Behunin Samantha M, Lopez-Pier Marissa A, Birch Camille L, McKee Laurel A K, Danilo Christiane, Khalpey Zain, Konhilas John P
Sarver Molecular Cardiovascular Research Program, Department of Physiology, University of Arizona, Tucson, Arizona.
Department of Surgery, University of Arizona, Tucson, Arizona.
Biophys J. 2015 Mar 24;108(6):1484-1494. doi: 10.1016/j.bpj.2015.02.012.
The myocardium undergoes extensive metabolic and energetic remodeling during the progression of cardiac disease. Central to remodeling are changes in the adenine nucleotide pool. Fluctuations in these pools can activate AMP-activated protein kinase (AMPK), the central regulator of cellular energetics. Binding of AMP to AMPK not only allosterically activates AMPK but also promotes phosphorylation of AMPK by an upstream kinase complex, LKB1/Mo25/STRAD (liver kinase B 1, mouse protein 25, STE-related adaptor protein). AMPK phosphorylation by the LKB1 complex results in a substantial increase in AMPK activity. Molecular targeting by the LKB1 complex depends on subcellular localization and transcriptional expression. Yet, little is known about the ability of the LKB1 complex to modulate targeting of AMPK after activation. Accordingly, we hypothesized that differing stoichiometric ratios of LKB1 activator complex to AMPK would uniquely impact myofilament function. Demembranated rat cardiac trabeculae were incubated with varying ratios of the LKB1 complex to AMPK or the LKB1 complex alone. After incubation, we measured the Ca(2+) sensitivity of tension, rate constant for tension redevelopment, maximum tension generation, length-dependent activation, cooperativity, and sarcomeric protein phosphorylation status. We found that the Ca(2+) sensitivity of tension and cross-bridge dynamics were dependent on the LKB1 complex/AMPK ratio. We also found that the LKB1 complex desensitizes and suppresses myofilament function independently of AMPK. A phospho-proteomic analysis of myofilament proteins revealed site-specific changes in cardiac Troponin I (cTnI) phosphorylation, as well as a unique distribution of cTnI phosphospecies that were dependent on the LKB1 complex/ AMPK ratio. Fibers treated with the LKB1 complex alone did not alter cTnI phosphorylation or phosphospecies distribution. However, LKB1 complex treatment independent of AMPK increased phosphorylation of myosin-binding protein C. Therefore, we conclude that the LKB1/AMPK signaling axis is able to alter muscle function through multiple mechanisms.
在心脏疾病进展过程中，心肌会经历广泛的代谢和能量重塑。重塑的核心是腺嘌呤核苷酸池的变化。这些池的波动可激活细胞能量代谢的核心调节因子——AMP激活的蛋白激酶（AMPK）。AMP与AMPK的结合不仅能变构激活AMPK，还能促进上游激酶复合物LKB1/Mo25/STRAD（肝激酶B1、小鼠蛋白25、STE相关衔接蛋白）对AMPK的磷酸化。LKB1复合物对AMPK的磷酸化导致AMPK活性大幅增加。LKB1复合物的分子靶向取决于亚细胞定位和转录表达。然而，关于LKB1复合物在激活后调节AMPK靶向的能力却知之甚少。因此，我们推测LKB1激活复合物与AMPK不同的化学计量比会对肌丝功能产生独特影响。将去膜的大鼠心脏小梁与不同比例的LKB1复合物与AMPK或单独的LKB1复合物一起孵育。孵育后，我们测量了张力的Ca(2+)敏感性、张力重新发展的速率常数、最大张力产生、长度依赖性激活、协同性以及肌节蛋白磷酸化状态。我们发现张力的Ca(2+)敏感性和横桥动力学取决于LKB1复合物/AMPK的比例。我们还发现LKB1复合物可使肌丝功能脱敏并抑制其功能，且与AMPK无关。对肌丝蛋白的磷酸蛋白质组分析揭示了心肌肌钙蛋白I（cTnI）磷酸化的位点特异性变化，以及取决于LKB1复合物/AMPK比例的cTnI磷酸化异构体的独特分布。单独用LKB1复合物处理的纤维不会改变cTnI磷酸化或磷酸化异构体分布。然而，独立于AMPK的LKB1复合物处理会增加肌球蛋白结合蛋白C的磷酸化。因此，我们得出结论，LKB1/AMPK信号轴能够通过多种机制改变肌肉功能。
