Aberrant myofibril assembly in tropomodulin1 null mice leads to aborted heart development and embryonic lethality

Abstract

Tropomodulin1 (Tmod1) caps thin filament pointed ends in striated muscle, where it controls filament lengths by regulating actin dynamics. Here, we investigated myofibril assembly and heart development in a Tmod1 knockout mouse. In the absence of Tmod1, embryonic development appeared normal up to embryonic day (E) 8.5. By E9.5, heart defects were evident, including aborted development of the myocardium and inability to pump, leading to embryonic lethality by E10.5. Confocal microscopy of hearts of E8-8.5 Tmod1 null embryos revealed structures resembling nascent myofibrils with continuous F-actin staining and periodic dots of alpha-actinin, indicating that I-Z-I complexes assembled in the absence of Tmod1. Myomesin, a thick filament component, was also assembled normally along these structures, indicating that thick filament assembly is independent of Tmod1. However, myofibrils did not become striated, and gaps in F-actin staining (H zones) were never observed. We conclude that Tmod1 is required for regulation of actin filament lengths and myofibril maturation; this is critical for heart morphogenesis during embryonic development.

Figure 1.

Targeted disruption of the Tmod1 gene. (A) Schematic of expected gene replacement at the Tmod1 locus. Restriction map of wild-type allele, targeting vector, and targeted allele are shown. Tmod1 exon 2 is represented as a black box, flanking isogenic genomic DNA as light gray boxes, targeting cassette by thick black arrows, and thymidine kinase gene (selectable marker) by dark gray arrow. Small black bars indicate Southern probes, and sizes of expected fragments after EcoRI digestion are indicated. Small black arrows indicate primers for PCR genotyping. (B) PCR analysis of E10.5 embryonic yolk sac DNA from representative wild-type (+/+), Tmod1 ^lacZ+/− (+/−), and Tmod1 ^lacZ−/− (−/−) embryo. The wild-type allele resulted in a 390-base fragment (open arrowhead), and the recombinant allele in a 1,100-base fragment (closed arrowhead). Left lane is a 200-bp DNA ladder. (C) Western blot of whole E10.5 embryos probed with anti-Tmod1 and GAPDH antibodies. Representative wild type (+/+), Tmod1 ^lacZ+/− (+/−), and Tmod1 ^lacZ−/− (−/−) embryos are shown. Arrowheads, Tmod1 and GAPDH polypeptides.

Figure 2.

Tmod1 expression is predominantly in the heart during early embryogenesis. (A) Whole mount X-gal staining of Tmod1 ^lacZ+/− embryos at stages E8.5–9.0, E9.5, and E13.5. Bars: (E8.5–9) 250 μm; (E9.5) 500 μm; (E13.5) 1 mm. Tmod1 ^lacZ expression (blue) is observed only in the heart of staged embryos at E8.5–9 and at E9.5. At E13.5, staining is also detected in the otic vesicle (arrow) and in somites (arrowheads). (B) Western blot of wild-type embryos at stages E8–8.5 to E12.5 probed with anti-Tmod1 antibodies, stripped, and then reprobed with a pan actin antibody as a measure of total protein. (C) Low magnification confocal micrographs of wild-type E8–8.5 embryos double stained for Tmod1 (left) and for F-actin with bodipy-phallacidin (right). Tmod1 protein staining is only in heart cells, identified by double staining with sarcomeric α-actinin (see Fig. 3). Location of heart cells is indicated by asterisks. Bar, 50 μm.

Figure 3.

Tmod1^{lacZ −/−} embryos appear normal at E8–8.5. Whole mounts and histological analysis of E8–8.5 wild-type (A, C, and E) and Tmod1 null (B, D, and F) embryos. Sagittal sections of wild-type (C and E) and null (D and F) embryos are shown. Note the seemingly normal overall morphological appearance of the knockout embryo at E8–8.5, as well as the normal early myocardium and matrix in the mutant heart tube (low and high magnification view in D and F, respectively), in comparison to wild type (low and high magnification view in C and E, respectively). V, ventricle; M, myocardium; MTX, matrix.

Figure 4.

Tmod1^{lacZ −/−} embryos display aberrant embryonic and cardiac development at E9.5–10. Whole mounts and histological analysis of E10 wild-type (A, C, and E) and E9.5 Tmod1 null (B, D, and F) embryos. Sagittal sections of wild-type (C and E) and null (D and F) embryos are shown. Severely underdeveloped myocardium and trabeculation (T) of the left ventricle are apparent in the null embryos (low and high magnification view in D and F, respectively) in comparison to wild type (low and high magnification view in C and E, respectively). Note, in boxed in regions, complete lack of cellularization within the AV canal in mutant (F), in comparison to wild type (E). V, ventricle; A, atrium; M, myocardium; DA, dorsal aorta; T, trabeculations; AV, AV canal; HT, heart.

Figure 5.

The hearts of Tmod1 null embryos fail to pump. Living E9.5 embryos were microinjected with India ink into the right ventricle, and the heart was allowed to pump the ink through the vasculature. (A) Ink injected into wild-type embryos (+/+) was pumped throughout the vasculature, while (B) ink injected into the Tmod1 ^lacZ−/− embryos (−/−) stayed within the heart tube, dispersing by passive diffusion over 6 h in culture. Bar, 1 mm.

Figure 6.

Tmod1 and sarcomeric α-actinin assembly into myofibrils in the heart during early embryogenesis of wild-type embryos. Confocal fluorescence micrographs of hearts of staged wild-type embryos at E8–8.5, E9.5, or E12.5 double stained with a rabbit antibody to Tmod1 and a mouse monoclonal antibody to sarcomeric α-actinin. Merge: Tmod1, green; α-actinin, red. Arrows, periodic α-actinin dots; brackets, α-actinin striations; arrowheads, Tmod1. Bar, 10 μm.

Figure 7.

Tmod1 and F-actin assembly into myofibrils in the heart of E8–8.5 and E9.5 wild-type embryos. Confocal fluorescence micrographs of relaxed embryonic hearts at stage E8–8.5 (first and second rows) or E9.5 (third row), double stained with a rabbit antibody to Tmod1 and bodipy-phallacidin for F-actin. Merge: F-actin, green; Tmod1, red. Arrowheads, Tmod1 concentrations at decreases or discontinuities in F-actin staining (E8–8.5) and at gaps in F-actin in E9.5 embryos. Inset, Tmod1 concentrations at gaps in F-actin staining in a myofibril from a different region of the same image of an E8–8.5 embryo. Note that the inset is at the same magnification as other panels. Bar, 5 μm.

Figure 8.

Myofibrils do not become striated in hearts of Tmod1^{lacZ −/−} embryos. Confocal fluorescence micrographs of hearts of wild-type (+/+) and Tmod1 ^lacZ−/− (−/−) embryos from stages E8–8.5 and E9.5, double stained with a monoclonal antibody to sarcomeric α-actinin and bodipy-phallacidin for F-actin. Merges: α-actinin, red; bodipy-phallacidin, green. Arrowheads, α-actinin dots along nascent myofibrils. Short brackets, α-actinin striations at Z discs. Long brackets, regions of continuous F-actin staining. Bar, 5 μm. Insets: 2× higher magnification images of representative nascent and mature myofibrils from the stages indicated. Open arrowheads in insets, gaps in F-actin staining.

Figure 9.

Myomesin assembles into myofibrils in Tmod1^{lacZ −/−} embryos. Confocal fluorescence micrographs of hearts of wild-type (+/+) and Tmod1 ^lacZ−/− (−/−) embryos from stages E8–8.5 and E9.5, double stained with a monoclonal antibody to myomesin and bodipy-phallacidin for F-actin. Merges: myomesin, red; bodipy-phallacidin, green. Arrowheads, periodic myomesin dots along assembling myofibrils. Short brackets, mature myomesin striations. Long brackets, regions of continuous F-actin staining. Bar, 5 μm.