Use of atorvastatin to inhibit hypoxia-induced myocardin expression
Chiung-Zuan Chiu
Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan
Division of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
Search for more papers by this authorBao-Wei Wang
Division of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
Search for more papers by this authorKou-Gi Shyu
Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
Division of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
Search for more papers by this authorChiung-Zuan Chiu
Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
School of Medicine, Fu-Jen Catholic University, New Taipei City, Taiwan
Division of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
Search for more papers by this authorBao-Wei Wang
Division of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
Search for more papers by this authorKou-Gi Shyu
Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
Division of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
Search for more papers by this authorAbstract
Eur J Clin Invest 2012; 42 (5): 564–571
Background Hypoxia induces the formation of reactive oxygen species (ROS), myocardin expression and cardiomyocyte hypertrophy. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been demonstrated to have both antioxidant and antihypertrophic effects. We evaluated the pathways of atorvastatin in repressing ROS and myocardin after hypoxia to prevent cardiomyocyte hypertrophy.
Materials and methods Cultured rat neonatal cardiomyocytes were subjected to hypoxia, and the expression of myocardin and ROS were evaluated. Different signal transduction inhibitors, atorvastatin and N-acetylcysteine (NAC) were used to identify the pathways that inhibited myocardin expression and ROS. Electrophoretic motility shift assay (EMSA) and luciferase assay were used to identify the binding of myocardin/serum response factor (SRF) and transcription to cardiomyocytes. Cardiomyocyte hypertrophy was assessed by 3H-proline incorporation assay.
Results Myocardin expression after hypoxia was inhibited by atorvastatin, RhoA/Rho kinase inhibitor (Y27632), extracellular signal-regulated kinase (ERK) small interfering RNA (siRNA)/ERK pathway inhibitor (PD98059), myocardin siRNA and NAC. Bindings of myocardin/SRF, transcription of myocardin/SRF to cardiomyocytes, presence of myocardin in the nuclei of cardiomyocytes and protein synthesis after hypoxia were identified by EMSA, luciferase assay, confocal microscopy and 3H-proline assay and were suppressed by atorvastatin, Y27632, PD98059 and NAC.
Conclusions Hypoxia in neonatal cardiomyocytes increases myocardin expression and ROS to cause cardiomyocyte hypertrophy, which can be prevented by atorvastatin by suppressing ROS and myocardin expression.
Supporting Information
Data S1. Materials and methods.
Figure S1. Hypoxia increases the expression of myocardin protein and mRNA in cultured rat neonatal cardiomyocytes.
Figure S2. Atorvastatin inhibits hypoxia-induced myocardin expression through a HMG-CoA reductase dependent pathway.
Figure S3. Myocardin expression under hypoxia is supressed by Rac1 inhibitor.
Figure S4. Effects of myocardin and ERK siRNAs expression in cardiomyocytes under 2·5% O2.
Figure S5. Confocal microscopy identifies the presence of myocardin (yellow; white arrow) in the nuclei (blue) of neonatal cardiomyocytes after hypoxia and Dp44mT, which is inhibited by atorvastatin, Y27632, PD98059, and NAC. (n = 3 per group).
Figure S6. Protein synthesis in neonatal cardiomyocytes increases after hypoxia.
Figure S7. Myocardin-related transcriptional factor-A (MRTF-A) increases expression after hypoxia in neonatal cardiomyocytes.
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References
- 1 Miano JM. Serum response factor: toggling between disparate programs of gene expression. J Mol Cell Cardiol 2003; 35: 577–93.
- 2 Wang D, Chang PS, Wang Z, Sutherland L, Richardson JA, Small E et al. Activation of cardiac gene expression by myocardin, a transcriptional cofactor for serum response factor. Cell 2001; 105: 851–62.
- 3 Zhang X, Azhar G, Zhong Y, Wei JY. Identification of a novel serum response factor cofactor in cardiac gene regulation. J Biol Chem 2004; 279: 55626–32.
- 4 Deindl E. Arteriogenesis: focus on signal transduction cascades and transcription factors. Thromb Haemost 2007; 98: 940–3.
- 5 Inamoto S, Hayashi T, Tazawa N, Mori T, Yamashita C, Nakano D et al. Angiotensin-II receptor blocker exerts cardioprotection in diabetic rats exposed to hypoxia. Circ J 2006; 70: 787–92.
- 6 Seta KA, Millhorn DE. Functional genomics approach to hypoxia signaling. J Appl Physiol 2004; 96: 765–73.
- 7 Wen JK, Han M. Myocardin. A novel potentiator of SRF-mediated transcription in cardiac muscle. Mol Cell 2002; 8: 1–2.
- 8 Firulli AB. Another hat for myocardin. J Mol Cell Cardiol 2002; 34: 1293–6.
- 9 Parmacek MS. Myocardin-related transcription factors: critical coactivators regulating cardiovascular development and adaptation. Circ Res 2007; 100: 633–44.
- 10 Miano JM. Channeling to myocardin. Circ Res 2004; 95: 340–2.
- 11 Reynolds PR, Mucenski ML, Le Cras TD, Nichols WC, Whitsett JA. Midkine is regulated by hypoxia and causes pulmonary vascular remodeling. J Biol Chem. 2004; 279: 37124–32.
- 12 Kuwahara K, Kinoshita H, Kuwabara Y, Nakagawa Y, Usami S, Minami T et al. Myocardin-related transcription factor A is a common mediator of mechanical stress- and neurohumoral stimulation-induced cardiac hypertrophic signaling leading to activation of brain natriuretic peptide gene expression. Mol Cell Biol 2010; 30: 4134–48.
- 13 Chiu CZ, Wang BW, Chung TH, Shyu KG. Angiotensin II and ERK pathway mediate the induction of myocardin by hypoxia in cultured rat neonatal cardiomyocytes. Clin Sci 2010; 109: 273–82.
- 14 Yamashita C, Hayashi T, Mori T, Tazawa N, Kwak CJ, Nakano D et al. Angiotensin II receptor blocker reduces oxidative stress and attenuates hypoxia-induced left ventricular remodeling in apolipoprotein E-knockout mice. Hypertens Res 2007; 30: 1219–30.
- 15 Yoshida T, Hoofnagle MH, Owens GK. Myocardin and Prx1 contribute to angiotensin II-induced expression of smooth muscle α-actin. Circ Res 2004; 94: 1075–82.
- 16 Sadoshima J, Malhotra R, Izumo S. The role of the cardiac renin-angiotensin system in load-induced cardiac hypertrophy. J Card Fail 1996; 2: S1–6.
- 17 Rikitake Y, Hirata K. Inhibition of RhoA or Rac1? Mechanisms of cholesterol-independent beneficial effects of statins Circ J 2009; 73: 231–2.
- 18 Ye Y, Hu SJ, Li L. Inhibition of farnesyl pyrophosphate synthase prevents angiotensin II-induced hypertrophic responses in rat neonatal cardiomyocytes: involvement of the RhoA/Rho kinase pathway. FEBS Lett 2009; 583: 2997–3003.
- 19 Saka M, Obata K, Ichihara S, Cheng XW, Kimata H, Noda A et al. Attenuation of ventricular hypertrophy and fibrosis in rats by pitavastatin: potential role of the RhoA-extracellular signal regulated kinase-serum response factor signaling pathway. Clin Exp Pharmacol Physiol 2006; 33: 1164–71.
- 20 Senthil V, Chen SN, Tsybouleva N, Halder T, Nagueh SF, Willerson JT et al. Prevention of cardiac hypertrophy by atorvastatin in a transgenic rabbit model of human hypertrophic cardiomyopathy. Circ Res 2005; 97: 285–92.
- 21 Adam O, Laufs U. Antioxidative effects of statins. Arch Toxicol 2008; 82: 885–92.
- 22 Shyu KG, Chen CC, Wang BW, Kuan P. Angiotensin II receptor antagonist blocks the expression of connexin 43 induced by cyclical mechanical stretch in cultured neonatal rat cardiac myocytes. J Mol Cell Cardiol 2001; 33: 691–8.
- 23 Brown JH, Del Re DP, Sussman MA. The Rac and Rho hall of fame: a decade of hypertrophic signaling hits. Circ Res 2006; 98: 730–42.
- 24 Baker KM, Chernin MI, Schreiber T, Sanghi S, Haiderzaidi S, Booz GW et al. Evidence of a novel intracrine mechanism in angiotensin II-induced cardiac hypertrophy. Regul Pept 2004; 120: 5–13.
- 25 Torrado M, Lopez E, Centeno A, Medrano C, Castro-Beiras A, Mikhailov AT. Myocardin mRNA is augmented in the failing myocardium: expression profiling in the porcine model and human dilated cardiomyopathy. J Mol Med 2008; 81: 566–77.
- 26 Pijnappels DA, Tuyn J, Vries AF, Grauss RW, Laarse A, Ypey DL et al. Resynchronization of separated rat cardiomyocyte fields with genetically modified human ventricular scar fibroblasts. Circulation 2007; 116: 2018–28.
- 27 Tuyn J, Pijnappels DA, Vries AF, Vries I, Dijke IV, Knan-Shanzer S et al. Fibroblasts from human postmyocardial infarction scars acquire properties of cardiomyocytes after transduction with a recombinant myocardin gene. FASEB J 2007; 21: 3369–79.
- 28 Xing W, Zhang TC, Cao D, Wang Z, Antos CL, Li S et al. Myocardin induces cardiomyocyte hypertrophy. Circ Res 2006; 98: 1089–97.
- 29 Hakoshima T, Shimizu T, Maesaki R. Structural basis of the Rho GTPase signaling. J Biochem J (Tokyo) 2003; 134: 327–31.
- 30 Hofer F. Activated Ras interacts with the Rac1 guanine nucleotide dissociation stimulator. Proc Natl Acad Sci U S A 1994; 91: 11089–93.
- 31 Schiller MR. Coupling receptor tyrosine kinases to Rho GTPases-GEFs: what’s the link. Cell Signal 2006; 18: 1834–43.
- 32 Sagara Y, Hirooka Y, Nozoe M, Ito K, Kimura Y, Sunagawa K. Pressor response induced by central angiotensin II is mediated by activation of Rho/Rho-kinase pathway via AT1 receptors. J Hypertens 2007; 25: 399–406.
- 33 Kimura K, Eguchi S. Angiotensin II type-1 receptor regulates RhoA and Rho-kinase/ROCK activation via multiple mechanisms. Am J Physiol Cell Physiol 2009; 297: C1059–61.
- 34 Nohria A, Prsic A, Liu PY, Okamoto R, Creager MA, Selwyn A et al. Statins inhibit Rho kinase activity in patients with atherosclerosis. Atherosclerosis 2009; 205: 517–21.
- 35 Hill CS, Wynne J, Treisman R. The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell 1995; 81: 1159–70.
- 36 Gineitis D, Treisman R. Differential usage of signal transduction pathways defines two types of serum response factor target gene. J Biol Chem 2001; 276: 24531–9.
- 37 Lui HW, Halayko AJ, Fernandes DJ, Harmon GS, McCauley JA, Kocieniewski P et al. The RhoA Rho kinase pathway regulates nuclear localization of serum response factor. Am J Respir Cell Mol Biol 2003; 29: 39–47.
