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Neuronal nitric oxide synthase splicing variants and their novel mechanisms of myocardial protection in hypertensive heart : 심근세포의 신경형 산화질소 합성효소 접합변이체 발현 양상과 고혈압에서 심근보호의 새로운 기전 연구
DC Field | Value | Language |
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dc.contributor.advisor | 장은화 | - |
dc.contributor.author | 장지현 | - |
dc.date.accessioned | 2017-07-14T01:47:12Z | - |
dc.date.available | 2017-07-14T01:47:12Z | - |
dc.date.issued | 2017-02 | - |
dc.identifier.other | 000000142153 | - |
dc.identifier.uri | https://hdl.handle.net/10371/122347 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 의과학과, 2017. 2. 장은화. | - |
dc.description.abstract | Cardiac nNOS is well established to be an intracellular Ca2+ modulator and regulates myocardial contractility in healthy and diseased hearts. Importantly, previous work from our research group has shown that nNOS is up-regulated in the cytosol/membrane of the myocardium in hypertension and promotes lusitropy through myofilament Ca2+ desensitiztion of cardiac myocytes. Until recently, the mechanisms mediating nNOS up-regulation by pathological stimuli and the mechanisms of cardiac protection in hypertension by nNOS are not fully understood. My recent work has shown that angiotensin II (Ang II, 1 M, 3 hrs) increased nNOS protein expression and activity in rat cardiac myocytes subsequent to AT1R/NADPH oxidase activation. Intriguingly, Ang II increased endothelial NOS (eNOS) Ser1177 and decreased eNOS Thr495 via NADPH oxidase-derived reactive oxygen species (ROS) | - |
dc.description.abstract | NOS inhibition (LNG-Nitroarginine Methyl Ester, L-NAME) or eNOS gene deletion (eNOS-/-) abolished AT2R translocation to plasma membrane and Ang II-induced nNOS protein expression. AT2R was S-nitrosated by NO, site-specific mutagenesis analysis and immunocytochemistry using confocal microscopy revealed the importance of C-terminal Cys349 residue in AT2R translocation to plasma membrane, suggesting that eNOS may have increased the S-nitrosation and activation of AT2R through Cys349.
Next, I aimed to investigate the molecular mechanisms mediating nNOS protection in the myocardium of hypertensive rats. Immunohistochemistry experiments confirmed that chronic inhibition of nNOS with the specific inhibitor, S-methyl-L-thiocitrulline (SMTC) in vivo induced cardiac hypertrophy and intermittent fibrosis in Ang II-induced hypertensive rat (SMTC+Ang II) with little effect in Sham. In echocardiography, LV septum, posterior wall thickness and the ejection fraction were increased but the end-diastolic and systolic chamber dimension were reduced in SMTC+Ang II compared to Sham, SMTC and Ang II. Transmission electron microscopy showed that the lengths of sarcomere and I-band but not the length of thick filament of the myocardium were significantly elongated in SMTC+Ang II. Fluorescent microscopic imaging confirmed the elongation of the sarcomere and Z-disc of LV myocytes from SMTC+Ang II. Immunoblotting experiments showed that the abundance of nebulette was increased but troponin I was reduced and the rest of the thick, thin and Z-disc proteins were not different after nNOS inhibition. Functionally, slack sarcomere lengths were increased before myocyte contraction in SMTC+Ang II. However, at steady-state contraction with field stimulation (2 Hz), the difference in sarcomere length was absent and the diastolic and systolic Ca2+ transient amplitudes were increased with nNOS inhibition. Myofilament Ca2+ sensitivity, which is reduced in hypertension by nNOS, was significantly increased in SMTC+Ang II. Furthermore, immunoblotting from the myofilament fraction of cardiac myocytes demonstrated the expression of nNOS (M.W. ~140 kDa) in LV myocytes from Sham and hypertensive rats (which was distinct from nNOS M.W. 155~165 kDa, in the plasma membrane/cytosol). Albeit with reduced intensity, nNOSprotein expression was detected in the myofilament of cardiac myocytes from nNOSnull mice. The protein expression of nNOS was significantly increased in SMTC and SMTC+Ang II in rats. Myofilament proteins were not affected by nNOS gene deletion. Taken together, I have revealed, for the first time, the cross talk between AT1R and AT2R on nNOS up-regulation via NADPH oxidase/ROS-stimulation of eNOS activity in cardiac myocytes. Using a hypertensive model with nNOS inhibition, I have demonstrated that nNOS is essential in maintaining sarcomere structure and function of cardiac myocyte, protects the heart from cardiac hypertrophy and fibrosis in hypertension. Importantly, I have identified a novel splice variant of nNOS, nNOS, in the myofilament fraction of cardiac myocyte. nNOS and nNOS-regulation of myofilament structure and kinetics will shed light on a new conceptual framework for better understanding of the hypertrophic progression and nNOS protection of the heart under stress. Some of the works are published in Basic Res Cardiol (2015) 110(3):21., and Nitric Oxide (2015) 11 | - |
dc.description.abstract | 50:20-27. | - |
dc.description.tableofcontents | General introduction 1
Materials and Methods 12 Chapter1 Mechanism leading to nNOS up-regulation by angiotensin II in rat cardiac myocyte 28 Introduction 29 Results 31 Discussion 49 Chapter2 Mechanism of nNOS-mediated cardiac protection in Ang II -induced hypertensive rat 55 Introduction 56 Results 58 PartI: Chronic inhibition of nNOS in vivo exacerbates cardiac hypertrophy and fibrosis in Ang II-induced hypertensive rat Results 58 PartII: Myofilament and sarcomere structure, intracellular Ca2+ level and myofilament Ca2+ sensitivity in cardiac myocyte following chronic inhibition of nNOS in normal and Ang II-induced hypertensive rats 67 PartIII: Identification of a novel splice variant of nNOS, nNOS, in myofilament fraction of cardiac myocytes from normal and Ang II-induced hypertensive rats with and without nNOS inhibition 82 PartIV: Myofilament and sarcomere structure in cardiac myocyte from nNOS null mice 91 Discussion 96 References 104 Abstract in Korean 118 | - |
dc.format | application/pdf | - |
dc.format.extent | 2774193 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | nNOS splice variant | - |
dc.subject | cardiac protection | - |
dc.subject | hypertension | - |
dc.subject | sarcomere | - |
dc.subject | myofilament | - |
dc.subject | heart | - |
dc.subject.ddc | 610 | - |
dc.title | Neuronal nitric oxide synthase splicing variants and their novel mechanisms of myocardial protection in hypertensive heart | - |
dc.title.alternative | 심근세포의 신경형 산화질소 합성효소 접합변이체 발현 양상과 고혈압에서 심근보호의 새로운 기전 연구 | - |
dc.type | Thesis | - |
dc.description.degree | Doctor | - |
dc.citation.pages | 120 | - |
dc.contributor.affiliation | 의과대학 의과학과 | - |
dc.date.awarded | 2017-02 | - |
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