The Function of Nonmuscle Myosin II Heavy Chains

Heart, Lung, And Blood Institute

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Abstract

De Lozanne and Spudich showed that ablation of nonmuscle myosin II in Dictyostelium resulted in a defect in cytokinesis, but not karyokinesis. Recently, we reported that ablation of NMHC II-B in mice results in a 70% decrease in the number of cardiac myocytes in the hearts of the ablated mice and an increase in binucleated cardiac myocyte. Cardiac myocytes do not contain NMHC II-A, but do contain NMHC II-C and normally contain II-B. Here we report on RNAi treatment of monkey COS-7 cells, a cell line that like cardiac myocytes lacks NMHC II-A, but contains NMHC II-B as well as II-C. We used 21 nucleotide small interfering RNA duplexes (siRNA) to specifically suppress the expression of NMHC II-B. Western blot analysis showed that compared to control cells, NMHC II-B expression levels decreased to 16% after 72h and 10.4% after 96h. There was no alteration in the expression level of NMHC II-A and II-C. Down-regulation of NMHC II-B expression significantly inhibited cell proliferation so that cell numbers were approximately 50% lower in the RNAi treated cells compared to control cells (72-168h). Whereas 8.73% of control cells were multi-nucleated, 62.4% of the RNAi treated cells were multi-nucleated (72h). Moreover, the RNAi treated cells had increased surface areas, and unlike control cells, lacked actin stress fibers. Confocal microscopy showed actin to be diffusely distributed throughout the cell. Other changes in cell cycle proteins include an increase in phospho-Rb and cyclin D3 in the mutant cells. These studies show that NMHC II-C alone is insufficient for normal cytokinesis and also suggest that similar to cardiac myocytes, there are other defects in the cell cycle of these cells. In a second project, we have replaced the NMHC II-B gene in mice with a construct encoding GFP-NMHC II-A. To date, we have generated both embryonic stem cells and chimeric mice. The purpose of this project is to see if NMHC II-A can rescue NMHC II-B knockout mice.