Expression of Tropomodulin1 (Tmod1) in the Heart Rescues Embryonic Lethality of Tmod1 Null Mice and Results in a Mild Hemolytic Anemia Due to Absence of Tmod1 in Red Blood Cells.

Tropomodulin1 (Tmod1) caps the pointed ends of actin filaments in the red blood cell (RBC) membrane skeleton and is proposed to regulate actin filament lengths and organization in the membrane skeleton. Tmod1 is first expressed at E7.5 of mouse embryogenesis in blood islands of the yolk sac and in the cardiac myocytes of the developing heart tube. Targeted deletion of the Tmod1 gene in mice leads to abnormal cardiac development and defective vasculogenesis and hematopoesis in the yolk sac, followed by embryonic lethality between E9.5–10. To investigate the function of Tmod1 in RBCs we rescued the embryonic lethality of the Tmod1 null mouse by crossing with a TOT mouse that expresses a Tmod1 transgene in the heart under the control of the α-myosin heavy chain promoter. Genotyping of litters from crosses of Tmod1-/+;TOT+ mice with Tmod1+/− mice demonstrates that embryos with no endogenous Tmod1 but expressing the Tmod1 transgene (Tmod1−/−;TOT+) develop to term with no apparent defects in vasculogenesis or hematopoeisis. Western blotting and immunofluorescence staining demonstrates that Tmod1 protein is present in the heart but absent from RBCs in Tmod1−/−;TOT+ rescued embryos or adult mice. Instead, the Tmod3 isoform that is normally present in embryonic RBCs, but not detected in adult RBCs of wild-type mice, is increased on the membranes of Tmod1-deficient adult RBCs. Hematological analyses reveal that these Tmod1-deficient mice exhibit a compensated mild hemolytic anemia, with decreased mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), increased red cell distribution width (RDW), and reticulocytosis. Quantitative western blotting demonstrates that major RBC membrane proteins are not altered in the absence of Tmod1, but that there is about a two-fold increase in levels of actin and tropomyosin on the membrane, suggesting that actin filaments may be longer than in normal RBCs. We conclude that 1) Tmod1 in RBCs is not required for embryonic development or viability, 2) yolk sac and hematopoetic defects in Tmod1 null mice are secondary to cardiac defects, 3) Tmod’s function in embryonic RBCs may be performed by the Tmod3 isoform that is normally expressed in these RBCs, and 4) the absence of Tmod1 in adult RBCs is incompletely compensated by up-regulation of Tmod3, leading to altered actin filaments, decreased membrane stability and a mild hemolytic anemia.