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Dopamine D2 receptors contribute to cardioprotection of ischemic post-conditioning via activating autophagy in isolated rat hearts
International Journal of Cardiology 203 (2016) 837–839
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Correspondence
Dopamine D2 receptors contribute to cardioprotection of ischemic post-conditioning via activating autophagy in isolated rat hearts Can Wei a, Jun Gao b, Meixiu Li a, Hong Li a, Yuehong Wang a, Hongzhu Li a,⁎, Changqing Xu a,⁎ a b
Department of Pathophysiology, Harbin Medical University, Harbin 150081, China Department of Orthopeadic Surgery, The First Hospital of Harbin, Harbin 150010, China
a r t i c l e
i n f o
Article history: Received 1 September 2015 Accepted 3 November 2015 Available online 9 November 2015 Keywords: Dopamine D2 receptors Post-conditioning Autophagy Heart Rats
Dopamine receptors (DR) belong to the family of seven transmembrane domain G-protein coupled receptors (GPCR) and are classified into D1-like (DR1) and D2-like receptor families (DR2) based on pharmacological properties, structure, and signal transduction system [1–7]. The DR1 families include D1- and D5receptor subtypes, which are generally coupled to GS proteins and stimulate the cAMP productions and increase the PKA activity. The DR2 families consist of D2-, D3-, and D4-receptor subtypes, which are coupled to Gi/o proteins and decrease the cAMP productions, the PKA activity and the concentration of intracellular calcium, and regulate potassium channels [1–7]. DR exist in many tissues and organs including the brain, kidney, heart and the peripheral nervous system and play the important physiological and pathological roles [2,5]. Recent studies found that DR2 inhibited hypertrophy and I/R injury and is involved in PC-induced protective effects in the cardiovascular system [2–4,7]. As everyone knows, PC reduces myocardial I/R injury and apoptosis [2,4,9,10]. Our data demonstrated that PC decreased I/R injury by preventing cardiomyocyte damages and apoptosis, reducing myocardial infarct size, and improving cardiac function. Bromocriptine (Bro,
⁎ Corresponding authors at: Department of Pathophysiology, Harbin Medical University, Baojian Road, Harbin 150081, China. E-mail addresses: [email protected] (H. Li), [email protected] (C. Xu).
http://dx.doi.org/10.1016/j.ijcard.2015.11.006 0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.
DR2 agonist) further enhanced the effects of PC, but Haloperidol (Hal, DR2 antagonist) canceled the Bro-caused beneficial effects during PC [2,4]. These results suggest that DR2 activation is an important factor in cardioprotection of PC. Our previous our researches indicated that PC increased DR2 expressions and Bro further enhanced the expression of DR2 during PC [2,4]. Increased DR2 expressions or activation is involved in PCinduced cardioprotection via inhibition of mPTP opening via activation of the ERK1/2, PI3K–Akt–GSK-3β and PKC-ε–mKATP pathways [2,4]. However, the involvement of autophagy in the mediation of DR2 activation in cardioprotection by PC is unclear. In the present study, we used isolated rat hearts exposed to PC as an experimental model to demonstrate the effect of DR2 activation on the PC-induced autophagy and relative mechanisms. Moreover, we found that (1) autophagy plays an important role in PC against myocardial I/R and (2) DR2 activation contributes to cardioprotective effects of PC through up-regulation of autophagy via inhibiting phosphorylation of mTOR. Autophagy is a key factor in keeping the balance between synthesis, degradation, and recycling of cellular components [11]. It allows the cell not only to recycle amino acids but also to remove damaged organelles, thereby eliminating oxidative stress and allowing cellular remodeling for survival [11]. In the heart, a number of stress conditions, including cardiomyopathy, pressure overload, heart failure and I/R injury, increase autophagic activity [8]. Nuclear disassembly and AVs of degenerated cardiomyocytes are observed in human hibernating myocardium [8,12]. LC3, the homolog of yeast Atg8, is as a marker for the detection of autophagosomes [8,13]. Endogenous LC3 is present in 2 forms, LC3-I and LC3-II. LC3-I in turn is modified to a membrane bound form LC3-II to prompt its localization to autophagosomes [8,13]. Then, autophagosomes fuse with lysosomes to form autolysosomes and promote autophagy [8,13]. The ratio of LC3-II/LC3-I is correlated with autophagosome formation [8,12]. The increase of LC3-II/ LC3-I shows the up-regulation of autophagy [8]. In contrast, the decrease of LC3-II/LC3-I shows the down-regulation of autophagy [8]. Beclin1 also is as a mammalian autophagic gene and plays an important role in the autophagosome formation [8,14]. The 3-MA (autophagy antagonist) inhibits autophagy by PI3K, which is important in the membrane dynamics process involved in AV trafficking [8]. In the present study, as shown in Figs. 1 and 2, PC increased the autophagic vesicles (AVs), the rate of LC3-II/LC3-I and Beclin 1
838
Correspondence
Fig. 1. Formation of AVs in ventricular tissue. The arrows: AVs (transmission electron microscopy: original magnification ×20,000). AVs were absent in the control, PC + Hal, PC + Hal + Bro, PC + 3-MA and PC + 3-MA + Bro ventricular tissue. In contrast, PC and PC + Bro ventricular tissue displayed AVs. There were AVs observed in PC and PC + Bro ventricular tissue.
expressions. Bro further enhanced the effects of PC on the AVs, the ratio of LC3-II/LC3-I and the expression of Beclin 1. Hal and 3-MA canceled the effect of Bro, respectively. Taken together, our findings indicated that the activation of DR2 is involved in PCinduced cardioprotection by increase of autophagy. mTOR, a protein kinase, regulates cell growth and metabolism and also is the best characterized regulator of autophagy [11,13]. mTOR is a down-regulator of autophagy [15,16]. A lot of mTOR up-stream activators activate/inhibit autophagy by the change of mTOR [16–17]. For instance, PI3K/Akt activation down-regulates autophagy through an increase of mTOR [16–17]. However, AMPK activation up-regulates autophagy via a decrease of mTOR [16, 17]. Therefore, mTOR is a central factor of autophagic signaling pathways and its activation suppresses autophagy. Our data showed that PC significantly decreased mTOR phosphorylation. Bro further decreased PC-induced phosphorylation of mTOR. Hal and 3-MA canceled the effect of Bro, respectively (Fig. 2). These results suggest that DR2 activation is involved in cardioprotection of PC through an increase of autophagy via down-regulation of mTOR.
In conclusion, our present study indicates that PC-induced cardioprotection is associated with the activation of autophagy through inhibiting the phosphorylation of mTOR. These findings, which are new ideas for researching mechanisms of PC-induced cardioprotection, provide new insight into the cardioprotective effects of DR2 and help develop new agents for the prevention and therapy of ischemic cardiomyopathy. Competing interests The authors report no relationships that could be construed as a conflict of interest. Acknowledgments This research is supported by the National Natural Science Foundation of China (no. 81000059, no. 81270273, no. 81270311) and the Natural Science Foundation of Heilongjiang Province (Heilongjiang Province Foundation for Returness, no. LC201430).
Correspondence
839
References [1] C. Missale, Dopamine receptors: from structure to function, Physiol. Rev. 78 (1998) 189–225. [2] H.Z. Li, C. Wei, J. Gao, et al., Mediation of dopamine D2 receptors activation in postconditioning-attenuated cardiomyocyte apoptosis, Exp. Cell Res. 323 (2014) 118–130. [3] H.Z. Li, J. Guo, et al., Role of dopamine D2 receptors in ischemia/reperfusion induced apoptosis of cultured neonatal rat cardiomyocytes, J. Biomed. Sci. 16 (18) (2011) 18. [4] J. Gao, J. Guo, H.X. Li, et al., Involvement of dopamine D2 receptors activation in ischemic post-conditioning-induced cardioprotection through promoting PKC-ε particulate translocation in isolated rat hearts, Mol. Cell. Biochem. 379 (2013) 267–276. [5] V. Narkar, O. Kunduzova, T. Hussain, et al., Dopamine D2-like receptor agonist bromocriptine protects against ischemia/reperfusion injury in rat kidney, Kidney Int. 66 (2004) 633–640. [6] A. Vegh, G.Y. Papp, C. Mraro, et al., Dopamine receptor agonist Z1046 reduces ischaemia severity in a canine model of coronary artery occlusion, Eur. J. Pharmacol. 344 (1998) 203–213. [7] H. Li, S. Shi, Y.H. Sun, et al., Dopamine D2 receptor stimulation inhibits angiotensin II-induced hypertrophy in cultured neonatal rat ventricular myocytes, Clin. Exp. Pharmacol. Physiol. 36 (2009) 312–318. [8] C. Wei, H. Li, L. Han, et al., Activation of autophagy in ischemic postconditioning contributes to cardioprotective effects against ischemia/reperfusion injury in rat hearts, J. Cardiovasc. Pharmacol. 61 (2013) 416–422. [9] Z.Q. Zhao, J.J. Vinten-Johansen, Postconditioning: reduction of reperfusion induced injury, Cardiovasc. Res. 70 (2006) 200–211. [10] P. Balakumar, A. Rohilla, M. Singh, Preconditioning and postconditioning to limit ischemia reperfusion-induced myocardial injury: what could be the next footstep? Pharmacol. Res. 57 (2008) 403–412. [11] C. Ouyang, J. You, Z. Xie, The interplay between autophagy and apoptosis in the diabetic heart, J. Mol. Cell. Cardiol. 71 (2014) 71–80. [12] A.M. Cuervo, Autophagy: many paths to the same end, Mol. Cell. Biochem. 263 (2004) 55–72. [13] Z.L. Zhang, Y. Fan, M.L. Liu, Ginsenoside Rg1 inhibits autophagy in H9c2 cardiomyocytes exposed to hypoxia/reoxygenation, Mol. Cell. Biochem. 365 (2012) 243–250. [14] I. Tanida, T. Ueno, E. Kominami, LC3 conjugation system in mammalian autophagy, Int. J. Biochem. Cell Biol. 36 (2004) 2503–2518. [15] T.P. Neufeld, TOR-dependent control of autophagy: biting the hand that feeds, Curr. Opin. Cell Biol. 22 (2010) 157–168. [16] P. Wang, Q.S. Guo, Z.W. Wang, et al., HBx induces HepG-2 cells autophagy through PI3K/Akt–mTOR pathway, Mol. Cell. Biochem. 372 (2013) 161–168. [17] D. Meley, C. Bauvy, J.H. Houben-Weerts, et al., AMP-activated protein kinase and the regulation of autophagic proteolysis, J Biol Chem 281 (2006) 34870–34879.
Fig. 2. The change of related factors with autophagy. The intensity of each band was quantified by densitometry, and data were normalized to the GAPDH or total mTOR signal. All data were from four independent experiments. #p b 0.05 vs. control group; &p b 0.05 vs. PC group; $p b 0.05 vs. PC + Bro group.
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Correspondence
Dopamine D2 receptors contribute to cardioprotection of ischemic post-conditioning via activating autophagy in isolated rat hearts Can Wei a, Jun Gao b, Meixiu Li a, Hong Li a, Yuehong Wang a, Hongzhu Li a,⁎, Changqing Xu a,⁎ a b
Department of Pathophysiology, Harbin Medical University, Harbin 150081, China Department of Orthopeadic Surgery, The First Hospital of Harbin, Harbin 150010, China
a r t i c l e
i n f o
Article history: Received 1 September 2015 Accepted 3 November 2015 Available online 9 November 2015 Keywords: Dopamine D2 receptors Post-conditioning Autophagy Heart Rats
Dopamine receptors (DR) belong to the family of seven transmembrane domain G-protein coupled receptors (GPCR) and are classified into D1-like (DR1) and D2-like receptor families (DR2) based on pharmacological properties, structure, and signal transduction system [1–7]. The DR1 families include D1- and D5receptor subtypes, which are generally coupled to GS proteins and stimulate the cAMP productions and increase the PKA activity. The DR2 families consist of D2-, D3-, and D4-receptor subtypes, which are coupled to Gi/o proteins and decrease the cAMP productions, the PKA activity and the concentration of intracellular calcium, and regulate potassium channels [1–7]. DR exist in many tissues and organs including the brain, kidney, heart and the peripheral nervous system and play the important physiological and pathological roles [2,5]. Recent studies found that DR2 inhibited hypertrophy and I/R injury and is involved in PC-induced protective effects in the cardiovascular system [2–4,7]. As everyone knows, PC reduces myocardial I/R injury and apoptosis [2,4,9,10]. Our data demonstrated that PC decreased I/R injury by preventing cardiomyocyte damages and apoptosis, reducing myocardial infarct size, and improving cardiac function. Bromocriptine (Bro,
⁎ Corresponding authors at: Department of Pathophysiology, Harbin Medical University, Baojian Road, Harbin 150081, China. E-mail addresses: [email protected] (H. Li), [email protected] (C. Xu).
http://dx.doi.org/10.1016/j.ijcard.2015.11.006 0167-5273/© 2015 Elsevier Ireland Ltd. All rights reserved.
DR2 agonist) further enhanced the effects of PC, but Haloperidol (Hal, DR2 antagonist) canceled the Bro-caused beneficial effects during PC [2,4]. These results suggest that DR2 activation is an important factor in cardioprotection of PC. Our previous our researches indicated that PC increased DR2 expressions and Bro further enhanced the expression of DR2 during PC [2,4]. Increased DR2 expressions or activation is involved in PCinduced cardioprotection via inhibition of mPTP opening via activation of the ERK1/2, PI3K–Akt–GSK-3β and PKC-ε–mKATP pathways [2,4]. However, the involvement of autophagy in the mediation of DR2 activation in cardioprotection by PC is unclear. In the present study, we used isolated rat hearts exposed to PC as an experimental model to demonstrate the effect of DR2 activation on the PC-induced autophagy and relative mechanisms. Moreover, we found that (1) autophagy plays an important role in PC against myocardial I/R and (2) DR2 activation contributes to cardioprotective effects of PC through up-regulation of autophagy via inhibiting phosphorylation of mTOR. Autophagy is a key factor in keeping the balance between synthesis, degradation, and recycling of cellular components [11]. It allows the cell not only to recycle amino acids but also to remove damaged organelles, thereby eliminating oxidative stress and allowing cellular remodeling for survival [11]. In the heart, a number of stress conditions, including cardiomyopathy, pressure overload, heart failure and I/R injury, increase autophagic activity [8]. Nuclear disassembly and AVs of degenerated cardiomyocytes are observed in human hibernating myocardium [8,12]. LC3, the homolog of yeast Atg8, is as a marker for the detection of autophagosomes [8,13]. Endogenous LC3 is present in 2 forms, LC3-I and LC3-II. LC3-I in turn is modified to a membrane bound form LC3-II to prompt its localization to autophagosomes [8,13]. Then, autophagosomes fuse with lysosomes to form autolysosomes and promote autophagy [8,13]. The ratio of LC3-II/LC3-I is correlated with autophagosome formation [8,12]. The increase of LC3-II/ LC3-I shows the up-regulation of autophagy [8]. In contrast, the decrease of LC3-II/LC3-I shows the down-regulation of autophagy [8]. Beclin1 also is as a mammalian autophagic gene and plays an important role in the autophagosome formation [8,14]. The 3-MA (autophagy antagonist) inhibits autophagy by PI3K, which is important in the membrane dynamics process involved in AV trafficking [8]. In the present study, as shown in Figs. 1 and 2, PC increased the autophagic vesicles (AVs), the rate of LC3-II/LC3-I and Beclin 1
838
Correspondence
Fig. 1. Formation of AVs in ventricular tissue. The arrows: AVs (transmission electron microscopy: original magnification ×20,000). AVs were absent in the control, PC + Hal, PC + Hal + Bro, PC + 3-MA and PC + 3-MA + Bro ventricular tissue. In contrast, PC and PC + Bro ventricular tissue displayed AVs. There were AVs observed in PC and PC + Bro ventricular tissue.
expressions. Bro further enhanced the effects of PC on the AVs, the ratio of LC3-II/LC3-I and the expression of Beclin 1. Hal and 3-MA canceled the effect of Bro, respectively. Taken together, our findings indicated that the activation of DR2 is involved in PCinduced cardioprotection by increase of autophagy. mTOR, a protein kinase, regulates cell growth and metabolism and also is the best characterized regulator of autophagy [11,13]. mTOR is a down-regulator of autophagy [15,16]. A lot of mTOR up-stream activators activate/inhibit autophagy by the change of mTOR [16–17]. For instance, PI3K/Akt activation down-regulates autophagy through an increase of mTOR [16–17]. However, AMPK activation up-regulates autophagy via a decrease of mTOR [16, 17]. Therefore, mTOR is a central factor of autophagic signaling pathways and its activation suppresses autophagy. Our data showed that PC significantly decreased mTOR phosphorylation. Bro further decreased PC-induced phosphorylation of mTOR. Hal and 3-MA canceled the effect of Bro, respectively (Fig. 2). These results suggest that DR2 activation is involved in cardioprotection of PC through an increase of autophagy via down-regulation of mTOR.
In conclusion, our present study indicates that PC-induced cardioprotection is associated with the activation of autophagy through inhibiting the phosphorylation of mTOR. These findings, which are new ideas for researching mechanisms of PC-induced cardioprotection, provide new insight into the cardioprotective effects of DR2 and help develop new agents for the prevention and therapy of ischemic cardiomyopathy. Competing interests The authors report no relationships that could be construed as a conflict of interest. Acknowledgments This research is supported by the National Natural Science Foundation of China (no. 81000059, no. 81270273, no. 81270311) and the Natural Science Foundation of Heilongjiang Province (Heilongjiang Province Foundation for Returness, no. LC201430).
Correspondence
839
References [1] C. Missale, Dopamine receptors: from structure to function, Physiol. Rev. 78 (1998) 189–225. [2] H.Z. Li, C. Wei, J. Gao, et al., Mediation of dopamine D2 receptors activation in postconditioning-attenuated cardiomyocyte apoptosis, Exp. Cell Res. 323 (2014) 118–130. [3] H.Z. Li, J. Guo, et al., Role of dopamine D2 receptors in ischemia/reperfusion induced apoptosis of cultured neonatal rat cardiomyocytes, J. Biomed. Sci. 16 (18) (2011) 18. [4] J. Gao, J. Guo, H.X. Li, et al., Involvement of dopamine D2 receptors activation in ischemic post-conditioning-induced cardioprotection through promoting PKC-ε particulate translocation in isolated rat hearts, Mol. Cell. Biochem. 379 (2013) 267–276. [5] V. Narkar, O. Kunduzova, T. Hussain, et al., Dopamine D2-like receptor agonist bromocriptine protects against ischemia/reperfusion injury in rat kidney, Kidney Int. 66 (2004) 633–640. [6] A. Vegh, G.Y. Papp, C. Mraro, et al., Dopamine receptor agonist Z1046 reduces ischaemia severity in a canine model of coronary artery occlusion, Eur. J. Pharmacol. 344 (1998) 203–213. [7] H. Li, S. Shi, Y.H. Sun, et al., Dopamine D2 receptor stimulation inhibits angiotensin II-induced hypertrophy in cultured neonatal rat ventricular myocytes, Clin. Exp. Pharmacol. Physiol. 36 (2009) 312–318. [8] C. Wei, H. Li, L. Han, et al., Activation of autophagy in ischemic postconditioning contributes to cardioprotective effects against ischemia/reperfusion injury in rat hearts, J. Cardiovasc. Pharmacol. 61 (2013) 416–422. [9] Z.Q. Zhao, J.J. Vinten-Johansen, Postconditioning: reduction of reperfusion induced injury, Cardiovasc. Res. 70 (2006) 200–211. [10] P. Balakumar, A. Rohilla, M. Singh, Preconditioning and postconditioning to limit ischemia reperfusion-induced myocardial injury: what could be the next footstep? Pharmacol. Res. 57 (2008) 403–412. [11] C. Ouyang, J. You, Z. Xie, The interplay between autophagy and apoptosis in the diabetic heart, J. Mol. Cell. Cardiol. 71 (2014) 71–80. [12] A.M. Cuervo, Autophagy: many paths to the same end, Mol. Cell. Biochem. 263 (2004) 55–72. [13] Z.L. Zhang, Y. Fan, M.L. Liu, Ginsenoside Rg1 inhibits autophagy in H9c2 cardiomyocytes exposed to hypoxia/reoxygenation, Mol. Cell. Biochem. 365 (2012) 243–250. [14] I. Tanida, T. Ueno, E. Kominami, LC3 conjugation system in mammalian autophagy, Int. J. Biochem. Cell Biol. 36 (2004) 2503–2518. [15] T.P. Neufeld, TOR-dependent control of autophagy: biting the hand that feeds, Curr. Opin. Cell Biol. 22 (2010) 157–168. [16] P. Wang, Q.S. Guo, Z.W. Wang, et al., HBx induces HepG-2 cells autophagy through PI3K/Akt–mTOR pathway, Mol. Cell. Biochem. 372 (2013) 161–168. [17] D. Meley, C. Bauvy, J.H. Houben-Weerts, et al., AMP-activated protein kinase and the regulation of autophagic proteolysis, J Biol Chem 281 (2006) 34870–34879.
Fig. 2. The change of related factors with autophagy. The intensity of each band was quantified by densitometry, and data were normalized to the GAPDH or total mTOR signal. All data were from four independent experiments. #p b 0.05 vs. control group; &p b 0.05 vs. PC group; $p b 0.05 vs. PC + Bro group.