The Function of Nonmuscle Myosin II Heavy Chains

切除非肌肉肌球蛋白重链（NMHC） II-B的小鼠在其心脏和大脑中出现主要结构异常，并在胚胎日死亡(E) 14.5。这些异常包括室间隔缺损（VSD）、源自右心室的主动脉错位和心脏细胞分裂缺陷。脑缺陷包括特定神经元群（面部和脑桥）的异常迁移和严重的脑积水。心肌细胞的独特之处在于它们不表达NMHC II-A，而神经元细胞则富含NMHC II-B，这可以解释观察到的表型。本实验的目的是观察NMHC II-A的表达是否可以挽救因NMHC II-B缺失而导致的缺陷。我们使用gfp标记的人NMHC II-A cDNA，将其插入II-B的第一个编码外显子，从而将GFP-NMHC II-A置于内源性II-B启动子的控制之下。有趣的是，我们发现20%的纯合子小鼠存活超过4个月，没有脑积水的迹象，也没有表现出明显的心脏缺陷。然而，通过组织学分析，他们确实表现出面部神经元和脑桥神经元的异常迁移。免疫印迹和免疫荧光分析证实，NMHC II-B在心脏、肺和脑中未表达，而GFP-NMHC II-A在这些器官中表达显著。对E12和出生之间小鼠的分析显示，大多数小鼠（80%）在E12和E16.5之间死亡，表现出与B-/B-小鼠相似的缺陷。这些包括室间隔缺损和双核和增大的心肌细胞。然而，与II-B消融小鼠相比，这些小鼠都没有起源于右心室的主动脉。少数存活到成年的小鼠在大约3个月大时开始出现扩张性心肌病并伴有肌纤维纤维化。异常包括心脏/体重比增加，肌纤维肥大和心脏胎儿蛋白如心房利钠肽和-心脏肌球蛋白重链的重新表达。因此，我们证明非肌肉肌凝蛋白II-B在小鼠发育过程中起着肌凝蛋白II-A无法替代的重要作用。

Mice ablated for nonmuscle myosin heavy chain (NMHC) II-B develop major structural abnormalities in their hearts and brains and die by embryonic day (E) 14.5. These abnormalities include a ventricular septal defect (VSD), mislocation of the aorta which emanates from the right ventricle and a defect in cytokinesis in the heart. The brain defects include abnormal migration of specific groups of neurons (facial and pontine) and a severe hydrocephalus. Cardiac myocytes are unique in that they do not express NMHC II-A, and neuronal cells are enriched for NMHC II-B, which could explain the phenotype observed. The purpose of the present experiments was to see whether expression of NMHC II-A could rescue the defects due to the loss of NMHC II-B. We used GFP-tagged human NMHC II-A cDNA to ablate NMHC II-B by inserting it into the first coding exon for II-B, thus placing GFP-NMHC II-A under control of the endogenous II-B promoter. Interestingly, we found that 20% of the homozygous mice survived beyond 4 months of age without evidence for hydrocephalus nor did they manifest obvious cardiac defects. However, they did show abnormal migration of facial neurons and pontine neurons by histological analysis. Immunoblot and immunofluorescence analyses confirmed no expression of NMHC II-B in the heart, lung and brain, and demonstrated significant expression of GFP-NMHC II-A in these organs. Analysis of mice between E12 and birth revealed that most of the mice (80%) died between E12 and E16.5 showing defects similar to those found in B-/B- mice. These included a ventricular septal defect and binucleated and enlarged cardiac myocytes. However, none of these mice had an aorta that originated in the right ventricle in contrast to II-B ablated mice. The few mice that survived to adulthood developed dilated cardiomyopathy accompanied by myofibrillar fibrosis starting about 3 months of age. Abnormalities included an increase in the heart/body weight ratio, myofibrillar hypertrophy and re-expression of cardiac fetal proteins such as atrial natriuretic peptide and beta-cardiac myosin heavy chain. Thus, we demonstrate that nonmuscle myosin II-B plays an important role during mouse development that cannot be substituted for by myosin II-A.