【作者】 牛晓琳；

【导师】 赵连友；

【作者基本信息】 第四军医大学 ， 内科学， 2010， 博士

【摘要】 研究背景:心肌肥厚是心脏对多种病理性刺激,如高血压,缺血性心脏病,瓣膜缺陷等引起压力负荷升高的一种代偿性反应。这种反应早期通过增加心脏收缩力有利于维持心功能。然而,持续的压力负荷,导致体内多种神经体液因子激活,这些神经体液因子最终会导致心肌损伤,心功能恶化,促进心脏衰竭的发生发展,严重威胁人类健康。然而心肌肥厚的发病机制目前尚不十分明确,并缺乏有效的治疗措施。因此,探索心肌肥厚的发生机制及其防治措施是心血管领域研究的热点问题。β肾上腺素能受体(β-AR)家族在心血管系统中发挥了重要的调节作用。近年来研究发现除了传统的β1/β2-AR之外,心脏还存在β3-AR。与β1/β2-AR相反,β3-AR的激活抑制心肌收缩力。已经在人体和动物模型中证实,心衰时β3-AR表达增加。然而β3-AR的增加在心衰中到底扮演了何种角色,是对儿茶酚胺浓度过高的代偿性保护反应还是心衰的促进因子目前尚不清楚。最新研究发现,一氧化氮合酶(NOS)解耦联是导致NO水平下降和活性氧(ROS)水平升高的重要机制,是高血压、糖尿病、胰岛素抵抗、肥胖、动脉粥样硬化、心力衰竭等疾病中发生的重要原因,纠正NOS解耦联有望为保护心血管系统提供有效途径。NOS解耦联是否参与了β3-AR对心脏的调控作用,目前尚未见报道。研究目的:本研究通过观察β3-AR基因敲除小鼠(β3-/-)压力负荷下心脏结构和功能的变化,以及压力负荷时给予野生型(WT)小鼠β3-AR激动剂治疗后心脏结构和功能的变化,从正反两方面研究β3-AR信号转导通路对压力负荷时心脏的调控作用,探讨NOS解耦联在β3-AR调节心脏功能中的作用,旨在阐明β3-AR通过调控NOS偶联发挥心脏保护作用,为心肌肥厚,心力衰竭的防治提供新的理论依据和治疗措施。研究方法:本研究通过结扎小鼠主动脉弓建立压力负荷心肌肥厚模型,采用心脏超声、组织学方法及多种分子生物学技术,(1)比较β3-AR基因敲除小鼠和野生型小鼠给予慢性压力负荷后,心脏结构和功能、NOS活性及蛋白表达,活性氧及BH4水平的变化。(2)观察给予β3-AR基因敲除小鼠BH4补充性治疗,对压力负荷所致心肌肥厚,心功能和活性氧的改变。(3)观察给予压力负荷野生型小鼠β3-AR激动剂BRL 37344治疗,心脏结构和功能,NOS活性及蛋白表达,活性氧水平的变化。研究结果:(1)8周大FVBβ3-/-小鼠体重,室壁厚度及超声测算的左室重量较FVB WT小鼠轻度增加,而心率,左室腔内径及收缩功能两组间没有统计学差别。14-18月时WT小鼠表现为轻度心肌肥厚(P<0.05 vs. 8周),而β3-/-小鼠则有明显的左室肥厚,其室壁厚度(1.30±0.14 vs. 0.86±0.07 mm, P<0.001)和左室重量(196±12 vs. 129±20 mg, P<0.05)均较WT小鼠显著增加。(2)分别给予β3-/-和WT小鼠轻度主动脉缩窄术(25G TAC)和假手术(sham)后,β3-/-小鼠TAC 9周后存活率较WT小鼠明显下降(38%vs. 85%,χ2=10.78, P<0.001)。(3)TAC 9周后,β3-/-小鼠较WT小鼠发生更为严重的心肌肥厚和心脏重构,表现为进一步增加的心脏重量/胫骨长度比值(HW/TL 175.2±17.8 vs. 123.3±4.0, P<0.01),心肌细胞直径(39.3±0.9 vs. 31.3±0.9μm, P<0.001)和心肌纤维化程度(2.7±0.3 vs. 1.2±0.1, P<0.05 vs. WT/TAC for all)。(4)心脏超声结果显示WT小鼠TAC后,LVEDD, LVESD和小轴缩短率与假手术组比较无明显变化,而β3-/-小鼠TAC后,LVEDD(3.90±0.26 vs. 2.91±0.04 mm, P<0.001)和LVESD(2.47±0.36 vs. 1.02±0.05 mm, P<0.001)较假手术组明显增加,小轴缩短率显著降低(38.2±5.0 vs. 64.9±1.8%, P<0.001)。(5)WT小鼠和β3-/-小鼠TAC后,室壁厚度(1.30±0.02 vs. 0.83±0.01 mm, P<0.001)明显增加,但β3-/-小鼠较WT小鼠的室壁厚度(1.43±0.03 vs. 1.02±0.03 mm, P<0.001 vs.β3-/-/sham; P<0.01 vs. WT/TAC)增加更多。(6)基础状态下β3-/-和WT小鼠心脏NOS活性无区别(26.9±0.4 vs. 27.6±0.4 A.U., P=NS)。TAC 9周后,WT小鼠NOS活性较sham组无明显变化(27.7±0.3 A.U., P=NS vs. WT/sham),而β3-/-小鼠心脏NOS活性较β3-/-/sham组明显下降(19.3±1.2 A.U., P<0.001 vs.β3-/-/sham)。(7)基础状态下,β3-/-和WT小鼠心脏超氧化物活性无差别(1145±146 vs. 1106±109 cpm/mg,P=NS)。TAC 9周后,WT和β3-/-小鼠心脏总O2-活性均较sham组增加,差异有统计学意义,但β3-/-小鼠O2-活性较WT小鼠升高近60% (2730±121 vs. 1719±52 cpm/mg,P<0.05 vs. sham, P<0.001 vs. WT/TAC)。(8)基础状态下β3-/-和WT小鼠心脏NOS依赖的O2-活性无差别(P=NS)。TAC 9周后,WT小鼠NOS依赖的O2-活性较sham组增加不到1倍,而β3-/-小鼠TAC 9周后NOS依赖的O2-活性较sham组升高2倍多,并且较WT/TAC小鼠进一步增加(P<0.01,β3-/-/TAC vs. WT/TAC)。(9)基础状态下β3-/-和WT小鼠心脏GTPCH-1的蛋白表达水平无区别。TAC 9周后,WT小鼠较sham组心脏GTPCH-1的蛋白表达水平无明显变化。而β3-/-小鼠TAC 9周后心肌GTPCH-1的蛋白表达量较sham组明显下降(P<0.05 vs.β3-/-/sham)。(10)各组间心肌组织BH4含量无明显变化,β3-/-小鼠TAC后BH4含量略有增高(35.6±1.9 vs. 27.0±0.9 pmol/mg protein,P<0.01)。而基础状态下β3-/-小鼠BH4/(BH2+生物嘌呤)比值较WT小鼠下降了25%(1.49±0.2 vs. 1.91±0.3,P<0.05),TAC后没有进一步下降。(11)给予FVBβ3-/-/ TAC小鼠BH4补充性治疗后,治疗组小鼠心肌收缩力较安慰剂组明显提高(-0.4±0.2 vs. -16.1±4.9%, P<0.05 vs.β3-/-/TAC),左室重量较安慰剂组显著降低(+15.0±6.8 vs. +81.8±13.7 %, P<0.01 vs.β3-/-/TAC)。(12)β3-/-/TAC BH4治疗组小鼠心肌NOS来源的O2-活性较安慰剂治疗组显著下降(P<0.05 vs.β3-/-/TAC+vehicle; P=NS vs.β3-/-/sham and WT/sham)。(13)C57BL/6 WT小鼠经27G TAC手术3周后心腔扩大,心肌收缩力下降。与sham组小鼠比较LVEDD增加82%(2.00±0.20 vs.1.10±0.03 mm; P<0.001),小轴缩短率降低36%(39.1±4.5 vs. 61.4±0.3 %;P<0.001)。左室重量(172±13 vs. 76±5 mg; P<0.001)和室壁厚度(1.21±0.04 vs. 0.84±0.02 mm; P<0.001)也较sham组小鼠增加。而通过皮下植入微量泵给予0.1 mg/kg/day BRL治疗完全防止了压力负荷所致的心腔扩大(LVESD 1.32±0.06 mm; P=NS vs. sham, P<0.01 vs. TAC)和心功能下降(FS% 57.8±1.4%; P=NS vs. sham, P<0.001 vs. TAC)。BRL治疗组小鼠左室重量和室壁厚度也较安慰剂组明显下降(P<0.001 vs. TAC)。(14)BRL治疗组小鼠与安慰剂组比较心肌肥厚的程度明显减轻(HW/TL 100±4 vs. 122±8 mg/cm; P<0.05),心肌细胞直径较安慰剂组明显降低(13.31±0.21 vs. 15.81±0.35μm, P<0.001)。然而BRL治疗对心肌纤维化无明显改善(1.50±0.35 vs. 1.67±0.33, P =NS)。(15)硝酸还原酶法测定的心肌NO终末产物硝酸盐和亚硝酸盐浓度在TAC 3周后较shan组小鼠降低50%(5.03±0.52 vs. 10.10±1.99μM, P<0.05)。BRL治疗组小鼠心肌NO活性恢复正常水平(13.73±1.84μM, P<0.01 vs. TAC; P=NS vs. sham)。(16)WT小鼠27G TAC 3周后心肌总O2-活性较对照组增加约2.5倍(21459±782.8 vs. 6099±1703 CPM/mg; P<0.001),BRL治疗组小鼠心肌总O2-活性较安慰剂治疗组明显下降(14017±838.2 CPM/mg; P<0.01)。更为重要的是,BRL对心肌超氧阴离子的抑制作用在nNOS特异性地拮抗剂L-VNIO的作用下完全消失(21992±75.68 vs. 21063±2930 CPM/mg; P=NS vs. TAC)。(17)TAC后,eNOS单体/二聚体比值显著增加(m/d 1.10±0.24 vs. 0.45±0.05; P<0.05),eNOS二聚体解离,然而BRL治疗并没有降低eNOS单体/二聚体比值(1.01±0.02; P=NS vs. TAC。(18)BRL对eNOS总蛋白表达量没有明显改变。但BRL治疗组小鼠心肌p-eNOSSer1177/eNOS比值较安慰剂治疗组明显降低(0.92±0.01 vs. 1.40±0.02; P<0.01)。而p-eNOSSer114/eNOS比值则较安慰剂治疗组增加了1倍(4.64±0.60 vs. 2.33±0.22; P<0.05)。p-eNOSThr495/eNOS比值在各组间也无明显变化。(19)压力负荷3周后小鼠心肌nNOS表达无明显变化,而BRL治疗组小鼠心肌nNOS蛋白表达量是安慰剂治疗组的3倍(1.11±0.22 vs. 0.39±0.17; P<0.05 vs. TAC)。iNOS蛋白表达在TAC后略有增加,但BRL对iNOS蛋白表达无影响(0.34±0.09; P=NS vs. TAC)。研究结论:(1)老年和轻度压力负荷后β3-/-较野生型小鼠发生了更为严重的心肌肥厚,心肌纤维化,心室腔扩大和心功能下降。(2)压力负荷时β3-AR基因敲除小鼠心肌发生NOS解耦联,NOS生成NO的水平下降,而生成活性氧水平增加。(3)压力负荷时β3-AR基因敲除小鼠心肌组织GTPCH- 1的蛋白表达量和BH4/(BH2+生物嘌呤)的比值下降可能是NOS解耦联的原因。外源性补充BH4治疗明显改善了压力负荷所致的心脏功能下降和左室肥厚。(4)给予野生型小鼠选择性β3-AR激动剂BRL治疗3周完全预防了压力负荷所致的心腔扩大,心脏功能下降,并部分地抑制了心肌肥厚的发展。(5)β3-AR激动剂治疗后心肌NO活性恢复正常,ROS活性降低,且BRL对ROS活性的抑制作用是通过nNOS实现的。(6)BRL治疗对eNOS二聚化水平,eNOS蛋白表达没有改变,但显著上调了nNOS的蛋白表达水平。综上所述,本研究从正反两面证实了β3-AR信号转导通路对压力负荷小鼠具有重要的保护作用。β3-AR通过调控NOS偶联发挥其心血管保护作用。这一研究为心肌肥厚和心力衰竭的防治提供了新的思路和理论依据,具有重要的临床应用价值。更多还原

【Abstract】 Background: Cardiac hypertrophy is a compensative response to multiple stessors, like hypertrophy, ischemic heart disease and valve defects. Initially, this response is benificial to maintain systolic function of the heart. However, sustained pressure overload may activate systematic nervous system and neuro-hormone which has deleterious effects on cardiac structure and performance, leading to cardiac de-compensation and heart failure progression. Thus, cardiac hypertrophy is a serious threat to human health. However, its pathogenesis is not yet very clear, and it still lacks effective therapeutic approaches. Therefore, to explore the mechanism and potential treatment of cardiac hypertrophy is currently a hot topic in the field of cardiovascular diseases. Recent studies revealed that besides traditionalβ1/β2-AR, there is another one,β3-AR expressed in the heart. Opposite toβ1/β2-AR,β3-AR stimulation induces a negative inotropic effect. It has been established thatβ3-AR was up-regulated in human heart failure and animal models. However, whether this is a protective response to catecholamine over-expression or it is a contributor to heart failure is still unclear. Recently, a number of studies reported that NOS uncoupling is the mechanism of NO deactivation and ROS activation in the pathophysiology of hypertension, diabeties, insulin resistance, obesity, Atherosclerosis and heart failure. Correcting NOS uncoupling is expected to provide an effective means to protect the cardiovascular system. Whether NOS uncoupling is involved in theβ3-AR regulation in heart has not been reported yet.Objectives: This study was therefore designed to observe the changes of cardiac structure and function inβ3-AR knockout (β3-/-) mice underwent pressure overload, andβ3-AR agonism effect on cardiac structure and function in pressure load wild-type mice; to studyβ3-AR signal transduction pathway on the regulation of cardiac pressure overload; to discuss the role of NOS uncoupling in theβ3-AR regulation of cardiac function, aims to clarify maintaining NOS coupling byβ3-AR plays a protective role in the heart, which provides new theoretical evidences and potential therapeutic prevention and treatment approaches for cardiac hypertrophy and heart failure.Methods: In this study, mice underwent transverse aortic constriction (TAC) to set the pressure over load induced cardiac hypertrophy and heart failure model as previously described. Transthoracic echocardiography, histology evaluation and a variety of molecular biology techniques were used to: (1) compare the changes of cardiac structure and function, NOS activity and protein expression, reactive oxygen species generation (ROS) and tetrahydrobiopterin (BH4) levels by chronic pressure overload inβ3-/- mice and wild-type (WT) mice; (2) observe the influence of BH4 supplement on cardiac hypertrophy, LV systolid function and ROS generation in pressure overloadedβ3-/-mice; (3) observe the effect of specificβ-3AR agonist BRL 37344 on cardiac structure and function, NOS activity and protein expression and superoxide generation in pressure-overloaded wild-type mice. Results: (1) At 8 weeks, body weight, ventricular wall thickness, and calculated left ventricular mass were slightly increased in FVBβ3-/- mice compared with FVB WT, while the heart rate, left ventricular cavity diameter and systolic function between the two groups were not statistically different. At 14-18 months,β3-/- mice had accentuated LV hypertrophy than WT mice, as evidenced by increased wall thickness (1.30±0.14 vs. 0.86±0.07 mm, P<0.001) and calculated LV mass (196±12 vs. 129±20 mg, P<0.05). (2)β3-/- mice had much greater mortality after mild transverse aortic constriction (25G TAC) than WT controls (38% vs. 85%,χ2 = 10.78, P < 0.001). (3) After 9 weeks of TAC,β3-/- mice also had greater LV hypertrophy and cardiac remodeling, with further increased heart weight to tibia length ratio (HW/TL 175.2±17.8 vs. 123.3±4.0, P<0.013), cardiac diameter (39.3±0.9 vs. 31.3±0.9μm, P<0.001) and enhanced fibrosis (2.7±0.3 vs. 1.2±0.1, P<0.05). (4) Echocardiography showed that 9 weeks of TAC didn’t induce any change of LVEDD, LVESD and fractional shortening in WT mice, whereas LVEDD (3.90±0.26 vs. 2.91±0.04 mm) and LVESD (2.47±0.36 vs. 1.02±0.05 mm) were significantly increased and FS% was markedly reduced inβ3-/- mice after TAC (38.2±5.0 vs. 64.9±1.8%, P<0.001 for all). (5) TAC induced increase in wall thickness (1.30±0.02 vs. 0.83±0.01 mm, P <0.001), which was further aggravated inβ3-/- mice (1.43±0.03 vs. 1.02±0.03 mm, P<0.001 vs.β3-/-/sham; P<0.01 vs. WT/TAC). (6) Cardiac Ca2+ dependent NOS activity was similar in WT andβ3-/- mice at baseline (26.9±0.4 vs. 27.6±0.4 A.U., P=NS). By 9 weeks of TAC, NOS activity was unchanged in WT (27.7±0.3 A.U., P=NS vs. WT/sham), but was significantly decreased inβ3-/- mice (19.3±1.2 A.U., P<0.001 vs.β3-/-/sham). (7) At baseline, superoxide generation was similar between WT andβ3-/- mice (145±146 vs. 1106±109 cpm/mg,P=NS). After 9 weeks of TAC, superoxide generation was increased in WT mice and was further raised 60% in inβ3-/- mice compared with WT mice (2730±121 vs. 1719±52 cpm/mg, P<0.05 vs. sham, P<0.001 vs. WT/TAC). (8) The increase of superoxide generation inβ3-/- mice after pressure overload is mainly due to the increase of NOS dependent superoxide. NOS dependent superoxide was similar in WT andβ3-/- mice at baseline (P=NS). However, after TAC, levels rose over 2 fold inβ3-/- mice vs.β3-/-/sham compared with less than 1 fold in WT mice vs. WT/sham. In addition, NOS dependent superoxide was higher inβ3-/-/TAC than in WT/TAC (P<0.05). (9) Cardiac GTPCH-1 protein expression was similar at baseline but declined significantly after 9 weeks of TAC inβ3-/-/TAC vs.β3-/-/sham (P<0.05). (10) Total BH4 level did not differ significantly between strains, although there was a slight increase inβ3-/-/TAC above baseline (35.6±1.9 vs. 27.0±0.9 pmol/mg protein, P<0.01). The ratio of BH4/(BH2+biopterin) was decreased by approximately 25% inβ3-/-mice at baseline compared to WT mice (1.49±0.2 vs. 1.91±0.3, P<0.05), yet was unchanged after TAC. (11) BH4 treated FVBβ3-/-/TAC mice had higher LV systolic function (-0.4±0.2 vs. -16.1±4.9%, P<0.05 vs.β3-/-/TAC), and lower calculated LV mass (+15.0±6.8 vs. +81.8±13.7 %, P<0.01 vs.β3-/-/TAC) compared to vehicle. (12) NOS-dependent superoxide production inβ3-/-/TAC with BH4 supplement was much lower compared to vehicle (P<0.05 vs.β3-/-/TAC+vehicle; P=NS vs.β3-/-/sham and WT/sham). (13) C57BL/6 mice developed increased LV chamber dilation and systolic dysfunction after 3 weeks of 27G TAC, as evidenced by 82% increased LVESD (2.00±0.20 vs.1.10±0.03 mm; P<0.001) and 36% reduced FS% (39.1±4.5 vs. 61.4±0.3 %; P<0.001) compared to sham mice assessed by echocardiography. Calculated LV mass (172±13 vs. 76±5 mg; P<0.001) and average wall thickness (1.21±0.04 vs. 0.84±0.02 mm; P<0.001) were increased as well. Three weeks of BRL treatment via subcutaneous osmotic pumps at 0.1 mg/kg/day totally prevented LV dilation (LVESD 1.32±0.06 mm; P=NS vs. sham, P<0.01 vs. TAC) and restores cardiac function back to normal (FS% 57.8±1.4%; P=NS vs. sham, P<0.001 vs. TAC). Calculated LV mass and average wall thickness were significantly lower in BRL treated mice compared to vehicle (P<0.001 vs. TAC) as well. (14) BRL treated mice developed less hypertrophy (HW/TL 100±4 vs. 122±8 mg/cm; P<0.05) and lower cardiomyocyte width (13.31±0.21 vs. 15.81±0.35μm, P<0.001) compared to vehicle. However, BRL had no effect on fibrosis scale (1.50±0.35 vs. 1.67±0.33, P =NS). (15) Nitrate plus Nitrite, the final production of NO which was examined by Griess assay was decreased 50% by 3 weeks of TAC (5.03±0.52 vs. 10.10±1.99μM, P<0.05). BRL treated mice had normal NO production as sham mice (13.73±1.84μM, P<0.01 vs. TAC; P=NS vs. sham). (16) LV superoxide production assayed by lucigenin-enhanced chemiluminescence was increased by 2.5 fold in TAC hearts over sham controls (21459±782.8 vs. 6099±1703 CPM/mg; P<0.001). BRL treated mice had less myocardial superoxide production compared to vehicle (14017±838.2 CPM/mg; P<0.01). More importantly, this suppression effect of BRL was abolished by acute inhibition with nNOS specific inhibitor L-VNIO (21992±75.68 vs. 21063±2930 CPM/mg; P=NS vs. TAC). (17) Three weeks of TAC resulted in increased eNOS monomer to dimmer ratio, which means more uncoupling of eNOS dimmer (1.10±0.24 vs. 0.45±0.05; P<0.05). BRL treatment didn’t have any influence on this ratio (1.01±0.02; P=NS vs. TAC). (18) Total eNOS protein expression was similar among groups. eNOSSer1177 phosphorylation, which is an indication of eNOS activation, was decreased by BRL treatment compared to vehicle (0.92±0.01 vs. 1.40±0.02; P<0.01), though there was no change between sham and TAC. In contrast, p-eNOSSer114 phosphorylation, an indication of eNOS deactivation, was increased 100% in BRL treated mice (4.64±0.60 vs. 2.33±0.22; P<0.05). eNOSThr495 phosphorylation was unchanged by BRL treatment. (19) nNOS protein expression was unchanged by TAC, however it was up-regulated to 3 fold by BRL treatment (1.11±0.22 vs. 0.39±0.17; P<0.05 vs. TAC). iNOS ptorein expression was slightly up-regulated by TAC while was unchanged by BRL treatment (0.34±0.09; P=NS vs. TAC).Conclusions: (1) Old and mild pressure overloadedβ3-/- mice developed worse cardiac hypertrophy, fibrosis, LV dilation and cardiac dysfunction than WT mice. (2) Pressure overload induced NOS uncoupling by decreased NOS activity and increased ROS inβ3-/- mice. (3) Depressed GTPCH-1 protein expression and lowered BH4 /(BH2+biopterin) ratio are part of the reason for NOS uncoupling inβ3-/- mice. BH4 supplement prevented LV dysfunction and heart hypertrophy induced by pressure overload. (4) Three weeks of specificβ3-AR agonist, BRL treatment totally prevented LV dilation and cardiac dysfunction and partially inhibited the development of myocardium in chronic pressure-overloaded WT mice. (5)β3-AR agonist treated mice had normal NO production and lower ROS activity. This suppression effect of BRL was abolished by nNOS specific inhibitor L-VNIO pretreatment,. (6) BRL had no influence on eNOS dimerization and protein expression while markedly up-regulated nNOS protein expression.Taken all these together, this study from both positive and negative sides revealed thatβ3-AR signal transduction pathway plays a vital important protective effect on pressure overloaded mice, which is associated with maintaining NOS coupling. This study provides a new direction and theoretical basis for the treatment of cardiac hypertrophy and heart failure which has important clinical potentials.更多还原