We have previously generated and characterized a Foxm1-null (Foxm1−/−
) mouse line in which mice die between embryonic days E13.5 and E16.5 due to severe defects in multiple organ systems including lungs, liver, heart and blood vessels 
. We further demonstrated that tissue-specific deletion of Foxm1 from hepatocytes 
, respiratory epithelium 
or smooth muscle cells 
was sufficient to cause lethality in utero
or shortly after birth. Therefore, Foxm1 is essential for organ morphogenesis in multiple organ systems. However, it remained to be determined whether cardiac malformation in Foxm1−/−
embryos was due to cardiomyocyte derived effects of Foxm1 signaling or if these Foxm1−/−
defects were indirect, resulting from abnormalities in other organ systems and altered embryonic homeostasis. To elucidate the cardiomyocyte-autonomous role of Foxm1 in embryonic heart development, we used the Cre-LoxP system to generate a conditional Foxm1 knockout mouse line in which Foxm1 is selectively deleted from cardiomyocytes under control of the Nkx2.5 promoter, one of the earliest known cardiac markers.
Deletion of Foxm1 from cardiomyocytes caused thinning and disorganization of the muscular walls of the heart, including both ventricles and the interventricular septum. Myocardial thinning was due to decreased cardiomyocyte proliferation accompanied by altered expression of multiple cell cycle regulatory genes. Ultimately, the myocardial hypoplasia in Nkx2.5-Cre/Foxm1fl/fl
embryos caused lethality in late gestation. Although the myocardial phenotype exhibited similarities to that observed in hearts from Foxm1−/−
mice, many unique features were observed in the conditional knockout model suggesting cardiomyocyte-autonomous roles for Foxm1 signaling. These include a delay in the onset of lethality, unaltered heart size, diminished cardiac capillary density and myocardial fibrosis. The decrease in cardiomyocyte proliferation in Nkx2.5-Cre/Foxm1fl/fl
embryos was significantly less than in Foxm1−/−
embryos suggesting that abnormalities in other cell types or tissues contributed to cardiac malformation in mice with complete deletion of Foxm1. Although we observed increased deposition of extracellular matrix in the atrio-ventricular valves of Nkx2.5-Cre/Foxm1fl/fl
hearts the size of the valves was unaltered. These results are in contrast to valve thickening in Foxm1−/−
and suggest that although Foxm1 does mediate atrio-ventricular valve formation, this signaling is not cardiomyocyte-dependent. Alternatively, valve defects Foxm1−/−
mice may result from altered blood pressure caused by structural abnormalities in blood vessels that were previously reported 
To date, embryonic lethality associated with ventricular hypoplasia and myocardial thinning has been linked to several signaling cascades including the transcription factor Hey2 
, members of the NFAT family 
or inactivation of serum response factor (SRF) 
. In this study we described a model of myocardial thinning owing to multiple factors and resulting in embryonic lethality. In addition to altered expression of various cell cycle regulatory genes, this study identified Hey2, myocardin and CaMKIIδ as novel targets of Foxm1 signaling in vivo
and as potential mediators of the thin ventricular phenotype.
We previously showed decreased expression of NFATc3 in Foxm1−/−
hearts and in Foxm1-depleted cardiomyocytes in vitro 
. This study confirmed that Foxm1 is a positive regulator of cardiac NFATc3 expression and further identified cardiomyocytes as the cell type responsible for Foxm1-regulated NFATc3 expression in vivo
. It has been previously shown that dual deletion of NFATc3 and NFATc4 causes thin ventricles, decreased proliferation of ventricular myocytes and pericardial effusion culminating in embryonic lethality 
. Therefore, decreased NFATc3 expression could be a contributing factor in myocardial thinning and embryonic lethality associated with Nkx2.5-Cre/Foxm1fl/fl
Decreased expression of Hey2 can contribute to the cardiac phenotype and embryonic lethality of Nkx2.5-Cre/Foxm1fl/fl
mice as evidenced by embryonic lethality and myocardial thinning in Hey2−/−
mice, a phenotype similar to Nkx2.5-Cre/Foxm1fl/fl
mice. Hey2 has also been shown to interact with the serum response factor (SRF) to inhibit activity of myocardin 
, which is essential for cardiogenesis, cardiomyocyte proliferation, migration and deposition of the extracellular matrix 
. Furthermore, deletion of SRF from cardiomyocytes resulted in lethality between E10.5–13.5 with thin myocardium, dilated chambers and disorganized IVS 
, a phenotype similar to that observed in the Nkx2.5-Cre/Foxm1fl/fl
hearts. Therefore, Foxm1 may directly influence myocardial development by decreasing expression of Hey2 and myocardin and possibly interfering with SRF-mediated signaling in cardiomyocytes.
CaMKIIδ deficiency caused augmented cardiac function in multiple heart injury models which manifested as severe alterations in cardiac structure. The finding of decreased CaMKIIδ mRNA in Nkx2.5-Cre/Foxm1fl/fl
hearts suggests a role for Foxm1 in regulating cardiac CaMKIIδ expression. Interestingly, interleukin-1β (IL-1β) mRNA was increased in Nkx2.5-Cre/Foxm1fl/fl
hearts. IL-1β has been reported to be a critical mediator of cardiac fibrosis 
. Since significant fibrosis was observed in the postnatal Nkx2.5-Cre/Foxm1fl/fl
heart, increased expression of IL-1β may contribute to fibrotic deposition.
In summary, we demonstrated that Foxm1 plays a cell-autonomous role in cardiomyocytes during cardiac development. Foxm1 deletion in developing cardiomyocytes caused embryonic lethality, decreased cardiomyocyte proliferation, diminished vascular density in the myocardium and induced cardiac fibrosis in the early postnatal period. This study further identified Hey2, myocardin, NFATc3, CaMKIIδ and various cell cycle regulatory genes as in vivo targets of Foxm1 signaling and potential mediators of the myocardial thinning and ventricular hypoplasia associated with the Nkx2.5-Cre/Foxm1fl/fl phenotype.