Myocardium-Specific Deletion of Rac1 Causes Ventricular Noncompaction and Outflow Tract Defects

Figure 1. Generation of mouse line with a myocardium specific deletion of Rac1 (Rac1Nkx2.5). A schema of the floxed Rac1 allele and Nkx2.5-Cre mediated generation of the Rac1 null allele (A). Rac1Nkx2.5 mice were generated by crossing Nkx2.5-Cre transgenic mice with Rac1f/f mice for 2 generations with the expected genotype frequencies indicated in brackets (B). Rac1 mRNA expression was significantly reduced in E12.5 Rac1Nkx2.5 ventricular myocardium compared to Rac1f/f littermates (C). Western blot analysis of E12.5 ventricular myocardium showed a significant decrease in Rac1 protein levels in Rac1Nkx2.5 hearts compared to controls (D). * p < 0.05, *** p < 0.001 by unpaired Student’s t-test.

Figure 2. Lineage tracing with Nkx2.5-Cre;mT/mG mice. GFP immunostaining of E12.5 Nkx2.5-Cre;mT/mG paraffin heart sections showed Cre recombinase activity in the atrial and ventricular myocardium (A), OFT (B) and aortic valve leaflets (C). Fluorescence imaging of cryosections of E18.5 Nkx2.5-Cre;mT/mG hearts showed that the epicardium (arrows) and endothelial cells (arrowhead) remain RFP+ in Nkx2.5-Cre;mT/mG hearts (D–F). RV, right ventricle; LV, left ventricle; PA, pulmonary artery; Ao, aorta; epi, epicardium. Scale bars: 250 µm (A–C), 10 µm (D–F).

Figure 3. Congenital heart defects in Rac1Nkx2.5. (A) Bifid cardiac apex in an E16.5 Rac1Nkx2.5 heart. Arrow indicates bifurcation between the RV and LV. (B,C) Ventricular septal defect (arrowhead) was found in E15.5 Rac1Nkx2.5 hearts with an arrow indicating the bifid cardiac apex. (D–G) Double outlet right ventricle (DORV) was found in E15.5 Rac1Nkx2.5 hearts. The pulmonary artery and aorta were connected to the RV and LV, respectively in Rac1f/f control hearts (D,F). However, both pulmonary artery and aorta were connected to the RV in Rac1Nkx2.5 hearts (E,G). RA, right atrium; LA, left atrium. Scale bars: 500 µm.

Figure 4. Ventricular myocardium abnormalities in Rac1Nkx2.5 hearts at E15.5. In comparison to Rac1f/f controls (A,C), the Rac1Nkx2.5 mice (B,D) show thin compact myocardium and hypertrabeculation in the RV and LV wall. The thickness of the compact myocardium in Rac1Nkx2.5 hearts (n = 5) was decreased while trabecular to compact myocardium ratio was increased as compared to littermate controls (n = 6) (E,F). Double-headed arrows in (A–D) indicate measurements of compact myocardium thickness. * p < 0.05, ** p < 0.01, *** p< 0.001 vs. Rac1f/f by Student’s t-test. Scale bars: 100 µm.

Figure 5. Disruption of F-actin organization and cardiomyocyte polarity in Rac1Nkx2.5 hearts. E18.5 cryosections of the heart were double stained for phalloidin and wheat germ agglutinin (WGA) to assess F-actin organization and cardiomyocyte cell polarity, respectively. The images were taken from the corresponding areas of LV myocardium free wall in Rac1f/f and Rac1Nkx2.5 mice. F-actin filament organization was disrupted in Rac1Nkx2.5 myocardium (B) compared to littermate controls (A). WGA staining shows rounded cardiomyocytes in Rac1Nkx2.5 ventricular myocardium (D) compared to the elongated cardiomyocytes in littermate controls (C). The cell borders of a cardiomyocyte in (C,D) are outlined. The short axis of cardiomyocyte diameter was significantly larger in Rac1Nkx2.5 compared to littermates (E). ** p < 0.01 by unpaired Student’s t-test. Scale bars: 10 µm.

Figure 6. Loss of Scrib expression in Rac1Nkx2.5 hearts. Scrib immunostaining was performed on E15.5 Rac1Nkx2.5 and Rac1f/f heart sections. The areas analyzed included the myocardium surrounding the aorta (A,B), RV (C,D) and the LV (E,F). Five sections per heart were used. The number of Scrib-expressing positive cells was significantly decreased in the myocardium surrounding the aorta (OFT), RV and LV of Rac1Nkx2.5 hearts (G). * p < 0.05, ** p < 0.01 by Student’s t-test. Scale bars: 50 µm.

Figure 7. Decreased proliferation rate in Rac1Nkx2.5 hearts. Phospho-histone H3 (pHH3) immunostaining to mark proliferating cells undergoing mitosis in ventricular myocardium (myo) of E9.5 Rac1Nkx2.5 and Rac1f/f hearts (A,B). Proliferation rate was significantly decreased in E9.5 Rac1Nkx2.5 ventricular myocardium compared to littermate controls (C). Cyclin D1 immunostaining in E9.5 Rac1f/f and Rac1Nkx2.5 ventricular myocardium marked cell progression through G1 (D,E). Cyclin D1 expression was significantly decreased in E9.5 Rac1Nkx2.5 ventricular myocardium compared to littermate controls (F). Cleaved caspase-3 (CC3) immunostaining to mark apoptotic cells in ventricular myocardium of E9.5 Rac1Nkx2.5 and Rac1f/f hearts. No apoptosis was detected in E9.5 Rac1Nkx2.5 and Rac1f/f ventricular myocardium (G,H). Apoptotic cells were detected in tissues outside of the heart in Rac1f/f and Rac1Nkx2.5 embryos (I,J). * p < 0.05, ** p < 0.01 by unpaired Student’s t-test. Scale bars: 10 µm (A,B), 50 µm (D–G).

Figure 8. Myocardial mRNA expression of transcription and growth factors in E12.5 Rac1f/f and Rac1Nkx2.5 hearts. (A) Scrib. (B) Nkx2.5. (C) Gata4. (D) Tbx5. (E) Tbx20. (F) Hand1. (G) Hand2. (H) Bmp10. (I) Mef2c. Data are expressed as mRNA to 28S ratios. n = 5–6 per group. * p < 0.05, ** p < 0.01 by unpaired Student’s t test.

Figure 9. Rac1 signaling promotes cardiac transcription/growth factor expression, cardiomyocyte polarity and proliferation, leading to normal development of the outflow tract and compact myocardium.