The Role of Nonmuscle Myosin II-B in Mouse Heart and Brain Development

非肌肉肌球蛋白 II-B 在小鼠心脏和大脑发育中的作用

基本信息

批准号：
7734989
负责人：
ROBERT ADELSTEIN
金额：
$ 37.61万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

Three isoforms of NM II: NM II-A, II-B and II-C are expressed in mammals. Ablation of NM II-A in mice (A-/A-) leads to embryonic lethality by E6.5 with disorganized germ layer formation. Mice ablated for NM II-B (B-/B-) die by E14.5 with defects in heart and brain development. The cardiac defects include a decrease in the number of cardiac myocytes which can be attributed in part to abnormalities in cytokinesis. To understand the function of NM II-C in vivo, we generated mice ablated for NM II-C (C-/C-). The C-/C- mice are viable and show no obvious abnormalities during development, but show mild phenotypic changes in the heart, including a decrease in cardiac function as measured by MRI at 6 months and moderate myocyte hypertrophy at one year. The presence of mild cardiac abnormalities in C-/C- mice prompted us to cross these mice with II-B hypomorphic mice which express only 12% of the wild type levels of NM II-B in the heart. The homozygous offspring of this mating (BIC-/BIC-) develop cardiac myocyte hypertrophy as well as interstitial fibrosis at 6 months, a time when both C-/C- and BI/BI mice show no such abnormalities. To further examine this effect in cardiac myocytes, we generated II-B and II-C double knockout mice (B-C-/B-C-). Similar to B-/B- mice, B-C-/B-C- mice died by E14.5 with a hypoplastic heart. In addition to a failure in cytokinesis, B-C-/B-C- cardiac myocytes also showed major defects in karyokinesis. During mitosis many B-C-/B-C- myocytes demonstrate defects in chromatid separation and spindle formation. While defective karyokinesis in the cardiac myocytes was occasionally observed (15%) in B-/B- mice, the frequency was increased to 90% in B-C-/B-C- mice. The requirement for NM II in karyokinesis was further demonstrated in cultured HL-1 atrial myocytes using siRNA knockdown of NM II or blebbistatin. Unlike the cardiac myocytes where NM II-B is the predominant isoform and II-A is not detected, HL-1 cells express 95% NM II-A and 5% II-B. An additive effect on karyokinesis in HL-1 cells was seen following siRNA knockdown of NM II-A and II-B. In summary we present evidence that NM II isoforms play a role in cardiac myocytes cytokinesis and karyokinesis.

NM II：NM II-A，II-B和II-C的三种同工型在哺乳动物中表达。 NM II-A在小鼠（A-/A-）中的消融导致E6.5的胚胎致死性，并形成混乱的生殖层形成。 E14.5死于NM II-B（B-/B-）的小鼠，心脏和脑发育缺陷。心脏缺陷包括减少心肌细胞的数量，这可能部分归因于细胞因子的异常。为了了解NM II-C在体内的功能，我们生成了用于NM II-C（C-/C-）的小鼠。 C-/c-小鼠是可行的，在发育过程中没有明显的异常，但是在心脏中显示出轻度的表型变化，包括在6个月时MRI测量的心脏功能降低以及一年中中度的心肌肥大。 C-/c-小鼠中有轻度心脏异常的存在促使我们与II-B型肌型小鼠穿越这些小鼠，这些小鼠仅表达心脏中NM II-B的12％。这种交配（BIC-/BIC-）的纯合后代在6个月时出现心肌肥大以及间质纤维化，这是C-/c/c/c/bi/bi鼠标均未表现出这种异常。为了进一步检查心肌细胞中的这种影响，我们产生了II-B和II-C双基因敲除小鼠（B-C-/B-C-）。与B-/B-小鼠相似，B-C-/B-C-小鼠死于E14.5，其心脏不良心脏死亡。除了细胞因子的衰竭外，B-C-/B-C-心肌细胞还显示出核苷酸的主要缺陷。在有丝分裂期间，许多B-C-/B-C-肌细胞表现出染色单体分离和纺锤体形成的缺陷。虽然偶尔在B-/B-小鼠中观察到心肌细胞中的核细胞缺陷（15％），但B-C-/B-C-小鼠中的频率增加到90％。在培养的HL-1心房肌细胞中，使用NM II或BLEBBISTATIN的siRNA敲低培养的HL-1心肌细胞中进一步证明了NM II的需求。与心肌细胞NM II-B是主要同工型，未检测到II-A的心肌细胞不同，HL-1细胞表达95％nm II-A和5％II-B。在NM II-A和II-B的siRNA敲低后，可以看到对HL-1细胞中核苷酸的添加作用。总而言之，我们提供的证据表明NM II同工型在心肌细胞细胞因子和核细胞中起作用。