Stachowski-Doll Marisa J, Papadaki Maria, Martin Thomas G, Ma Weikang, Gong Henry M, Shao Stephanie, Shen Shi, Muntu Nitha Aima, Kumar Mohit, Perez Edith, Martin Jody L, Moravec Christine S, Sadayappan Sakthivel, Campbell Stuart G, Irving Thomas, Kirk Jonathan A
Department of Cell and Molecular Physiology, Loyola University Stritch School of Medicine, Maywood, IL (M.J.S.-D., M.P., T.G.M., N.A.M., E.P., J.A.K.).
Center for Synchrotron Radiation Research and Instrumentation and Department of Biological Sciences, Illinois Institute of Technology, Chicago (W.M., H.M.G., T.I.).
Circ Res. 2022 Mar 18;130(6):871-886. doi: 10.1161/CIRCRESAHA.121.319491. Epub 2022 Feb 16.
Altered kinase localization is gaining appreciation as a mechanism of cardiovascular disease. Previous work suggests GSK-3β (glycogen synthase kinase 3β) localizes to and regulates contractile function of the myofilament. We aimed to discover GSK-3β's in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance.
Inducible cardiomyocyte-specific GSK-3β knockout mice and left ventricular myocardium from nonfailing and failing human hearts were studied.
Skinned cardiomyocytes from knockout mice failed to exhibit calcium sensitization with stretch indicating a loss of length-dependent activation (LDA), the mechanism underlying the Frank-Starling Law. Titin acts as a length sensor for LDA, and knockout mice had decreased titin stiffness compared with control mice, explaining the lack of LDA. Knockout mice exhibited no changes in titin isoforms, titin phosphorylation, or other thin filament phosphorylation sites known to affect passive tension or LDA. Mass spectrometry identified several z-disc proteins as myofilament phospho-substrates of GSK-3β. Agreeing with the localization of its targets, GSK-3β that is phosphorylated at Y216 binds to the z-disc. We showed pY216 was necessary and sufficient for z-disc binding using adenoviruses for wild-type, Y216F, and Y216E GSK-3β in neonatal rat ventricular cardiomyocytes. One of GSK-3β's z-disc targets, abLIM-1 (actin-binding LIM protein 1), binds to the z-disc domains of titin that are important for maintaining passive tension. Genetic knockdown of abLIM-1 via siRNA in human engineered heart tissues resulted in enhancement of LDA, indicating abLIM-1 may act as a negative regulator that is modulated by GSK-3β. Last, GSK-3β myofilament localization was reduced in left ventricular myocardium from failing human hearts, which correlated with depressed LDA.
We identified a novel mechanism by which GSK-3β localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3β levels were reduced in patients with heart failure, indicating z-disc localized GSK-3β is a possible therapeutic target to restore the Frank-Starling mechanism in patients with heart failure.
激酶定位改变作为心血管疾病的一种机制正逐渐受到重视。先前的研究表明糖原合酶激酶3β（GSK-3β）定位于肌丝并调节其收缩功能。我们旨在探究GSK-3β在体内调节肌丝功能的作用、相关机制以及转化意义。
对诱导型心肌细胞特异性GSK-3β基因敲除小鼠以及非衰竭和衰竭人类心脏的左心室心肌进行研究。
基因敲除小鼠的脱细胞心肌细胞在拉伸时未表现出钙敏化，表明长度依赖性激活（LDA）丧失，而LDA是Frank-Starling定律的基础机制。肌联蛋白作为LDA的长度传感器，与对照小鼠相比，基因敲除小鼠的肌联蛋白刚度降低，这解释了LDA的缺失。基因敲除小鼠的肌联蛋白异构体、肌联蛋白磷酸化或其他已知影响被动张力或LDA的细肌丝磷酸化位点均无变化。质谱分析确定了几种z盘蛋白为GSK-3β的肌丝磷酸化底物。与其靶点的定位一致，在Y216位点磷酸化的GSK-3β与z盘结合。我们使用野生型、Y216F和Y216E GSK-3β腺病毒在新生大鼠心室心肌细胞中证明pY216对于z盘结合是必要且充分的。GSK-3β的z盘靶点之一，肌动蛋白结合LIM蛋白1（abLIM-1），与肌联蛋白的z盘结构域结合，而该结构域对于维持被动张力很重要。在人类工程心脏组织中通过小干扰RNA对abLIM-1进行基因敲低导致LDA增强，表明abLIM-1可能作为一种受GSK-3β调节的负调节因子。最后，在衰竭人类心脏的左心室心肌中，GSK-3β的肌丝定位减少，这与LDA降低相关。
我们确定了一种新机制，通过该机制GSK-3β定位于肌丝以调节LDA。重要的是，心力衰竭患者的z盘GSK-3β水平降低，表明z盘定位的GSK-3β可能是恢复心力衰竭患者Frank-Starling机制的治疗靶点。
