Nonmuscle myosin IIB regulates epicardial integrity and epicardium-derived mesenchymal cell maturation

Abstract

Nonmuscle myosin IIB (NMIIB; heavy chain encoded by MYH10) is essential for cardiac myocyte cytokinesis. The role of NMIIB in other cardiac cells is not known. Here, we show that NMIIB is required in epicardial formation and functions to support myocardial proliferation and coronary vessel development. Ablation of NMIIB in epicardial cells results in disruption of epicardial integrity with a loss of E-cadherin at cell-cell junctions and a focal detachment of epicardial cells from the myocardium. NMIIB-knockout and blebbistatin-treated epicardial explants demonstrate impaired mesenchymal cell maturation during epicardial epithelial-mesenchymal transition. This is manifested by an impaired invasion of collagen gels by the epicardium-derived mesenchymal cells and the reorganization of the cytoskeletal structure. Although there is a marked decrease in the expression of mesenchymal genes, there is no change in Snail (also known as Snai1) or E-cadherin expression. Studies from epicardiumspecific NMIIB-knockout mice confirm the importance of NMIIB for epicardial integrity and epicardial functions in promoting cardiac myocyte proliferation and coronary vessel formation during heart development. Our findings provide a novel mechanism linking epicardial formation and epicardial function to the activity of the cytoplasmic motor protein NMIIB.

Non-muscle myosin IIB (Myh10) is required for epicardial function and coronary vessel formation during mammalian development

“…As the formation of cardiac valve structures is highly dependent upon a functional endocardium [ 13 , 52 ], the presence of these structures in EHC mutants supports the hypothesis that endocardial function is not significantly impaired following loss of NMHC IIB. A recently published complementary study has demonstrated that coronary vessel development is impaired when NMHC IIB is deleted specifically within the epicardium [ 53 ], supporting our findings that epicardial abnormalities in EHC mutant mice contribute to defective coronary vessel development.…”

“…Indeed, formation of the atrioventricular valves, a process dependent upon endocardial cushion EMT [ 13 ], is evident in EHC mutant hearts ( S5 Fig ). We have demonstrated that NMHC IIB is the predominant NMHC II isoform in epicardial cells both in vivo and in vitro , and that NMHC IIB is localised to distinct subcellular regions (in agreement with the findings of Lo et al, [ 28 ], and Ma et al, [ 53 ]). Therefore, it may be hypothesised that NMHC IIB plays critical roles in epicardial and cardiac development, which cannot be replaced by other NMHC II isoforms when NMHC IIB function is globally ablated.…”

“…The absence of mature coronary vessels in EHC mutants suggests that in vivo these dysfunctional EPDCs are incapable of contributing to the vascular network. A complementary study has demonstrated that coronary vessel development is impaired when Myh10 is deleted specifically in the epicardium [ 53 ], consistent with our conclusion that epicardial defects underpin disrupted coronary vessel development in EHC mutant mice.…”

“…This finding suggests that the severe morphological defects present in NMHC IIB null hearts are caused by loss of NMHC IIB from other cardiac cell populations. It has recently been demonstrated that epicardial-specific deletion of Myh10 does impair coronary vessel development [ 53 ], although the reported defects are not as severe as we have documented here for the EHC mutant, EHC/Myh10∆ compound heterozygote, or Myh10∆ homozygous mutant embryos. Further investigation is required to determine if these phenotypic differences are due to differences in assays used or due to requirements for NMHC IIB in multiple cell types during coronary vessel development.…”

“…The detection of abnormalities in EMT signaling, indicated by increased epicardial cell proliferation and reduced Snail expression in EHC mutants, is surprising, as NMHC IIB would be expected to be a downstream effector of cell motility in EMT. Although EMT defects have been detected in the epicardial-specific Myh10 knock out [ 53 ], comparison of the results of that study with ours is complicated by the use of different assay methods and the potential for requirements for NMHC IIB in non-epicardial cells to influence our findings. However, our results suggest that the loss of NMHC IIB disrupts processes required for EMT signaling, through the NF-κB pathway acting downstream of TGFβ and PDGF inputs [ 70 ].…”