Preferred mitotic orientation in pattern formation by vascular mesenchymal cells.

Cellular self-organization is essential to physiological tissue and organ development. We previously observed that vascular mesenchymal cells, a multipotent subpopulation of aortic smooth muscle cells, self-organize into macroscopic, periodic patterns in culture. The patterns are produced by cells gathering into raised aggregates in the shape of nodules or ridges. To determine whether these patterns are accounted for by an oriented pattern of cell divisions or postmitotic relocation of cells, we acquired time-lapse, videomicrographic phase-contrast, and fluorescence images during self-organization. Cell division events were analyzed for orientation of daughter cells in mitoses during separation and their angle relative to local cell alignment, and frequency distribution of the mitotic angles was analyzed by both histographic and bin-free statistical methods. Results showed a statistically significant preferential orientation of daughter cells along the axis of local cell alignment as early as day 8, just before aggregate formation. This alignment of mitotic axes was also statistically significant at the time of aggregate development (day 11) and after aggregate formation was complete (day 15). Treatment with the nonmuscle myosin II inhibitor, blebbistatin, attenuated alignment of mitotic orientation, whereas Rho kinase inhibition eliminated local cell alignment, suggesting a role for stress fiber orientation in this self-organization. Inhibition of cell division using mitomycin C reduced the macroscopic pattern formation. Time-lapse monitoring of individual cells expressing green fluorescent protein showed postmitotic movement of cells into neighboring aggregates. These findings suggest that polarization of mitoses and postmitotic migration of cells both contribute to self-organization into periodic, macroscopic patterns in vascular stem cells.

Critical role of nuclear calcium/calmodulin-dependent protein kinase IIdeltaB in cardiomyocyte survival in cardiomyopathy.

Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in cardiac contractility and heart disease. However, the specific role of alternatively spliced variants of CaMKII in cardiac disease and apoptosis remains poorly explored. Here we report that the deltaB subunit of CaMKII (CaMKIIdeltaB), which is the predominant nuclear isoform of calcium/calmodulin-dependent protein kinases in heart muscle, acts as an anti-apoptotic factor and is a novel target of the antineoplastic and cardiomyopathic drug doxorubicin (Dox (adriamycin)). Hearts of rats that develop cardiomyopathy following chronic treatment with Dox also show down-regulation of CaMKIIdeltaB mRNA, which correlates with decreased cardiac function in vivo, reduced expression of sarcomeric proteins, and increased tissue damage associated with Dox cardiotoxicity. Overexpression of CaMKIIdeltaB in primary cardiac cells inhibits Dox-mediated apoptosis and prevents the loss of the anti-apoptotic protein Bcl-2. Specific silencing of CaMKIIdeltaB by small interfering RNA prevents the formation of organized sarcomeres and decreases the expression of Bcl-2, which all mimic the effect of Dox. CaMKIIdeltaB is required for GATA-4-mediated co-activation and binding to the Bcl-2 promoter. These results reveal that CaMKIIdeltaB plays an essential role in cardiomyocyte survival and provide a mechanism for the protective role of CaMKIIdeltaB. These results suggest that selective targeting of CaMKII in the nuclear compartment might represent a strategy to regulate cardiac apoptosis and to reduce Dox-mediated cardiotoxicity.