β3-Adrenergic Receptor Modulates NOS Coupling In Pressure Overloaded Cardiac Hypertrophy

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.