International Journal of Cardiology 178 (2015) 165–167
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Mitofusin 2 ameliorates aortic remodeling by suppressing ras/raf/ERK pathway and regulating mitochondrial function in vascular smooth muscle cells Zuoguang Wang a, Qiuli Niu a,1, Xiaoyun Peng b, Mei Li a, Ya Liu a, Jielin Liu a, Shaojun Wen a, Yongxiang Wei a,⁎ a b
Department of Hypertension, Beijing Anzhen Hospital, Capital Medical University, Beijing Institute of Heart, Lung, Blood Vessel Diseases, Beijing 100029, PR China Institute of Westnorth Plateau Biology, Chinese Academy of Sciences, 650032, PR China
a r t i c l e
i n f o
Article history: Received 12 September 2014 Accepted 21 October 2014 Available online 22 October 2014 Keywords: Mitofusin 2 Aortic remodeling Antihypertensive treatment Mitochondria Oxidative stress
Mitofusin 2 (Mfn2) is a critical negative regulator of vascular smooth muscle cell (VSMC) hypertrophy and proliferation by regulating mitochondrial fusion, ras/raf/ERK signal transduction, oxidative stress, calcium infusion, and apoptosis [1,2]. The purpose of this study was to investigate if Mfn2 was related with aortic remodeling and the potential mechanism. Eight-week-old spontaneously hypertensive (SHR) and WKY rats were randomly divided into seven groups (n = 9 for each group): positive control group, SHR group; SHR + dihydralazine (3 mg/kg/day), Dih group; SHR + carvedilol (30 mg/kg/day), Car group; SHR + amlodipine besylate (5 mg/kg/day), Aml group; SHR + perindopril (2 mg/kg/day), Per group; SHR + candesartan cilexetil (2 mg/kg/day), Can group; negative control group, WKY group. Rats were treated with antihypertensive drugs daily for 8 weeks. Blood pressure was determined every week. At the beginning of this study, SBP was higher in SHR (153.12 ± 6.53 mm Hg) than in WKY rats (104.33 ± 4.71 mm Hg, P b 0.01), and reached a maximum about the fourth treated week and remained elevated until the end of the study (195.33 ± 7.00; 128.50 ± 2.66 mm Hg, P b 0.01). Morphological changes became apparent in SHR compared to WKY: the cell alignment was irregular; VSMC fibers became thick, irregular ⁎ Corresponding author at: No. 2, Anzhenlu, Beijing 100029, PR China. E-mail address: [email protected] (Y. Wei). 1 Co-first author.
http://dx.doi.org/10.1016/j.ijcard.2014.10.122 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
and enlarged; and cell density was reduced. These changes were gradually attenuated in the Dih, Car, Aml, Per and Can groups after antihypertensive treatment (Fig. 1A). Media thickness (MT)/lumen diameter (LD) ratio and vascular remodeling were also reduced after antihypertensive treatment (Fig. 1B). By a transmission electron microscope, we found that the SHR group showed the worst mitochondrial phenotype, including irregularly arranged and spherical-shaped mitochondria swollen with electronlucent matrix, and the most increased number of mitochondria compared to the treated SHR groups and WKY group (P b 0.01). When detecting by confocal laser scanning microscopy, mitochondrial membrane potential in the Per and Can groups was relatively higher than in the Dih, Car and Aml groups but lower than in the WKY group (P b 0.01) [3]. In order to investigate the effect of ROS on VSMCs, we detected malondialdehyde and O2− levels which were obviously higher in the SHR group than in the WKY group (P b 0.01) and decreased in antihypertensive treated SHR groups, but still higher than in the WKY group (P b 0.01 for all). Generally, superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels were lower in the SHR group than in the WKY group and increased after antihypertensive treatment (P b 0.01 for all). Adventitial tissue and endothelium removed aortic tissue were homogenized, and RNA and protein were purified for qRT-PCR and western blotting detection of Mfn2/ras/raf gene/protein expression. The results showed that Mfn2 protein-expression level in Hyd, Car, Aml, Per and Can groups was significantly lower than in the WKY group (P b 0.01 for all), but higher than in the SHR group (P b 0.01 for all). ras protein-expression level in antihypertensive treatment groups was lowered than in the SHR group (P b 0.01 for all, except the Car group). raf protein-expression level was significantly decreased in antihypertensive treatment groups than in the SHR group, but still higher than the WKY group (P b 0.01 for all) (Fig. 1C, D, E, F). As to gene-expression, Mfn2, ras and raf genes in the aorta media tissue were similar to that of protein-expression trend for all the seven groups [4]. Pearson correlation analysis showed that MT/LD was closely related with Mfn2 protein-expression (P b 0.01, R = −0.724), and other indexes such as malondialdehyde, SOD, or GPx were also related with MT/LD weakly.
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Z. Wang et al. / International Journal of Cardiology 178 (2015) 165–167
Fig. 1. Aortic morphologic changes and protein expression level of different groups. ⁎P b 0.05, ⁎⁎P b 0.01, compared to SHR group; †P b 0.05, ††P b 0.01, compared to WKY group.
Fig. 2. Diagraph of mitofusin 2 affects aortic remodeling and possible mechanisms. ROS, reactive oxygen species; CCB, calcium channel antagonist; ARB, angiotensin II Type 1 receptor blocker; ACEI, angiotensin converting enzyme inhibitor; CaMKII, calmodulin-dependent kinase II; +, promotion; −, inhibition.
Z. Wang et al. / International Journal of Cardiology 178 (2015) 165–167
In total, aortic remodeling of SHR is different depending on antihypertensive agents. Generally, candesartan cilexetil and perindopril are two best antihypertenisve agents, amlodipine besylate is also a good one, while dihydralazine and carvedilol are not. These are consistent with mitochondrial numbers, structure and functional state in VSMCs, and mitochondrial oxidative factor level in the aortic media tissue. Particularly, Mfn2 protein-expression level in VSMC is more closely related with aortic remodeling than indexes. The potential mechanism is that Mfn2 can suppress ras/raf/ERK pathway and regulate mitochondrial structure and function in VSMCs, which play important roles in affecting VSMC hypotrophy, proliferation, differentiation and apoptosis, and contribute to aortic remodeling (Fig. 2). Therefore, Mfn2 maybe a potential marker for target organ damage and a target for antihypertensive treatment [5,6]. Grants This work was supported by grants from the National Natural Science Foundation of China (Nos. 81270216, 81370229) and Beijing Natural Science Foundation (Nos. 7102045, 7133232).
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References [1] K.H. Chen, X. Guo, D. Ma, et al., Dysregulation of HSG triggers vascular proliferative disorders, Nat. Cell Biol. 6 (2004) 872–883. [2] K.H. Chen, A. Dasgupta, J. Ding, et al., Role of mitofusin 2 in controlling cellular proliferation, FASEB J. 28 (2014) 382–394. [3] H.X. Li, Y.F. Zhou, X. Zhao, et al., GATA-4 protects against hypoxia-induced cardiomyocyte injury: effects on mitochondrial membrane potential, Can. J. Physiol. Pharmacol. 11 (2014) 1–10. [4] B.W. Li, A.C. Rush, J. Tan, et al., Quantitative analysis of gender-regulated transcripts in the filarial nematode Brugia malayi by real-time RT-PCR, Mol. Biochem. Parasitol. 137 (2004) 329–337. [5] F. Chang, L.S. Steelman, J.T. Lee, et al., Signal transduction mediated by the Ras/Raf/ MEK/ERK pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention, Leukemia 17 (2003) 1263–1293. [6] J.K. Salabei, B.G. Hill, Mitochondrial fission induced by platelet-derived growth factor regulates vascular smooth muscle cell bioenergetics and cell proliferation, Redox Biol. 1 (2013) 542–551.
Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Letter to the Editor
Mitofusin 2 ameliorates aortic remodeling by suppressing ras/raf/ERK pathway and regulating mitochondrial function in vascular smooth muscle cells Zuoguang Wang a, Qiuli Niu a,1, Xiaoyun Peng b, Mei Li a, Ya Liu a, Jielin Liu a, Shaojun Wen a, Yongxiang Wei a,⁎ a b
Department of Hypertension, Beijing Anzhen Hospital, Capital Medical University, Beijing Institute of Heart, Lung, Blood Vessel Diseases, Beijing 100029, PR China Institute of Westnorth Plateau Biology, Chinese Academy of Sciences, 650032, PR China
a r t i c l e
i n f o
Article history: Received 12 September 2014 Accepted 21 October 2014 Available online 22 October 2014 Keywords: Mitofusin 2 Aortic remodeling Antihypertensive treatment Mitochondria Oxidative stress
Mitofusin 2 (Mfn2) is a critical negative regulator of vascular smooth muscle cell (VSMC) hypertrophy and proliferation by regulating mitochondrial fusion, ras/raf/ERK signal transduction, oxidative stress, calcium infusion, and apoptosis [1,2]. The purpose of this study was to investigate if Mfn2 was related with aortic remodeling and the potential mechanism. Eight-week-old spontaneously hypertensive (SHR) and WKY rats were randomly divided into seven groups (n = 9 for each group): positive control group, SHR group; SHR + dihydralazine (3 mg/kg/day), Dih group; SHR + carvedilol (30 mg/kg/day), Car group; SHR + amlodipine besylate (5 mg/kg/day), Aml group; SHR + perindopril (2 mg/kg/day), Per group; SHR + candesartan cilexetil (2 mg/kg/day), Can group; negative control group, WKY group. Rats were treated with antihypertensive drugs daily for 8 weeks. Blood pressure was determined every week. At the beginning of this study, SBP was higher in SHR (153.12 ± 6.53 mm Hg) than in WKY rats (104.33 ± 4.71 mm Hg, P b 0.01), and reached a maximum about the fourth treated week and remained elevated until the end of the study (195.33 ± 7.00; 128.50 ± 2.66 mm Hg, P b 0.01). Morphological changes became apparent in SHR compared to WKY: the cell alignment was irregular; VSMC fibers became thick, irregular ⁎ Corresponding author at: No. 2, Anzhenlu, Beijing 100029, PR China. E-mail address: [email protected] (Y. Wei). 1 Co-first author.
http://dx.doi.org/10.1016/j.ijcard.2014.10.122 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
and enlarged; and cell density was reduced. These changes were gradually attenuated in the Dih, Car, Aml, Per and Can groups after antihypertensive treatment (Fig. 1A). Media thickness (MT)/lumen diameter (LD) ratio and vascular remodeling were also reduced after antihypertensive treatment (Fig. 1B). By a transmission electron microscope, we found that the SHR group showed the worst mitochondrial phenotype, including irregularly arranged and spherical-shaped mitochondria swollen with electronlucent matrix, and the most increased number of mitochondria compared to the treated SHR groups and WKY group (P b 0.01). When detecting by confocal laser scanning microscopy, mitochondrial membrane potential in the Per and Can groups was relatively higher than in the Dih, Car and Aml groups but lower than in the WKY group (P b 0.01) [3]. In order to investigate the effect of ROS on VSMCs, we detected malondialdehyde and O2− levels which were obviously higher in the SHR group than in the WKY group (P b 0.01) and decreased in antihypertensive treated SHR groups, but still higher than in the WKY group (P b 0.01 for all). Generally, superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels were lower in the SHR group than in the WKY group and increased after antihypertensive treatment (P b 0.01 for all). Adventitial tissue and endothelium removed aortic tissue were homogenized, and RNA and protein were purified for qRT-PCR and western blotting detection of Mfn2/ras/raf gene/protein expression. The results showed that Mfn2 protein-expression level in Hyd, Car, Aml, Per and Can groups was significantly lower than in the WKY group (P b 0.01 for all), but higher than in the SHR group (P b 0.01 for all). ras protein-expression level in antihypertensive treatment groups was lowered than in the SHR group (P b 0.01 for all, except the Car group). raf protein-expression level was significantly decreased in antihypertensive treatment groups than in the SHR group, but still higher than the WKY group (P b 0.01 for all) (Fig. 1C, D, E, F). As to gene-expression, Mfn2, ras and raf genes in the aorta media tissue were similar to that of protein-expression trend for all the seven groups [4]. Pearson correlation analysis showed that MT/LD was closely related with Mfn2 protein-expression (P b 0.01, R = −0.724), and other indexes such as malondialdehyde, SOD, or GPx were also related with MT/LD weakly.
166
Z. Wang et al. / International Journal of Cardiology 178 (2015) 165–167
Fig. 1. Aortic morphologic changes and protein expression level of different groups. ⁎P b 0.05, ⁎⁎P b 0.01, compared to SHR group; †P b 0.05, ††P b 0.01, compared to WKY group.
Fig. 2. Diagraph of mitofusin 2 affects aortic remodeling and possible mechanisms. ROS, reactive oxygen species; CCB, calcium channel antagonist; ARB, angiotensin II Type 1 receptor blocker; ACEI, angiotensin converting enzyme inhibitor; CaMKII, calmodulin-dependent kinase II; +, promotion; −, inhibition.
Z. Wang et al. / International Journal of Cardiology 178 (2015) 165–167
In total, aortic remodeling of SHR is different depending on antihypertensive agents. Generally, candesartan cilexetil and perindopril are two best antihypertenisve agents, amlodipine besylate is also a good one, while dihydralazine and carvedilol are not. These are consistent with mitochondrial numbers, structure and functional state in VSMCs, and mitochondrial oxidative factor level in the aortic media tissue. Particularly, Mfn2 protein-expression level in VSMC is more closely related with aortic remodeling than indexes. The potential mechanism is that Mfn2 can suppress ras/raf/ERK pathway and regulate mitochondrial structure and function in VSMCs, which play important roles in affecting VSMC hypotrophy, proliferation, differentiation and apoptosis, and contribute to aortic remodeling (Fig. 2). Therefore, Mfn2 maybe a potential marker for target organ damage and a target for antihypertensive treatment [5,6]. Grants This work was supported by grants from the National Natural Science Foundation of China (Nos. 81270216, 81370229) and Beijing Natural Science Foundation (Nos. 7102045, 7133232).
167
References [1] K.H. Chen, X. Guo, D. Ma, et al., Dysregulation of HSG triggers vascular proliferative disorders, Nat. Cell Biol. 6 (2004) 872–883. [2] K.H. Chen, A. Dasgupta, J. Ding, et al., Role of mitofusin 2 in controlling cellular proliferation, FASEB J. 28 (2014) 382–394. [3] H.X. Li, Y.F. Zhou, X. Zhao, et al., GATA-4 protects against hypoxia-induced cardiomyocyte injury: effects on mitochondrial membrane potential, Can. J. Physiol. Pharmacol. 11 (2014) 1–10. [4] B.W. Li, A.C. Rush, J. Tan, et al., Quantitative analysis of gender-regulated transcripts in the filarial nematode Brugia malayi by real-time RT-PCR, Mol. Biochem. Parasitol. 137 (2004) 329–337. [5] F. Chang, L.S. Steelman, J.T. Lee, et al., Signal transduction mediated by the Ras/Raf/ MEK/ERK pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention, Leukemia 17 (2003) 1263–1293. [6] J.K. Salabei, B.G. Hill, Mitochondrial fission induced by platelet-derived growth factor regulates vascular smooth muscle cell bioenergetics and cell proliferation, Redox Biol. 1 (2013) 542–551.
