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Nitric oxide synthases and diabetic cardiomyopathy
ARTICLE IN PRESS Nitric Oxide ■■ (2014) ■■–■■
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Nitric Oxide j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n i o x
Nitric oxide synthases and diabetic cardiomyopathy Sanskriti Khanna, Gurinder Bir Singh, Madhu Khullar * Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
A R T I C L E
I N F O
Article history: Received 27 May 2014 Revised 22 July 2014 Available online Keywords: NOS Diabetic cardiomyopathy Epigenetic
A B S T R A C T
Cardiovascular complications associated with diabetes significantly contribute to high mortality and morbidity worldwide. The pathophysiology of diabetic cardiomyopathy (DCM), although extensively researched upon, is partially understood. Impairment in various signaling pathways including nitric oxide (NO) signaling has been implicated in the pathogenesis of diabetes induced myocardial damage. Nitric oxide synthases (NOS), the enzymes responsible for NO generation, play an important role in various physiological processes. Altered expression and activity of NOS have been implicated in cardiovascular diseases, however, the role of NOS and their regulation in the pathogenesis of DCM remain poorly understood. In the present review, we focus on the role of myocardial NOS in the development of DCM. Since epigenetic modifications play an important role in regulation of gene expression, this review also describes the epigenetic regulation of NOS. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Cardiovascular disease is a significant contributor to the high mortality and morbidity associated with diabetes. Nearly 80% of the deaths associated with diabetes are attributed to cardiac complications [1]. It has been observed that diabetic patients tend to develop heart failure in the absence of risk factors such as hypertension or coronary artery disease. The cardiovascular complications in diabetic patients in the absence or out of proportion to their underlying vascular disease have been termed as “diabetic cardiomyopathy” (DCM) [2]. It has been suggested that DCM is a frequently unrecognized pathological process in asymptomatic diabetic patients [3]. The pathophysiology of diabetic cardiomyopathy is partially understood, but both hyperglycemia and changes in cardiac metabolism contribute to the disease process. Dysregulation of multiple biochemical pathways such as enhanced reactive oxygen production, nonenzymatic glycation, polyol pathway and advanced glycation end products pathway, activation of the diacylglycerol–protein kinase C (PKC) pathway and NO signaling pathway has been proposed to link the adverse effects of persistent hyperglycemia with DCM (Fig. 1). Geraldes and King [4] have proposed that hyperglycemia may lead to alterations in cellular signaling pathways resulting in cellular dysfunction and heart failure in diabetic patients. Impaired NO signaling has been implicated in the genesis of diabetes induced myocardial damage, however, molecular mechanisms of NO signaling leading to pathogenesis of DCM remain poorly understood. In the
* Corresponding author. Fax: +91 1722744401. E-mail address: [email protected] (M. Khullar).
present review, we focus on the role of myocardial NOS in the development of DCM. Since epigenetic modifications play an important role in regulation of gene expression, this review also describes the epigenetic regulation of NOS.
2. NOS in heart NO is constitutively or inductively synthesized during a reaction catalyzed by NOS which exists in three isoforms. All the three NOS isoforms, namely endothelial (eNOS), neuronal (nNOS), and inducible (iNOS), are expressed in the heart although at spatially confined sub-cellular locations [5] (Fig. 2). In cardiomyocytes, eNOS localizes at the caveolae where it associates with caveolin-3 and modulates several signal transduction pathways [5]. nNOS, however, is confined to the sarcoplasmic reticulum of cardiac myocyte [6] where it regulates the activity of ryanodine receptor, sarcoplasmic reticulum Ca2+-ATPase (SERCA) or the L-type Ca2+ channel [7]. Whereas, iNOS is expressed during pathological states and is localized throughout the cytoplasm [8]. The functions of NOS in heart under normal physiological conditions have been reviewed by Balligand and Cannon [9]. NO is a free radical which reacts with various molecules to cause multiple biological effects. Under normal circumstances, the controlled production of NO (by eNOS) conveys antiapoptotic signal, inhibits platelet aggregation and adhesion to the vascular wall, besides playing an important role in cardiac contractile function. The vasodilatory function of NO is mediated via the activation of guanylate cyclase. NO also has cardio-protective effects [10]. Depending upon its source and level of output, NO can either play a cardio-protective or a detrimental role [9]. iNOS is absent in the healthy heart but, as the name indicates, it is induced in cells
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Diabetes
Hyperglycemia Defective Insulin Signaling iNOS
Free Fatty Acids Triglyceride
RNS GLUT
ROS PKCβ2
AGE
Fatty Acid Uptake
Glucose Oxidation Altered Calcium Homeostasis
Cardiac Lipid Accumulation
Fatty Acid Oxidation
Glucotoxicity Lipotoxicity Fibrosis Hypertrophy Mitochondrial Dysfunction Altered Calcium Homeostasis
DIABETIC CARDIOMYOPATHY Fig. 1. Pathophysiology of diabetic cardiomyopathy: Both hyperglycemia and changes in cardiac metabolism contribute to alterations in multiple biochemical pathways such as enhanced reactive oxygen species (ROS) production, reactive nitrogen species (RNS) production, advanced glycation end products (AGE) pathway, activation of the diacylglycerol–protein kinase C (PKC) pathway and NO signaling pathway resulting in diabetic cardiomyopathy. Impaired NO signaling has been implicated in the genesis of diabetes induced myocardial damage.
under stress conditions, including hyperglycemia, oxidative stress and hyperinsulinemia. An increased myocardial NO production can cause nitration of actin and other cytoskeletal proteins in the myocardium. This alters their structure and may have damaging effects on the contractile function of myofilaments. NO also interacts with oxygen radicals and leads to the formation of peroxynitrite, a potent oxidant, which in high concentration causes tissue damage [9].
caveolae
eNOS
NO
Fig. 2. Subcellular localization of NOS isoforms in cardiomyocytes: In cardiomyocytes, eNOS localizes at the caveolae where it controls heart rate, contraction, diastolic relaxation and oxygen consumption; nNOS is confined to the sarcoplasmic reticulum of cardiac myocyte where it regulates the activity of sarcoplasmic reticulum Ca2+ATPase (SERCA) or the L-type Ca2+ channel (LCC); whereas, iNOS is expressed during pathological states and is localized throughout the cytoplasm. iNOS, on being induced, continuously produces NO until the enzyme is degraded, leading to the formation of peroxynitrile (ONOO−) which contributes to contractile dysfunction and even apoptosis.
3. NOS in cardiovascular diseases Dysregulated expression and activity of NOS isoforms have been implicated in pathophysiology of several cardiovascular diseases including atherosclerosis, hypertension and diabetes. In general, eNOS has been found to play a protective role and nNOS has been shown to play iNOS a deterimental role in heart failure [11]. For example, Barouch et al. [5] reported that nNOS and eNOS-deficient mice developed age-related increase in LV wall thickness and mass and n/eNOS double knockout mice developed phenotype which was similar to that seen in hypertensive hypertrophic cardiomyopathy in humans, suggesting that both these isoforms play an important role in maintaining the architecture and function of heart. The protective role of eNOS was further confirmed by Janssens et al. [12], who observed that cardiomyocyte-restricted eNOS overexpression was protective against adverse left ventricular (LV) remodeling after coronary artery ligation in transgenic mice. Consistent with these findings, an increased mortality, exacerbation of LV remodeling, and LV dysfunction has been reported in eNOS-knockout mice with heart failure due to myocardial infarction [13] or to pressure overload [14]. Similarly, deficiency of nNOS was shown to accelerate adverse LV remodeling in myocardial injury-induced heart failure in nNOSdeficient mice [15,16] indicating that nNOS has a protective role in myocardial infarction induced HF. In contrast, an increased cardiac iNOS expression has been linked with volume-overload–induced HF [8] and in isoproterenol-induced heart failure [17]. Although cardiomyocyte-specific overexpression of iNOS did not lead to heart failure [18], iNOS-knockout mice showed improved survival during myocardial infarction-induced [19,20] or pressure overloadinduced heart failure [21]. The role of different NOS isoforms in the pathogenesis of heart failure was recently confirmed by Shibata et al. [22] who generated triple negative (n/i/eNOS−/−) mice in which all three NOS isoforms were completely disrupted. Their results
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confirmed a protective role of eNOS and nNOS in heart failure whereas iNOS was found to exert unfavorable role. The molecular mechanisms of contrasting roles of NOS isoforms in heart failure are unclear, but have been proposed to be due to differences in spatial localization, differential regulation and NO-generating capacity of different isoforms [23–25].
4. NOS in diabetic cardiomyopathy Endothelial dysfunction is an important feature of DCM and has been attributed to dysregulation of NO generation and bioavailabilty. Felaco et al. [26] reported decreased levels of eNOS protein and of its co-enzyme, NADPH-d, in the endothelium and vascular wall (but not in the myocardium) of BB/W diabetic rat hearts as compared to healthy control rats suggesting that dysregulation of NOS pathway was involved in pathogenesis of diabetes and associated cardiovascular complications. This was further confirmed by Hink et al. [27] who found increased expression of eNOS in diabetic hearts and by Sampaio et al. [28] who observed that cardiac abnormalities in diabetic rats resembled those found in L-NAME–induced cardiomyopathy suggesting a role for the NO system in the pathophysiology of DCM. iNOS is an inducible enzyme whose expression can be induced in all cells and tissues by cytokines and other agents [29]. Besides eNOS, altered expression and activity of iNOS have been shown to contribute to diabetes associated cardiovascular complications. For example, an increased expression of iNOS was seen in mesenteric arteries (SMA) of streptozotocin-induced diabetic rats [30]. An increased expression of myocardial iNOS along with a concurrent downregulation of eNOS has been reported in streptozotocin induced diabetic rats, indicating differential regulation of eNOS and iNOS isoforms in diabetic hearts [31]. However, other studies have observed an increased protein expression of both iNOS and eNOS in the left ventricular tissues of streptozotocin-induced diabetic rats [32–34]. A recent study carried out in streptozotocin-induced diabetic rats showed decreased protein levels of phosphorylatedeNOS but increased levels of iNOS protein [35]. The role of iNOS in cardiomyocyte cell death associated with DCM was suggested by Puthanveetil et al. [36] who reported that iNOS, on being induced by foxo1, mediated the nitrosylation of GAPDH and caspase-3 which subsequently caused cardiomyocyte cell death. iNOS, on being induced, continuously produces NO until the enzyme is degraded, and this leads to excessive NO formation, which contributes to contractile dysfunction [37]. Moreover, due to substrate limitation, iNOS may become uncoupled and produce reactive oxygen species (ROS) and contribute to oxidative stress, resulting in contractile dysfunction and heart failure (HF). In fact Jo et al. [38] showed that oral administration of sepiapterin, which inhibited iNOS uncoupling improved left ventricular function in streptozotocininduced diabetic mice confirming a role for iNOS uncoupling in the pathogenesis of DCM. eNOS uncoupling has also been reported in DCM; administration of L-NAME, an NOS inhibitor was shown to reduce the levels of NO, nitrotyrosine and ROS in diabetic animals, indicating the occurrence of eNOS uncoupling in diabetic hearts [39,40]. There are few studies on the status of nNOS in diabetic cardiomyopathy. Bardell et al. [30] found no change in the expression or activity of nNOS in GK (Goto-Kakizaki rats) diabetic rats. However, Jesmin et al. [33] reported a reduced expression of nNOS in the hearts of streptozotocin-induced diabetic rats. Saraiva et al. [41] also reported a reduced expression of cardiac nNOS at mRNA and protein level in leptin deficient (ob/ob) mice with an associated increase in oxidative-stress. However, Amour et al. [42] reported an increased expression of nNOS which was suggested to play a role in decreased positive inotropic response to β3-adrenoceptor
3
stimulation during DCM. Specific nNOS inhibition was able to restore the positive inotropic response. The mechanisms regulating expression of different NOS forms in DCM are not well known (Fig. 3). Nagareddy et al. [43] have proposed that hyperglycemia induced increase in iNOS may be mediated by PKC-β2. They observed increased expression of PKC-β2 and iNOS in high glucose treated cardiomyocytes and in the hearts of streptozotocin-induced diabetic rats and these effects could be attenuated by inhibiting PKC-β2 either by an inhibitor (LY333531) or by siRNA. Further, PKC-β2 inhibition also corrected the cardiovascular abnormalities in streptozotocin-induced diabetic rats suggesting that diabetes induced dysregulation of iNOS may be mediated by PKC-β2. Soliman et al. [44] have proposed that PKC induced upregulation of iNOS in DCM may be mediated by RhoA/ROCK pathway. They showed that inhibition of PKCβ2, iNOS, RhoA, or ROCK disrupted the expression of each other and suggested that RhoA/ROCK, PKCβ2 and iNOS interact with each other and form a positive feedback loop sustaining their activation in cardiomyocytes isolated from diabetic rat hearts. In a recent study, Lei et al. [45] showed a link between PKCβ2, caveolin-3 and Akt/eNOS/NO signaling and cardiac dysfunction associated with diabetes. They reported an increased expression of cardiac PKCβ2 along with a decrease in caveolin-3 expression in diabetic rats and in high-glucose treated cardiomyocytes. Further, inhibition of PKCβ2 resulted in increased caveolin-3 expression and phosphorylation of Akt and eNOS and decreased iNOS expression and improved diastolic dysfunction in diabetic rats, suggesting PKC-β inhibition to be a potential therapeutic target for DCM. 5. Epigenetic regulation of NOS Epigenetics refers to heritable changes in gene function that occur without a change in the nucleotide sequence [46]. Epigenetic modifications such as DNA methylations, histone modifications, and microRNAs are important regulators of gene expression, function and have been implicated in pathophysiology of diverse diseases including cardiovascular diseases. Current literature shows that epigenetic alterations of pathways play an important role in pathophysiology of DCM [47,48]. Since, altered cardiac NOS expression is involved in the pathophysiology of DCM; in this section, we review the role of epigenetics in regulation of NOS cardiovascular diseases and in DCM. Epigenetic regulation of NOS isoforms can have important role in cardiovascular diseases. Expression of eNOS mRNA is restricted to the endothelial cell layer of arterial blood vessels. Chan et al. [49] showed that this restricted expression of eNOS is controlled by promoter DNA methylation of constitutively expressed gene in the vascular endothelium. Besides DNA methylation, histone modifications have been also found to regulate eNOS expression; endothelial cells were found to have increased histone H3 lysine 9, histone H4 lysine 12 acetylation, and di- and tri-methylated lysine 4 of histone H3 at the core promoter [50]. These reports suggest that NOS promoter methylation and histone modifications regulate cell specific eNOS expression. Breton et al. [51] reported that altered DNA methylation of nNOS may be involved in the pathogenesis of atherogenesis through regulation of NO production. Rao et al. [52] observed that increased methylation of promoter regions of vascular endothelial growth factor receptor 2 (VEGF-R2) and eNOS is critical for angiogenesis. Methylated CpG sites lead to methyl-CpG–binding domain 2 (MBD2) mediated promoter suppression of VEGF-R2 and eNOS in angiogenesis. Apart from promoter methylation and histone modifications, microRNA have been shown to play an important role in negatively controlling gene expression by hybridization to the 3′UTR of their
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HYPERGLYCEMIA Caveolae
eNOS
NO
Akt Cav-3
PI3K
PKC-β2
iNOS
NO
ROCK
RhoA
Cardiomyocyte
Damage Fig. 3. Mechanisms regulating expression of eNOS and iNOS in DCM: Hyperglycemia induced increase in iNOS may be mediated by PKC-β2 and these molecules are in positive feedback loop with RhoA and ROCK. This figure also shows a link between PKCβ2, caveolin-3 (Cav-3), Akt/eNOS/NO signaling and cardiac dysfunction associated with diabetes.
target mRNA, inhibiting mRNA translation or promoting its degradation [53]. Recent studies have shown that microRNAs may play an important role in pathophysiology of various cardiovascular diseases including DCM [48,54,55]. The role of microRNAs in regulating NOS expression is not well studied, however few recent studies suggest that microRNAs may regulate expression of eNOS and iNOS. For example, Liu et al. [56] have reported regulation of eNOS expression by miR-30c in the regulation of the cardiovascular system. Weber et al. [57] evaluated the role of microRNAs in response to shear stress given to endothelial cells. They found that miR-21 upregulation in response to the mechanical stress led to downregulation of PTEN, a known antagonist of PI3K/Akt/eNOS pathway, suggesting that miR-21 may regulate the expression of eNOS via PTEN under shear stress. Similarly, Guo et al. [58] reported that miR-939 directly targeted iNOS in cytokine-stimulated human hepatocytes and inhibited its expression. Meloni et al. [59] have suggested a protective role of miR-24 in myocardial infarction (MI). They reported an increased expression of miR-24 in endothelial cells (ECs) and lower expression in the peri-infarct tissue, its resident cardiomyocytes and fibroblasts after myocardial infarction (MI). eNOS was identified as direct target of miR-24 in human cultured ECs and miR-24 delivery reduced infarct size, induced fibroblast apoptosis and increased angiogenesis and blood perfusion in the peri-infarct myocardium and overall improved cardiac function [59]. The role of microRNA in regulating NOS expression in DCM is not well studied. In a recent study, Patella et al. [60] have shown increased expression of miR-492 in high-glucose treated HUVEC cells which was associated with downregulation of eNOS. They observed that miR-492 indirectly targeted eNOS under hyperglycemic condition via suppression of SP1 and PDPK1, the known regulators of eNOS expression.
6. Conclusion The nitric oxide pathway plays an important role in maintaining normal cardiac functioning. Dysregulated expression and activity of NOS isoforms appear to contribute to contractile dysfunction, endothelial dysfunction, cardiomyocyte cell death and dysfunction in inotropic response seen in DCM. However, mechanisms regulating the expression and activity of NOS isoforms in DCM are not well studied. Various NOS mediated molecular mechanisms, such as increased nitrosative stress, NOS uncoupling leading to oxidative stress have been proposed to play a role in the pathophysiology of diabetesassociated cardiomyopathy. The experimental findings attained so far highlight the importance of maintaining normal NOS expression and function in the myocardium. Thus, an in-depth knowledge regarding the regulation of the NOS isoforms in diabetic cardiomyopathy is needed and could provide newer therapeutic targets for treating DCM. Conflict of interest The authors declare that they have no conflict of interest. References [1] S.A. Hayat, B. Patel, R.S. Khattar, R.A. Malik, Diabetic cardiomyopathy: mechanisms, diagnosis and treatment, Clin. Sci. 107 (6) (2004) 539–557. [2] J.G. Duncan, Mitochondrial dysfunction in diabetic cardiomyopathy, Biochim. Biophys. Acta 1813 (7) (2011) 1351–1359. [3] T. Miki, S. Yuda, H. Kouzu, T. Miura, Diabetic cardiomyopathy: pathophysiology and clinical features, Heart Fail. Rev. 18 (2) (2013) 149–166. [4] P. Geraldes, G.L. King, Activation of protein kinase C isoforms and its impact on diabetic complications, Circ. Res. 106 (8) (2010) 1319–1331.
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[5] L.A. Barouch, R.W. Harrison, M.W. Skaf, G.O. Rosas, T.P. Cappola, Z.A. Kobeissi, et al., Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms, Nature 416 (6878) (2002) 337–339. [6] I.N. Mungrue, D.S. Bredt, nNOS at a glance: implications for brain and brawn, J. Cell Sci. 117 (Pt 13) (2004) 2627–2629. [7] C.E. Sears, S.M. Bryant, E.A. Ashley, C.A. Lygate, S. Rakovic, H.L. Wallis, et al., Cardiac neuronal nitric oxide synthase isoform regulates myocardial contraction and calcium handling, Circ. Res. 92 (5) (2003) e52–e59. [8] O. Gealekman, Z. Abassi, I. Rubinstein, J. Winaver, O. Binah, Role of myocardial inducible nitric oxide synthase in contractile dysfunction and beta-adrenergic hyporesponsiveness in rats with experimental volume-overload heart failure, Circulation 105 (2) (2002) 236–243. [9] J.L. Balligand, P.J. Cannon, Nitric oxide synthases and cardiac muscle. Autocrine and paracrine influences, Arterioscler. Thromb. Vasc. Biol. 17 (10) (1997) 1846–1858. [10] S. Moncada, R.M. Palmer, E.A. Higgs, Nitric oxide: physiology, pathophysiology, and pharmacology, Pharmacol. Rev. 43 (2) (1991) 109–142. [11] K.M. Naseem, The role of nitric oxide in cardiovascular diseases, Mol. Aspects Med. 26 (1–2) (2005) 33–65. [12] S. Janssens, P. Pokreisz, L. Schoonjans, M. Pellens, P. Vermeersch, M. Tjwa, et al., Cardiomyocyte-specific overexpression of nitric oxide synthase 3 improves left ventricular performance and reduces compensatory hypertrophy after myocardial infarction, Circ. Res. 94 (9) (2004) 1256–1262. [13] M. Scherrer-Crosbie, R. Ullrich, K.D. Bloch, H. Nakajima, B. Nasseri, H.T. Aretz, et al., Endothelial nitric oxide synthase limits left ventricular remodeling after myocardial infarction in mice, Circulation 104 (11) (2001) 1286–1291. [14] F. Ichinose, K.D. Bloch, J.C. Wu, R. Hataishi, H.T. Aretz, M.H. Picard, et al., Pressure overload-induced LV hypertrophy and dysfunction in mice are exacerbated by congenital NOS3 deficiency, Am. J. Physiol. Heart Circ. Physiol. 286 (3) (2004) H1070–H1075. [15] R.M. Saraiva, K.M. Minhas, S.V.Y. Raju, L.A. Barouch, E. Pitz, K.H. Schuleri, et al., Deficiency of neuronal nitric oxide synthase increases mortality and cardiac remodeling after myocardial infarction: role of nitroso-redox equilibrium, Circulation 112 (22) (2005) 3415–3422. [16] D. Dawson, C.A. Lygate, M.-H. Zhang, K. Hulbert, S. Neubauer, B. Casadei, nNOS gene deletion exacerbates pathological left ventricular remodeling and functional deterioration after myocardial infarction, Circulation 112 (24) (2005) 3729–3737. [17] P. Krenek, J. Kmecova, D. Kucerova, Z. Bajuszova, P. Musil, A. Gazova, et al., Isoproterenol-induced heart failure in the rat is associated with nitric oxidedependent functional alterations of cardiac function, Eur. J. Heart Fail. 11 (2) (2009) 140–146. [18] J. Heger, A. Gödecke, U. Flögel, M.W. Merx, A. Molojavyi, W.N. Kühn-Velten, et al., Cardiac-specific overexpression of inducible nitric oxide synthase does not result in severe cardiac dysfunction, Circ. Res. 90 (1) (2002) 93–99. [19] F. Sam, D.B. Sawyer, Z. Xie, D.L. Chang, S. Ngoy, D.A. Brenner, et al., Mice lacking inducible nitric oxide synthase have improved left ventricular contractile function and reduced apoptotic cell death late after myocardial infarction, Circ. Res. 89 (4) (2001) 351–356. [20] Y.-H. Liu, O.A. Carretero, O.H. Cingolani, T.-D. Liao, Y. Sun, J. Xu, et al., Role of inducible nitric oxide synthase in cardiac function and remodeling in mice with heart failure due to myocardial infarction, Am. J. Physiol. Heart Circ. Physiol. 289 (6) (2005) H2616–H2623. [21] P. Zhang, X. Xu, X. Hu, E.D. van Deel, G. Zhu, Y. Chen, Inducible nitric oxide synthase deficiency protects the heart from systolic overload-induced ventricular hypertrophy and congestive heart failure, Circ. Res. 100 (7) (2007) 1089–1098. [22] K. Shibata, H. Shimokawa, N. Yanagihara, Y. Otsuji, M. Tsutsui, Nitric oxide synthases and heart failure – lessons from genetically manipulated mice, J. UOEH 35 (2) (2013) 147–158. [23] S.D. Prabhu, Nitric oxide protects against pathological ventricular remodeling: reconsideration of the role of NO in the failing heart, Circ. Res. 94 (9) (2004) 1155–1157. [24] I.N. Mungrue, M. Husain, D.J. Stewart, The role of NOS in heart failure: lessons from murine genetic models, Heart Fail. Rev. 7 (4) (2002) 407–422. [25] R.M. Saraiva, J.M. Hare, Nitric oxide signaling in the cardiovascular system: implications for heart failure, Curr. Opin. Cardiol. 21 (3) (2006) 221– 228. [26] M. Felaco, A. Grilli, M.A. De Lutiis, A. Patruno, N. Libertini, A.A. Taccardi, et al., Endothelial nitric oxide synthase (eNOS) expression and localization in healthy and diabetic rat hearts, Ann. Clin. Lab. Sci. 31 (2) (2001) 179–186. [27] U. Hink, H. Li, H. Mollnau, M. Oelze, E. Matheis, M. Hartmann, et al., Mechanisms underlying endothelial dysfunction in diabetes mellitus, Circ. Res. 88 (2) (2001) E14–E22. [28] R.C. Sampaio, J.E. Tanus-Santos, S.E.S.F.C. Melo, S. Hyslop, K.G. Franchini, I.M. Luca, et al., Hypertension plus diabetes mimics the cardiomyopathy induced by nitric oxide inhibition in rats, Chest 122 (4) (2002) 1412–1420. [29] J. MacMicking, Q.W. Xie, C. Nathan, Nitric oxide and macrophage function, Annu. Rev. Immunol. 15 (1997) 323–350. [30] A.L. Bardell, K.M. MacLeod, Evidence for inducible nitric-oxide synthase expression and activity in vascular smooth muscle of streptozotocin-diabetic rats, J. Pharmacol. Exp. Ther. 296 (2) (2001) 252–259. [31] P.R. Nagareddy, Z. Xia, J.H. McNeill, K.M. MacLeod, Increased expression of iNOS is associated with endothelial dysfunction and impaired pressor responsiveness in streptozotocin-induced diabetes, Am. J. Physiol. Heart Circ. Physiol. 289 (5) (2005) H2144–H2152.
5
[32] H. Farhangkhoee, Z.A. Khan, S. Chen, S. Chakrabarti, Differential effects of curcumin on vasoactive factors in the diabetic rat heart, Nutr. Metab. 3 (2006) 27. [33] S. Jesmin, S. Zaedi, S. Maeda, I. Yamaguchi, K. Goto, T. Miyauchi, Effects of a selective endothelin a receptor antagonist on the expressions of iNOS and eNOS in the heart of early streptozotocin-induced diabetic rats, Exp. Biol. Med. (Maywood) 231 (6) (2006) 925–931. [34] M.J. Crespo, J. Zalacaín, D.C. Dunbar, N. Cruz, L. Arocho, Cardiac oxidative stress is elevated at the onset of dilated cardiomyopathy in streptozotocin-diabetic rats, J. Cardiovasc. Pharmacol. Ther. 13 (1) (2008) 64–71. [35] J. Cao, C. Vecoli, D. Neglia, B. Tavazzi, G. Lazzarino, M. Novelli, et al., Cobaltprotoporphyrin improves heart function by blunting oxidative stress and restoring NO synthase equilibrium in an animal model of experimental diabetes, Front. Physiol 3 (2012) 160. [36] P. Puthanveetil, D. Zhang, Y. Wang, F. Wang, A. Wan, A. Abrahani, et al., Diabetes triggers a PARP1 mediated death pathway in the heart through participation of FoxO1, J. Mol. Cell. Cardiol. 53 (5) (2012) 677–686. [37] A. Pautz, J. Art, S. Hahn, S. Nowag, C. Voss, H. Kleinert, Regulation of the expression of inducible nitric oxide synthase, Nitric Oxide 23 (2) (2010) 75– 93. [38] H. Jo, H. Otani, F. Jo, T. Shimazu, T. Okazaki, K. Yoshioka, et al., Inhibition of nitric oxide synthase uncoupling by sepiapterin improves left ventricular function in streptozotocin-induced diabetic mice, Clin. Exp. Pharmacol. Physiol. 38 (8) (2011) 485–493. [39] J. Ren, J. Duan, D.P. Thomas, X. Yang, N. Sreejayan, J.R. Sowers, et al., IGF-I alleviates diabetes-induced RhoA activation, eNOS uncoupling, and myocardial dysfunction, Am. J. Physiol. Regul. Integr. Comp. Physiol. 294 (3) (2008) R793–R802. [40] N.D. Roe, D.P. Thomas, J. Ren, Inhibition of NADPH oxidase alleviates experimental diabetes-induced myocardial contractile dysfunction, Diabetes Obes. Metab. 13 (5) (2011) 465–473. [41] R.M. Saraiva, K.M. Minhas, M. Zheng, E. Pitz, A. Treuer, D. Gonzalez, et al., Reduced neuronal nitric oxide synthase expression contributes to cardiac oxidative stress and nitroso-redox imbalance in ob/ob mice, Nitric Oxide 16 (3) (2007) 331–338. [42] J. Amour, X. Loyer, M. Le Guen, N. Mabrouk, J.-S. David, E. Camors, et al., Altered contractile response due to increased beta3-adrenoceptor stimulation in diabetic cardiomyopathy: the role of nitric oxide synthase 1-derived nitric oxide, Anesthesiology 107 (3) (2007) 452–460. [43] P.R. Nagareddy, H. Soliman, G. Lin, P.S. Rajput, U. Kumar, J.H. McNeill, et al., Selective inhibition of protein kinase C beta(2) attenuates inducible nitric oxide synthase-mediated cardiovascular abnormalities in streptozotocin-induced diabetic rats, Diabetes 58 (10) (2009) 2355–2364. [44] H. Soliman, A. Gador, Y.-H. Lu, G. Lin, G. Bankar, K.M. MacLeod, Diabetes-induced increased oxidative stress in cardiomyocytes is sustained by a positive feedback loop involving Rho kinase and PKC 2, Am. J. Physiol. Heart Circ. Physiol. 303 (8) (2012) H989–H1000. [45] S. Lei, H. Li, J. Xu, Y. Liu, X. Gao, J. Wang, et al., Hyperglycemia-induced protein kinase C β2 activation induces diastolic cardiac dysfunction in diabetic rats by impairing caveolin-3 expression and Akt/eNOS signaling, Diabetes 62 (7) (2013) 2318–2328. [46] P. Cubas, C. Vincent, E. Coen, An epigenetic mutation responsible for natural variation in floral symmetry, Nature 401 (6749) (1999) 157–161. [47] G.B. Singh, R. Sharma, M. Khullar, Epigenetics and diabetic cardiomyopathy, Diabetes Res. Clin. Pract. 94 (1) (2011) 14–21. [48] M. Asrih, S. Steffens, Emerging role of epigenetics and miRNA in diabetic cardiomyopathy, Cardiovasc. Pathol. 22 (2) (2013) 117–125. [49] Y. Chan, J.E. Fish, C. D’Abreo, S. Lin, G.B. Robb, A.-M. Teichert, et al., The cell-specific expression of endothelial nitric-oxide synthase: a role for DNA methylation, J. Biol. Chem. 279 (33) (2004) 35087–35100. [50] J.E. Fish, C.C. Matouk, A. Rachlis, S. Lin, S.C. Tai, C. D’Abreo, et al., The expression of endothelial nitric-oxide synthase is controlled by a cell-specific histone code, J. Biol. Chem. 280 (26) (2005) 24824–24838. [51] C.V. Breton, C. Park, K. Siegmund, W.J. Gauderman, L. Whitfield-Maxwell, H.N. Hodis, et al., NOS1 methylation and carotid artery intima-media thickness in children, Circ. Cardiovasc. Genet. 7 (2) (2014) 116–122. [52] X. Rao, J. Zhong, S. Zhang, Y. Zhang, Q. Yu, P. Yang, et al., Loss of methyl-CpGbinding domain protein 2 enhances endothelial angiogenesis and protects mice against hind-limb ischemic injury, Circulation 123 (25) (2011) 2964– 2974. [53] A. Rodriguez, S. Griffiths-Jones, J.L. Ashurst, A. Bradley, Identification of mammalian microRNA host genes and transcription units, Genome Res. 14 (10A) (2004) 1902–1910. [54] B. Feng, S. Chen, B. George, Q. Feng, S. Chakrabarti, miR133a regulates cardiomyocyte hypertrophy in diabetes, Diabetes Metab. Res. Rev. 26 (1) (2010) 40–49. [55] S. Chen, P. Puthanveetil, B. Feng, S.J. Matkovich, G.W. Dorn 2nd, S. Chakrabarti, Cardiac miR-133a overexpression prevents early cardiac fibrosis in diabetes, J. Cell. Mol. Med. 18 (3) (2014) 415–421. [56] B. Liu, J. Hewinson, H. Xu, F. Montero, C.R. Sunico, F. Portillo, et al., NOS antagonism using viral vectors as an experimental strategy: implications for in vivo studies of cardiovascular control and peripheral neuropathies, Meth. Mol. Biol. 704 (2011) 197–223. [57] M. Weber, M.B. Baker, J.P. Moore, C.D. Searles, miR-21 is induced in endothelial cells by shear stress and modulates apoptosis and eNOS activity, Biochem. Biophys. Res. Commun. 393 (4) (2010) 643–648.
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[58] Z. Guo, L. Shao, L. Zheng, Q. Du, P. Li, B. John, et al., miRNA-939 regulates human inducible nitric oxide synthase posttranscriptional gene expression in human hepatocytes, Proc. Natl Acad. Sci. U.S.A. 109 (15) (2012) 5826–5831. [59] M. Meloni, M. Marchetti, K. Garner, B. Littlejohns, G. Sala-Newby, N. Xenophontos, et al., Local inhibition of microRNA-24 improves reparative
angiogenesis and left ventricle remodeling and function in mice with myocardial infarction, Mol. Ther. 21 (7) (2013) 1390–1402. [60] F. Patella, E. Leucci, M. Evangelista, B. Parker, J. Wen, A. Mercatanti, et al., MiR-492 impairs the angiogenic potential of endothelial cells, J. Cell. Mol. Med. 17 (8) (2013) 1006–1015.
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Nitric Oxide j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n i o x
Nitric oxide synthases and diabetic cardiomyopathy Sanskriti Khanna, Gurinder Bir Singh, Madhu Khullar * Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
A R T I C L E
I N F O
Article history: Received 27 May 2014 Revised 22 July 2014 Available online Keywords: NOS Diabetic cardiomyopathy Epigenetic
A B S T R A C T
Cardiovascular complications associated with diabetes significantly contribute to high mortality and morbidity worldwide. The pathophysiology of diabetic cardiomyopathy (DCM), although extensively researched upon, is partially understood. Impairment in various signaling pathways including nitric oxide (NO) signaling has been implicated in the pathogenesis of diabetes induced myocardial damage. Nitric oxide synthases (NOS), the enzymes responsible for NO generation, play an important role in various physiological processes. Altered expression and activity of NOS have been implicated in cardiovascular diseases, however, the role of NOS and their regulation in the pathogenesis of DCM remain poorly understood. In the present review, we focus on the role of myocardial NOS in the development of DCM. Since epigenetic modifications play an important role in regulation of gene expression, this review also describes the epigenetic regulation of NOS. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Cardiovascular disease is a significant contributor to the high mortality and morbidity associated with diabetes. Nearly 80% of the deaths associated with diabetes are attributed to cardiac complications [1]. It has been observed that diabetic patients tend to develop heart failure in the absence of risk factors such as hypertension or coronary artery disease. The cardiovascular complications in diabetic patients in the absence or out of proportion to their underlying vascular disease have been termed as “diabetic cardiomyopathy” (DCM) [2]. It has been suggested that DCM is a frequently unrecognized pathological process in asymptomatic diabetic patients [3]. The pathophysiology of diabetic cardiomyopathy is partially understood, but both hyperglycemia and changes in cardiac metabolism contribute to the disease process. Dysregulation of multiple biochemical pathways such as enhanced reactive oxygen production, nonenzymatic glycation, polyol pathway and advanced glycation end products pathway, activation of the diacylglycerol–protein kinase C (PKC) pathway and NO signaling pathway has been proposed to link the adverse effects of persistent hyperglycemia with DCM (Fig. 1). Geraldes and King [4] have proposed that hyperglycemia may lead to alterations in cellular signaling pathways resulting in cellular dysfunction and heart failure in diabetic patients. Impaired NO signaling has been implicated in the genesis of diabetes induced myocardial damage, however, molecular mechanisms of NO signaling leading to pathogenesis of DCM remain poorly understood. In the
* Corresponding author. Fax: +91 1722744401. E-mail address: [email protected] (M. Khullar).
present review, we focus on the role of myocardial NOS in the development of DCM. Since epigenetic modifications play an important role in regulation of gene expression, this review also describes the epigenetic regulation of NOS.
2. NOS in heart NO is constitutively or inductively synthesized during a reaction catalyzed by NOS which exists in three isoforms. All the three NOS isoforms, namely endothelial (eNOS), neuronal (nNOS), and inducible (iNOS), are expressed in the heart although at spatially confined sub-cellular locations [5] (Fig. 2). In cardiomyocytes, eNOS localizes at the caveolae where it associates with caveolin-3 and modulates several signal transduction pathways [5]. nNOS, however, is confined to the sarcoplasmic reticulum of cardiac myocyte [6] where it regulates the activity of ryanodine receptor, sarcoplasmic reticulum Ca2+-ATPase (SERCA) or the L-type Ca2+ channel [7]. Whereas, iNOS is expressed during pathological states and is localized throughout the cytoplasm [8]. The functions of NOS in heart under normal physiological conditions have been reviewed by Balligand and Cannon [9]. NO is a free radical which reacts with various molecules to cause multiple biological effects. Under normal circumstances, the controlled production of NO (by eNOS) conveys antiapoptotic signal, inhibits platelet aggregation and adhesion to the vascular wall, besides playing an important role in cardiac contractile function. The vasodilatory function of NO is mediated via the activation of guanylate cyclase. NO also has cardio-protective effects [10]. Depending upon its source and level of output, NO can either play a cardio-protective or a detrimental role [9]. iNOS is absent in the healthy heart but, as the name indicates, it is induced in cells
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2
Diabetes
Hyperglycemia Defective Insulin Signaling iNOS
Free Fatty Acids Triglyceride
RNS GLUT
ROS PKCβ2
AGE
Fatty Acid Uptake
Glucose Oxidation Altered Calcium Homeostasis
Cardiac Lipid Accumulation
Fatty Acid Oxidation
Glucotoxicity Lipotoxicity Fibrosis Hypertrophy Mitochondrial Dysfunction Altered Calcium Homeostasis
DIABETIC CARDIOMYOPATHY Fig. 1. Pathophysiology of diabetic cardiomyopathy: Both hyperglycemia and changes in cardiac metabolism contribute to alterations in multiple biochemical pathways such as enhanced reactive oxygen species (ROS) production, reactive nitrogen species (RNS) production, advanced glycation end products (AGE) pathway, activation of the diacylglycerol–protein kinase C (PKC) pathway and NO signaling pathway resulting in diabetic cardiomyopathy. Impaired NO signaling has been implicated in the genesis of diabetes induced myocardial damage.
under stress conditions, including hyperglycemia, oxidative stress and hyperinsulinemia. An increased myocardial NO production can cause nitration of actin and other cytoskeletal proteins in the myocardium. This alters their structure and may have damaging effects on the contractile function of myofilaments. NO also interacts with oxygen radicals and leads to the formation of peroxynitrite, a potent oxidant, which in high concentration causes tissue damage [9].
caveolae
eNOS
NO
Fig. 2. Subcellular localization of NOS isoforms in cardiomyocytes: In cardiomyocytes, eNOS localizes at the caveolae where it controls heart rate, contraction, diastolic relaxation and oxygen consumption; nNOS is confined to the sarcoplasmic reticulum of cardiac myocyte where it regulates the activity of sarcoplasmic reticulum Ca2+ATPase (SERCA) or the L-type Ca2+ channel (LCC); whereas, iNOS is expressed during pathological states and is localized throughout the cytoplasm. iNOS, on being induced, continuously produces NO until the enzyme is degraded, leading to the formation of peroxynitrile (ONOO−) which contributes to contractile dysfunction and even apoptosis.
3. NOS in cardiovascular diseases Dysregulated expression and activity of NOS isoforms have been implicated in pathophysiology of several cardiovascular diseases including atherosclerosis, hypertension and diabetes. In general, eNOS has been found to play a protective role and nNOS has been shown to play iNOS a deterimental role in heart failure [11]. For example, Barouch et al. [5] reported that nNOS and eNOS-deficient mice developed age-related increase in LV wall thickness and mass and n/eNOS double knockout mice developed phenotype which was similar to that seen in hypertensive hypertrophic cardiomyopathy in humans, suggesting that both these isoforms play an important role in maintaining the architecture and function of heart. The protective role of eNOS was further confirmed by Janssens et al. [12], who observed that cardiomyocyte-restricted eNOS overexpression was protective against adverse left ventricular (LV) remodeling after coronary artery ligation in transgenic mice. Consistent with these findings, an increased mortality, exacerbation of LV remodeling, and LV dysfunction has been reported in eNOS-knockout mice with heart failure due to myocardial infarction [13] or to pressure overload [14]. Similarly, deficiency of nNOS was shown to accelerate adverse LV remodeling in myocardial injury-induced heart failure in nNOSdeficient mice [15,16] indicating that nNOS has a protective role in myocardial infarction induced HF. In contrast, an increased cardiac iNOS expression has been linked with volume-overload–induced HF [8] and in isoproterenol-induced heart failure [17]. Although cardiomyocyte-specific overexpression of iNOS did not lead to heart failure [18], iNOS-knockout mice showed improved survival during myocardial infarction-induced [19,20] or pressure overloadinduced heart failure [21]. The role of different NOS isoforms in the pathogenesis of heart failure was recently confirmed by Shibata et al. [22] who generated triple negative (n/i/eNOS−/−) mice in which all three NOS isoforms were completely disrupted. Their results
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confirmed a protective role of eNOS and nNOS in heart failure whereas iNOS was found to exert unfavorable role. The molecular mechanisms of contrasting roles of NOS isoforms in heart failure are unclear, but have been proposed to be due to differences in spatial localization, differential regulation and NO-generating capacity of different isoforms [23–25].
4. NOS in diabetic cardiomyopathy Endothelial dysfunction is an important feature of DCM and has been attributed to dysregulation of NO generation and bioavailabilty. Felaco et al. [26] reported decreased levels of eNOS protein and of its co-enzyme, NADPH-d, in the endothelium and vascular wall (but not in the myocardium) of BB/W diabetic rat hearts as compared to healthy control rats suggesting that dysregulation of NOS pathway was involved in pathogenesis of diabetes and associated cardiovascular complications. This was further confirmed by Hink et al. [27] who found increased expression of eNOS in diabetic hearts and by Sampaio et al. [28] who observed that cardiac abnormalities in diabetic rats resembled those found in L-NAME–induced cardiomyopathy suggesting a role for the NO system in the pathophysiology of DCM. iNOS is an inducible enzyme whose expression can be induced in all cells and tissues by cytokines and other agents [29]. Besides eNOS, altered expression and activity of iNOS have been shown to contribute to diabetes associated cardiovascular complications. For example, an increased expression of iNOS was seen in mesenteric arteries (SMA) of streptozotocin-induced diabetic rats [30]. An increased expression of myocardial iNOS along with a concurrent downregulation of eNOS has been reported in streptozotocin induced diabetic rats, indicating differential regulation of eNOS and iNOS isoforms in diabetic hearts [31]. However, other studies have observed an increased protein expression of both iNOS and eNOS in the left ventricular tissues of streptozotocin-induced diabetic rats [32–34]. A recent study carried out in streptozotocin-induced diabetic rats showed decreased protein levels of phosphorylatedeNOS but increased levels of iNOS protein [35]. The role of iNOS in cardiomyocyte cell death associated with DCM was suggested by Puthanveetil et al. [36] who reported that iNOS, on being induced by foxo1, mediated the nitrosylation of GAPDH and caspase-3 which subsequently caused cardiomyocyte cell death. iNOS, on being induced, continuously produces NO until the enzyme is degraded, and this leads to excessive NO formation, which contributes to contractile dysfunction [37]. Moreover, due to substrate limitation, iNOS may become uncoupled and produce reactive oxygen species (ROS) and contribute to oxidative stress, resulting in contractile dysfunction and heart failure (HF). In fact Jo et al. [38] showed that oral administration of sepiapterin, which inhibited iNOS uncoupling improved left ventricular function in streptozotocininduced diabetic mice confirming a role for iNOS uncoupling in the pathogenesis of DCM. eNOS uncoupling has also been reported in DCM; administration of L-NAME, an NOS inhibitor was shown to reduce the levels of NO, nitrotyrosine and ROS in diabetic animals, indicating the occurrence of eNOS uncoupling in diabetic hearts [39,40]. There are few studies on the status of nNOS in diabetic cardiomyopathy. Bardell et al. [30] found no change in the expression or activity of nNOS in GK (Goto-Kakizaki rats) diabetic rats. However, Jesmin et al. [33] reported a reduced expression of nNOS in the hearts of streptozotocin-induced diabetic rats. Saraiva et al. [41] also reported a reduced expression of cardiac nNOS at mRNA and protein level in leptin deficient (ob/ob) mice with an associated increase in oxidative-stress. However, Amour et al. [42] reported an increased expression of nNOS which was suggested to play a role in decreased positive inotropic response to β3-adrenoceptor
3
stimulation during DCM. Specific nNOS inhibition was able to restore the positive inotropic response. The mechanisms regulating expression of different NOS forms in DCM are not well known (Fig. 3). Nagareddy et al. [43] have proposed that hyperglycemia induced increase in iNOS may be mediated by PKC-β2. They observed increased expression of PKC-β2 and iNOS in high glucose treated cardiomyocytes and in the hearts of streptozotocin-induced diabetic rats and these effects could be attenuated by inhibiting PKC-β2 either by an inhibitor (LY333531) or by siRNA. Further, PKC-β2 inhibition also corrected the cardiovascular abnormalities in streptozotocin-induced diabetic rats suggesting that diabetes induced dysregulation of iNOS may be mediated by PKC-β2. Soliman et al. [44] have proposed that PKC induced upregulation of iNOS in DCM may be mediated by RhoA/ROCK pathway. They showed that inhibition of PKCβ2, iNOS, RhoA, or ROCK disrupted the expression of each other and suggested that RhoA/ROCK, PKCβ2 and iNOS interact with each other and form a positive feedback loop sustaining their activation in cardiomyocytes isolated from diabetic rat hearts. In a recent study, Lei et al. [45] showed a link between PKCβ2, caveolin-3 and Akt/eNOS/NO signaling and cardiac dysfunction associated with diabetes. They reported an increased expression of cardiac PKCβ2 along with a decrease in caveolin-3 expression in diabetic rats and in high-glucose treated cardiomyocytes. Further, inhibition of PKCβ2 resulted in increased caveolin-3 expression and phosphorylation of Akt and eNOS and decreased iNOS expression and improved diastolic dysfunction in diabetic rats, suggesting PKC-β inhibition to be a potential therapeutic target for DCM. 5. Epigenetic regulation of NOS Epigenetics refers to heritable changes in gene function that occur without a change in the nucleotide sequence [46]. Epigenetic modifications such as DNA methylations, histone modifications, and microRNAs are important regulators of gene expression, function and have been implicated in pathophysiology of diverse diseases including cardiovascular diseases. Current literature shows that epigenetic alterations of pathways play an important role in pathophysiology of DCM [47,48]. Since, altered cardiac NOS expression is involved in the pathophysiology of DCM; in this section, we review the role of epigenetics in regulation of NOS cardiovascular diseases and in DCM. Epigenetic regulation of NOS isoforms can have important role in cardiovascular diseases. Expression of eNOS mRNA is restricted to the endothelial cell layer of arterial blood vessels. Chan et al. [49] showed that this restricted expression of eNOS is controlled by promoter DNA methylation of constitutively expressed gene in the vascular endothelium. Besides DNA methylation, histone modifications have been also found to regulate eNOS expression; endothelial cells were found to have increased histone H3 lysine 9, histone H4 lysine 12 acetylation, and di- and tri-methylated lysine 4 of histone H3 at the core promoter [50]. These reports suggest that NOS promoter methylation and histone modifications regulate cell specific eNOS expression. Breton et al. [51] reported that altered DNA methylation of nNOS may be involved in the pathogenesis of atherogenesis through regulation of NO production. Rao et al. [52] observed that increased methylation of promoter regions of vascular endothelial growth factor receptor 2 (VEGF-R2) and eNOS is critical for angiogenesis. Methylated CpG sites lead to methyl-CpG–binding domain 2 (MBD2) mediated promoter suppression of VEGF-R2 and eNOS in angiogenesis. Apart from promoter methylation and histone modifications, microRNA have been shown to play an important role in negatively controlling gene expression by hybridization to the 3′UTR of their
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HYPERGLYCEMIA Caveolae
eNOS
NO
Akt Cav-3
PI3K
PKC-β2
iNOS
NO
ROCK
RhoA
Cardiomyocyte
Damage Fig. 3. Mechanisms regulating expression of eNOS and iNOS in DCM: Hyperglycemia induced increase in iNOS may be mediated by PKC-β2 and these molecules are in positive feedback loop with RhoA and ROCK. This figure also shows a link between PKCβ2, caveolin-3 (Cav-3), Akt/eNOS/NO signaling and cardiac dysfunction associated with diabetes.
target mRNA, inhibiting mRNA translation or promoting its degradation [53]. Recent studies have shown that microRNAs may play an important role in pathophysiology of various cardiovascular diseases including DCM [48,54,55]. The role of microRNAs in regulating NOS expression is not well studied, however few recent studies suggest that microRNAs may regulate expression of eNOS and iNOS. For example, Liu et al. [56] have reported regulation of eNOS expression by miR-30c in the regulation of the cardiovascular system. Weber et al. [57] evaluated the role of microRNAs in response to shear stress given to endothelial cells. They found that miR-21 upregulation in response to the mechanical stress led to downregulation of PTEN, a known antagonist of PI3K/Akt/eNOS pathway, suggesting that miR-21 may regulate the expression of eNOS via PTEN under shear stress. Similarly, Guo et al. [58] reported that miR-939 directly targeted iNOS in cytokine-stimulated human hepatocytes and inhibited its expression. Meloni et al. [59] have suggested a protective role of miR-24 in myocardial infarction (MI). They reported an increased expression of miR-24 in endothelial cells (ECs) and lower expression in the peri-infarct tissue, its resident cardiomyocytes and fibroblasts after myocardial infarction (MI). eNOS was identified as direct target of miR-24 in human cultured ECs and miR-24 delivery reduced infarct size, induced fibroblast apoptosis and increased angiogenesis and blood perfusion in the peri-infarct myocardium and overall improved cardiac function [59]. The role of microRNA in regulating NOS expression in DCM is not well studied. In a recent study, Patella et al. [60] have shown increased expression of miR-492 in high-glucose treated HUVEC cells which was associated with downregulation of eNOS. They observed that miR-492 indirectly targeted eNOS under hyperglycemic condition via suppression of SP1 and PDPK1, the known regulators of eNOS expression.
6. Conclusion The nitric oxide pathway plays an important role in maintaining normal cardiac functioning. Dysregulated expression and activity of NOS isoforms appear to contribute to contractile dysfunction, endothelial dysfunction, cardiomyocyte cell death and dysfunction in inotropic response seen in DCM. However, mechanisms regulating the expression and activity of NOS isoforms in DCM are not well studied. Various NOS mediated molecular mechanisms, such as increased nitrosative stress, NOS uncoupling leading to oxidative stress have been proposed to play a role in the pathophysiology of diabetesassociated cardiomyopathy. The experimental findings attained so far highlight the importance of maintaining normal NOS expression and function in the myocardium. Thus, an in-depth knowledge regarding the regulation of the NOS isoforms in diabetic cardiomyopathy is needed and could provide newer therapeutic targets for treating DCM. Conflict of interest The authors declare that they have no conflict of interest. References [1] S.A. Hayat, B. Patel, R.S. Khattar, R.A. Malik, Diabetic cardiomyopathy: mechanisms, diagnosis and treatment, Clin. Sci. 107 (6) (2004) 539–557. [2] J.G. Duncan, Mitochondrial dysfunction in diabetic cardiomyopathy, Biochim. Biophys. Acta 1813 (7) (2011) 1351–1359. [3] T. Miki, S. Yuda, H. Kouzu, T. Miura, Diabetic cardiomyopathy: pathophysiology and clinical features, Heart Fail. Rev. 18 (2) (2013) 149–166. [4] P. Geraldes, G.L. King, Activation of protein kinase C isoforms and its impact on diabetic complications, Circ. Res. 106 (8) (2010) 1319–1331.
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[5] L.A. Barouch, R.W. Harrison, M.W. Skaf, G.O. Rosas, T.P. Cappola, Z.A. Kobeissi, et al., Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms, Nature 416 (6878) (2002) 337–339. [6] I.N. Mungrue, D.S. Bredt, nNOS at a glance: implications for brain and brawn, J. Cell Sci. 117 (Pt 13) (2004) 2627–2629. [7] C.E. Sears, S.M. Bryant, E.A. Ashley, C.A. Lygate, S. Rakovic, H.L. Wallis, et al., Cardiac neuronal nitric oxide synthase isoform regulates myocardial contraction and calcium handling, Circ. Res. 92 (5) (2003) e52–e59. [8] O. Gealekman, Z. Abassi, I. Rubinstein, J. Winaver, O. Binah, Role of myocardial inducible nitric oxide synthase in contractile dysfunction and beta-adrenergic hyporesponsiveness in rats with experimental volume-overload heart failure, Circulation 105 (2) (2002) 236–243. [9] J.L. Balligand, P.J. Cannon, Nitric oxide synthases and cardiac muscle. Autocrine and paracrine influences, Arterioscler. Thromb. Vasc. Biol. 17 (10) (1997) 1846–1858. [10] S. Moncada, R.M. Palmer, E.A. Higgs, Nitric oxide: physiology, pathophysiology, and pharmacology, Pharmacol. Rev. 43 (2) (1991) 109–142. [11] K.M. Naseem, The role of nitric oxide in cardiovascular diseases, Mol. Aspects Med. 26 (1–2) (2005) 33–65. [12] S. Janssens, P. Pokreisz, L. Schoonjans, M. Pellens, P. Vermeersch, M. Tjwa, et al., Cardiomyocyte-specific overexpression of nitric oxide synthase 3 improves left ventricular performance and reduces compensatory hypertrophy after myocardial infarction, Circ. Res. 94 (9) (2004) 1256–1262. [13] M. Scherrer-Crosbie, R. Ullrich, K.D. Bloch, H. Nakajima, B. Nasseri, H.T. Aretz, et al., Endothelial nitric oxide synthase limits left ventricular remodeling after myocardial infarction in mice, Circulation 104 (11) (2001) 1286–1291. [14] F. Ichinose, K.D. Bloch, J.C. Wu, R. Hataishi, H.T. Aretz, M.H. Picard, et al., Pressure overload-induced LV hypertrophy and dysfunction in mice are exacerbated by congenital NOS3 deficiency, Am. J. Physiol. Heart Circ. Physiol. 286 (3) (2004) H1070–H1075. [15] R.M. Saraiva, K.M. Minhas, S.V.Y. Raju, L.A. Barouch, E. Pitz, K.H. Schuleri, et al., Deficiency of neuronal nitric oxide synthase increases mortality and cardiac remodeling after myocardial infarction: role of nitroso-redox equilibrium, Circulation 112 (22) (2005) 3415–3422. [16] D. Dawson, C.A. Lygate, M.-H. Zhang, K. Hulbert, S. Neubauer, B. Casadei, nNOS gene deletion exacerbates pathological left ventricular remodeling and functional deterioration after myocardial infarction, Circulation 112 (24) (2005) 3729–3737. [17] P. Krenek, J. Kmecova, D. Kucerova, Z. Bajuszova, P. Musil, A. Gazova, et al., Isoproterenol-induced heart failure in the rat is associated with nitric oxidedependent functional alterations of cardiac function, Eur. J. Heart Fail. 11 (2) (2009) 140–146. [18] J. Heger, A. Gödecke, U. Flögel, M.W. Merx, A. Molojavyi, W.N. Kühn-Velten, et al., Cardiac-specific overexpression of inducible nitric oxide synthase does not result in severe cardiac dysfunction, Circ. Res. 90 (1) (2002) 93–99. [19] F. Sam, D.B. Sawyer, Z. Xie, D.L. Chang, S. Ngoy, D.A. Brenner, et al., Mice lacking inducible nitric oxide synthase have improved left ventricular contractile function and reduced apoptotic cell death late after myocardial infarction, Circ. Res. 89 (4) (2001) 351–356. [20] Y.-H. Liu, O.A. Carretero, O.H. Cingolani, T.-D. Liao, Y. Sun, J. Xu, et al., Role of inducible nitric oxide synthase in cardiac function and remodeling in mice with heart failure due to myocardial infarction, Am. J. Physiol. Heart Circ. Physiol. 289 (6) (2005) H2616–H2623. [21] P. Zhang, X. Xu, X. Hu, E.D. van Deel, G. Zhu, Y. Chen, Inducible nitric oxide synthase deficiency protects the heart from systolic overload-induced ventricular hypertrophy and congestive heart failure, Circ. Res. 100 (7) (2007) 1089–1098. [22] K. Shibata, H. Shimokawa, N. Yanagihara, Y. Otsuji, M. Tsutsui, Nitric oxide synthases and heart failure – lessons from genetically manipulated mice, J. UOEH 35 (2) (2013) 147–158. [23] S.D. Prabhu, Nitric oxide protects against pathological ventricular remodeling: reconsideration of the role of NO in the failing heart, Circ. Res. 94 (9) (2004) 1155–1157. [24] I.N. Mungrue, M. Husain, D.J. Stewart, The role of NOS in heart failure: lessons from murine genetic models, Heart Fail. Rev. 7 (4) (2002) 407–422. [25] R.M. Saraiva, J.M. Hare, Nitric oxide signaling in the cardiovascular system: implications for heart failure, Curr. Opin. Cardiol. 21 (3) (2006) 221– 228. [26] M. Felaco, A. Grilli, M.A. De Lutiis, A. Patruno, N. Libertini, A.A. Taccardi, et al., Endothelial nitric oxide synthase (eNOS) expression and localization in healthy and diabetic rat hearts, Ann. Clin. Lab. Sci. 31 (2) (2001) 179–186. [27] U. Hink, H. Li, H. Mollnau, M. Oelze, E. Matheis, M. Hartmann, et al., Mechanisms underlying endothelial dysfunction in diabetes mellitus, Circ. Res. 88 (2) (2001) E14–E22. [28] R.C. Sampaio, J.E. Tanus-Santos, S.E.S.F.C. Melo, S. Hyslop, K.G. Franchini, I.M. Luca, et al., Hypertension plus diabetes mimics the cardiomyopathy induced by nitric oxide inhibition in rats, Chest 122 (4) (2002) 1412–1420. [29] J. MacMicking, Q.W. Xie, C. Nathan, Nitric oxide and macrophage function, Annu. Rev. Immunol. 15 (1997) 323–350. [30] A.L. Bardell, K.M. MacLeod, Evidence for inducible nitric-oxide synthase expression and activity in vascular smooth muscle of streptozotocin-diabetic rats, J. Pharmacol. Exp. Ther. 296 (2) (2001) 252–259. [31] P.R. Nagareddy, Z. Xia, J.H. McNeill, K.M. MacLeod, Increased expression of iNOS is associated with endothelial dysfunction and impaired pressor responsiveness in streptozotocin-induced diabetes, Am. J. Physiol. Heart Circ. Physiol. 289 (5) (2005) H2144–H2152.
5
[32] H. Farhangkhoee, Z.A. Khan, S. Chen, S. Chakrabarti, Differential effects of curcumin on vasoactive factors in the diabetic rat heart, Nutr. Metab. 3 (2006) 27. [33] S. Jesmin, S. Zaedi, S. Maeda, I. Yamaguchi, K. Goto, T. Miyauchi, Effects of a selective endothelin a receptor antagonist on the expressions of iNOS and eNOS in the heart of early streptozotocin-induced diabetic rats, Exp. Biol. Med. (Maywood) 231 (6) (2006) 925–931. [34] M.J. Crespo, J. Zalacaín, D.C. Dunbar, N. Cruz, L. Arocho, Cardiac oxidative stress is elevated at the onset of dilated cardiomyopathy in streptozotocin-diabetic rats, J. Cardiovasc. Pharmacol. Ther. 13 (1) (2008) 64–71. [35] J. Cao, C. Vecoli, D. Neglia, B. Tavazzi, G. Lazzarino, M. Novelli, et al., Cobaltprotoporphyrin improves heart function by blunting oxidative stress and restoring NO synthase equilibrium in an animal model of experimental diabetes, Front. Physiol 3 (2012) 160. [36] P. Puthanveetil, D. Zhang, Y. Wang, F. Wang, A. Wan, A. Abrahani, et al., Diabetes triggers a PARP1 mediated death pathway in the heart through participation of FoxO1, J. Mol. Cell. Cardiol. 53 (5) (2012) 677–686. [37] A. Pautz, J. Art, S. Hahn, S. Nowag, C. Voss, H. Kleinert, Regulation of the expression of inducible nitric oxide synthase, Nitric Oxide 23 (2) (2010) 75– 93. [38] H. Jo, H. Otani, F. Jo, T. Shimazu, T. Okazaki, K. Yoshioka, et al., Inhibition of nitric oxide synthase uncoupling by sepiapterin improves left ventricular function in streptozotocin-induced diabetic mice, Clin. Exp. Pharmacol. Physiol. 38 (8) (2011) 485–493. [39] J. Ren, J. Duan, D.P. Thomas, X. Yang, N. Sreejayan, J.R. Sowers, et al., IGF-I alleviates diabetes-induced RhoA activation, eNOS uncoupling, and myocardial dysfunction, Am. J. Physiol. Regul. Integr. Comp. Physiol. 294 (3) (2008) R793–R802. [40] N.D. Roe, D.P. Thomas, J. Ren, Inhibition of NADPH oxidase alleviates experimental diabetes-induced myocardial contractile dysfunction, Diabetes Obes. Metab. 13 (5) (2011) 465–473. [41] R.M. Saraiva, K.M. Minhas, M. Zheng, E. Pitz, A. Treuer, D. Gonzalez, et al., Reduced neuronal nitric oxide synthase expression contributes to cardiac oxidative stress and nitroso-redox imbalance in ob/ob mice, Nitric Oxide 16 (3) (2007) 331–338. [42] J. Amour, X. Loyer, M. Le Guen, N. Mabrouk, J.-S. David, E. Camors, et al., Altered contractile response due to increased beta3-adrenoceptor stimulation in diabetic cardiomyopathy: the role of nitric oxide synthase 1-derived nitric oxide, Anesthesiology 107 (3) (2007) 452–460. [43] P.R. Nagareddy, H. Soliman, G. Lin, P.S. Rajput, U. Kumar, J.H. McNeill, et al., Selective inhibition of protein kinase C beta(2) attenuates inducible nitric oxide synthase-mediated cardiovascular abnormalities in streptozotocin-induced diabetic rats, Diabetes 58 (10) (2009) 2355–2364. [44] H. Soliman, A. Gador, Y.-H. Lu, G. Lin, G. Bankar, K.M. MacLeod, Diabetes-induced increased oxidative stress in cardiomyocytes is sustained by a positive feedback loop involving Rho kinase and PKC 2, Am. J. Physiol. Heart Circ. Physiol. 303 (8) (2012) H989–H1000. [45] S. Lei, H. Li, J. Xu, Y. Liu, X. Gao, J. Wang, et al., Hyperglycemia-induced protein kinase C β2 activation induces diastolic cardiac dysfunction in diabetic rats by impairing caveolin-3 expression and Akt/eNOS signaling, Diabetes 62 (7) (2013) 2318–2328. [46] P. Cubas, C. Vincent, E. Coen, An epigenetic mutation responsible for natural variation in floral symmetry, Nature 401 (6749) (1999) 157–161. [47] G.B. Singh, R. Sharma, M. Khullar, Epigenetics and diabetic cardiomyopathy, Diabetes Res. Clin. Pract. 94 (1) (2011) 14–21. [48] M. Asrih, S. Steffens, Emerging role of epigenetics and miRNA in diabetic cardiomyopathy, Cardiovasc. Pathol. 22 (2) (2013) 117–125. [49] Y. Chan, J.E. Fish, C. D’Abreo, S. Lin, G.B. Robb, A.-M. Teichert, et al., The cell-specific expression of endothelial nitric-oxide synthase: a role for DNA methylation, J. Biol. Chem. 279 (33) (2004) 35087–35100. [50] J.E. Fish, C.C. Matouk, A. Rachlis, S. Lin, S.C. Tai, C. D’Abreo, et al., The expression of endothelial nitric-oxide synthase is controlled by a cell-specific histone code, J. Biol. Chem. 280 (26) (2005) 24824–24838. [51] C.V. Breton, C. Park, K. Siegmund, W.J. Gauderman, L. Whitfield-Maxwell, H.N. Hodis, et al., NOS1 methylation and carotid artery intima-media thickness in children, Circ. Cardiovasc. Genet. 7 (2) (2014) 116–122. [52] X. Rao, J. Zhong, S. Zhang, Y. Zhang, Q. Yu, P. Yang, et al., Loss of methyl-CpGbinding domain protein 2 enhances endothelial angiogenesis and protects mice against hind-limb ischemic injury, Circulation 123 (25) (2011) 2964– 2974. [53] A. Rodriguez, S. Griffiths-Jones, J.L. Ashurst, A. Bradley, Identification of mammalian microRNA host genes and transcription units, Genome Res. 14 (10A) (2004) 1902–1910. [54] B. Feng, S. Chen, B. George, Q. Feng, S. Chakrabarti, miR133a regulates cardiomyocyte hypertrophy in diabetes, Diabetes Metab. Res. Rev. 26 (1) (2010) 40–49. [55] S. Chen, P. Puthanveetil, B. Feng, S.J. Matkovich, G.W. Dorn 2nd, S. Chakrabarti, Cardiac miR-133a overexpression prevents early cardiac fibrosis in diabetes, J. Cell. Mol. Med. 18 (3) (2014) 415–421. [56] B. Liu, J. Hewinson, H. Xu, F. Montero, C.R. Sunico, F. Portillo, et al., NOS antagonism using viral vectors as an experimental strategy: implications for in vivo studies of cardiovascular control and peripheral neuropathies, Meth. Mol. Biol. 704 (2011) 197–223. [57] M. Weber, M.B. Baker, J.P. Moore, C.D. Searles, miR-21 is induced in endothelial cells by shear stress and modulates apoptosis and eNOS activity, Biochem. Biophys. Res. Commun. 393 (4) (2010) 643–648.
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[58] Z. Guo, L. Shao, L. Zheng, Q. Du, P. Li, B. John, et al., miRNA-939 regulates human inducible nitric oxide synthase posttranscriptional gene expression in human hepatocytes, Proc. Natl Acad. Sci. U.S.A. 109 (15) (2012) 5826–5831. [59] M. Meloni, M. Marchetti, K. Garner, B. Littlejohns, G. Sala-Newby, N. Xenophontos, et al., Local inhibition of microRNA-24 improves reparative
angiogenesis and left ventricle remodeling and function in mice with myocardial infarction, Mol. Ther. 21 (7) (2013) 1390–1402. [60] F. Patella, E. Leucci, M. Evangelista, B. Parker, J. Wen, A. Mercatanti, et al., MiR-492 impairs the angiogenic potential of endothelial cells, J. Cell. Mol. Med. 17 (8) (2013) 1006–1015.
Please cite this article in press as: Sanskriti Khanna, Gurinder Bir Singh, Madhu Khullar, Nitric oxide synthases and diabetic cardiomyopathy, Nitric Oxide (2014), doi: 10.1016/ j.niox.2014.08.004