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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

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Authors
장지현
Advisor
장은화
Major
의과대학 의과학과
Issue Date
2017-02
Publisher
서울대학교 대학원
Keywords
nNOS splice variantcardiac protectionhypertensionsarcomeremyofilamentheart
Description
학위논문 (박사)-- 서울대학교 대학원 : 의과학과, 2017. 2. 장은화.
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)
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, nNOSprotein expression was detected in the myofilament of cardiac myocytes from nNOSnull 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
50:20-27.
Language
English
URI
https://hdl.handle.net/10371/122347
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College of Medicine/School of Medicine (의과대학/대학원)Dept. of Biomedical Sciences (대학원 의과학과)Theses (Ph.D. / Sc.D._의과학과)
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