Expression and Subcellular Localization of Mammalian Formin Fhod3 in the Embryonic and Adult Heart

The formin family proteins play pivotal roles in actin filament assembly via the FH2 domain. The mammalian formin Fhod3 is highly expressed in the heart, and its mRNA in the adult heart contains exons 11, 12, and 25, which are absent from non-muscle Fhod3 isoforms. In cultured neonatal cardiomyocytes, Fhod3 localizes to the middle of the sarcomere and appears to function in its organization, although it is suggested that Fhod3 localizes differently in the adult heart. Here we show, using immunohistochemical analysis with three different antibodies, each recognizing distinct regions of Fhod3, that Fhod3 localizes as two closely spaced bands in middle of the sarcomere in both embryonic and adult hearts. The bands are adjacent to the M-line that crosslinks thick myosin filaments at the center of a sarcomere but distant from the Z-line that forms the boundary of the sarcomere, which localization is the same as that observed in cultured cardiomyocytes. Detailed immunohistochemical and immuno-electron microscopic analyses reveal that Fhod3 localizes not at the pointed ends of thin actin filaments but to a more peripheral zone, where thin filaments overlap with thick myosin filaments. We also demonstrate that the embryonic heart of mice specifically expresses the Fhod3 mRNA isoform harboring the three alternative exons, and that the characteristic localization of Fhod3 in the sarcomere does not require a region encoded by exon 25, in contrast to an essential role of exons 11 and 12. Furthermore, the exon 25-encoded region appears to be dispensable for actin-organizing activities both in vivo and in vitro, albeit it is inserted in the catalytic FH2 domain

Cardiovascular Function in Mice During Normal Pregnancy and in the Absence of Endothelial NO Synthase

In humans, the increased cardiovascular demands of pregnancy are met by increases in cardiac output (CO), stroke volume (SV), plasma volume (PV), and cardiac and aortic inner dimensions and a concurrent decrease in arterial pressure that indicates a fall in total peripheral vascular resistance. The mechanisms responsible for these changes are incompletely understood, but NO synthase (NOS) is believed to play a central role. We assessed whether C57Bl/6J (B6) mice show similar changes and whether these changes are altered in mice lacking the gene for endothelial NOS (eNOS). The CO of B6 mice increased 28% by day 9.5 of gestation because of a 25% increase in SV, and increased 48% by day 17.5 because of a 41% increase in SV. The increase in SV at day 17.5 was associated with a 27% increase in PV, a 15% decrease in arterial pressure, and 10% to 15% increases in aortic and left-ventricular inner dimensions. In the absence of eNOS, CO increased 22% by day 9.5 because of increases in SV (14%) and heart rate (9%), but increased no further by day 17.5. SV near term was lower than B6 mice despite similar 26% increases in PV and 14% decreases in arterial pressure in association with blunted left-ventricular chamber enlargement. All reported changes are P<0.05. We conclude that cardiovascular changes during pregnancy are similar in B6 mice and humans. eNOS plays a critical role in increasing stroke volume in late gestation by promoting cardiac remodeling

3D Visualisation and Quantification by Microcomputed Tomography of Late Gestational Changes in the Arterial and Venous Feto-Placental Vasculature of the Mouse

This study evaluates microcomputed tomography (micro-CT) as a method to obtain quantitative three-dimensional (3D) information on the arterial and venous vasculature of the mouse placenta. Surface renderings at embryonic days (E) 13.5, 15.5, and 18.5 (full term) revealed that the arterial and venous vasculature branched within the chorionic plate whereas only the arterial vasculature deeply penetrated the placenta. Umbilical vessel diameters measured by micro-CT did not significantly differ from those measured non-invasively in vivo by ultrasound biomicroscopy. Variability in umbilical diameters, and surface area and volume measurements of arterial and venous vascular trees due to experimental error was low relative to biological variability, and significant inter-litter differences within gestational ages were detected. Furthermore, umbilical vessel diameter increased significantly and incrementally to an arterial diameter of 0.631 ± 0.009 mm and a venous diameter of 0.690 ± 0.018 mm at E18.5. Umbilical vein diameter was 3–9% greater than the artery, and both were significantly correlated with embryonic body weight ( R ≥ 0.96). Surface area and volume were determined for vessels greater than the minimum resolvable diameter of 0.03 mm which therefore excluded capillaries. Arterial surface area and volume were unchanged from E13.5–15.5 but then more than doubled at E18.5 (to 170 ± 13 mm 2 and 7.2 ± 0.8 mm 3, respectively). Venous surface areas and volumes changed similarly with development although surface areas were lower than their arterial counterparts. We conclude that micro-CT has sufficient accuracy and precision to quantify late gestational changes in the 3D structure of the arterial and venous vasculature of the mouse placenta

Endothelial NO Synthase Augments Fetoplacental Blood Flow, Placental Vascularization, and Fetal Growth in Mice

It is not known whether eNOS deficiency in the mother or the conceptus (ie, placenta and fetus) causes fetal growth restriction in mice lacking the endothelial NO synthase gene (eNOS knockout [KO]). We hypothesized that eNOS sustains fetal growth by maintaining low fetoplacental vascular tone and promoting fetoplacental vascularity and that this is a conceptus effect and is independent of maternal genotype. We found that eNOS deficiency blunted fetal growth, and blunted the normal increase in umbilical blood flow and umbilical venous diameter and the decrease in umbilical arterial Resistance Index in late gestation (14.5–17.5 days) in eNOS KO relative to C57Bl/6J controls. On day 17.5, fetoplacental capillary lobule length and capillary density in vascular corrosion casts were reduced in eNOS KO placentas. Reduced vascularization may be a result of decreased vascular endothelial growth factor mRNA and protein expression in eNOS KO placentas at this stage. These factors, combined with significant anemia found in eNOS KO fetuses, would be anticipated to reduce fetal oxygen delivery and contribute to the fetal tissue hypoxia that was detected in the heart, lung, kidney, and liver by immunohistochemistry using pimonidazole. Although maternal eNOS deficiency impairs uteroplacental adaptations to pregnancy, maternal genotype was not a significant factor affecting growth in heterozygous conceptuses. This indicates that fetal growth restriction was primarily caused by conceptus eNOS deficiency. In mice, placental hemodynamic and vascular changes with gestation and growth restriction showed strong parallels with human pregnancy. Thus, the eNOS KO model could provide insights into the pathogenesis of human intrauterine growth restriction