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Inhibition of Rho family GTPases by Rho GDP dissociation inhibitor disrupts cardiac morphogenesis and inhibits cardiomyocyte proliferation

¹ Department of Molecular and Cellular Biology,
² Section of Cardiovascular Sciences,
³ Center for Cardiovascular Development,
⁴ Department of Medicine, Baylor College of Medicine, Houston, Texas, USA
⁵ Department of Pathology, Mie University, School of Medicine, Tsu, Mie, Japan

View larger version (41K): [in a new window]   Fig. 1. Generation of Rho GDI transgenic founder lines. (A) Schematic diagram of transgenic vector showing insertion of Rho GDI cDNA under the MHC promoter and locations of the primers used for genotyping by PCR. (B,C) Representative Southern blot and PCR screening of nontransgenic (NTG) and transgenic (TG) mice using tail DNA. ß-casein was also amplified as a control. (D) Quantitative DNA dot blot analysis was performed as described in the Materials and Methods, and identified six transgenic founder lines (L1, L2, M1, M2, H1 and H2) with copy number ranging from 1 to 20.

View larger version (9K): [in a new window]   Fig. 2. F1 heretozygotes of high-copy number founders were embryonic lethal. (A) Lateral view of E9.5 nontransgenic and transgenic embryos from the H1 founder. All E9.5 transgenic embryos were markedly growth-retarded with severe cardiac defect with a dilated pericardium. E9.5 transgenic embryos from the H1 and H2 founders were morphologically undistinguishable. A, atrium; V, ventricle; PS, pericardial sac. (B) Genotypic and phenotypic analysis of F1 embryos from the H1 founder. PCR screening was performed with DNA extracted from the yolk sac of the embryos at E8.5, E9.5, E10.5, E11.5 and from the tail of weaned mice. Some of the E10.5 and all of the E11.5 transgenic embryos were dead and in the process of being resorbed (dead). (C) Genotypic and phenotypic analysis of F1 embryos from the H2 founder.

View larger version (36K): [in a new window]   Fig. 3. Homozygotes of the middle-copy number line M2 were embryonic lethal. (A) Lateral view of E10.5 nontransgenic, heterozygous and homozygous embryos. All heretozygous embryos were phenotypically indistinguishable from the nontransgenic littermates. All E10.5 homozygous embryos were markedly growth-retarded with severe cardiac defect. (B) Genotypic and phenotypic analysis of embryos from heterozygous matings, as described in the legend of Fig. 2.

View larger version (57K): [in a new window]   Fig. 4. Cardiac-specific expression of Rho GDI transgene. (A) Whole-mount in situ hybridization of nontransgenic (NTG) and transgenic F1 embryos at E9.5 from the H2 founder (TG) with an antisense probe generated from the polyadenylation sequences of the transgene (human G-CSF cDNA). No signal was observed with a sense probe (data not shown). (B) Expression of the transgene was markedly increased in the adult mouse heart. Western blot analysis was performed with cardiac proteins extracted from nontransgenic and heterozygotes of the M2 founder line at E12.5 or 4 weeks after birth. The blot was probed with an anti-Rho GDI antibody recognizing both endogenous Rho GDI and the transgene. (C) The expression level of the transgene was proportional to the copy number. Western blot analysis was performed with cardiac proteins extracted from adult nontransgenic and transgenic heterozygotes of L1, L2, M1 and M2 founder lines using an anti-Rho GDI polyclonal antibody. An anti-Myc antibody recognizes only the transgene (data not shown).

View larger version (48K): [in a new window]   Fig. 5. Increased cardiac expression of Rho GDI inhibited the activity of Rho family members. (A) RhoA, Rac1, Cdc42 and RhoGDI were expressed in the early developing hearts. Western blot analysis of RhoA, Rac1, Cdc42 and Rho GDI was performed with protein samples prepared from a pool of 10 hearts from E9.5 or E11.5 embryos. (B) Membrane translocation of RhoA, Rac1 and Cdc42 was inhibited in the transgenic hearts. Western blot analysis of RhoA, Rac1 and Cdc42 was performed with the cytosolic and membrane fractions of cardiac and skeletal proteins extracted from heterozygotes and nontransgenic littermates of the M2 line.

View larger version (82K): [in a new window]   Fig. 6. Increased levels of Rho GDI interfered with cardiac looping morphogenesis and ventricular maturation. (A-F) Whole-mount in situ hybridization of E9.5 nontransgenic embryos (A,C,E) and F1 heterozygotes of the H2 founder (B,D,F) with a cardiac -actin probe. (A,B) Right side, (C,D) frontal and (E,F) left side views of the hearts. The transgenic hearts did not show rightward looping and there was no evidence for the demarcation of future right and left ventricles. (G-H) Transverse sections of whole-mount in situ hybridized E9.5 nontransgenic and F1 heterozygous embryos of the H2 founder showing cardiac -actin expression, visualized by phase-contrast microscopy. Transgenic embryos had thin ventricular wall, absence of trabeculation, and lack of chamber demarcation. (I-J) Enlargement of the boxed areas in G and H. (K-L) Sections of E10.5 nontransgenic and heterozygous hearts of the M2 line. No morphological defect was detected in the M2 heterozygous embryos. A, atrium; V, ventricle; LV, left ventricle; RV, right ventricle.

View larger version (52K): [in a new window]   Fig. 7. Transgenic Rho GDI expression inhibited cardiac cell proliferation. (A,B) Immunofluorescent staining for phosphohistone H3, visualized by fluorescent microscopy. The number of nuclei stained positive in the heart fields (indicated by small arrows) was markedly higher in nontransgenic embryos (A) than in transgenic embryos (B). The number of positive nuclei was also markedly higher in non-cardiac tissues than in cardiac tissue in the transgenic embryos (B). (C,D) Phosphohistone H3 (C) and DAPI (D) were visualized in the same fields and were seen to colocalize in mitotic subepicardial cells (indicated by small arrows). The majority of nuclei that stained positive were located in the subepicardial cell layer (SM) immediately under the epicardium (E). (E) The number of phosphohistone H3-positive nuclei (FITC, green) was determined as a percentage of the total nuclei (DAPI, blue) within the subepicardial cell layer as shown in C and D. *P<0.05 vs nontransgenic mice. A, atrium; V, ventricle.

View larger version (71K): [in a new window]   Fig. 8. Increased Rho GDI cardiac expression stimulated accumulation of p21 transcripts and repressed cyclin A mRNA content. RNA was isolated from E9.5 embryonic hearts of the nontransgenic (NTG) or transgenic F1 embryos from the H2 founder. Each line represents PCR reactions performed with the same cDNA synthesized from a RNA sample pooled from ten hearts using gene-specific primers for the indicated transcripts. PCR reactions were separated by polyacrylamide gel electrophoresis and quantitated by PhosphorImage analysis. For each primer set, two or three cycle-numbers were tested to be certain that PCR product accumulates within a linear range. The level for each amplified transcript was normalized to that of GAPDH. The normalized level for each transcript in the transgenic heart sample was expressed relative that in the nontransgenic heart sample.

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