Identification of a novel splice variant of neuronal nitric oxide synthase, nNOSβ, in myofilament fraction of murine cardiomyocytes
Identification of a novel splice variant of neuronal nitric oxide synthase, nNOSβ, in myofilament fraction of murine cardiomyocytes
复制标题
鉴定神经元一氧化氮合酶的新剪接变体，nNOSβ，在小鼠心肌细胞的肌丝部分。
DOI：
10.1016/j.niox.2015.07.005
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发表时间：
2015-11-15
影响因子：
3.9
通讯作者：
Zhang, Yin Hua
中科院分区：
文献类型：
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作者：
Jang, Ji Hyun;Kang, Min Jueng;Zhang, Yin Hua
神经元型一氧化氮合酶（nNOS或NOS 1）、nNOS α和nNOS μ的剪接变体形式被充分确立为在心肌细胞的离散区室（例如肌浆网、SR、质膜或线粒体中的小窝）中功能性表达。迄今为止，nNOS是否在心肌细胞的肌丝组分中表达以及nNOS的剪接变体形式尚不清楚。使用两种nNOS特异性抗体（BD Transduction Laboratories aa 1095-1289和圣克鲁斯生物技术aa 2-300）的免疫印迹结果清楚地表明，nNOS在心肌细胞的肌营养素富集部分中大量表达。虽然膜/胞质溶胶组分中nNOS的分子量类似于165 kDa，但肌丝中的nNOS低于140 kDa，表明肌丝中nNOS的主要剪接变体是nNOS β。RT-PCR结果证实nNOS α和nNOS β mRNA在大鼠心肌细胞中均有表达。类似地，使用心肌细胞的肌丝裂解物的免疫沉淀实验鉴定了具有低分子量的nNOS（M.W.类似于140 kDa），证实了nNOS β。有趣的是，nNOS的所有三种剪接变体在来自nNOS（-/-）小鼠（其缺乏nNOS α/mu）的心肌细胞（包括肌丝部分）的裂解物中均检测不到。此外，高血压大鼠心肌细胞肌丝中的nNOS β表达与假手术组相比无差异。基于iTRAQ的定量蛋白质组学分析显示，nNOS调节心肌肌丝中类似于20种蛋白质的磷酸化。总的来说，我们提供了直接的证据表明，不同的剪接变异体的nNOS表达在心肌细胞的肌丝和膜/胞质溶胶部分。不同剪接变异体在心肌细胞的不同隔室中的离散表达表明nNOS在健康和患病心脏中发挥不同的作用。(C)2015 Elsevier Inc. All rights reserved.
Splice variant forms of neuronal nitric oxide synthase (nNOS or NOS1), nNOS alpha and nNOS mu are well established to be functionally expressed in discrete compartments in cardiomyocytes (e.g. sarcoplasmic reticulum, SR, caveolae in plasma membrane or mitochondria). So far, whether nNOS is expressed in myofilament fraction of cardiomyocytes and the splice variant form of nNOS are unknown. Immunoblotting results using two nNOS specific antibodies (BD Transduction Laboratories aa 1095-1289 and Santa Cruz Biotechnology aa 2-300) clearly demonstrated that nNOS was abundantly expressed in myofilament-enriched fraction of cardiomyocytes. Whilst the molecular weight of nNOS in membrane/cytosol fractions was similar to 165 kDa, nNOS in myofilament was below 140 kDa, suggesting that the predominant splice variant of nNOS in myofilament is nNOS beta. RT-PCR results confirmed the expressions of both nNOS alpha and nNOS beta mRNAs in rat cardiomyocytes. Similarly, immunoprecipitation experiments using myofilament lysates of cardiomyocytes identified nNOS with low molecular weight (M.W. similar to 140 kDa), confirming nNOS beta. Intriguingly, all three splice variants of nNOS were undetectable in the lysates of cardiomyocytes (including myofilament fractions) from nNOS(-/-) mice (which lacks nNOS alpha/mu). Furthermore, nNOS beta expression in myofilament of cardiomyocytes was not different in hypertensive rats compared to the level expressed in sham. iTRAQ-based quantitative proteomics analysis revealed that nNOS regulates phosphorylations of similar to 20 proteins in cardiac myofilaments. Collectively, we provide direct evidence that different splice variants of nNOS are expressed in myofilament and membrane/cytosol fractions of cardiomyocytes. Discrete expressions of various splice variants in different compartments of cardiomyocytes suggest diverse roles nNOS play in healthy and diseased heart. (C) 2015 Elsevier Inc. All rights reserved.