Mammalian formin Fhod3 plays an essential role in cardiogenesis by organizing myofibrillogenesis

Abstract

Heart development requires organized integration of actin filaments into the sarcomere, the contractile unit of myofibrils, although it remains largely unknown how actin filaments are assembled during myofibrillogenesis. Here we show that Fhod3, a member of the formin family of proteins that play pivotal roles in actin filament assembly, is essential for myofibrillogenesis at an early stage of heart development. Fhod3(-/-) mice appear normal up to embryonic day (E) 8.5, when the developing heart, composed of premyofibrils, initiates spontaneous contraction. However, these premyofibrils fail to mature and myocardial development does not continue, leading to embryonic lethality by E11.5. Transgenic expression of wild-type Fhod3 in the heart restores myofibril maturation and cardiomyogenesis, which allow Fhod3(-/-) embryos to develop further. Moreover, cardiomyopathic changes with immature myofibrils are caused in mice overexpressing a mutant Fhod3, defective in binding to actin. These findings indicate that actin dynamics, regulated by Fhod3, participate in sarcomere organization during myofibrillogenesis and thus play a crucial role in heart development.

Keywords: Actin; Fhod3; Formin; Myofibrillogenesis; Sarcomere.

Fig. 1.. Targeted disruption of the Fhod3 gene.

(A) Schematic representation of the Fhod3 gene-targeting strategy. Exon 1 of the Fhod3 gene is represented as a box in black; flanking isogenic genomic DNAs in light gray; and the targeting cassette in white. Green bars indicate probes used for Southern blot analysis, and expected sizes of fragments obtained after NcoI digestion are indicated in base pair. Small black arrowheads indicate primers for PCR genotyping. (B) PCR analysis of the embryonic yolk sac DNAs from wild-type (+/+), Fhod3^+/− (+/−), and Fhod3^−/− (−/−) embryos. The 655-bp and 862-bp fragments were produced with the wild-type and recombinant alleles, respectively. (C) Southern blot analysis of NcoI-digested tail DNAs. The wild-type and recombinant alleles were detected as 13.4-kbp and 5.3-kbp fragments, respectively. (D) Detection of Fhod3 protein by immunoblot analysis. Proteins prepared from the whole embryo of wild-type (+/+), Fhod3^+/− (+/−), and Fhod3^−/− (−/−) mice at E9.5 were analyzed by immunoblot with anti-Fhod3 and anti-GAPDH antibodies.

Fig. 2.. Cardiac development in Fhod3^−/− embryos at E9.5–11.5.

(A) Whole-mount lacZ staining of Fhod3^+/+ (+/+), Fhod3^+/− (+/−), and Fhod3^−/− (−/−) embryos at E7.5–11.5. Bars: (E7.5) 250 µm; (E8.5/E9.5) 500 µm; (E10.5/E11.5) 1 mm. (B) Looping of the heart tube between E8.5 and E11.5 (front view). Bars: (E8.5/E9.5) 250 µm; (E10.5/E11.5) 500 µm. The contour of the wild-type heart is indicated by dotted lines. (C) Histological analysis of embryonic hearts between E8.5 and E10.5. Transverse sections (E8.5) and longitudinal sections (E9.5/E10.5) of hearts were stained with hematoxylin and eosin. Bars, 100 µm. (D) Kymographic analysis of beating of embryonic hearts between E8.5 and E10.5. The kymographs were generated from supplementary material Movies 1–6, and oriented so that the right lateral and left lateral walls are upward and downward, respectively (E8.5), or the anterior and inferoposterior walls are upward and downward, respectively (E9.5 and E10.5).

Fig. 3.. Myofibrils in the embryonic heart.

(A–C) Confocal fluorescence micrographs of hearts of wild-type (+/+) and Fhod3^−/− (−/−) embryos. Sections of embryonic hearts at E 8.5 (A), E9.5 (B), and E10.5 (C) were subjected to immunofluorescent staining for α-actinin (green) and phalloidin staining for F-actin (red). Bars, 2 µm. (D) Electron micrographs of thin sections of wild-type (+/+) and Fhod3^−/− (−/−) hearts at E9.5 and E10.5. Magenta and yellow arrowheads indicate Z-lines and Z-body-like electron-dense spots, respectively. Bar, 500 nm.

Fig. 4.. Effect of transgenic expression of Fhod3 in the heart of Fhod3^−/− mice.

(A,B) Whole-mount (left panels) and histological (right panels) analyses of wild-type (Fhod3^+/+) and Fhod3^{−/−Tg(α-MHC-Fhod3)} (Fhod3^−/−;Tg⁺) embryos at E11.5 (A) and E17.5 (B). V, ventricle; A, atrium; LV, left ventricle; RV, right ventricle; LA, left atrium; RA, right atrium; Ao, aorta. Bars in A: (left) 1 mm; (right) 200 µm. Bars in B: (left) 5 mm; (right) 1 mm. (C,D) Confocal fluorescence micrographs of hearts of wild-type (+/+) and Fhod3^{−/−Tg(α-MHC-Fhod3)} (Fhod3^−/−;Tg⁺) embryos at E10.0 (C) and E17.5 (D). Sections of embryonic hearts were subjected to immunofluorescent staining for α-actinin (green) and phalloidin staining for F-actin (red). Bars, 2 µm.

Fig. 5.. Transgenic expression of Fhod3 (I1127A) in the heart.

(A) Genotypes of offspring from the mating of Fhod3^+/− × Fhod3^{+/+Tg(α-MHC-Fhod3IA)}. The number in parentheses indicates the percentage of the total number of offspring. (B) Histological analysis of hearts from Fhod3^+/− and Fhod3^{+/−Tg(α-MHC-Fhod3IA)} (Fhod3^+/−Tg(IA)) mice at age of 8 day. Bars, 100 µm. (C) Confocal fluorescence micrographs of hearts from Fhod3^+/− and Fhod3^{+/−Tg(α-MHC-Fhod3IA)} (Fhod3^+/−Tg(IA)) mice at P8. Sections of hearts were subjected to immunofluorescent staining for α-actinin (green) and phalloidin staining for F-actin (red). Bars, 2 µm. (D) Kaplan-Meier survival curves versus days after TAC. TAC-Fhod3^+/+ (n = 24); TAC-Fhod3^+/− (n = 31); sham-Fhod3^+/+ (n = 4); and sham-Fhod3^+/− (n = 4) mice.