Exercise and H2S mitigate homocysteine-mediated beta2-adrenergic receptor dysfunc

描述（由申请人提供）：具体目的：缺乏运动动力是肥胖和2型糖尿病（T2D）的标志。高脂肪饮食（HFD）是肥胖和T2D的主要原因，后者损害心肌细胞的收缩性，导致心力衰竭。刺激- 2-肾上腺素能受体（- 2-AR）可诱导心肌收缩，减轻心功能障碍。高同型半胱氨酸（Hcy）水平升高称为高同型半胱氨酸血症（HHcy，一种合并症）与糖尿病心力衰竭有关。Hcy与- 2-AR竞争并拮抗。该项目的长期目标是了解2- AR和Hcy轴在糖尿病中的作用。半胱甘氨酸合成酶（CBS）和半胱甘氨酸合成酶可将Hcy转化为H2S（一种抗高血压、血管舒张和抗氧化气体），从而改善HHcy。裂合酶(CSE)。在糖尿病中，CBS和CSE的水平被抑制，从而诱导HHcy，从而损害H2S的形成。H2S是AKT（一种参与葡萄糖代谢的抗氧化剂）的诱导剂，而AKT在糖尿病中被下调。我们已经证明H2S在糖尿病心肌细胞中诱导一种叫做Gs （2-AR诱导剂）的刺激性g蛋白偶联受体。另一方面，HHcy会衰减g。然而，运动可以减轻hcy，并上调T2D中的β 2-AR。此外，运动和沙丁胺醇（2-AR激动剂）在减轻糖尿病心肌细胞收缩功能障碍方面具有协同作用。然而，潜在的机制尚不清楚。我们已经证明，在糖尿病中，HHcy激活MMP9，诱导心脏纤维化并损害心肌收缩性。此外，调节心脏纤维化和肥厚的microRNA-133a （miR-133a）在糖尿病心脏中减弱。我们的初步研究表明，HHcy （CBS+/-小鼠）和HFD治疗会减弱心肌2-AR和miR-133a，而运动和H2S则会减轻这种衰减。运动和沙丁胺醇对糖尿病患者AKT和Gs的诱导具有协同作用。2-AR （2-ARTg）的转基因表达可减轻HFD诱导的心脏纤维化。此外，消融MMP9基因可改善HFD诱导的收缩功能障碍。HFD治疗的WT和CBS+/-小鼠都有左心室功能障碍和收缩力受损，这可以通过运动训练减轻。HHcy和HFD还下调肌内质网atp酶2a （serca2a；调节肌肉收缩时的钙通量），运动和H2S可改善这一作用。该提议的中心假设是，在HFD诱导的糖尿病中，运动不耐受部分是由于HHcy抑制- 2-AR和Gs，下调AKT和miR-133a，诱导MMP9导致肌细胞收缩功能障碍。运动和H2S通过减轻HHcy和改善糖尿病患者的收缩功能障碍诱导2-AR和Gs（图1）。我们将通过以下三个具体目标来验证这一假设：具体目标#1：确定糖尿病患者的高同型半胱氨酸血症是否会减弱β 2-AR，运动和H2S是否会减轻这种减弱。假设：糖尿病患者2-AR下调，Hcy升高，运动和H2S诱导2-AR，降低Hcy水平。特异性目的#2：确定糖尿病患者高同型半胱氨酸血症和运动是否会导致AKT和miR-133a的减弱和MMP9的诱导，而H2S可以改善这种减弱和诱导。假设：在糖尿病中，Hcy水平升高抑制AKT和miR-133a，诱导MMP9。运动和H2S通过降低糖尿病心脏的Hcy水平诱导AKT，减弱MMP9。具体目的#3：确定糖尿病患者高同型半胱氨酸血症是否损害心肌细胞的收缩功能，运动和H2S是否减轻收缩功能障碍。假设：在糖尿病中，Hcy和MMP9升高，而β - 2-AR和AKT减弱，导致收缩功能障碍，运动和H2S改善这些水平，减轻收缩功能障碍。这些研究将阐明同型半胱氨酸介导的2-AR在糖尿病中的衰减机制，以及2-AR激动剂、运动和H2S在糖尿病并发症中的心脏保护作用。

DESCRIPTION (provided by applicant): Specific Aims: Lack of motivation to exercise is the hallmark of obesity and type2 diabetes (T2D). High fat diet (HFD) is the major cause of obesity and T2D that impair contractility of cardiomyocytes leading to heart failure. The stimulation of �2-adrenergic receptors (�2-AR) induces contractility and mitigates cardiac dysfunction. Elevated level of homocysteine (Hcy) called hyperhomocysteinemia (HHcy, a co-morbid condition) is associated with heart failure in diabetes. Hcy competes with and antagonizes �2-AR. The long term goal of the project is to understand the role of �2- AR and Hcy axis in diabetes. HHcy is ameliorated by conversion of Hcy to H2S (an anti-hypertensive, vasorelaxing, and anti-oxidant gas) by cystathionine � synthase (CBS) and cystathionine ? lyase (CSE). In diabetes, the levels of CBS and CSE are suppressed that induces HHcy and thereby impairs formation of H2S. H2S is an inducer of AKT (an anti-oxidant involved in glucose metabolism) and AKT is down regulated in diabetes. We have shown that H2S induces a stimulatory G-protein couple receptor called Gs (inducer of �2-AR) in diabetic cardiomyocytes. On the other hand, HHcy attenuates Gs. However, exercise mitigates HHcy and up regulates �2-AR in T2D. Also, exercise and salbutamol (�2-AR agonist) have synergistic effect on mitigation of contractile dysfunction in diabetic cardiomyocytes. However, the underlying mechanism is unclear. We have shown that in diabetes, HHcy activates MMP9 that induces cardiac fibrosis and impairs myocardial contractility. Also, microRNA-133a (miR-133a) that regulates cardiac fibrosis and hypertrophy is attenuated in diabetic hearts. Our preliminary studies show that HHcy (CBS+/- mice) and HFD treatment attenuate myocardial �2-AR and miR-133a, whereas exercise and H2S mitigate this attenuation. Exercise and salbutamol have synergistic effect on induction of AKT and Gs in diabetes. The transgenic expression of �2-AR (�2-ARTg) mitigates HFD induced cardiac fibrosis. Also, ablation of MMP9 gene ameliorates HFD induced contractile dysfunction. Both HFD treated WT and CBS+/- mice have left ventricle dysfunction and impaired contractility, which is mitigated by exercise training. HHcy and HFD also down regulates sarco-endoplasmic reticulum ATPase 2a (serca2a; regulates calcium flux during muscle contraction), which is improved by exercise and H2S. The central hypothesis of the proposal is that in HFD induced diabetes, the intolerance to exercise is, in part, due to attenuation of �2-AR and Gs by HHcy that down regulates AKT and miR-133a and induces MMP9 causing myocytes contractile dysfunction. The exercise and H2S induce �2-AR and Gs by mitigating HHcy and ameliorate contractile dysfunction in diabetes (Figure 1). We will test this hypothesis by following three specific aims: Specific Aim#1: To determine whether the �2-AR is attenuated by hyperhomocysteinemia in diabetes and exercise and H2S mitigate this attenuation. Hypothesis: In diabetes, �2-AR is down regulated and Hcy is elevated, and exercise and H2S induce �2-AR and decrease Hcy level. Specific Aim#2: To determine whether the AKT and miR-133a are attenuated and MMP9 is induced by hyperhomocysteinemia in diabetes and exercise and H2S ameliorate this attenuation and induction. Hypothesis: In diabetes, the elevated level of Hcy inhibits AKT and miR-133a and induces MMP9. Exercise and H2S induce AKT and attenuate MMP9 by decreasing Hcy level in diabetic hearts. Specific Aim#3: To determine whether the contractility of cardiomyocytes is impaired by hyperhomocysteinemia in diabetes and exercise and H2S mitigate the contractile dysfunction. Hypothesis: In diabetes, Hcy and MMP9 are elevated while �2-AR and AKT are attenuated leading to contractile dysfunction, and exercise and H2S ameliorate these levels and mitigate contractile dysfunction. These studies will elucidate the mechanism of homocysteine-mediated attenuation of �2-AR in diabetes and cardio-protective role of �2-AR agonist, exercise and H2S in diabetic complications.