Rodriques Aaron, Liu Guoxia, Katchman Alex, Zakharov Sergey, Wan Elaine, Kalocsay Marian, Eisert Robyn, Bradshaw Gary A, Chen Bi-Xing, Yang Lin, Reiken Steven, Lai Xianghai, Ji Ruiping, Saadallah Najla, Topkara Veli, Fanourakis Stavros, Yuan Qi, Kushner Jared
Department of Medicine, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA.
Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
bioRxiv. 2025 May 13:2025.05.08.652977. doi: 10.1101/2025.05.08.652977.
Maladaptive changes in the function, expression and localization of proteins involved in calcium-handling worsen the impaired contractility of systolic heart failure (HF). Standard proteomics techniques require cell lysis and so are unable to characterize changes specific to the critical subcellular domain bounded by the T-tubule and the sarcoplasmic reticulum, known as the cardiac dyad. Traditional approaches are also less likely to capture low-affinity protein-protein interactions on lipid membranes. To improve our understanding of heart failure pathophysiology, we applied proximity proteomics to the cardiac dyad of mice with ischemic cardiomyopathy.
Using two lines of transgenic mice expressing fusion proteins of the engineered ascorbate peroxidase APEX2 with subunits of the cardiac voltage gated calcium channel, Ca1.2, we labeled the dyad proteome of live, intact myocytes from healthy mice (N=6) and mice with coronary artery ligation HF (N=5) by peroxidase-catalyzed biotinylation. Quantitative mass spectrometry with isobaric tandem mass tags (TMT) was used to assess alterations in the local dyad proteome in myocytes from mice with chronic, remodeled HF. We subsequently generated a mouse with inducible cardiac overexpression of Galectin-1 to examine the effects of this protein on cardiac function and more specifically on the calcium-handling properties of mature myocytes, using cellular electrophysiology, calcium imaging, and echocardiography.
From mice with HF, we found significant enrichment of 43 proteins defined by their abundance and proximity to transgenic Ca1.2 α channels, and a significant reduction in 22 proteins, out a of a total of 2326 proteins quantified. We also significantly enriched 286 proteins, and saw a reduction in 13 proteins, defined by proximity and abundance to Ca1.2 β-subunits, out of a total 2236 proteins identified. Pathway analysis revealed HF is associated with increased abundance of components of the 26s proteasome and microtubules in the dyad, as well as the dimerizing, carbohydrate binding protein Galectin-1. Cardiac specific overexpression of Galectin-1 in healthy mice increases activation of Ca1.2 and the Ryanodine receptor and accelerates myocyte relaxation through phosphorylation of Phospholamban.
Using proximity proteomics to examine the effects of HF , we find increased localization of Galectin-1 to the cardiac dyad, and that overexpression of Galectin-1 accelerates calcium kinetics in the heart.
参与钙处理的蛋白质在功能、表达和定位上的适应性改变会加剧收缩性心力衰竭（HF）受损的收缩力。标准蛋白质组学技术需要细胞裂解，因此无法表征由T小管和肌浆网界定的关键亚细胞结构域（称为心脏二联体）特有的变化。传统方法也不太可能捕获脂质膜上低亲和力的蛋白质-蛋白质相互作用。为了更好地理解心力衰竭的病理生理学，我们将邻近蛋白质组学应用于缺血性心肌病小鼠的心脏二联体。
我们使用两系转基因小鼠，其表达工程化抗坏血酸过氧化物酶APEX2与心脏电压门控钙通道Ca1.2亚基的融合蛋白，通过过氧化物酶催化的生物素化标记来自健康小鼠（N = 6）和冠状动脉结扎HF小鼠（N = 5）的活的完整心肌细胞的二联体蛋白质组。使用带有等压串联质谱标签（TMT）的定量质谱法评估慢性重塑HF小鼠心肌细胞中二联体局部蛋白质组的变化。随后，我们构建了一种可诱导心脏过表达半乳糖凝集素-1的小鼠，以使用细胞电生理学、钙成像和超声心动图检查该蛋白对心脏功能的影响，更具体地说是对成熟心肌细胞钙处理特性的影响。
在HF小鼠中，我们发现，在总共定量的2326种蛋白质中，有43种蛋白质因其与转基因Ca1.2α通道的丰度和接近程度而显著富集，22种蛋白质显著减少。在总共鉴定的2236种蛋白质中，我们还发现有286种蛋白质因与Ca1.2β亚基的接近程度和丰度而显著富集，13种蛋白质减少。通路分析显示，HF与二联体中26S蛋白酶体和微管成分以及二聚化的碳水化合物结合蛋白半乳糖凝集素-1丰度增加有关。健康小鼠心脏特异性过表达半乳糖凝集素-1会增加Ca1.2和兰尼碱受体的激活，并通过磷酸化受磷蛋白加速心肌细胞松弛。
使用邻近蛋白质组学研究HF的影响，我们发现半乳糖凝集素-1在心脏二联体中的定位增加，并且半乳糖凝集素-1的过表达会加速心脏中的钙动力学。
