Original Research Received: January 14, 2014 Accepted after revision: May 22, 2014 Published online: August 19, 2014
Cardiology 2014;129:75–83 DOI: 10.1159/000364779
Effects of Advanced Glycation End Products on Calcium Handling in Cardiomyocytes Dewen Yan a Xinping Luo b Yali Li c Wenjuan Liu b Jianxin Deng a, d Na Zheng b Kaiping Gao b Qiaobing Huang d Jie Liu b
a Department of Endocrinology, The First Affiliated Hospital of Shenzhen University, Shenzhen, b Department of Pathophysiology, School of Medicine, Shenzhen University, Shenzhen, c Department of Anesthesiology, Shenzhen People’s Hospital, 2nd Clinical Medical College of Jinan University, Shenzhen, and d Department of Pathophysiology, Southern Medical School, Guangzhou, China
Abstract Background and Aims: Advanced glycation end products (AGEs) accumulate in diabetes and the engagement of receptor for AGE (RAGE) by AGEs contributes to the pathogenesis of diabetic cardiomyopathy. This study aims to investigate the effects of AGE/RAGE on ryanodine receptor (RyR) activity and Ca2+ handling in cardiomyocytes to elucidate the possible mechanism underlying cardiac dysfunction in diabetic cardiomypathy. Methods and Results: Confocal imaging Ca2+ spark, the elementary Ca2+ release event reflecting RyR activity in intact cell, as well as SR Ca2+ content and systolic Ca2+ transient were performed in cultured neonatal rat ventricular myocytes. The results show that 50 mg/ml AGE increased the frequency of Ca2+ sparks by 160%, while 150 mg/ml AGE increased it by 53%. AGE decreased the amplitude, width and duration of Ca2+ sparks. Blocking RAGE with anti-RAGE IgG completely abolished the alteration of Ca2+ sparks. The SR Ca2+ content indicated by the amplitude (ΔF/F0) of 20 mM caffeine-elicited Ca2+ transient was significantly decreased by 150 mg/ml AGE. In parallel, the amplitude of systolic Ca2+ transient evoked by 1 Hz-field stimula-
© 2014 S. Karger AG, Basel 0008–6312/14/1292–0075$39.50/0 E-Mail [email protected] www.karger.com/crd
tion was remarkably decreased by 150 mg/ml AGE. The antiRAGE antibody completely restored the impaired SR load and systolic Ca2+ transient. Conclusion: AGE/RAGE signal enhanced Ca2+ spark-mediated SR Ca2+ leak, causing partial depletion of SR Ca2+ content and consequently decreasing systolic Ca2+ transient, which may contribute to contractile dysfunction in diabetic cardiomyopathy. © 2014 S. Karger AG, Basel
Introduction
Advanced glycation end products (AGE), the products of nonenzymatic glycation and oxidation of proteins and lipids, accumulate in diverse biological settings, such as diabetes, inflammation, renal failure and aging. The receptor for AGE (RAGE) is the best characterized cell surface molecule that recognizes AGE and has been shown to be expressed in various organs including the heart [1, 2]. The engagement of RAGE by AGE in a variety of settings triggers rapid generation of reactive oxygen species (ROS) and the upregulation of inflammatory pathways [3]. Recent studies indicate that accumulation of AGE in heart tissue and AGE/RAGE
D.Y., X.L. and Y.L. contributed equally to this work.
Jie Liu, MD, PhD School of Medicine Shenzhen University Shenzhen, Guangdong 518060 (China) E-Mail ljljz @ yahoo.com
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Key Words Advanced glycation end products · Cardiac myocyte · Calcium spark · Excitation-contraction coupling · Calcium transient
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Methods Culture of Neonatal Rat Ventricular Myocytes and Treatment Protocol Sprague-Dawley rats (2 days old) were purchased from the Animal Center of Southern Medical University and handled according to a protocol approved by the Institutional Care and Use Committee of Shenzhen University that conforms 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). Neonatal rat ventricular myocytes (NRVMs) were isolated and cultured as previously described [16]. After culture for 24 h, the cells were starved for 2 h before being stimulated with 50 or 150 μg/ml AGE-modified bovine serum albumin (AGE-BSA) for 8 h. The choice of effective concentrations of AGE-BSA was based on previous studies [12, 17]. In the case of anti-RAGE antibody treatment, NRVMs were pretreated with 50 or 150 μg/ml anti-RAGE IgG for 1 h then cultured in fresh complete medium with 50 or 150 μg/ml AGEBSA for 8 h before the subsequent experiments. Monoclonal antibodies against the different epitopes of RAGE (anti-RAGE IgG) were prepared and characterized as previously described [17]. Preparation of AGE-BSA AGE-BSA was prepared according to the protocol of Hou et al. [18, 19]. Briefly, BSA (150 mmol/l, pH 7.4) was incubated in PBS with D-glucose (250 mmol/l) at 37 ° C for 8 weeks. Control albumin was incubated without glucose. At the end of the incubation period, both solutions were extensively dialyzed against PBS and purified. The endotoxin content was detected by a Limulus amebocyte lysate assay (Sigma) and was found to be 0.05; fig. 3a, b). In contrast, 150 μg/ml AGE significantly decreased ΔF/F0 (1.28 ± 0.10, n = 67, p < 0.01 vs. control; fig. 3a, b). The rise time of the Ca2+ transient indicated by the time to peak was prolonged by 150 μg/ml AGE (fig. 3c), suggesting that the synchrony of SR Ca2+ release was impaired. Neither 50 nor 150 μg/ml AGE had any significant effect on the half time of decay of the Ca2+ transient (fig. 3c), suggesting that SR Ca2+ recycling was uncompromised. Blocking RAGE Abolished AGE-Induced Alterations in Ca2+ Sparks We next explored whether AGE regulated the occurrence of Ca2+ sparks via RAGE. For this purpose, antiRAGE IgG (50 or 150 μg/ml for AGE50 or AGE150) was applied to NRVMs 1 h before AGE addition to block binding of AGE to cell surface RAGE. The results show that anti-RAGE IgG (Ab) completely abolished the increase in spark frequency stimulated by 50 or 150 μg/ml AGE (n = 169 and 80 in the AGE50 + Ab and AGE150 + Ab groups, respectively; fig. 4a). Furthermore, AGE-induced alterations of the amplitude, FDHM and FWHW of Ca2+ sparks were completely reversed by the treatment with anti-RAGE antibody (fig. 4b–d). Yan/Luo/Li/Liu/Deng/Zheng/Gao/ Huang/Liu
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Fig. 2. AGE decreased SR Ca2+ content in cardiomyocytes. a Representative time
courses of caffeine-elicited Ca transients in control and AGE-stimulated cells. b Average of the SR Ca2+ content indicated by the amplitude of the caffeine-elicited Ca2+ transient (ΔF/F0) in the control, AGE50 and AGE150 groups. c Resting calcium levels (F0) in control and AGE-stimulated cells. * p < 0.05, ** p < 0.01 versus control.
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Fig. 3. AGE decreased the systolic Ca2+ transient in cardiomyocytes. a Typical confocal line-scan images of Ca2+ transients (upper panels)
along with the spatial average (lower panels) in control and AGEstimulated cells. The Ca2+ transient was elicited by 1-Hz field stimulation. b–d Average of the amplitude (ΔF/F0; b), time to peak (TTP; c) and half time of decay (T50; d) of the Ca2+ transient in the control, AGE50 and AGE150 groups. * p < 0.05, ** p < 0.01 versus control.
Fig. 4. Effects of blocking RAGE on AGE-mediated modulation of Ca2+ sparks. Anti-RAGE IgG (Ab) was added 1 h before AGE was applied. a–d Statistical analysis of the frequency (a), amplitude (F/ F0; b), FDHM (c) and FWHM (d) in the control, AGE50, AGE50 + Ab (50 g/ml anti-RAGE IgG), AGE150 and AGE150 + Ab (150 μg/ ml anti-RAGE IgG) groups. The values in each group were normalized by the value in the control. * p < 0.05, ** p < 0.01.
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Fig. 5. Blocking RAGE restored SR Ca2+ content and the systolic Ca2+ transient reduced by AGE. a–c Statistics of the amplitude of the caffeine-elicited Ca2+ transient (a) and the amplitude (b) and time to peak (TTP; c) of the systolic Ca2+ transient normalized by
control values in the control, AGE50, AGE50 + Ab, AGE150 and AGE150 + Ab groups. * p < 0.05, ** p < 0.01 versus control; ## p < 0.01 versus AGE150.
Blocking RAGE Restored AGE-Induced Reduction of SR Ca2+ Content and the Systolic Ca2+ Transient The role of RAGE in the AGE-induced reduction of SR load and the systolic Ca2+ transient was investigated by blocking RAGE with anti-RAGE IgG. Figure 5a shows that anti-RAGE IgG (Ab) completely restored SR Ca2+ content, which was partially depleted by AGE (n = 13 and 6 in the AGE50 + Ab and AGE150 + Ab groups, respectively; fig. 5a). Meanwhile, the alterations in the amplitude and rise time (time to peak) of the systolic Ca2+ transient were completely abolished by anti-RAGE antibody (n = 19 and 16 in the AGE50 + Ab and AGE150 + Ab groups, respectively; fig. 5b, c). 80
Cardiology 2014;129:75–83 DOI: 10.1159/000364779
AGE Increased Intracellular ROS Production and RyR2 Oxidation It has been suggested that oxidative modification of RyR2 increases the channel activity. We thus investigated whether AGE increased intracellular ROS levels and enhanced oxidative stress in RyR2. The results show that AGE (50 μg/ml) significantly increased intracellular ROS levels, indexed by the intensity of DCFDA fluorescence (fig. 6a, b), and the oxidative level of RyR2, indexed by the intensity of mBB fluorescence (fig. 6c–e). Furthermore, the antioxidant Manganese (III) 5,10,15,20-tetrakis (4-benzoic acid) porphyrin (MnTBAP) (100 μmol/l) largely inhibited the effects of AGE on intracellular ROS production and RyR2 oxidation (fig. 6). The data collectively indicate that AGE increased intracellular ROS production and RyR2 oxidation. The Antioxidant MnTBAP Inhibited the AGE-Induced Increase in Ca2+ Spark Frequency We next examined the effect of MnTBAP on AGEinduced high-frequency Ca2+ sparks. As shown in figure 7, MnTBAP significantly decreased the frequency of Ca2+ sparks, which was increased by AGE. Moreover, AGEinduced alterations in the amplitude, FDHM and FWHM of Ca2+ sparks were largely corrected by MnTBAP treatment. Therefore, AGE increased RyR activity through oxidation of RyR2.
Discussion
In this study, we found that the AGE/RAGE signal reduced the systolic Ca2+ transient, which is in agreement with a previous study [12]. Importantly, we revealed that AGE reduced SR Ca2+ content through interaction with RAGE, which accounts, at least partially, for the decrease in the systolic Ca2+ transient. Furthermore, AGE/RAGE interaction increased RyR activity, and the enhancement of the Ca2+ spark-mediated SR leak contributes to the reduction of SR Ca2+ content. Two sources of Ca2+ contribute to the systolic Ca2+ transient, namely Ca2+ influx through L-type Ca2+ channels and SR Ca2+ release through RyRs. In rodent heart, the former contributes to 10–30% while the latter contributes to more than 70% of the systolic Ca2+ transient. In this study, we showed that AGE at 150 μg/ml significantly decreased SR Ca2+ content, which was in accordance with the alteration of the magnitude of the systolic Ca2+ transient. Furthermore, we found that blocking RAGE with anti-RAGE IgG completely restored SR load Yan/Luo/Li/Liu/Deng/Zheng/Gao/ Huang/Liu
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Fig. 6. AGE increased intracellular ROS production and oxidative stress in RyR2 in cardiomyocytes. a Representative images of ROSsensitive indicator DCFDA-loaded cells in the control, AGE50, MnTBAP and AGE50 + MnTBAP groups. b Averages of DCFDA fluorescence in different groups. c Representative images of mBB
fluorescence intensity and Coomassie blue-stained gels in parallel. d, e Statistics of RyR2 content (d) and relative free thiol content of RyR2 (e) measured by normalizing mBB fluorescence to the RyR2 level. ** p < 0.01 versus control; ## p < 0.01 versus AGE50; † p < 0.05 versus MnTBAP. OD = Optical density.
and consequently increased the systolic Ca2+ transient. Therefore, the study revealed for the first time that reduction in SR Ca2+ content is a major reason for the AGE/ RAGE-induced decrease in the systolic Ca2+ transient. SR Ca2+ content is finely tuned by SR Ca2+ release and SR Ca2+ recycling [13]. A previous study demonstrated that 200 μg/ml AGE impaired SR Ca2+ recycling [12]. In this study, we found that neither 50 nor 150 μg/ml AGE had any effect on SR Ca2+ recycling, whereby the half time of decay of the systolic Ca2+ transient remained unaltered. The difference between this and the previous study may be due to the differences in AGE concentration and experimental conditions. Evidence from this study suggests that reduction of SR Ca2+ content in our experimental condition is unrelated to impairment of SR Ca2+ recycling. It has been shown that spontaneous Ca2+ sparks
mediate diastolic SR Ca2+ release, which is known to play an important role in setting SR Ca2+ content in resting states [21]. Enhanced diastolic SR Ca2+ leak is a potential cause of reduction of SR Ca2+ content [8]. The causal relationship between enhancement of SR Ca2+ leak and reduction of SR load has been demonstrated in cardiac hypertrophy and heart failure [20, 22, 23], as well as in burn trauma-induced cardiac dysfunction [13]. In this study, we found that 50 μg/ml AGE remarkably increased the frequency of Ca2+ sparks by 160%, while 150 μg/ml AGE only caused a 53% increase. In contrast, 50 μg/ml AGE caused a slight decrease in SR Ca2+ content by 17%, whereas 150 μg/ml AGE significantly decreased SR Ca2+ content by 27%. A similar phenomenon was observed in our previous study [24], in which burn serum (BS; increasing RyR activity) stimulation for 20 min in cardio-
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However, the resulting enhancement of SR leak caused a more severe depletion of SR Ca2+ content. The decrease in SR Ca2+ content inhibited the occurrence of Ca2+ sparks. Therefore, the frequency of Ca2+ sparks in the AGE50 group was higher than that in the AGE150 group. The dose-dependent decrease in the amplitude of Ca2+ sparks in response to AGE stimulation also implicates the inhibition of reduced SR load in the formation of Ca2+ sparks. Taking these results together, we propose that a hyperactive RyR-mediated SR leak underlies SR Ca2+ depletion in response to AGE stimulation. Furthermore, AGE-induced hyperactive RyRs were related to intracellular ROS overproduction and enhancement of oxidative stress in RyRs. The hyperactive RyR-mediated SR leak could be prevented by antioxidants. Our present study demonstrated for the first time that AGE/RAGE interaction impaired intracellular Ca2+ handling by enhancing SR Ca2+ leak, which is an important mechanism for AGE/RAGE-induced cardiac dysfunction. Although previous studies have demonstrated that the Yan/Luo/Li/Liu/Deng/Zheng/Gao/ Huang/Liu
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Cardiology 2014;129:75–83 DOI: 10.1159/000364779
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myocytes caused a dramatic increase in Ca2+ spark frequency while SR Ca2+ content remained normal, whereas Ca2+ spark frequency was significantly decreased (still higher than normal) and SR Ca2+ content was remarkably reduced 60 min after BS stimulation. The contradictory alterations in Ca2+ sparks and SR load were attributed to hyperactive RyRs; a short-lived SR leak due to hyperactive RyRs had no significant effect on SR Ca2+ content. With the prolongation of BS stimulation, a hyperactive RyRmediated SR Ca2+ leak gradually depleted SR Ca2+ content. The decrease in SR Ca2+ content reduced the occurrence of Ca2+ sparks and consequently prevented further depletion of SR load. A balance between SR Ca2+ content and the occurrence of Ca2+ sparks was finally reached under reduced SR load. As in the case of AGE stimulation, high-frequency Ca2+ sparks occurred without an increase in SR Ca2+ content, indicating that RyR activity was increased. AGE at 50 μg/ml increased Ca2+ spark frequency but caused only a slight depletion of SR Ca2+ content. AGE at 150 μg/ml should increase RyR activity strongly. 82
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Fig. 7. The antioxidant MnTBAP inhibited the occurrence of high-frequency Ca2+ sparks induced by AGE. a Typical Ca2+ spark images in the control, AGE50, MnTBAP and AGE50 + MnTBAP groups. b–e Statistical analysis of the frequency (b), amplitude (F/F0; c), FDHM (d) and FWHM (e) of Ca2+ sparks in the four groups. ** p < 0.01 versus control; # p < 0.05, ## p < 0.01 versus AGE50; † p < 0.05 versus MnTBAP.
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AGE/RAGE signal contributes to cardiac dysfunction in diabetic cardiomyopathy, we should be cautious to translate our present findings into the pathogenesis of diabetic cardiomyopathy because our data were obtained in cultured cardiomyocytes rather than diabetic animal models. In summary, this study demonstrated for the first time that AGE/RAGE interaction increased Ca2+ spark frequency due to increasing RyR activity in cardiomyocytes. The enhanced Ca2+ spark-mediated SR Ca2+ leak partially depleted SR Ca2+ content, resulting in a decrease in the
systolic Ca2+ transient. The findings suggest the potential therapeutic effect of preventing SR Ca2+ leak on the treatment of diabetic cardiac dysfunction. Acknowledgments This work was supported by the National Science Foundation of China (No. 31171096, 30973122 and 81200122) and the Basic Research Foundation of Shenzhen (No. JC201005250059A, JCYJ20120613115535998 and 20130326112207234).
1 Neeper M, Schmidt AM, Brett J, Yan SD, Wang F, Pan YC, Elliston K, Stern D, Shaw A: Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins. J Biol Chem 1992;267:14998– 15004. 2 Schmidt AM, Vianna M, Gerlach M, Brett J, Ryan J, Kao J, Esposito C, Hegarty H, Hurley W, Clauss M, et al: Isolation and characterization of two binding proteins for advanced glycosylation end products from bovine lung which are present on the endothelial cell surface. J Biol Chem 1992;267:14987–14997. 3 Ramasamy R, Vannucci SJ, Yan SS, Herold K, Yan SF, Schmidt AM: Advanced glycation end products and RAGE: a common thread in aging, diabetes, neurodegeneration, and inflammation. Glycobiology 2005;15:16R–28R. 4 Ma H, Li SY, Xu P, Babcock SA, Dolence EK, Brownlee M, Li J, Ren J: Advanced glycation endproduct (AGE) accumulation and AGE receptor (RAGE) up-regulation contribute to the onset of diabetic cardiomyopathy. J Cell Mol Med 2009;13:1751–1764. 5 Nielsen JM, Kristiansen SB, Norregaard R, Andersen CL, Denner L, Nielsen TT, Flyvbjerg A, Botker HE: Blockage of receptor for advanced glycation end products prevents development of cardiac dysfunction in db/db type 2 diabetic mice. Eur J Heart Fail 2009;11: 638–647. 6 Bers DM: Cardiac excitation-contraction coupling. Nature 2002;415:198–205. 7 Fauconnier J, Meli AC, Thireau J, Roberge S, Shan J, Sassi Y, Reiken SR, Rauzier JM, Marchand A, Chauvier D, Cassan C, Crozier C, Bideaux P, Lompre AM, Jacotot E, Marks AR, Lacampagne A: Ryanodine receptor leak mediated by caspase-8 activation leads to left ventricular injury after myocardial ischemiareperfusion. Proc Natl Acad Sci USA 2011; 108:13258–13263. 8 Jiang X, Liu W, Deng J, Lan L, Xue X, Zhang C, Cai G, Luo X, Liu J: Polydatin protects cardiac function against burn injury by inhibiting sarcoplasmic reticulum Ca2+ leak by reducing oxidative modification of ryanodine receptors. Free Radic Biol Med 2013;60:292–299.
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9 Zima AV, Bovo E, Bers DM, Blatter LA: Ca2+ spark-dependent and -independent sarcoplasmic reticulum Ca2+ leak in normal and failing rabbit ventricular myocytes. J Physiol 2010;588:4743–4757. 10 Lyon AR, Bannister ML, Collins T, Pearce E, Sepehripour AH, Dubb SS, Garcia E, O’Gara P, Liang L, Kohlbrenner E, Hajjar RJ, Peters NS, Poole-Wilson PA, Macleod KT, Harding SE: SERCA2a gene transfer decreases sarcoplasmic reticulum calcium leak and reduces ventricular arrhythmias in a model of chronic heart failure. Circ Arrhythm Electrophysiol 2011;4:362–372. 11 Belke DD, Swanson EA, Dillmann WH: Decreased sarcoplasmic reticulum activity and contractility in diabetic db/db mouse heart. Diabetes 2004;53:3201–3208. 12 Petrova R, Yamamoto Y, Muraki K, Yonekura H, Sakurai S, Watanabe T, Li H, Takeuchi M, Makita Z, Kato I, Takasawa S, Okamoto H, Imaizumi Y, Yamamoto H: Advanced glycation endproduct-induced calcium handling impairment in mouse cardiac myocytes. J Mol Cell Cardiol 2002;34:1425–1431. 13 Deng J, Wang G, Huang Q, Yan Y, Li K, Tan W, Jin C, Wang Y, Liu J: Oxidative stress-induced leaky sarcoplasmic reticulum underlying acute heart failure in severe burn trauma. Free Radic Biol Med 2008;44:375–385. 14 Yano M, Okuda S, Oda T, Tokuhisa T, Tateishi H, Mochizuki M, Noma T, Doi M, Kobayashi S, Yamamoto T, Ikeda Y, Ohkusa T, Ikemoto N, Matsuzaki M: Correction of defective interdomain interaction within ryanodine receptor by antioxidant is a new therapeutic strategy against heart failure. Circulation 2005;112:3633–3643. 15 Hidalgo C, Donoso P, Carrasco MA: The ryanodine receptors Ca2+ release channels: cellular redox sensors? IUBMB Life 2005; 57: 315–322. 16 Liu W, Deng J, Xu J, Wang H, Yuan M, Liu N, Jiang Y, Liu J: High-mobility group box 1 (HMGB1) downregulates cardiac transient outward potassium current (Ito) through downregulation of Kv4.2 and Kv4.3 channel transcripts and proteins. J Mol Cell Cardiol 2010;49:438–448.
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References
Cardiology 2014;129:75–83 DOI: 10.1159/000364779
Effects of Advanced Glycation End Products on Calcium Handling in Cardiomyocytes Dewen Yan a Xinping Luo b Yali Li c Wenjuan Liu b Jianxin Deng a, d Na Zheng b Kaiping Gao b Qiaobing Huang d Jie Liu b
a Department of Endocrinology, The First Affiliated Hospital of Shenzhen University, Shenzhen, b Department of Pathophysiology, School of Medicine, Shenzhen University, Shenzhen, c Department of Anesthesiology, Shenzhen People’s Hospital, 2nd Clinical Medical College of Jinan University, Shenzhen, and d Department of Pathophysiology, Southern Medical School, Guangzhou, China
Abstract Background and Aims: Advanced glycation end products (AGEs) accumulate in diabetes and the engagement of receptor for AGE (RAGE) by AGEs contributes to the pathogenesis of diabetic cardiomyopathy. This study aims to investigate the effects of AGE/RAGE on ryanodine receptor (RyR) activity and Ca2+ handling in cardiomyocytes to elucidate the possible mechanism underlying cardiac dysfunction in diabetic cardiomypathy. Methods and Results: Confocal imaging Ca2+ spark, the elementary Ca2+ release event reflecting RyR activity in intact cell, as well as SR Ca2+ content and systolic Ca2+ transient were performed in cultured neonatal rat ventricular myocytes. The results show that 50 mg/ml AGE increased the frequency of Ca2+ sparks by 160%, while 150 mg/ml AGE increased it by 53%. AGE decreased the amplitude, width and duration of Ca2+ sparks. Blocking RAGE with anti-RAGE IgG completely abolished the alteration of Ca2+ sparks. The SR Ca2+ content indicated by the amplitude (ΔF/F0) of 20 mM caffeine-elicited Ca2+ transient was significantly decreased by 150 mg/ml AGE. In parallel, the amplitude of systolic Ca2+ transient evoked by 1 Hz-field stimula-
© 2014 S. Karger AG, Basel 0008–6312/14/1292–0075$39.50/0 E-Mail [email protected] www.karger.com/crd
tion was remarkably decreased by 150 mg/ml AGE. The antiRAGE antibody completely restored the impaired SR load and systolic Ca2+ transient. Conclusion: AGE/RAGE signal enhanced Ca2+ spark-mediated SR Ca2+ leak, causing partial depletion of SR Ca2+ content and consequently decreasing systolic Ca2+ transient, which may contribute to contractile dysfunction in diabetic cardiomyopathy. © 2014 S. Karger AG, Basel
Introduction
Advanced glycation end products (AGE), the products of nonenzymatic glycation and oxidation of proteins and lipids, accumulate in diverse biological settings, such as diabetes, inflammation, renal failure and aging. The receptor for AGE (RAGE) is the best characterized cell surface molecule that recognizes AGE and has been shown to be expressed in various organs including the heart [1, 2]. The engagement of RAGE by AGE in a variety of settings triggers rapid generation of reactive oxygen species (ROS) and the upregulation of inflammatory pathways [3]. Recent studies indicate that accumulation of AGE in heart tissue and AGE/RAGE
D.Y., X.L. and Y.L. contributed equally to this work.
Jie Liu, MD, PhD School of Medicine Shenzhen University Shenzhen, Guangdong 518060 (China) E-Mail ljljz @ yahoo.com
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Key Words Advanced glycation end products · Cardiac myocyte · Calcium spark · Excitation-contraction coupling · Calcium transient
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Methods Culture of Neonatal Rat Ventricular Myocytes and Treatment Protocol Sprague-Dawley rats (2 days old) were purchased from the Animal Center of Southern Medical University and handled according to a protocol approved by the Institutional Care and Use Committee of Shenzhen University that conforms 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). Neonatal rat ventricular myocytes (NRVMs) were isolated and cultured as previously described [16]. After culture for 24 h, the cells were starved for 2 h before being stimulated with 50 or 150 μg/ml AGE-modified bovine serum albumin (AGE-BSA) for 8 h. The choice of effective concentrations of AGE-BSA was based on previous studies [12, 17]. In the case of anti-RAGE antibody treatment, NRVMs were pretreated with 50 or 150 μg/ml anti-RAGE IgG for 1 h then cultured in fresh complete medium with 50 or 150 μg/ml AGEBSA for 8 h before the subsequent experiments. Monoclonal antibodies against the different epitopes of RAGE (anti-RAGE IgG) were prepared and characterized as previously described [17]. Preparation of AGE-BSA AGE-BSA was prepared according to the protocol of Hou et al. [18, 19]. Briefly, BSA (150 mmol/l, pH 7.4) was incubated in PBS with D-glucose (250 mmol/l) at 37 ° C for 8 weeks. Control albumin was incubated without glucose. At the end of the incubation period, both solutions were extensively dialyzed against PBS and purified. The endotoxin content was detected by a Limulus amebocyte lysate assay (Sigma) and was found to be 0.05; fig. 3a, b). In contrast, 150 μg/ml AGE significantly decreased ΔF/F0 (1.28 ± 0.10, n = 67, p < 0.01 vs. control; fig. 3a, b). The rise time of the Ca2+ transient indicated by the time to peak was prolonged by 150 μg/ml AGE (fig. 3c), suggesting that the synchrony of SR Ca2+ release was impaired. Neither 50 nor 150 μg/ml AGE had any significant effect on the half time of decay of the Ca2+ transient (fig. 3c), suggesting that SR Ca2+ recycling was uncompromised. Blocking RAGE Abolished AGE-Induced Alterations in Ca2+ Sparks We next explored whether AGE regulated the occurrence of Ca2+ sparks via RAGE. For this purpose, antiRAGE IgG (50 or 150 μg/ml for AGE50 or AGE150) was applied to NRVMs 1 h before AGE addition to block binding of AGE to cell surface RAGE. The results show that anti-RAGE IgG (Ab) completely abolished the increase in spark frequency stimulated by 50 or 150 μg/ml AGE (n = 169 and 80 in the AGE50 + Ab and AGE150 + Ab groups, respectively; fig. 4a). Furthermore, AGE-induced alterations of the amplitude, FDHM and FWHW of Ca2+ sparks were completely reversed by the treatment with anti-RAGE antibody (fig. 4b–d). Yan/Luo/Li/Liu/Deng/Zheng/Gao/ Huang/Liu
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Fig. 2. AGE decreased SR Ca2+ content in cardiomyocytes. a Representative time
courses of caffeine-elicited Ca transients in control and AGE-stimulated cells. b Average of the SR Ca2+ content indicated by the amplitude of the caffeine-elicited Ca2+ transient (ΔF/F0) in the control, AGE50 and AGE150 groups. c Resting calcium levels (F0) in control and AGE-stimulated cells. * p < 0.05, ** p < 0.01 versus control.
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Fig. 3. AGE decreased the systolic Ca2+ transient in cardiomyocytes. a Typical confocal line-scan images of Ca2+ transients (upper panels)
along with the spatial average (lower panels) in control and AGEstimulated cells. The Ca2+ transient was elicited by 1-Hz field stimulation. b–d Average of the amplitude (ΔF/F0; b), time to peak (TTP; c) and half time of decay (T50; d) of the Ca2+ transient in the control, AGE50 and AGE150 groups. * p < 0.05, ** p < 0.01 versus control.
Fig. 4. Effects of blocking RAGE on AGE-mediated modulation of Ca2+ sparks. Anti-RAGE IgG (Ab) was added 1 h before AGE was applied. a–d Statistical analysis of the frequency (a), amplitude (F/ F0; b), FDHM (c) and FWHM (d) in the control, AGE50, AGE50 + Ab (50 g/ml anti-RAGE IgG), AGE150 and AGE150 + Ab (150 μg/ ml anti-RAGE IgG) groups. The values in each group were normalized by the value in the control. * p < 0.05, ** p < 0.01.
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Fig. 5. Blocking RAGE restored SR Ca2+ content and the systolic Ca2+ transient reduced by AGE. a–c Statistics of the amplitude of the caffeine-elicited Ca2+ transient (a) and the amplitude (b) and time to peak (TTP; c) of the systolic Ca2+ transient normalized by
control values in the control, AGE50, AGE50 + Ab, AGE150 and AGE150 + Ab groups. * p < 0.05, ** p < 0.01 versus control; ## p < 0.01 versus AGE150.
Blocking RAGE Restored AGE-Induced Reduction of SR Ca2+ Content and the Systolic Ca2+ Transient The role of RAGE in the AGE-induced reduction of SR load and the systolic Ca2+ transient was investigated by blocking RAGE with anti-RAGE IgG. Figure 5a shows that anti-RAGE IgG (Ab) completely restored SR Ca2+ content, which was partially depleted by AGE (n = 13 and 6 in the AGE50 + Ab and AGE150 + Ab groups, respectively; fig. 5a). Meanwhile, the alterations in the amplitude and rise time (time to peak) of the systolic Ca2+ transient were completely abolished by anti-RAGE antibody (n = 19 and 16 in the AGE50 + Ab and AGE150 + Ab groups, respectively; fig. 5b, c). 80
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AGE Increased Intracellular ROS Production and RyR2 Oxidation It has been suggested that oxidative modification of RyR2 increases the channel activity. We thus investigated whether AGE increased intracellular ROS levels and enhanced oxidative stress in RyR2. The results show that AGE (50 μg/ml) significantly increased intracellular ROS levels, indexed by the intensity of DCFDA fluorescence (fig. 6a, b), and the oxidative level of RyR2, indexed by the intensity of mBB fluorescence (fig. 6c–e). Furthermore, the antioxidant Manganese (III) 5,10,15,20-tetrakis (4-benzoic acid) porphyrin (MnTBAP) (100 μmol/l) largely inhibited the effects of AGE on intracellular ROS production and RyR2 oxidation (fig. 6). The data collectively indicate that AGE increased intracellular ROS production and RyR2 oxidation. The Antioxidant MnTBAP Inhibited the AGE-Induced Increase in Ca2+ Spark Frequency We next examined the effect of MnTBAP on AGEinduced high-frequency Ca2+ sparks. As shown in figure 7, MnTBAP significantly decreased the frequency of Ca2+ sparks, which was increased by AGE. Moreover, AGEinduced alterations in the amplitude, FDHM and FWHM of Ca2+ sparks were largely corrected by MnTBAP treatment. Therefore, AGE increased RyR activity through oxidation of RyR2.
Discussion
In this study, we found that the AGE/RAGE signal reduced the systolic Ca2+ transient, which is in agreement with a previous study [12]. Importantly, we revealed that AGE reduced SR Ca2+ content through interaction with RAGE, which accounts, at least partially, for the decrease in the systolic Ca2+ transient. Furthermore, AGE/RAGE interaction increased RyR activity, and the enhancement of the Ca2+ spark-mediated SR leak contributes to the reduction of SR Ca2+ content. Two sources of Ca2+ contribute to the systolic Ca2+ transient, namely Ca2+ influx through L-type Ca2+ channels and SR Ca2+ release through RyRs. In rodent heart, the former contributes to 10–30% while the latter contributes to more than 70% of the systolic Ca2+ transient. In this study, we showed that AGE at 150 μg/ml significantly decreased SR Ca2+ content, which was in accordance with the alteration of the magnitude of the systolic Ca2+ transient. Furthermore, we found that blocking RAGE with anti-RAGE IgG completely restored SR load Yan/Luo/Li/Liu/Deng/Zheng/Gao/ Huang/Liu
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Fig. 6. AGE increased intracellular ROS production and oxidative stress in RyR2 in cardiomyocytes. a Representative images of ROSsensitive indicator DCFDA-loaded cells in the control, AGE50, MnTBAP and AGE50 + MnTBAP groups. b Averages of DCFDA fluorescence in different groups. c Representative images of mBB
fluorescence intensity and Coomassie blue-stained gels in parallel. d, e Statistics of RyR2 content (d) and relative free thiol content of RyR2 (e) measured by normalizing mBB fluorescence to the RyR2 level. ** p < 0.01 versus control; ## p < 0.01 versus AGE50; † p < 0.05 versus MnTBAP. OD = Optical density.
and consequently increased the systolic Ca2+ transient. Therefore, the study revealed for the first time that reduction in SR Ca2+ content is a major reason for the AGE/ RAGE-induced decrease in the systolic Ca2+ transient. SR Ca2+ content is finely tuned by SR Ca2+ release and SR Ca2+ recycling [13]. A previous study demonstrated that 200 μg/ml AGE impaired SR Ca2+ recycling [12]. In this study, we found that neither 50 nor 150 μg/ml AGE had any effect on SR Ca2+ recycling, whereby the half time of decay of the systolic Ca2+ transient remained unaltered. The difference between this and the previous study may be due to the differences in AGE concentration and experimental conditions. Evidence from this study suggests that reduction of SR Ca2+ content in our experimental condition is unrelated to impairment of SR Ca2+ recycling. It has been shown that spontaneous Ca2+ sparks
mediate diastolic SR Ca2+ release, which is known to play an important role in setting SR Ca2+ content in resting states [21]. Enhanced diastolic SR Ca2+ leak is a potential cause of reduction of SR Ca2+ content [8]. The causal relationship between enhancement of SR Ca2+ leak and reduction of SR load has been demonstrated in cardiac hypertrophy and heart failure [20, 22, 23], as well as in burn trauma-induced cardiac dysfunction [13]. In this study, we found that 50 μg/ml AGE remarkably increased the frequency of Ca2+ sparks by 160%, while 150 μg/ml AGE only caused a 53% increase. In contrast, 50 μg/ml AGE caused a slight decrease in SR Ca2+ content by 17%, whereas 150 μg/ml AGE significantly decreased SR Ca2+ content by 27%. A similar phenomenon was observed in our previous study [24], in which burn serum (BS; increasing RyR activity) stimulation for 20 min in cardio-
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However, the resulting enhancement of SR leak caused a more severe depletion of SR Ca2+ content. The decrease in SR Ca2+ content inhibited the occurrence of Ca2+ sparks. Therefore, the frequency of Ca2+ sparks in the AGE50 group was higher than that in the AGE150 group. The dose-dependent decrease in the amplitude of Ca2+ sparks in response to AGE stimulation also implicates the inhibition of reduced SR load in the formation of Ca2+ sparks. Taking these results together, we propose that a hyperactive RyR-mediated SR leak underlies SR Ca2+ depletion in response to AGE stimulation. Furthermore, AGE-induced hyperactive RyRs were related to intracellular ROS overproduction and enhancement of oxidative stress in RyRs. The hyperactive RyR-mediated SR leak could be prevented by antioxidants. Our present study demonstrated for the first time that AGE/RAGE interaction impaired intracellular Ca2+ handling by enhancing SR Ca2+ leak, which is an important mechanism for AGE/RAGE-induced cardiac dysfunction. Although previous studies have demonstrated that the Yan/Luo/Li/Liu/Deng/Zheng/Gao/ Huang/Liu
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myocytes caused a dramatic increase in Ca2+ spark frequency while SR Ca2+ content remained normal, whereas Ca2+ spark frequency was significantly decreased (still higher than normal) and SR Ca2+ content was remarkably reduced 60 min after BS stimulation. The contradictory alterations in Ca2+ sparks and SR load were attributed to hyperactive RyRs; a short-lived SR leak due to hyperactive RyRs had no significant effect on SR Ca2+ content. With the prolongation of BS stimulation, a hyperactive RyRmediated SR Ca2+ leak gradually depleted SR Ca2+ content. The decrease in SR Ca2+ content reduced the occurrence of Ca2+ sparks and consequently prevented further depletion of SR load. A balance between SR Ca2+ content and the occurrence of Ca2+ sparks was finally reached under reduced SR load. As in the case of AGE stimulation, high-frequency Ca2+ sparks occurred without an increase in SR Ca2+ content, indicating that RyR activity was increased. AGE at 50 μg/ml increased Ca2+ spark frequency but caused only a slight depletion of SR Ca2+ content. AGE at 150 μg/ml should increase RyR activity strongly. 82
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Fig. 7. The antioxidant MnTBAP inhibited the occurrence of high-frequency Ca2+ sparks induced by AGE. a Typical Ca2+ spark images in the control, AGE50, MnTBAP and AGE50 + MnTBAP groups. b–e Statistical analysis of the frequency (b), amplitude (F/F0; c), FDHM (d) and FWHM (e) of Ca2+ sparks in the four groups. ** p < 0.01 versus control; # p < 0.05, ## p < 0.01 versus AGE50; † p < 0.05 versus MnTBAP.
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AGE/RAGE signal contributes to cardiac dysfunction in diabetic cardiomyopathy, we should be cautious to translate our present findings into the pathogenesis of diabetic cardiomyopathy because our data were obtained in cultured cardiomyocytes rather than diabetic animal models. In summary, this study demonstrated for the first time that AGE/RAGE interaction increased Ca2+ spark frequency due to increasing RyR activity in cardiomyocytes. The enhanced Ca2+ spark-mediated SR Ca2+ leak partially depleted SR Ca2+ content, resulting in a decrease in the
systolic Ca2+ transient. The findings suggest the potential therapeutic effect of preventing SR Ca2+ leak on the treatment of diabetic cardiac dysfunction. Acknowledgments This work was supported by the National Science Foundation of China (No. 31171096, 30973122 and 81200122) and the Basic Research Foundation of Shenzhen (No. JC201005250059A, JCYJ20120613115535998 and 20130326112207234).
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