S-Space College of Medicine/School of Medicine (의과대학/대학원) Dept. of Biomedical Sciences (대학원 의과학과) Theses (Ph.D. / Sc.D._의과학과)
Novel Regulatory Mechanisms of Myocardial Contraction Mediated by Neuronal Nitric Oxide Synthase in Angiotension II-induced Hypertensive Rats : 안지오텐신 II로 유발된 고혈압 백서에서 신경형 산화질소 합성효소를 통한 심근수축 변화의 새로운 기전
- 의과대학 의과학과
- Issue Date
- 서울대학교 대학원
- 학위논문 (박사)-- 서울대학교 대학원 : 의과학과 의과학 전공, 2013. 2. 장은화.
- Renin-angiotensin-system has been implicated in hypertension, cardiac hypertrophy and fatal heart failure. Angiotensin II (Ang II) is an important mediator of myocardial pathogenesis, mostly via inducing oxidative stress secondary to NADPH oxidase (NOX) activation and adverse remodeling leading to contractile dysfunction. In contrast, neuronal nitric oxide synthase (nNOS) has been shown to reduce intracellular reactive oxygen species (ROS) by modulating cardiac oxidases and facilitates myocardial relaxation by promoting Ca2+ reuptake into the sarcoplasmic reticulum (SR) through increasing PKA-dependent phospholamban phosphorylation (PLN-Ser16). Importantly, nNOS protein expression and activity are up-regulated in hypertrophic and failing myocardium which protects the heart from disease progression. So far, the mechanisms mediating nNOS regulation of intracellular ROS, signaling and contractility in hypertensive heart are unknown. Therefore, the aim of the present study is to examine whether nNOS protein expression and activity are affected by Ang II treatment (both in vitro and in vivo via osmotic minipumps, 125ng/min/kg, 4 wks) in left ventricular (LV) myocytes and the cellular mechanisms mediating nNOS regulation of myocyte contraction and relaxation in normal and Ang II-induced hypertensive rats.
The results showed that nNOS protein expression and activity were increased by Ang II treatment both in vitro (3h) and in vivo (hypertension). At 3h, nNOS reduced NOX activity and facilitated LV myocyte relaxation. Interestingly, nNOS increased PLN-Ser16 via cGMP/PKG-dependent (but PKA, CaMKII or peroxynitrite-independent) signaling. Similarly, nNOS up-regulation was associated with faster myocyte relaxation, increased PLN-Ser16 and quicker decay kinetics of Ca2+ transients (tau) in hypertensive rat heart. However, faster tau and relaxation were independent of nNOS, PKA, CaMKII or cGMP/PKG. Instead, nNOS increased troponin I phosphorylation (TnI-Ser23/24) and reduced myofilament Ca2+ sensitivity, which in turn, increased diastolic Ca2+ level and promoted Ca2+ reuptake into the SR. In summary, the results suggest that nNOS shifts its target proteins and signaling during disease progression: from PKA-dependent PLN-Ser16 in normal to PKG-dependent PLN-Ser16 at early stage with pathogenic stress (Ang II 3h) to cGMP/PKG-dependent phosphorylation of myofilament proteins (e.g. TnI) in hypertensive myocardium. These novel findings are important in better understandings of the endogenous cardiac protective mechanisms of nNOS during disease progression.
Fatty acids (FA) are preferential substrates of cardiac metabolism to provide fuels for myocardial contraction. NO has been implicated in FA uptake and utilization in the myocardium from normal heart
conversely, reduced bioavailability of NO has been associated with impaired FA metabolism and cardiac performance in failing heart. So far, functional roles of nNOS in FA regulation of myocardial contractility in normal and hypertensive myocardium remain elusive. Hence, nNOS regulation of FA-dependent LV myocyte contraction in sham and Ang II-induced hypertensive rats and the potential mechanisms mediating the function of nNOS were explored.
Supplementation of palmitic acid (PA) to the perfusate increased LV myocyte contraction in shams but not in hypertension. Carnitine palmitoyltransperase 1 and nNOS mediated the inotropic effect of PA in shams. Surprisingly, nNOS was responsible for impaired PA-dependent inotropic effect in hypertensive rats. nNOS increased s-nitrosylation of transglutaminase II (TG2) and reduced its activity and attenuated PA-increase in LV myocyte contraction, possibly via reducing ATP production in hypertensive myocardium. Exercise training restored PA-dependent LV myocyte contraction in hypertensive heart, an effect that was independent of nNOS activity. These results suggest that nNOS plays contrasting roles in FA-dependent metabolism and LV myocyte contraction in healthy and hypertensive hearts.
Taken together, my research revealed several novel mechanisms of nNOS in posttranscriptional regulation of intracellular signaling pathways, Ca2+ handling processes, metabolism and myocyte contraction in both healthy and diseased heart.