The Role of Nonmuscle Myosins in Cardiac Development

先前的工作表明，非肌肉肌球蛋白重链II-B(NMHC II-B)消融的小鼠会发展出一种不寻常的心肌细胞增大形式，出现在胚胎发育期间，涉及心房和心室。心肌细胞不同于心脏中的非心肌细胞(成纤维细胞、内皮细胞和平滑肌细胞等)。不含NMHC II-A。最近，我们用免疫荧光法证明了人和小鼠心肌细胞中存在第三种形式的非肌肉肌球蛋白II，即NMHC II-C。因此，由于NMHC II-A和II-B都被证明参与了这一过程，因此NMHC II-B消融的心肌细胞可能会出现胞质分裂缺陷。为了研究这种可能性，我们在胚胎14天对B-/B-小鼠心脏与对照心脏(B+/B-，B+/B+)的心肌细胞数量进行了定量比较。我们发现B-/B-心脏每心脏切面的心肌细胞数量减少了50%以上，但非心肌细胞的数量在B-/B-心脏和对照心脏中是相同的，所有非心肌细胞都含有NMHC II-A。作为细胞质分裂而不是核分裂缺陷的进一步证据，我们对双核的程度进行了定量，发现在E12.5，23%的B-/B-心肌细胞双核，而对照组心肌细胞的双核比例为1%。在B-/B-心脏的非心肌细胞中没有双核的证据。我们通过测量胚胎14天的BrdU标记指数(BrdU标记的肌细胞百分比)来比较心肌细胞的增殖程度，发现B-/B-心肌细胞的BrdU标记指数显著降低，而心脏和其他器官的B-/B-非心肌细胞的BrdU标记指数无明显变化。我们将这些数据解释为一种先前未被描述的心肌细胞增大原因的证据，在这种情况下，胞质分裂而不是核分裂部分缺陷，从而在胚胎发育期间损害心肌细胞的增殖。我们将胞质分裂的部分失败而不是完全失败归因于NMHC II-C的存在。然而，仅有NMHC II-C似乎不足以进行正常的胞质分裂。相反，胚胎12岁时，心肌中仅表达正常NMHC II-B的7%的低形态小鼠的心肌细胞没有显示出双核和肥大的证据，这与正常胞质分裂的恢复一致。有趣的是，我们还发现，携带单一NMHC II-B突变R709C的亚型纯合子小鼠的心肌细胞数量也减少了，其中许多显示出双核和增大的证据。

Previous work has shown that nonmuscle myosin heavy chain II-B (NMHC II-B) ablated mice develop an unusual form of myocyte enlargement which appears during embryonic development and involves both atria and ventricles. Cardiac myocytes differ from the non-myocytes in the heart (fibroblasts, endothelial and smooth muscle cells, etc.) in not containing NMHC II-A. Recently, we have demonstrated that a third form of nonmuscle myosin II, NMHC II-C, exists in human and murine cardiac myocytes using immunofluorescence. Thus, cardiac myocytes ablated for NMHC II-B might be expected to show a defect in cytokinesis, since both NMHC II-A and II-B have been shown to participate in this process. To study this possibility, we quantitated the number of cardiac myocytes in B-/B- mouse hearts compared to control hearts (B+/B-, B+/B+) at E14. We found that the number of cardiac myocytes per heart section was decreased by more than 50% in the B-/B- heart, but that the number of non-myocytes, all of which contain NMHC II-A, was the same for both B-/B- and control hearts. As further evidence for a defect in cytokinesis, but not karyokinesis, we quantitated the extent of binucleation and found that, at E12.5, 23% of the B-/B- cardiac myocytes were binucleated compared to 1% of the cardiac myocytes in control hearts. There was no evidence for binucleation in the non-myocytes of the B-/B- hearts. We compared the extent of myocyte proliferation by measuring the BrdU labeling index (% myocytes labeled with BrdU) at E14 and found that it was significantly reduced for B-/B- cardiac myocytes, but not for B-/B- non-myocytes in the heart nor for other organs. We interpret these data as evidence for a previously undescribed cause of cardiac myocyte enlargement, in which cytokinesis, but not karyokinesis, is partially defective, thereby impairing myocyte proliferation during embryonic development. We attribute the partial, rather than complete, failure of cytokinesis to the presence of NMHC II-C. However, NMHC II-C alone does not appear to be sufficient for normal cytokinesis. In contrast, the cardiac myocytes of hypomorphic mice expressing only 7% of the normal amount of NMHC II-B in the heart at E12 showed no evidence of binucleation and hypertrophy, consistent with the restoration of normal cytokinesis. Interestingly, we also found that hypomorphic homozygous mice harboring the single NMHC II-B mutation R709C also have a decreased number of cardiac myocytes, many of which show evidence for binucleation and enlargement.