazine (10 mg/kg). Rats were anaesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (8 mg/kg). 2.2. Measurement of plasma concentration of glycine Glycine was injected into mice and the plasma was collected for further use at different time points. The plasma was blown dry by nitrogen and then dissolved in mobile phase. Agilent HPLC was coupled to a Micromass Quattro Ultima mass spectrometer with an ESI (Electrospray Ionisation) source, together with a Discovery C18 column (50 mm 2.1 mm 5 lm) for study. The mobile phase solvents were H2O containing 0.1% TDFHA and ACN containing 0.1% TDFHA. Gradient composition started from 10% ACN and was maintained for 1 min. It was rapidly raised to 15% at 3 min, to 20% at 5 min, to 25% at 6 min, to 40% at 7 min, to 75% at 8.90 min, and finally to 98% at 9 min till the end of the analysis. Analysis was performed by using HPLC–ESI–MS/MS for a more specific and sensitive detection [12,13]. 2.3. TAC surgery Cardiac hypertrophy was induced in mice and rats by constricting to transverse aorta. Briefly, a small midline skin cut was made just above the sternum of the animal. Muscles were gently separated until trachea was visible. Partial left side thoracotomy to the second rib was performed with blunt ended spring scissor and the sternum was retracted using a chest retractor. Blunt tip 45° angled forceps were used to gently separate the two lobes of thymus and to clean fat tissue from the aortic arch. A small piece of a 7-0 silk suture (presoaked in sterile saline) was placed between the innominate and left carotid arteries using a 90 degree curved forceps. Two loose knots were tied around the transverse aorta and a small piece of a 27 gauge blunt needle was placed parallel to the transverse aorta. The first knot was quickly tied against the needle, followed by the second one. The needle was promptly removed in order to yield a constriction of 0.4 mm in diameter. 2.4. Ang II pump implantation Mini-osmotic pumps (model 2004, ALZET technical services, USA) filled with Ang II (1.5 mg/kg/day) or saline were dorsally implanted into mice for 21 days. Glycine (700 mg/kg/day) was peritoneally injected into mice a week before the surgery. 2.5. Cardiac imaging Transthoracic 2-dimensional M-mode echocardiography was performed with Vevo 770 (VisualSonics, Toronto, Canada) equipped with a 30-MHz transducer. Percentage of fractional shortening, LV wall thickness, and LV mass and ejection fraction (EF) was calculated as described [14].
41
2.6. Antibodies and reagents Antibody against glyR a2 and GAPDH were from Abcam Biotech (San Francisco, USA). Glycine, Ang II and antibody against a-actinin were obtained from Sigma. Antibodies against cRaf, p-cRaf, mitogen-activated protein kinase kinase1/2 (MEK1/2), p-MEK1/2, ERK1/2, p-ERK1/2, p38, p-p38, c-Jun N-terminal kinase (JNK) and p-JNK were obtained from Cell Signaling Technology (Boston, USA). Plasma concentrations of TGF-b and TNF-a were determined by using mouse TGF-b ELISA and rat TNF-a ELISA Kit (Excell, Shanghai, China). Plasma concentrations of ammonia and urea were determined by using blood ammonia assay kit and blood urea assay kit (Jiangcheng, Nanjing, China). 2.7. Isolation of neonatal rat cardiomyocytes and fibroblasts Neonatal rat cardiomyocytes (NRCMs) were prepared by enzymatic digestion of hearts obtained from newborn (0–3 day old) Sprague–Dawley rat pups by using percoll gradient centrifugation. For in vitro experiments NRCMs and fibroblasts were pre-treated with glycine (5 mM) for 30 min followed by addition of Ang II (1 lM). Cells were harvested and used for the mRNA and protein studies. 3.3. Protective effect of glycine on Ang II induced cardiac hypertrophy We also examined the effect of glycine on the Ang II induced cardiac hypertrophy in mice. Consistent with the results from the TAC model, we found that glycine could protect the mouse heart against Ang II induced hypertrophy by echocardiography measurements and heart appearance analysis (Fig. 3a–c). Histomorphometric analysis showed an inhibitive effect of glycine on cardiac fibrosis in Ang II administrated mice (Fig. 3d–g). Furthermore, pretreatment with glycine could inhibit Ang II induced phosphorylation of cRaf-MEK1/2-ERK1/2 in the mouse myocardium (Fig. 3h and i). Antagonistic effect of glycine on Ang II induced cardiac hypertrophy was further investigated by using NRCMs in vitro. We found that treatment with Ang II (1 lM) resulted in a significant upregulation of hypertrophic genes including ANP, BNP, and b-MHC in NRCMs. This action could be markedly blocked by pretreatment of NRCMs with glycine dose-dependently (Fig. 4a). Treatment with 5 mM glycine significantly decreased the NRCMs surface area in the presence of Ang II (Fig. 4b and c). Glycine pre-treated NRCMs also exhibited a suppressive phosphorylation of cRaf-MEK1/2ERK1/2 in the presence of Ang II (Fig. 4d and e). As such, these data demonstrated that glycine could prevent Ang II induced murine cardiac hypertrophy in vivo and in vitro. 3.4. Cardiac protection of glycine is mediated via a glycine receptor It is known that glyR plays an important role in mediation of cytoprotection of glycine [6,19,20]. GlyR is an ionotropic or ligand-gated receptor consisting with different sub-units including glyR a1, a2, a3, and a4. To investigate the role of glyR in glycine antagonizing cardiac hypertrophy, we first examined the expression of glyR in the heart. It is interesting that only glyR a2 was expressed in the cardiomyocyte but not in the cardiofibroblast in rats as measured by RT-PCR (Fig. 5a), immunofluorescence staining (Fig. 5b), and western blot (Fig. 5c). Moreover, we found that expression of glyR a2 was increased in adult rat cardiomyocytes compared with NRCMs (Fig. 5c). When endogenous glyR a2 was knocked down (70%) in cultured NRCMs by using a siRNA (Fig. 5d), the antagonistic effect of glycine on Ang II induced up-regulation of ANP, BNP and b-MHC disappeared (Fig. 5e). Consistently, inhibitive effects of glycine on cRaf-MEK1/2-ERK1/2 phosphorylation were also abolished in
42
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
Fig. 1. Glycine attenuates TAC induced LV fibrosis in mice. (a and b) Mice were pre-treated with glycine (G) (150, 300 or 700 mg/kg) or vehicle (V). Six days later, mice received TAC surgery for 4 weeks. (a) Representative images of whole hearts from mice. (b) Measurements of HW/BW and HW/TL (n = 6–10). (c) Plasma glycine levels 1 day and 6 days after glycine injection (n = 4–6). (d) Plasma concentration of ammonia after glycine injection (n = 4–6). (e) Plasma concentration of urea after glycine injection (n = 4–6). (f–l) Mice were pre-treated with 700 mg/kg glycine (G) or vehicle (V). Six days later, mice received TAC surgery for 4 weeks. (f) Masson’s trichomeand. (g) Picrosirius red staining for collagen deposition in LV tissue sections. (h) Stained sections were analyzed with Image-Pro Plus software (n = 4–6). (i and j) mRNA levels of collagen I, collagen III, ANP and BNP in mouse heart (n = 6). (k) Representative immunoblot of csapase-3 and GAPDH in whole heart lysates. (l) Quantification of band intensity normalized by corresponding GAPDH levels (n = 3). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
NRCMs (Fig. 5f and g). We also injected directly an adenoassociated virus coated anti-glyR a2 sequence into the rat heart to determine the role of glyR a2 in vivo [21,22]. This caused that
glyR a2 expression was down-regulated by 80% to 70% from 1 to 2 weeks after TAC (Fig. 6a). The cardiac protection of glycine disappeared in this conditions as measurements of LV mass, LVPW,
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
43
Fig. 2. Glycine attenuates TAC induced cardiac hypertrophy in mice. (a–g) Mice were pre-treated with 700 mg/kg glycine (G) or vehicle (V). Six days later, mice received TAC surgery for 4 weeks. (a) Analysis of LV mass, LVPW and IVS by echocardiography (n = 5–7). (b) Hematoxylin and eosin (H&E) staining of mouse hearts. (c) Analysis of EF by echocardiography (n = 5–7). (d) Representative immunoblot of p-cRaf, cRaf, p-MEK1/2, MEK1/2, p-ERK1/2 and ERK1/2 in whole heart lysates. (e) Quantification of band intensity (n = 3). (f) Representative immunoblot of p-p38, p38, p-JNK and JNK in whole heart lysates. (g) Quantification of band intensity (n = 3). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
44
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
Fig. 3. Glycine attenuates Ang II induced cardiac hypertrophy in mice. (a–i) Mice were pre-treated with 700 mg/kg glycine (G) or vehicle (V). Six days later, mice received Ang II treatment for 21 days. (a) Analysis of LV mass, LVPW and IVS by echocardiography (n = 6–8). (b) Representative images of whole hearts. (c) Measurements of HW/BW and HW/TL (n = 6–8). (d) H&E staining. (e) Masson’s trichome staining. (f) Picro-sirius red staining of myocardium sections. (g) Stained sections were analyzed with Image-Pro Plus software (n = 4–6). (h) Representative immunoblot of p-cRaf, cRaf, p-MEK1/2, MEK1/2, p-ERK1/2, and ERK1/2 in whole heart lysates. (i) Quantification of band intensity (n = 3). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
45
Fig. 4. Glycine attenuates Ang II induced hypertrophy in NRCMs. (a) NRCMs were pre-treated with glycine (1, 5 or 10 mM) (G) for 30 min followed by addition of Ang II (1 lM) for 24 h. mRNA levels of ANP, BNP, and b-MHC in NRCMs were measured (n = 6–8). (b) NRCMs were pre-treated with glycine (5 mM) (G) for 30 min followed by addition of Ang II (1 lM) for 24 h. Representative images of a-actinin immunostaining in NRCMs. (c) Quantification of cell surface area (n = 3. Fifty cells were counted per experiment). (d) NRCMs were pre-treated with glycine (5 mM) (G) for 30 min followed by addition of Ang II (1 lM) for 1 h. Representative immunoblot of p-cRaf, cRaf, pMEK1/2, MEK1/2, p-ERK1/2, and ERK1/2 in NRCMs lysates. (e) Quantification of band intensity (n = 3). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
IVS, HW/BW, and HW/TL 2 weeks post TAC, while glycine still exerted its effects in the scram sequence group (Fig. 6b and c). Same as the in vitro observations, inhibitive effects of glycine on cRaf-MEK1/2-ERK1/2 phosphorylation were also abolished in the anti-glyR a2 sequence group (Fig. 6d and e). Taken together, these results indicate that glyR a2 might be required for glycine’s cardiac protection in rats.
3.5. Glycine inhibits Ang II induced fibrotic response by a cardiomyocyte-dependent mechanism Since no glyR expression has been found in the fibroblast, how does glycine exert its anti-cardiac fibrosis effect by a glyR reliable mechanism? This question promoted us to perform co-culture studies with neonatal rat fibroblasts and NRCMs. Measurements
46
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
Fig. 5. GlyR a2 is required for the effect of glycine on NRCMs. (a) mRNA expression of glyRs in rat spinal, NRCMs (CM) and neonatal rat fibroblasts (CF). (b) Representative images of NRCMs triple staining. (c) Immunoblot of glyR a1, glyR a2 and GAPDH in spinal, and adult rat cardiomyocytes lysates and NRCMs. (d) NRCMs were transfected with siRNA targeting glyR a2 (SiRNA-GlyR) or scrambled siRNA (SiRNA-( )) and expression of glyR a2 and GAPDH were analyzed by immunoblot assay. GAPDH was blotted as a loading control (n = 3). (e) NRCMs were transfected with siRNA targeting glyR a2 (SiRNA-GlyR) or scrambled siRNA (SiRNA-( )). After 3 days, NRCMs were pre-treated with glycine (5 mM) (G) for 30 min followed by addition of Ang II (1 lM) for 24 h. mRNA levels of ANP, BNP and b-MHC in NRCMs (n = 6–8). (f) NRCMs were transfected with siRNA targeting glyR a2 (SiRNA-GlyR) or scrambled siRNA (SiRNA-( )). After 3 days, NRCMs were pre-treated with glycine (5 mM) (G) for 30 min followed by addition of Ang II (1 lM) for 1 h. Representative immunoblot of p-cRaf, cRaf, p-MEK1/2, MEK1/2, p-ERK1/2, and ERK1/2 in NRCMs lysates. (g) Quantification of band intensity (n = 3). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01.
of mRNA levels revealed that glycine inhibited Ang II induced overproduction of collagen I and collagen III in neonatal rat fibroblasts only in the presence of NRCMs (Fig. 7a). The Ang II insulted NRCMs produced much more TGF-b and ET-1 which was significantly inhibited by treatment with glycine (Fig. 7b). No significant
changes were found in the production of angiotensinogen and TNF-a in the glycine treated NRCMs. The effect of glycine on fibrotic response was also examined by in vivo experiments. We found that glycine significantly inhibited TAC induced increases in TGF-b, ET-1, and angiotensinogen gene expression in mice hearts (Fig. 7c).
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
47
Fig. 6. Activating glyR a2 prevents TAC induced cardiac hypertrophy in rats. (a) After injection of the anti-glyR a2 sequence containing adeno-associated virus (SiRNA-GlyR) for one or two weeks. Immunoblot of glyR a2 and GAPDH in rat LV. GAPDH was blotted as a loading control (n = 5–7). (b–e) After injection of the anti-glyR a2 sequence containing adeno-associated virus (SiRNA-GlyR) for one week, TAC surgery was performed together with treatment with glycine (700 mg/kg) (G) or vehicle (V). (b) Measurements of HW/BW and HW/TL in rats (n = 5–7). (c) Analysis of LV mass, LVPW and IVS in rats by echocardiography. (d) Representative immunoblot of p-cRaf, cRaf, pMEK1/2, MEK1/2, p-ERK1/2 and ERK1/2 in rat whole heart lysates. (e) Quantification of band intensity (n = 3). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, *** P < 0.001.
48
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
Fig. 7. Glycine inhibits Ang II stimulated overproduction of collagens in rat fibroblasts. (a) Cells were pre-treated with glycine (5 mM) (G) for 30 min followed by addition of Ang II (1 lM) for 24 h. mRNA levels of collagen I and collagen III in neonatal rat fibroblasts co-culturing with NRCMs. (n = 6–8). (b) NRCMs were pre-treated with glycine (5 mM) (G) for 30 min followed by addition of Ang II (1 lM) for 24 h. mRNA levels of TGF-b, ET-1, angiotensinogen and TNF-a in NRCMs. (n = 6–8). (c and d) Mice were pretreated with 700 mg/kg glycine (G) or vehicle (V). Six days later, mice received TAC surgery for 4 weeks. (c) mRNA levels of TGF-b, ET-1, angiotensinogen and TNF-a in the mice hearts (n = 6–8). (d) Plasma levels of TGF-b and TNF-a in mice (n = 6–8). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
Similar reaction pattern was also found in plasma TGF-b in mice (Fig. 7d). Thus, these data suggest that glycine inhibit TAC induced overproduction of collagens in fibroblasts via preventing release of TGF-b and ET-1 by stressed cardiomyocytes.
4. Discussion Heart hypertrophy is initially physiological to compensate for the loss of heart functions. However, sustained stresses lead to a pathological status, in which myocardium becomes stiffened by interstitial fibrosis and thereby diastolic dysfunction induces global remodeling of the heart, dilated cardiomyopathy and heart failure [23,24]. Heart failure is one of the most devastating diseases in which cardiac hypertrophy is a determinant of the clinical course [1,25]. The lifetime risk of heart failure is 1 in 5 among both men and women. Despite the advances in the development of new therapies in past decades, the survival rate after the onset of heart failure remains very low [26,27]. Therefore, a novel anti-hypertrophic drug or a potential therapeutic target for the treatment of cardiac hypertrophy and heart failure is extremely needed. The present study demonstrates for the first time that glycine may antagonize pressure overload induced cardiac hypertrophy and fibrosis in rodents. Glycine has been regarded as a ‘‘nutritionally nonessential amino acid’’ for humans due to the presence of its endogenous synthesis in the body [28,29]. Plasma level of glycine is normally about 300 lM in adult humans [30,31], which is similar to the mouse level measured by the present study. However, it has been reported that the amount of glycine synthesized in vivo would be insufficient to meet metabolic demands in humans and animals [32]. Moreover, accumulative lines of evidence have revealed that glycine is associated with the cytoprotection, anti-inflammatory responses, and animal development [33–35]. Implication with high doses of glycine exhibits the beneficial effects on gabexatemesilate induced lung endothelial cells injury [36], I/R injury in cultured cells, perfused organs, and in vivo models including heart, liver, kidney, skeletal muscle, and small intestine [7,37,38]. In the present study, the plasma concentrations of glycine were higher than 0.5 mM, the effective level for protection against I/R injury in rat small intestine [31], for more than 8 h/day after intraperitoneal injection of 700 mg/kg glycine. Meanwhile, ammonia, one of the catabolic products of glycine, was sustained at the normal level for more than 22 h/day. The administered glycine may be excreted in the urine only in a minor amount. The majority is taken up by cells and metabolized via a variety of other routes. Therefore, the observed improvements in the cardiac structures and functions would be attributed to the implanted glycine. The doses of 800 mg/kg body weight would be acceptable for intraperitoneal injection of glycine [39]. A medical record for five years on oral administration of 50–66 g/day glycine has been tracked for the treatment of refractory obsessive-compulsive disorder and body dysmorphic disorder. No obvious side-effect has been found [40]. However, high doses of glycine can cause serious adverse effects. Intravenous infusion of 1.5% glycine causes the cardiotoxic effects in rabbits [41]. Rat myocardial cells are supposed to die in more than 1.0% (133 mM) glycine in vitro [42]. In humans, the threshold concentration of plasma glycine would be 5 mM at which overt adverse effects may start to develop. A significant finding of this study is that the protection of glycine might be mediated by glyR a2. It is interesting that only glyR a2 among the four family members is expressed in the cardiomyocyte. GlyR a2 is highly expressed at the embryonic and neonatal stages but postnatally is largely replaced by glyR a1 in central nervous system [43]. The definite role of glyR a2 in the heart is not
49
fully known yet. GlyR a2 has been reported to be less sensitive to ethanol, tropisetron and Zinc than glyR a1 [19]. It might be that the presence of glyR a2 endows the myocardium with special functions in the development and metabolism, although a glyR a2 deficiency mouse model seems to be phenotypically normal [43,44]. It has been reported that extrasynaptic activation of glyRs containing the a2 subunit in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx, which further modulates actomyosin contractility to fine-tune nuclear translocation during migration [45]. Nobles et al. reported that selective glyR a2 subunit control of crossover inhibition between the on and off retinal pathways and is involved in regulation of interneuron differentiation during spinal cord development [46]. Moreover, our results indicate that glyR a2 in the myocardium may be a novel target to antagonize heart failure. The cytoprotective effects of glyRs have been reported to associate with the MAPK (JNK, ERK1/2 and p38) signaling pathways [47,48]. The present study reveals that suppressive phosphorylation of cRaf-MEK1/2-ERK1/2 is initiated by glycine – glyR a2 interactions in cardiomyocytes. As a central regulator, the MEK1/2ERK1/2 signaling promotes cardiac hypertrophy in the body. This is, in part, by enhancing the transcriptional activity of nuclear factor of activated T cells (NFAT) [49]. The p38 and JNK signaling is reported to negatively regulate the cardiac growth response by directly phosphorylating NFAT in the heart [50]. The signaling pathways integrate specific stimuli at the cell membrane and transmit it to intracellular target proteins that are involved in transcription, protein synthesis and protein stability in the myocardium. Therefore, inhibition of MEK1/2- ERK1/2 and activation of p38 and/or JNK might be benefit to the prevention and treatment of maladaptive hypertrophy. Fibrosis is fundamental to pathological remodeling of myocardium in the diseased heart. It is intriguing that there is no glyR in fibroblasts but the expression of glyR a2 in the heart is requisite for antagonistic effect of glycine on the cardiac fibrosis. This might be explained by that glycine itself would not directly impact the production of collagens by fibroblasts. Its direct target for antifibrosis seems to be the cardiomyocytes. Glycine could block the stress, such as Ang II, stimulated release of TGF-b and ET-1 by cardiomyocytes. Both TGF-b and ET-1 can stimulate the production of collagens by fibroblasts. Furthermore, it has been reported that TGF-b also can promote cardiomyocyte hypertrophy [51]. Thus, via an indirect suppression on the production of collagens by fibroblasts and direct effect of TGF-b on cardiomyocytes, glycine may prevent the cardiac fibrosis and development of myocardial remodeling process which is critical in the progression of heart failure [52,53]. Beyond the inhibition of fibrotic responses, glyR mediated cardiac protection of glycine may have other mechanisms. Glycine can inhibit calcium fluxes in lymphocytes, macrophages, and neutrophils, leading to a decreased secretion of cytokines (e.g. TNF-a). These anti-inflammatory effects are also likely mediated by functional glyRs [6,54]. A modulating effect of glycine on endothelial cells has been reported via glyRs. [55]. We have found that glycine antagonizes cerebral I/R induced injury by inhibiting extrinsic and intrinsic apoptotic pathways, which is associated with the presence of glyRs [48]. However, similar effect of glycine is not found in the present study. Discovery of the new pharmacological roles of glycine may pave a path to generate a novel therapy differing from the angiotensin-converting enzyme inhibitor and angiotensin type 1 receptor antagonist for treatment of heart failure.
Conflict of interest statement None declared.
50
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
Acknowledgements This work was supported by National Natural Science Foundation of China [81230070 and 91339202 to Qi Chen, 81300211 to Xudong Zhu, 81670263 to Xiaoyu Li, 81370005 to Jingjing Ben]; College Natural Science Foundation of Jiangsu [13KJB310005 to Xudong Zhu]; Jiangsu Province Education Office of the major basic research projects [15KJA310001 to Xiaoyu Li] and the Collaborative Innovation Center For Cardiovascular Disease Translational Medicine of Jiangsu Province. References [1] J.O. Mudd, D.A. Kass, Tackling heart failure in the twenty-first century, Nature 451 (2008) 919–928. [2] I. Rachmin, S. Tshori, Y. Smith, A. Oppenheim, S. Marchetto, G. Kay, R.S. Foo, N. Dagan, E. Golomb, D. Gilon, J.P. Borg, E. Razin, Erbin is a negative modulator of cardiac hypertrophy, Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 5902–5907. [3] J.A. Towbin, N.E. Bowles, The failing heart, Nature 415 (2002) 227–233. [4] O. Gjesdal, D.A. Bluemke, J.A. Lima, Cardiac remodeling at the population level– risk factors, screening, and outcomes, Nat. Rev. Cardiol. 8 (2011) 673–685. [5] R.Y. Gundersen, P. Vaagenes, T. Breivik, F. Fonnum, P.K. Opstad, Glycine–an important neurotransmitter and cytoprotective agent, Acta Anaesthesiol. Scand. 49 (2005) 1108–1116. [6] J.V. den Eynden, S.S. Ali, N. Horwood, S. Carmans, B. Brone, N. Hellings, P. Steels, R.J. Harvey, J.M. Rigo, Glycine and glycine receptor signalling in non-neuronal cells, Front. Mol. Neurosci. 2 (2009) 9. [7] M. Ruiz-Meana, P. Pina, D. Garcia-Dorado, A. Rodriguez-Sinovas, I. Barba, E. Miro-Casas, M. Mirabet, J. Soler-Soler, Glycine protects cardiomyocytes against lethal reoxygenation injury by inhibiting mitochondrial permeability transition, J. Physiol. 558 (2004) 873–882. [8] G. Warnecke, B. Schulze, T. Steinkamp, A. Haverich, U. Klima, Glycine application and right heart function in a porcine heart transplantation model, Transpl. Int. 19 (2006) 218–224. [9] H.D. Wang, X.X. Lu, D.X. Lu, R.B. Qi, Y.P. Wang, Y.M. Fu, L.W. Wang, Glycine inhibits the LPS-induced increase in cytosolic Ca2+ concentration and TNFalpha production in cardiomyocytes by activating a glycine receptor, Acta Pharmacol. Sin. 30 (2009) 1107–1114. [10] M.O. Date, T. Morita, N. Yamashita, K. Nishida, O. Yamaguchi, Y. Higuchi, S. Hirotani, Y. Matsumura, M. Hori, M. Tada, K. Otsu, The antioxidant N-2mercaptopropionyl glycine attenuates left ventricular hypertrophy in in vivo murine pressure-overload model, J. Am. Coll. Cardiol. 39 (2002) 907–912. [11] M. Takefuji, A. Wirth, M. Lukasova, S. Takefuji, T. Boettger, T. Braun, T. Althoff, S. Offermanns, N. Wettschureck, G(13)-mediated signaling pathway is required for pressure overload-induced cardiac remodeling and heart failure, Circulation 126 (2012) 1972–1982. [12] Y. Ohashi, Y. Nakai, H. Sakamoto, Y. Ohno, Y. Sugiura, H. Okamoto, A. Tanaka, Y. Kakinoki, K. Kishimoto, M. Hayashi, Serum levels of soluble interleukin-2 receptor in patients with perennial allergic rhinitis before and after immunotherapy, Ann. Allergy Asthma Immunol. 77 (1996) 203–208. [13] J.M. Malinovsky, F. Servin, A. Cozian, J.Y. Lepage, M. Pinaud, Ketamine and norketamine plasma concentrations after i.v., nasal and rectal administration in children, Br. J. Anaesth. 77 (1996) 203–207. [14] W. Liu, M. Zi, R. Naumann, S. Ulm, J. Jin, D.M. Taglieri, S. Prehar, J. Gui, H. Tsui, R.P. Xiao, L. Neyses, R.J. Solaro, Y. Ke, E.J. Cartwright, M. Lei, X. Wang, Pak1 as a novel therapeutic target for antihypertrophic treatment in the heart, Circulation 124 (2011) 2702–2715. [15] A. Ghallab, G. Celliere, S.G. Henkel, D. Driesch, S. Hoehme, U. Hofmann, S. Zellmer, P. Godoy, A. Sachinidis, M. Blaszkewicz, R. Reif, R. Marchan, L. Kuepfer, D. Haussinger, D. Drasdo, R. Gebhardt, J.G. Hengstler, Model-guided identification of a therapeutic strategy to reduce hyperammonemia in liver diseases, J. Hepatol. 64 (2016) 860–871. [16] X. Wang, M.J. Karau, K.E. Greenwood-Quaintance, D.R. Block, J.N. Mandrekar, S. A. Cunningham, R. Patel, Ureaplasma urealyticum causes hyperammonemia in an experimental immunocompromised murine model, PLoS One 11 (2016) e0161214. [17] B.A. Rose, T. Force, Y. Wang, Mitogen-activated protein kinase signaling in the heart: angels versus demons in a heart-breaking tale, Physiol. Rev. 90 (2010) 1507–1546. [18] J.H. van Berlo, M. Maillet, J.D. Molkentin, Signaling effectors underlying pathologic growth and remodeling of the heart, J. Clin. Invest. 123 (2013) 37– 45. [19] G.E. Yevenes, H.U. Zeilhofer, Allosteric modulation of glycine receptors, Br. J. Pharmacol. 164 (2011) 224–236. [20] C. Pan, X. Bai, L. Fan, Y. Ji, X. Li, Q. Chen, Cytoprotection by glycine against ATPdepletion-induced injury is mediated by glycine receptor in renal cells, Biochem. J. 390 (2005) 447–453. [21] R.J. Hajjar, U. Schmidt, T. Matsui, J.L. Guerrero, K.H. Lee, J.K. Gwathmey, G.W. Dec, M.J. Semigran, A. Rosenzweig, Modulation of ventricular function through gene transfer in vivo, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 5251–5256. [22] R.J. Hajjar, F. del Monte, T. Matsui, A. Rosenzweig, Prospects for gene therapy for heart failure, Circ. Res. 86 (2000) 616–621.
[23] M. Maillet, J.H. van Berlo, J.D. Molkentin, Molecular basis of physiological heart growth: fundamental concepts and new players, Nat. Rev. Mol. Cell Biol. 14 (2013) 38–48. [24] E. Dirkx, M.M. Gladka, L.E. Philippen, A.S. Armand, V. Kinet, S. Leptidis, H. El Azzouzi, K. Salic, M. Bourajjaj, G.J. da Silva, S. Olieslagers, R. van der Nagel, R. de Weger, N. Bitsch, N. Kisters, S. Seyen, Y. Morikawa, C. Chanoine, S. Heymans, P. G. Volders, T. Thum, S. Dimmeler, P. Cserjesi, T. Eschenhagen, P.A. da Costa Martins, L.J. De Windt, Nfat and miR-25 cooperate to reactivate the transcription factor Hand2 in heart failure, Nat. Cell Biol. 15 (2013) 1282– 1293. [25] N. Koitabashi, D.A. Kass, Reverse remodeling in heart failure–mechanisms and therapeutic opportunities, Nat. Rev. Cardiol. 9 (2012) 147–157. [26] J.K. Rogers, S.J. Pocock, J.J. McMurray, C.B. Granger, E.L. Michelson, J. Ostergren, M.A. Pfeffer, S.D. Solomon, K. Swedberg, S. Yusuf, Analysing recurrent hospitalizations in heart failure: a review of statistical methodology, with application to CHARM-Preserved, Eur. J. Heart Fail. 16 (2014) 33–40. [27] D.S. Lee, J.A. Ezekowitz, Risk stratification in acute heart failure, Can. J. Cardiol. 30 (2014) 312–319. [28] G. Wu, Functional amino acids in nutrition and health, Amino Acids 45 (2013) 407–411. [29] W. Wang, Z. Wu, Z. Dai, Y. Yang, J. Wang, G. Wu, Glycine metabolism in animals and humans: implications for nutrition and health, Amino Acids 45 (2013) 463–477. [30] Y. Lamers, J. Williamson, M. Ralat, E.P. Quinlivan, L.R. Gilbert, C. Keeling, R.D. Stevens, C.B. Newgard, P.M. Ueland, K. Meyer, A. Fredriksen, P.W. Stacpoole, J.F. Gregory 3rd, Moderate dietary vitamin B-6 restriction raises plasma glycine and cystathionine concentrations while minimally affecting the rates of glycine turnover and glycine cleavage in healthy men and women, J. Nutr. 139 (2009) 452–460. [31] F. Petrat, J. Drowatzky, K. Boengler, B. Finckh, K.J. Schmitz, R. Schulz, H. de Groot, Protection from glycine at low doses in ischemia-reperfusion injury of the rat small intestine, Eur. Surg. Res. 46 (2011) 180–187. [32] R. Rezaei, W. Wang, Z. Wu, Z. Dai, J. Wang, G. Wu, Biochemical and physiological bases for utilization of dietary amino acids by young Pigs, J. Anim. Sci. Biotechnol. 4 (2013) 7. [33] S.P. Sommer, S. Sommer, B. Sinha, R.G. Leyh, Glycine preconditioning to ameliorate pulmonary ischemia reperfusion injury in rats, Interact. Cardiovasc. Thorac. Surg. 14 (2012) 521–525. [34] D.H. Baker, Advances in protein-amino acid nutrition of poultry, Amino Acids 37 (2009) 29–41. [35] G. Wu, Z. Wu, Z. Dai, Y. Yang, W. Wang, C. Liu, B. Wang, J. Wang, Y. Yin, Dietary requirements of ‘‘nutritionally non-essential amino acids” by animals and humans, Amino Acids 44 (2013) 1107–1113. [36] T. Aki, N. Egashira, Y. Yamauchi, M. Hama, T. Yano, Y. Itoh, T. Yamada, R. Oishi, Protective effects of amino acids against gabexate mesilate-induced cell injury in porcine aorta endothelial cells, J. Pharmacol. Sci. 107 (2008) 238–245. [37] S. Kallakuri, E. Ascher, M. Pagala, P. Gade, A. Hingorani, M. Scheinman, K. Mehraein, T. Jacob, Protective effect of glycine in mesenteric ischemia and reperfusion injury in a rat model, J. Vasc. Surg. 38 (2003) 1113–1120. [38] M.M. Habib, H.J. Hodgson, B.R. Davidson, The role of glycine in hepatic ischemia-reperfusion injury, Curr. Pharm. Des. 12 (2006) 2953–2967. [39] Y. Liu, T.P. Wong, M. Aarts, A. Rooyakkers, L. Liu, T.W. Lai, D.C. Wu, J. Lu, M. Tymianski, A.M. Craig, Y.T. Wang, NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo, J. Neurosci. 27 (2007) 2846–2857. [40] W.L. Cleveland, R.L. DeLaPaz, R.A. Fawwaz, R.S. Challop, High-dose glycine treatment of refractory obsessive-compulsive disorder and body dysmorphic disorder in a 5-year period, Neural Plast. 2009 (2009) 768398. [41] W.B. Zhang, R.G. Hahn, ‘Double toxicity’ of glycine solution in the mouse, Br. J. Urol. 77 (1996) 203–206. [42] W. Zhang, B.S. Andersson, R.G. Hahn, Effect of irrigating fluids and prostate tissue extracts on isolated cardiomyocytes, Urology 46 (1995) 821–824. [43] S. Dutertre, C.M. Becker, H. Betz, Inhibitory glycine receptors: an update, J. Biol. Chem. 287 (2012) 40216–40223. [44] T.L. Young-Pearse, L. Ivic, A.R. Kriegstein, C.L. Cepko, Characterization of mice with targeted deletion of glycine receptor alpha 2, Mol. Cell. Biol. 26 (2006) 5728–5734. [45] A. Avila, P.M. Vidal, T.N. Dear, R.J. Harvey, J.M. Rigo, L. Nguyen, Glycine receptor alpha2 subunit activation promotes cortical interneuron migration, Cell Rep. 4 (2013) 738–750. [46] R.D. Nobles, C. Zhang, U. Muller, H. Betz, M.A. McCall, Selective glycine receptor alpha2 subunit control of crossover inhibition between the on and off retinal pathways, J. Neurosci. 32 (2012) 3321–3332. [47] L. Jiang, X. Qin, X. Zhong, L. Liu, L. Jiang, Y. Lu, L. Fan, Z. He, Q. Chen, Glycineinduced cytoprotection is mediated by ERK1/2 and AKT in renal cells with ATP depletion, Eur. J. Cell Biol. 90 (2011) 333–341. [48] Y. Lu, J. Zhang, B. Ma, K. Li, X. Li, H. Bai, Q. Yang, X. Zhu, J. Ben, Q. Chen, Glycine attenuates cerebral ischemia/reperfusion injury by inhibiting neuronal apoptosis in mice, Neurochem. Int. 61 (2012) 649–658. [49] B. Sanna, O.F. Bueno, Y.S. Dai, B.J. Wilkins, J.D. Molkentin, Direct and indirect interactions between calcineurin-NFAT and MEK1-extracellular signalregulated kinase 1/2 signaling pathways regulate cardiac gene expression and cellular growth, Mol. Cell. Biol. 25 (2005) 865–878. [50] J. Heineke, J.D. Molkentin, Regulation of cardiac hypertrophy by intracellular signalling pathways, Nat. Rev. Mol. Cell Biol. 7 (2006) 589–600.
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51 [51] J.E. Cartledge, C. Kane, P. Dias, M. Tesfom, L. Clarke, B. McKee, S. Al Ayoubi, A. Chester, M.H. Yacoub, P. Camelliti, C.M. Terracciano, Functional crosstalk between cardiac fibroblasts and adult cardiomyocytes by soluble mediators, Cardiovasc. Res. 105 (2015) 260–270. [52] K.E. Porter, N.A. Turner, Cardiac fibroblasts: at the heart of myocardial remodeling, Pharmacol. Ther. 123 (2009) 255–278. [53] B.C. Berk, K. Fujiwara, S. Lehoux, ECM remodeling in hypertensive heart disease, J. Clin. Invest. 117 (2007) 568–575.
51
[54] M.D. Wheeler, R.G. Thurman, Production of superoxide and TNF-alpha from alveolar macrophages is blunted by glycine, Am. J. Physiol. 277 (1999) L952– L959. [55] S. Yamashina, A. Konno, M.D. Wheeler, I. Rusyn, E.V. Rusyn, A.D. Cox, R.G. Thurman, Endothelial cells contain a glycine-gated chloride channel, Nutr. Cancer 40 (2001) 197–204.
Contents lists available at ScienceDirect
Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm
Glycine prevents pressure overload induced cardiac hypertrophy mediated by glycine receptor Yan Lu a,b,1, Xudong Zhu a,1, Jinjie Li a, Ru Fang a, Zhuoyun Wang a, Jing Zhang a, Kexue Li a, Xiaoyu Li a, Hui Bai a, Qing Yang a, Jingjing Ben a, Hanwen Zhang a, Qi Chen a,⇑ a b
Atherosclerosis Research Center, Key Laboratory of Cardiovascular Disease and Molecular Intervention, Nanjing Medical University, Nanjing 210029, People’s Republic of China Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 28 June 2016 Accepted 4 November 2016 Available online 9 November 2016 Chemical compounds studied in this article: Glycine (Pubchem CID: 750) Angiotensin II (Pubchem CID: 172198) Keywords: Cardiac hypertrophy Glycine Glycine receptor ERK1/2 signaling
a b s t r a c t As a major amino acid, glycine has multiple functions in metabolism, growth, immunity, cytoprotection, and survival. The aim of this study was to determine the effects of glycine on pathologic cardiac hypertrophy and the mechanism underlying it. Pre-treatment with glycine significantly attenuated murine cardiac hypertrophy induced by transverse aortic constriction or by administration of angiotensin II (Ang II). This action was associated with a suppressive extracellular signal-regulated kinase 1/2 phosphorylation in myocardium. The cardioprotective effect of glycine disappeared when endogenous glycine receptor a2 was knocked down by mRNA interference in rats. Co-culture experiments revealed that glycine could also antagonize Ang II stimulated release of transforming growth factor b and endothelin-1 by cardiomyocytes, which prevented an over-production of collagens in rat fibroblasts. These results, for the first time, demonstrate that glycine may be a novel cardioprotector against pressure overload induced cardiac hypertrophy. Thus, glycine would be useful in the prevention of cardiac hypertrophy and heart failure. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Despite great advances in the understanding and treatment of heart failure, the disease remains a leading cause of death worldwide [1,2]. Heart failure is structurally characterized by pathologic hypertrophy of the myocardium which temporarily preserves pump function and reduces ventricular wall stress. However, prolonged cardiac hypertrophy can cause arrhythmias, dilated cardiomyopathy and heart failure [3,4]. In contrast to physiological hypertrophy, pathological hypertrophy is characterized by accumulation of interstitial collagen and cell death, both of which contribute to increased risk for myocardial infarction, arrhythmia and sudden death. Therefore, it would be of great therapeutic interest to prevent pathological hypertrophy. Glycine is a major amino acid in mammals and other animals. It plays an important role in metabolism, growth, development, immunity, cytoprotection, and survival [5,6]. Recent studies have shown a few beneficial effects of glycine on cardiomyocytes under ischemia-reperfusion (I/R) conditions. For example, 3 mM glycine ⇑ Corresponding author at: Atherosclerosis Research Center, Key Laboratory of Cardiovascular Disease and Molecular Intervention, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, People’s Republic of China. E-mail address: [email protected] (Q. Chen). 1 Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.bcp.2016.11.008 0006-2952/Ó 2016 Elsevier Inc. All rights reserved.
increases the cell viability of isolated rat hearts after I/R [7]. Infusion of glycine into animal donor hearts is good for right ventricular function after transplantation [8]. Glycine can inhibit the LPS induced increase in cytosolic Ca2+ concentration and tumor necrosis factor-a (TNF-a) production in cardiomyocytes by activating a glycine receptor (glyR) [9]. The antioxidant N-2mercaptopropionyl glycine has been reported to attenuate cardiac hypertrophy induced by TAC in mice [10]. However, whether glycine has an impact on cardiac hypertrophy is unknown. In the current study, we demonstrate that glycine significantly attenuates murine left ventricular (LV) hypertrophy and cardiac fibrosis induced by either transverse aortic constriction (TAC) or angiotensin II (Ang II) administration. Mechanistically, we show that the cardioprotective effect of glycine may be via glyR a2 coupling to inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and preventing production of transforming growth factor-b (TGF-b) and endothelin-1 (ET-1) by cardiomyocytes. 2. Materials and methods 2.1. Animals and treatments All aspects of the animal care and experimental protocols were in accordance with the Guide for the Care and Use of Laboratory
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
Animals (NIH publication, 8th edition, 2011) and approved by the Experimental Animal Care and Use Committee of Nanjing Medical University. Male 8-week-old C57BL/6J mice, male Sprague-Dawley rats each weighing 250 ± 30 g, and neonatal Sprague-Dawley rats (0–3 day old) were obtained from the Animal Center of Nanjing Medical University. All animal experiments performed in this study adhered to the protocols approved by the Institutional Animal Care and Use Committee of the Nanjing Medical University. Cardiac hypertrophy and heart failure were induced by TAC surgery or by Ang II administration by using an osmotic pump as described [11]. Glycine (Sigma-Aldrich, St Louis, USA) was suspended in saline for intraperitoneal injection. Mice were anaesthetized with an intraperitoneal injection of ketamine (100 mg/ kg) and xylazine (10 mg/kg). Rats were anaesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (8 mg/kg). 2.2. Measurement of plasma concentration of glycine Glycine was injected into mice and the plasma was collected for further use at different time points. The plasma was blown dry by nitrogen and then dissolved in mobile phase. Agilent HPLC was coupled to a Micromass Quattro Ultima mass spectrometer with an ESI (Electrospray Ionisation) source, together with a Discovery C18 column (50 mm 2.1 mm 5 lm) for study. The mobile phase solvents were H2O containing 0.1% TDFHA and ACN containing 0.1% TDFHA. Gradient composition started from 10% ACN and was maintained for 1 min. It was rapidly raised to 15% at 3 min, to 20% at 5 min, to 25% at 6 min, to 40% at 7 min, to 75% at 8.90 min, and finally to 98% at 9 min till the end of the analysis. Analysis was performed by using HPLC–ESI–MS/MS for a more specific and sensitive detection [12,13]. 2.3. TAC surgery Cardiac hypertrophy was induced in mice and rats by constricting to transverse aorta. Briefly, a small midline skin cut was made just above the sternum of the animal. Muscles were gently separated until trachea was visible. Partial left side thoracotomy to the second rib was performed with blunt ended spring scissor and the sternum was retracted using a chest retractor. Blunt tip 45° angled forceps were used to gently separate the two lobes of thymus and to clean fat tissue from the aortic arch. A small piece of a 7-0 silk suture (presoaked in sterile saline) was placed between the innominate and left carotid arteries using a 90 degree curved forceps. Two loose knots were tied around the transverse aorta and a small piece of a 27 gauge blunt needle was placed parallel to the transverse aorta. The first knot was quickly tied against the needle, followed by the second one. The needle was promptly removed in order to yield a constriction of 0.4 mm in diameter. 2.4. Ang II pump implantation Mini-osmotic pumps (model 2004, ALZET technical services, USA) filled with Ang II (1.5 mg/kg/day) or saline were dorsally implanted into mice for 21 days. Glycine (700 mg/kg/day) was peritoneally injected into mice a week before the surgery. 2.5. Cardiac imaging Transthoracic 2-dimensional M-mode echocardiography was performed with Vevo 770 (VisualSonics, Toronto, Canada) equipped with a 30-MHz transducer. Percentage of fractional shortening, LV wall thickness, and LV mass and ejection fraction (EF) was calculated as described [14].
41
2.6. Antibodies and reagents Antibody against glyR a2 and GAPDH were from Abcam Biotech (San Francisco, USA). Glycine, Ang II and antibody against a-actinin were obtained from Sigma. Antibodies against cRaf, p-cRaf, mitogen-activated protein kinase kinase1/2 (MEK1/2), p-MEK1/2, ERK1/2, p-ERK1/2, p38, p-p38, c-Jun N-terminal kinase (JNK) and p-JNK were obtained from Cell Signaling Technology (Boston, USA). Plasma concentrations of TGF-b and TNF-a were determined by using mouse TGF-b ELISA and rat TNF-a ELISA Kit (Excell, Shanghai, China). Plasma concentrations of ammonia and urea were determined by using blood ammonia assay kit and blood urea assay kit (Jiangcheng, Nanjing, China). 2.7. Isolation of neonatal rat cardiomyocytes and fibroblasts Neonatal rat cardiomyocytes (NRCMs) were prepared by enzymatic digestion of hearts obtained from newborn (0–3 day old) Sprague–Dawley rat pups by using percoll gradient centrifugation. For in vitro experiments NRCMs and fibroblasts were pre-treated with glycine (5 mM) for 30 min followed by addition of Ang II (1 lM). Cells were harvested and used for the mRNA and protein studies. 3.3. Protective effect of glycine on Ang II induced cardiac hypertrophy We also examined the effect of glycine on the Ang II induced cardiac hypertrophy in mice. Consistent with the results from the TAC model, we found that glycine could protect the mouse heart against Ang II induced hypertrophy by echocardiography measurements and heart appearance analysis (Fig. 3a–c). Histomorphometric analysis showed an inhibitive effect of glycine on cardiac fibrosis in Ang II administrated mice (Fig. 3d–g). Furthermore, pretreatment with glycine could inhibit Ang II induced phosphorylation of cRaf-MEK1/2-ERK1/2 in the mouse myocardium (Fig. 3h and i). Antagonistic effect of glycine on Ang II induced cardiac hypertrophy was further investigated by using NRCMs in vitro. We found that treatment with Ang II (1 lM) resulted in a significant upregulation of hypertrophic genes including ANP, BNP, and b-MHC in NRCMs. This action could be markedly blocked by pretreatment of NRCMs with glycine dose-dependently (Fig. 4a). Treatment with 5 mM glycine significantly decreased the NRCMs surface area in the presence of Ang II (Fig. 4b and c). Glycine pre-treated NRCMs also exhibited a suppressive phosphorylation of cRaf-MEK1/2ERK1/2 in the presence of Ang II (Fig. 4d and e). As such, these data demonstrated that glycine could prevent Ang II induced murine cardiac hypertrophy in vivo and in vitro. 3.4. Cardiac protection of glycine is mediated via a glycine receptor It is known that glyR plays an important role in mediation of cytoprotection of glycine [6,19,20]. GlyR is an ionotropic or ligand-gated receptor consisting with different sub-units including glyR a1, a2, a3, and a4. To investigate the role of glyR in glycine antagonizing cardiac hypertrophy, we first examined the expression of glyR in the heart. It is interesting that only glyR a2 was expressed in the cardiomyocyte but not in the cardiofibroblast in rats as measured by RT-PCR (Fig. 5a), immunofluorescence staining (Fig. 5b), and western blot (Fig. 5c). Moreover, we found that expression of glyR a2 was increased in adult rat cardiomyocytes compared with NRCMs (Fig. 5c). When endogenous glyR a2 was knocked down (70%) in cultured NRCMs by using a siRNA (Fig. 5d), the antagonistic effect of glycine on Ang II induced up-regulation of ANP, BNP and b-MHC disappeared (Fig. 5e). Consistently, inhibitive effects of glycine on cRaf-MEK1/2-ERK1/2 phosphorylation were also abolished in
42
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
Fig. 1. Glycine attenuates TAC induced LV fibrosis in mice. (a and b) Mice were pre-treated with glycine (G) (150, 300 or 700 mg/kg) or vehicle (V). Six days later, mice received TAC surgery for 4 weeks. (a) Representative images of whole hearts from mice. (b) Measurements of HW/BW and HW/TL (n = 6–10). (c) Plasma glycine levels 1 day and 6 days after glycine injection (n = 4–6). (d) Plasma concentration of ammonia after glycine injection (n = 4–6). (e) Plasma concentration of urea after glycine injection (n = 4–6). (f–l) Mice were pre-treated with 700 mg/kg glycine (G) or vehicle (V). Six days later, mice received TAC surgery for 4 weeks. (f) Masson’s trichomeand. (g) Picrosirius red staining for collagen deposition in LV tissue sections. (h) Stained sections were analyzed with Image-Pro Plus software (n = 4–6). (i and j) mRNA levels of collagen I, collagen III, ANP and BNP in mouse heart (n = 6). (k) Representative immunoblot of csapase-3 and GAPDH in whole heart lysates. (l) Quantification of band intensity normalized by corresponding GAPDH levels (n = 3). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
NRCMs (Fig. 5f and g). We also injected directly an adenoassociated virus coated anti-glyR a2 sequence into the rat heart to determine the role of glyR a2 in vivo [21,22]. This caused that
glyR a2 expression was down-regulated by 80% to 70% from 1 to 2 weeks after TAC (Fig. 6a). The cardiac protection of glycine disappeared in this conditions as measurements of LV mass, LVPW,
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
43
Fig. 2. Glycine attenuates TAC induced cardiac hypertrophy in mice. (a–g) Mice were pre-treated with 700 mg/kg glycine (G) or vehicle (V). Six days later, mice received TAC surgery for 4 weeks. (a) Analysis of LV mass, LVPW and IVS by echocardiography (n = 5–7). (b) Hematoxylin and eosin (H&E) staining of mouse hearts. (c) Analysis of EF by echocardiography (n = 5–7). (d) Representative immunoblot of p-cRaf, cRaf, p-MEK1/2, MEK1/2, p-ERK1/2 and ERK1/2 in whole heart lysates. (e) Quantification of band intensity (n = 3). (f) Representative immunoblot of p-p38, p38, p-JNK and JNK in whole heart lysates. (g) Quantification of band intensity (n = 3). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
44
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
Fig. 3. Glycine attenuates Ang II induced cardiac hypertrophy in mice. (a–i) Mice were pre-treated with 700 mg/kg glycine (G) or vehicle (V). Six days later, mice received Ang II treatment for 21 days. (a) Analysis of LV mass, LVPW and IVS by echocardiography (n = 6–8). (b) Representative images of whole hearts. (c) Measurements of HW/BW and HW/TL (n = 6–8). (d) H&E staining. (e) Masson’s trichome staining. (f) Picro-sirius red staining of myocardium sections. (g) Stained sections were analyzed with Image-Pro Plus software (n = 4–6). (h) Representative immunoblot of p-cRaf, cRaf, p-MEK1/2, MEK1/2, p-ERK1/2, and ERK1/2 in whole heart lysates. (i) Quantification of band intensity (n = 3). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
45
Fig. 4. Glycine attenuates Ang II induced hypertrophy in NRCMs. (a) NRCMs were pre-treated with glycine (1, 5 or 10 mM) (G) for 30 min followed by addition of Ang II (1 lM) for 24 h. mRNA levels of ANP, BNP, and b-MHC in NRCMs were measured (n = 6–8). (b) NRCMs were pre-treated with glycine (5 mM) (G) for 30 min followed by addition of Ang II (1 lM) for 24 h. Representative images of a-actinin immunostaining in NRCMs. (c) Quantification of cell surface area (n = 3. Fifty cells were counted per experiment). (d) NRCMs were pre-treated with glycine (5 mM) (G) for 30 min followed by addition of Ang II (1 lM) for 1 h. Representative immunoblot of p-cRaf, cRaf, pMEK1/2, MEK1/2, p-ERK1/2, and ERK1/2 in NRCMs lysates. (e) Quantification of band intensity (n = 3). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
IVS, HW/BW, and HW/TL 2 weeks post TAC, while glycine still exerted its effects in the scram sequence group (Fig. 6b and c). Same as the in vitro observations, inhibitive effects of glycine on cRaf-MEK1/2-ERK1/2 phosphorylation were also abolished in the anti-glyR a2 sequence group (Fig. 6d and e). Taken together, these results indicate that glyR a2 might be required for glycine’s cardiac protection in rats.
3.5. Glycine inhibits Ang II induced fibrotic response by a cardiomyocyte-dependent mechanism Since no glyR expression has been found in the fibroblast, how does glycine exert its anti-cardiac fibrosis effect by a glyR reliable mechanism? This question promoted us to perform co-culture studies with neonatal rat fibroblasts and NRCMs. Measurements
46
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
Fig. 5. GlyR a2 is required for the effect of glycine on NRCMs. (a) mRNA expression of glyRs in rat spinal, NRCMs (CM) and neonatal rat fibroblasts (CF). (b) Representative images of NRCMs triple staining. (c) Immunoblot of glyR a1, glyR a2 and GAPDH in spinal, and adult rat cardiomyocytes lysates and NRCMs. (d) NRCMs were transfected with siRNA targeting glyR a2 (SiRNA-GlyR) or scrambled siRNA (SiRNA-( )) and expression of glyR a2 and GAPDH were analyzed by immunoblot assay. GAPDH was blotted as a loading control (n = 3). (e) NRCMs were transfected with siRNA targeting glyR a2 (SiRNA-GlyR) or scrambled siRNA (SiRNA-( )). After 3 days, NRCMs were pre-treated with glycine (5 mM) (G) for 30 min followed by addition of Ang II (1 lM) for 24 h. mRNA levels of ANP, BNP and b-MHC in NRCMs (n = 6–8). (f) NRCMs were transfected with siRNA targeting glyR a2 (SiRNA-GlyR) or scrambled siRNA (SiRNA-( )). After 3 days, NRCMs were pre-treated with glycine (5 mM) (G) for 30 min followed by addition of Ang II (1 lM) for 1 h. Representative immunoblot of p-cRaf, cRaf, p-MEK1/2, MEK1/2, p-ERK1/2, and ERK1/2 in NRCMs lysates. (g) Quantification of band intensity (n = 3). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01.
of mRNA levels revealed that glycine inhibited Ang II induced overproduction of collagen I and collagen III in neonatal rat fibroblasts only in the presence of NRCMs (Fig. 7a). The Ang II insulted NRCMs produced much more TGF-b and ET-1 which was significantly inhibited by treatment with glycine (Fig. 7b). No significant
changes were found in the production of angiotensinogen and TNF-a in the glycine treated NRCMs. The effect of glycine on fibrotic response was also examined by in vivo experiments. We found that glycine significantly inhibited TAC induced increases in TGF-b, ET-1, and angiotensinogen gene expression in mice hearts (Fig. 7c).
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
47
Fig. 6. Activating glyR a2 prevents TAC induced cardiac hypertrophy in rats. (a) After injection of the anti-glyR a2 sequence containing adeno-associated virus (SiRNA-GlyR) for one or two weeks. Immunoblot of glyR a2 and GAPDH in rat LV. GAPDH was blotted as a loading control (n = 5–7). (b–e) After injection of the anti-glyR a2 sequence containing adeno-associated virus (SiRNA-GlyR) for one week, TAC surgery was performed together with treatment with glycine (700 mg/kg) (G) or vehicle (V). (b) Measurements of HW/BW and HW/TL in rats (n = 5–7). (c) Analysis of LV mass, LVPW and IVS in rats by echocardiography. (d) Representative immunoblot of p-cRaf, cRaf, pMEK1/2, MEK1/2, p-ERK1/2 and ERK1/2 in rat whole heart lysates. (e) Quantification of band intensity (n = 3). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, *** P < 0.001.
48
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
Fig. 7. Glycine inhibits Ang II stimulated overproduction of collagens in rat fibroblasts. (a) Cells were pre-treated with glycine (5 mM) (G) for 30 min followed by addition of Ang II (1 lM) for 24 h. mRNA levels of collagen I and collagen III in neonatal rat fibroblasts co-culturing with NRCMs. (n = 6–8). (b) NRCMs were pre-treated with glycine (5 mM) (G) for 30 min followed by addition of Ang II (1 lM) for 24 h. mRNA levels of TGF-b, ET-1, angiotensinogen and TNF-a in NRCMs. (n = 6–8). (c and d) Mice were pretreated with 700 mg/kg glycine (G) or vehicle (V). Six days later, mice received TAC surgery for 4 weeks. (c) mRNA levels of TGF-b, ET-1, angiotensinogen and TNF-a in the mice hearts (n = 6–8). (d) Plasma levels of TGF-b and TNF-a in mice (n = 6–8). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
Similar reaction pattern was also found in plasma TGF-b in mice (Fig. 7d). Thus, these data suggest that glycine inhibit TAC induced overproduction of collagens in fibroblasts via preventing release of TGF-b and ET-1 by stressed cardiomyocytes.
4. Discussion Heart hypertrophy is initially physiological to compensate for the loss of heart functions. However, sustained stresses lead to a pathological status, in which myocardium becomes stiffened by interstitial fibrosis and thereby diastolic dysfunction induces global remodeling of the heart, dilated cardiomyopathy and heart failure [23,24]. Heart failure is one of the most devastating diseases in which cardiac hypertrophy is a determinant of the clinical course [1,25]. The lifetime risk of heart failure is 1 in 5 among both men and women. Despite the advances in the development of new therapies in past decades, the survival rate after the onset of heart failure remains very low [26,27]. Therefore, a novel anti-hypertrophic drug or a potential therapeutic target for the treatment of cardiac hypertrophy and heart failure is extremely needed. The present study demonstrates for the first time that glycine may antagonize pressure overload induced cardiac hypertrophy and fibrosis in rodents. Glycine has been regarded as a ‘‘nutritionally nonessential amino acid’’ for humans due to the presence of its endogenous synthesis in the body [28,29]. Plasma level of glycine is normally about 300 lM in adult humans [30,31], which is similar to the mouse level measured by the present study. However, it has been reported that the amount of glycine synthesized in vivo would be insufficient to meet metabolic demands in humans and animals [32]. Moreover, accumulative lines of evidence have revealed that glycine is associated with the cytoprotection, anti-inflammatory responses, and animal development [33–35]. Implication with high doses of glycine exhibits the beneficial effects on gabexatemesilate induced lung endothelial cells injury [36], I/R injury in cultured cells, perfused organs, and in vivo models including heart, liver, kidney, skeletal muscle, and small intestine [7,37,38]. In the present study, the plasma concentrations of glycine were higher than 0.5 mM, the effective level for protection against I/R injury in rat small intestine [31], for more than 8 h/day after intraperitoneal injection of 700 mg/kg glycine. Meanwhile, ammonia, one of the catabolic products of glycine, was sustained at the normal level for more than 22 h/day. The administered glycine may be excreted in the urine only in a minor amount. The majority is taken up by cells and metabolized via a variety of other routes. Therefore, the observed improvements in the cardiac structures and functions would be attributed to the implanted glycine. The doses of 800 mg/kg body weight would be acceptable for intraperitoneal injection of glycine [39]. A medical record for five years on oral administration of 50–66 g/day glycine has been tracked for the treatment of refractory obsessive-compulsive disorder and body dysmorphic disorder. No obvious side-effect has been found [40]. However, high doses of glycine can cause serious adverse effects. Intravenous infusion of 1.5% glycine causes the cardiotoxic effects in rabbits [41]. Rat myocardial cells are supposed to die in more than 1.0% (133 mM) glycine in vitro [42]. In humans, the threshold concentration of plasma glycine would be 5 mM at which overt adverse effects may start to develop. A significant finding of this study is that the protection of glycine might be mediated by glyR a2. It is interesting that only glyR a2 among the four family members is expressed in the cardiomyocyte. GlyR a2 is highly expressed at the embryonic and neonatal stages but postnatally is largely replaced by glyR a1 in central nervous system [43]. The definite role of glyR a2 in the heart is not
49
fully known yet. GlyR a2 has been reported to be less sensitive to ethanol, tropisetron and Zinc than glyR a1 [19]. It might be that the presence of glyR a2 endows the myocardium with special functions in the development and metabolism, although a glyR a2 deficiency mouse model seems to be phenotypically normal [43,44]. It has been reported that extrasynaptic activation of glyRs containing the a2 subunit in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx, which further modulates actomyosin contractility to fine-tune nuclear translocation during migration [45]. Nobles et al. reported that selective glyR a2 subunit control of crossover inhibition between the on and off retinal pathways and is involved in regulation of interneuron differentiation during spinal cord development [46]. Moreover, our results indicate that glyR a2 in the myocardium may be a novel target to antagonize heart failure. The cytoprotective effects of glyRs have been reported to associate with the MAPK (JNK, ERK1/2 and p38) signaling pathways [47,48]. The present study reveals that suppressive phosphorylation of cRaf-MEK1/2-ERK1/2 is initiated by glycine – glyR a2 interactions in cardiomyocytes. As a central regulator, the MEK1/2ERK1/2 signaling promotes cardiac hypertrophy in the body. This is, in part, by enhancing the transcriptional activity of nuclear factor of activated T cells (NFAT) [49]. The p38 and JNK signaling is reported to negatively regulate the cardiac growth response by directly phosphorylating NFAT in the heart [50]. The signaling pathways integrate specific stimuli at the cell membrane and transmit it to intracellular target proteins that are involved in transcription, protein synthesis and protein stability in the myocardium. Therefore, inhibition of MEK1/2- ERK1/2 and activation of p38 and/or JNK might be benefit to the prevention and treatment of maladaptive hypertrophy. Fibrosis is fundamental to pathological remodeling of myocardium in the diseased heart. It is intriguing that there is no glyR in fibroblasts but the expression of glyR a2 in the heart is requisite for antagonistic effect of glycine on the cardiac fibrosis. This might be explained by that glycine itself would not directly impact the production of collagens by fibroblasts. Its direct target for antifibrosis seems to be the cardiomyocytes. Glycine could block the stress, such as Ang II, stimulated release of TGF-b and ET-1 by cardiomyocytes. Both TGF-b and ET-1 can stimulate the production of collagens by fibroblasts. Furthermore, it has been reported that TGF-b also can promote cardiomyocyte hypertrophy [51]. Thus, via an indirect suppression on the production of collagens by fibroblasts and direct effect of TGF-b on cardiomyocytes, glycine may prevent the cardiac fibrosis and development of myocardial remodeling process which is critical in the progression of heart failure [52,53]. Beyond the inhibition of fibrotic responses, glyR mediated cardiac protection of glycine may have other mechanisms. Glycine can inhibit calcium fluxes in lymphocytes, macrophages, and neutrophils, leading to a decreased secretion of cytokines (e.g. TNF-a). These anti-inflammatory effects are also likely mediated by functional glyRs [6,54]. A modulating effect of glycine on endothelial cells has been reported via glyRs. [55]. We have found that glycine antagonizes cerebral I/R induced injury by inhibiting extrinsic and intrinsic apoptotic pathways, which is associated with the presence of glyRs [48]. However, similar effect of glycine is not found in the present study. Discovery of the new pharmacological roles of glycine may pave a path to generate a novel therapy differing from the angiotensin-converting enzyme inhibitor and angiotensin type 1 receptor antagonist for treatment of heart failure.
Conflict of interest statement None declared.
50
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51
Acknowledgements This work was supported by National Natural Science Foundation of China [81230070 and 91339202 to Qi Chen, 81300211 to Xudong Zhu, 81670263 to Xiaoyu Li, 81370005 to Jingjing Ben]; College Natural Science Foundation of Jiangsu [13KJB310005 to Xudong Zhu]; Jiangsu Province Education Office of the major basic research projects [15KJA310001 to Xiaoyu Li] and the Collaborative Innovation Center For Cardiovascular Disease Translational Medicine of Jiangsu Province. References [1] J.O. Mudd, D.A. Kass, Tackling heart failure in the twenty-first century, Nature 451 (2008) 919–928. [2] I. Rachmin, S. Tshori, Y. Smith, A. Oppenheim, S. Marchetto, G. Kay, R.S. Foo, N. Dagan, E. Golomb, D. Gilon, J.P. Borg, E. Razin, Erbin is a negative modulator of cardiac hypertrophy, Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 5902–5907. [3] J.A. Towbin, N.E. Bowles, The failing heart, Nature 415 (2002) 227–233. [4] O. Gjesdal, D.A. Bluemke, J.A. Lima, Cardiac remodeling at the population level– risk factors, screening, and outcomes, Nat. Rev. Cardiol. 8 (2011) 673–685. [5] R.Y. Gundersen, P. Vaagenes, T. Breivik, F. Fonnum, P.K. Opstad, Glycine–an important neurotransmitter and cytoprotective agent, Acta Anaesthesiol. Scand. 49 (2005) 1108–1116. [6] J.V. den Eynden, S.S. Ali, N. Horwood, S. Carmans, B. Brone, N. Hellings, P. Steels, R.J. Harvey, J.M. Rigo, Glycine and glycine receptor signalling in non-neuronal cells, Front. Mol. Neurosci. 2 (2009) 9. [7] M. Ruiz-Meana, P. Pina, D. Garcia-Dorado, A. Rodriguez-Sinovas, I. Barba, E. Miro-Casas, M. Mirabet, J. Soler-Soler, Glycine protects cardiomyocytes against lethal reoxygenation injury by inhibiting mitochondrial permeability transition, J. Physiol. 558 (2004) 873–882. [8] G. Warnecke, B. Schulze, T. Steinkamp, A. Haverich, U. Klima, Glycine application and right heart function in a porcine heart transplantation model, Transpl. Int. 19 (2006) 218–224. [9] H.D. Wang, X.X. Lu, D.X. Lu, R.B. Qi, Y.P. Wang, Y.M. Fu, L.W. Wang, Glycine inhibits the LPS-induced increase in cytosolic Ca2+ concentration and TNFalpha production in cardiomyocytes by activating a glycine receptor, Acta Pharmacol. Sin. 30 (2009) 1107–1114. [10] M.O. Date, T. Morita, N. Yamashita, K. Nishida, O. Yamaguchi, Y. Higuchi, S. Hirotani, Y. Matsumura, M. Hori, M. Tada, K. Otsu, The antioxidant N-2mercaptopropionyl glycine attenuates left ventricular hypertrophy in in vivo murine pressure-overload model, J. Am. Coll. Cardiol. 39 (2002) 907–912. [11] M. Takefuji, A. Wirth, M. Lukasova, S. Takefuji, T. Boettger, T. Braun, T. Althoff, S. Offermanns, N. Wettschureck, G(13)-mediated signaling pathway is required for pressure overload-induced cardiac remodeling and heart failure, Circulation 126 (2012) 1972–1982. [12] Y. Ohashi, Y. Nakai, H. Sakamoto, Y. Ohno, Y. Sugiura, H. Okamoto, A. Tanaka, Y. Kakinoki, K. Kishimoto, M. Hayashi, Serum levels of soluble interleukin-2 receptor in patients with perennial allergic rhinitis before and after immunotherapy, Ann. Allergy Asthma Immunol. 77 (1996) 203–208. [13] J.M. Malinovsky, F. Servin, A. Cozian, J.Y. Lepage, M. Pinaud, Ketamine and norketamine plasma concentrations after i.v., nasal and rectal administration in children, Br. J. Anaesth. 77 (1996) 203–207. [14] W. Liu, M. Zi, R. Naumann, S. Ulm, J. Jin, D.M. Taglieri, S. Prehar, J. Gui, H. Tsui, R.P. Xiao, L. Neyses, R.J. Solaro, Y. Ke, E.J. Cartwright, M. Lei, X. Wang, Pak1 as a novel therapeutic target for antihypertrophic treatment in the heart, Circulation 124 (2011) 2702–2715. [15] A. Ghallab, G. Celliere, S.G. Henkel, D. Driesch, S. Hoehme, U. Hofmann, S. Zellmer, P. Godoy, A. Sachinidis, M. Blaszkewicz, R. Reif, R. Marchan, L. Kuepfer, D. Haussinger, D. Drasdo, R. Gebhardt, J.G. Hengstler, Model-guided identification of a therapeutic strategy to reduce hyperammonemia in liver diseases, J. Hepatol. 64 (2016) 860–871. [16] X. Wang, M.J. Karau, K.E. Greenwood-Quaintance, D.R. Block, J.N. Mandrekar, S. A. Cunningham, R. Patel, Ureaplasma urealyticum causes hyperammonemia in an experimental immunocompromised murine model, PLoS One 11 (2016) e0161214. [17] B.A. Rose, T. Force, Y. Wang, Mitogen-activated protein kinase signaling in the heart: angels versus demons in a heart-breaking tale, Physiol. Rev. 90 (2010) 1507–1546. [18] J.H. van Berlo, M. Maillet, J.D. Molkentin, Signaling effectors underlying pathologic growth and remodeling of the heart, J. Clin. Invest. 123 (2013) 37– 45. [19] G.E. Yevenes, H.U. Zeilhofer, Allosteric modulation of glycine receptors, Br. J. Pharmacol. 164 (2011) 224–236. [20] C. Pan, X. Bai, L. Fan, Y. Ji, X. Li, Q. Chen, Cytoprotection by glycine against ATPdepletion-induced injury is mediated by glycine receptor in renal cells, Biochem. J. 390 (2005) 447–453. [21] R.J. Hajjar, U. Schmidt, T. Matsui, J.L. Guerrero, K.H. Lee, J.K. Gwathmey, G.W. Dec, M.J. Semigran, A. Rosenzweig, Modulation of ventricular function through gene transfer in vivo, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 5251–5256. [22] R.J. Hajjar, F. del Monte, T. Matsui, A. Rosenzweig, Prospects for gene therapy for heart failure, Circ. Res. 86 (2000) 616–621.
[23] M. Maillet, J.H. van Berlo, J.D. Molkentin, Molecular basis of physiological heart growth: fundamental concepts and new players, Nat. Rev. Mol. Cell Biol. 14 (2013) 38–48. [24] E. Dirkx, M.M. Gladka, L.E. Philippen, A.S. Armand, V. Kinet, S. Leptidis, H. El Azzouzi, K. Salic, M. Bourajjaj, G.J. da Silva, S. Olieslagers, R. van der Nagel, R. de Weger, N. Bitsch, N. Kisters, S. Seyen, Y. Morikawa, C. Chanoine, S. Heymans, P. G. Volders, T. Thum, S. Dimmeler, P. Cserjesi, T. Eschenhagen, P.A. da Costa Martins, L.J. De Windt, Nfat and miR-25 cooperate to reactivate the transcription factor Hand2 in heart failure, Nat. Cell Biol. 15 (2013) 1282– 1293. [25] N. Koitabashi, D.A. Kass, Reverse remodeling in heart failure–mechanisms and therapeutic opportunities, Nat. Rev. Cardiol. 9 (2012) 147–157. [26] J.K. Rogers, S.J. Pocock, J.J. McMurray, C.B. Granger, E.L. Michelson, J. Ostergren, M.A. Pfeffer, S.D. Solomon, K. Swedberg, S. Yusuf, Analysing recurrent hospitalizations in heart failure: a review of statistical methodology, with application to CHARM-Preserved, Eur. J. Heart Fail. 16 (2014) 33–40. [27] D.S. Lee, J.A. Ezekowitz, Risk stratification in acute heart failure, Can. J. Cardiol. 30 (2014) 312–319. [28] G. Wu, Functional amino acids in nutrition and health, Amino Acids 45 (2013) 407–411. [29] W. Wang, Z. Wu, Z. Dai, Y. Yang, J. Wang, G. Wu, Glycine metabolism in animals and humans: implications for nutrition and health, Amino Acids 45 (2013) 463–477. [30] Y. Lamers, J. Williamson, M. Ralat, E.P. Quinlivan, L.R. Gilbert, C. Keeling, R.D. Stevens, C.B. Newgard, P.M. Ueland, K. Meyer, A. Fredriksen, P.W. Stacpoole, J.F. Gregory 3rd, Moderate dietary vitamin B-6 restriction raises plasma glycine and cystathionine concentrations while minimally affecting the rates of glycine turnover and glycine cleavage in healthy men and women, J. Nutr. 139 (2009) 452–460. [31] F. Petrat, J. Drowatzky, K. Boengler, B. Finckh, K.J. Schmitz, R. Schulz, H. de Groot, Protection from glycine at low doses in ischemia-reperfusion injury of the rat small intestine, Eur. Surg. Res. 46 (2011) 180–187. [32] R. Rezaei, W. Wang, Z. Wu, Z. Dai, J. Wang, G. Wu, Biochemical and physiological bases for utilization of dietary amino acids by young Pigs, J. Anim. Sci. Biotechnol. 4 (2013) 7. [33] S.P. Sommer, S. Sommer, B. Sinha, R.G. Leyh, Glycine preconditioning to ameliorate pulmonary ischemia reperfusion injury in rats, Interact. Cardiovasc. Thorac. Surg. 14 (2012) 521–525. [34] D.H. Baker, Advances in protein-amino acid nutrition of poultry, Amino Acids 37 (2009) 29–41. [35] G. Wu, Z. Wu, Z. Dai, Y. Yang, W. Wang, C. Liu, B. Wang, J. Wang, Y. Yin, Dietary requirements of ‘‘nutritionally non-essential amino acids” by animals and humans, Amino Acids 44 (2013) 1107–1113. [36] T. Aki, N. Egashira, Y. Yamauchi, M. Hama, T. Yano, Y. Itoh, T. Yamada, R. Oishi, Protective effects of amino acids against gabexate mesilate-induced cell injury in porcine aorta endothelial cells, J. Pharmacol. Sci. 107 (2008) 238–245. [37] S. Kallakuri, E. Ascher, M. Pagala, P. Gade, A. Hingorani, M. Scheinman, K. Mehraein, T. Jacob, Protective effect of glycine in mesenteric ischemia and reperfusion injury in a rat model, J. Vasc. Surg. 38 (2003) 1113–1120. [38] M.M. Habib, H.J. Hodgson, B.R. Davidson, The role of glycine in hepatic ischemia-reperfusion injury, Curr. Pharm. Des. 12 (2006) 2953–2967. [39] Y. Liu, T.P. Wong, M. Aarts, A. Rooyakkers, L. Liu, T.W. Lai, D.C. Wu, J. Lu, M. Tymianski, A.M. Craig, Y.T. Wang, NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo, J. Neurosci. 27 (2007) 2846–2857. [40] W.L. Cleveland, R.L. DeLaPaz, R.A. Fawwaz, R.S. Challop, High-dose glycine treatment of refractory obsessive-compulsive disorder and body dysmorphic disorder in a 5-year period, Neural Plast. 2009 (2009) 768398. [41] W.B. Zhang, R.G. Hahn, ‘Double toxicity’ of glycine solution in the mouse, Br. J. Urol. 77 (1996) 203–206. [42] W. Zhang, B.S. Andersson, R.G. Hahn, Effect of irrigating fluids and prostate tissue extracts on isolated cardiomyocytes, Urology 46 (1995) 821–824. [43] S. Dutertre, C.M. Becker, H. Betz, Inhibitory glycine receptors: an update, J. Biol. Chem. 287 (2012) 40216–40223. [44] T.L. Young-Pearse, L. Ivic, A.R. Kriegstein, C.L. Cepko, Characterization of mice with targeted deletion of glycine receptor alpha 2, Mol. Cell. Biol. 26 (2006) 5728–5734. [45] A. Avila, P.M. Vidal, T.N. Dear, R.J. Harvey, J.M. Rigo, L. Nguyen, Glycine receptor alpha2 subunit activation promotes cortical interneuron migration, Cell Rep. 4 (2013) 738–750. [46] R.D. Nobles, C. Zhang, U. Muller, H. Betz, M.A. McCall, Selective glycine receptor alpha2 subunit control of crossover inhibition between the on and off retinal pathways, J. Neurosci. 32 (2012) 3321–3332. [47] L. Jiang, X. Qin, X. Zhong, L. Liu, L. Jiang, Y. Lu, L. Fan, Z. He, Q. Chen, Glycineinduced cytoprotection is mediated by ERK1/2 and AKT in renal cells with ATP depletion, Eur. J. Cell Biol. 90 (2011) 333–341. [48] Y. Lu, J. Zhang, B. Ma, K. Li, X. Li, H. Bai, Q. Yang, X. Zhu, J. Ben, Q. Chen, Glycine attenuates cerebral ischemia/reperfusion injury by inhibiting neuronal apoptosis in mice, Neurochem. Int. 61 (2012) 649–658. [49] B. Sanna, O.F. Bueno, Y.S. Dai, B.J. Wilkins, J.D. Molkentin, Direct and indirect interactions between calcineurin-NFAT and MEK1-extracellular signalregulated kinase 1/2 signaling pathways regulate cardiac gene expression and cellular growth, Mol. Cell. Biol. 25 (2005) 865–878. [50] J. Heineke, J.D. Molkentin, Regulation of cardiac hypertrophy by intracellular signalling pathways, Nat. Rev. Mol. Cell Biol. 7 (2006) 589–600.
Y. Lu et al. / Biochemical Pharmacology 123 (2017) 40–51 [51] J.E. Cartledge, C. Kane, P. Dias, M. Tesfom, L. Clarke, B. McKee, S. Al Ayoubi, A. Chester, M.H. Yacoub, P. Camelliti, C.M. Terracciano, Functional crosstalk between cardiac fibroblasts and adult cardiomyocytes by soluble mediators, Cardiovasc. Res. 105 (2015) 260–270. [52] K.E. Porter, N.A. Turner, Cardiac fibroblasts: at the heart of myocardial remodeling, Pharmacol. Ther. 123 (2009) 255–278. [53] B.C. Berk, K. Fujiwara, S. Lehoux, ECM remodeling in hypertensive heart disease, J. Clin. Invest. 117 (2007) 568–575.
51
[54] M.D. Wheeler, R.G. Thurman, Production of superoxide and TNF-alpha from alveolar macrophages is blunted by glycine, Am. J. Physiol. 277 (1999) L952– L959. [55] S. Yamashina, A. Konno, M.D. Wheeler, I. Rusyn, E.V. Rusyn, A.D. Cox, R.G. Thurman, Endothelial cells contain a glycine-gated chloride channel, Nutr. Cancer 40 (2001) 197–204.