Congenital Heart Disease–Causing Gata4 Mutation Displays Functional Deficits In Vivo

Defects of atrial and ventricular septation are the most frequent form of congenital heart disease, accounting for almost 50% of all cases. We previously reported that a heterozygous G296S missense mutation of GATA4 caused atrial and ventricular septal defects and pulmonary valve stenosis in humans. GATA4 encodes a cardiac transcription factor, and when deleted in mice it results in cardiac bifida and lethality by embryonic day (E)9.5. In vitro, the mutant GATA4 protein has a reduced DNA binding affinity and transcriptional activity and abolishes a physical interaction with TBX5, a transcription factor critical for normal heart formation. To characterize the mutation in vivo, we generated mice harboring the same mutation, Gata4 G295S. Mice homozygous for the Gata4 G295S mutant allele have normal ventral body patterning and heart looping, but have a thin ventricular myocardium, single ventricular chamber, and lethality by E11.5. While heterozygous Gata4 G295S mutant mice are viable, a subset of these mice have semilunar valve stenosis and small defects of the atrial septum. Gene expression studies of homozygous mutant mice suggest the G295S protein can sufficiently activate downstream targets of Gata4 in the endoderm but not in the developing heart. Cardiomyocyte proliferation deficits and decreased cardiac expression of CCND2, a member of the cyclin family and a direct target of Gata4, were found in embryos both homozygous and heterozygous for the Gata4 G295S allele. To further define functions of the Gata4 G295S mutation in vivo, compound mutant mice were generated in which specific cell lineages harbored both the Gata4 G295S mutant and Gata4 null alleles. Examination of these mice demonstrated that the Gata4 G295S protein has functional deficits in early myocardial development. In summary, the Gata4 G295S mutation functions as a hypomorph in vivo and leads to defects in cardiomyocyte proliferation during embryogenesis, which may contribute to the development of congenital heart defects in humans

Chronic NF-κB blockade reduces cytosolic and mitochondrial oxidative stress and attenuates renal injury and hypertension in SHR

Nuclear factor-κB (NF-κB) plays an important role in hypertensive renal injury; however, its roles in perpetuating mitochondrial oxidative stress and renal dysfunction remain unclear. In this study, we assessed the effects of chronic NF-κB blockade with pyrrolidine dithiocarbamate (PDTC) on renal dysfunction and mitochondrial redox status in spontaneously hypertensive rats (SHR). PDTC (150 mg·kg body wt−1·day−1) or vehicle was administered orally to 8-wk-old SHR and their respective controls for 15 wk. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography at the start of and at every third week throughout the study. After 15 wk of treatment, anesthetized rats underwent acute renal experiments to determine renal blood flow and glomerular filtration rate using PAH and inulin clearance techniques, respectively. Following renal experiments, kidneys were excised from killed rats, and cortical mitochondria were isolated for reactive oxygen species (ROS) measurements using electron paramagnetic resonance. Tissue mRNA and protein levels of NF-κB and oxidative stress genes were determined using real-time PCR and immunofluorescence or Western blotting, respectively. PDTC treatment partially attenuated the increase in SBP (196.4 ± 9.76 vs. 151.4 ± 2.12; P < 0.05) and normalized renal hemodynamic and excretory parameters and ATP production rates in SHR. PDTC treatment also attenuated the higher levels of cytosolic and mitochondrial ROS generation and tissue mRNA and protein expression levels of NF-κB and oxidative stress genes in SHR without any comparable responses in control rats. These findings suggest that NF-κB activation by ROS induces the cytosolic and mitochondrial oxidative stress and tissue injury that contribute to renal dysfunction observed in SHR

Congenital heart disease-causing Gata4 mutation displays functional deficits in vivo.

Defects of atrial and ventricular septation are the most frequent form of congenital heart disease, accounting for almost 50% of all cases. We previously reported that a heterozygous G296S missense mutation of GATA4 caused atrial and ventricular septal defects and pulmonary valve stenosis in humans. GATA4 encodes a cardiac transcription factor, and when deleted in mice it results in cardiac bifida and lethality by embryonic day (E)9.5. In vitro, the mutant GATA4 protein has a reduced DNA binding affinity and transcriptional activity and abolishes a physical interaction with TBX5, a transcription factor critical for normal heart formation. To characterize the mutation in vivo, we generated mice harboring the same mutation, Gata4 G295S. Mice homozygous for the Gata4 G295S mutant allele have normal ventral body patterning and heart looping, but have a thin ventricular myocardium, single ventricular chamber, and lethality by E11.5. While heterozygous Gata4 G295S mutant mice are viable, a subset of these mice have semilunar valve stenosis and small defects of the atrial septum. Gene expression studies of homozygous mutant mice suggest the G295S protein can sufficiently activate downstream targets of Gata4 in the endoderm but not in the developing heart. Cardiomyocyte proliferation deficits and decreased cardiac expression of CCND2, a member of the cyclin family and a direct target of Gata4, were found in embryos both homozygous and heterozygous for the Gata4 G295S allele. To further define functions of the Gata4 G295S mutation in vivo, compound mutant mice were generated in which specific cell lineages harbored both the Gata4 G295S mutant and Gata4 null alleles. Examination of these mice demonstrated that the Gata4 G295S protein has functional deficits in early myocardial development. In summary, the Gata4 G295S mutation functions as a hypomorph in vivo and leads to defects in cardiomyocyte proliferation during embryogenesis, which may contribute to the development of congenital heart defects in humans

<i>Gata4 G295S</i> mutation has <i>in vivo</i> functional deficits in the early embryonic myocardium.

<p>(A) Embryonic lethality by E10.5 was found in compound heterozygote mice expressing only a <i>Gata4 G295S</i> mutant allele in early myocardium with the <i>Nkx2-5-Cre</i>, but normal Mendelian ratios were seen when the <i>Gata4 G295S</i> mutant allele was expressed in endocardium and late myocardium using <i>Tie2-Cre</i> and α-<i>MHC-Cre</i>, respectively. Images (B, D, F, H, J, L) and histologic sections (C, E, G, I, K, M) of E10.5 embryos generated with <i>Tie2-Cre</i>, which is specific for endocardium (B–E); α<i>-MHC-Cre</i>, which is specific for late embryonic myocardium (F–I); and <i>Nkx2.5-Cre</i>, which is specific for early embryonic myocardium (J–M), are shown. (L,M) Growth retardation and myocardial thinning were seen in <i>Gata4 G295S^ki/flox</i>; <i>Nkx2-5-Cre⁺</i> E10.5 embryos similar to the phenotype of the <i>Gata4 G295S^ki/ki</i> embryo. (H,I) The hearts of <i>Gata4 G295S^ki/flox</i>; α<i>-MHC-Cre⁺</i> appeared normal at E10.5. (D,E) While the <i>Gata4 G295S^ki/flox</i>; <i>Tie2-Cre⁺</i> did not show growth retardation or myocardial thinning, hypocellular endocardial cushions were noted (*). A, atria; V, ventricle; scale bars indicate 200 µm.</p

G295S mutation in <i>Gata4</i> results in cardiomyocyte proliferation defects.

<p>Immunofluorescent staining for phosphohistone H3 (red) along with desmin staining for cardiomyocytes (green) demonstrates decreased cardiomyocyte proliferation in <i>Gata4 G295S^ki/ki</i> (B) as compared to wildtype (A) in histologic sections of E9.5 hearts. V, ventricle. (C) Quantification of phosphohistone H3 (PH3) staining of cardiomyocytes is decreased in <i>Gata4 G295S^ki/ki</i> embryonic hearts when compared to wildtype. *, p value<0.05. (D) Quantitative RT-PCR demonstrates decreased cyclin D2 expression in the E9.5 hearts of both <i>Gata4 G295S^ki/wt</i> and <i>Gata4 G295S^ki/ki</i> embryos.*, p value<0.05. (E) Cyclin D2 β-gal reporter assays using wildtype Gata4 or Gata4 G295S mutant plasmids demonstrates that the G295S mutant protein has decreased transactivation ability as compared to wildtype Gata4. Error bars represent the standard deviation at least three independent experiments each performed in triplicate. *, p value<0.05. Scale bars indicate 200 µm.</p

<i>Gata4 G295S^ki/ki</i> mice display growth retardation and a thin myocardium.

<p>Right (A,D,G), left (B,E,H) and frontal (C,F,I) views of embryos are shown. Growth retardation of <i>Gata4 G295S^ki/ki</i> E9.5 embryos (G,H) when compared to wildtype (A,B) and heterozygote littermates (D,E). A fused heart tube with proper looping is found in <i>Gata4 G295S^ki/ki</i> embryos (I) simlar to wildtype (C) and <i>Gata4 G295S^ki/wt</i> (F) littermates. Coronal sections through <i>Gata4 G295S^ki/wt</i> (J,K) and <i>Gata4 G295S^ki/ki</i> (L,M) embryos at E9.5 (J,L) and E10.5 (K,M). Normal myocardial thickness is found at E9.5 in homozygous mutant embryos (L) while thin myocardium is seen at E10.5 (M) when compared to heterozygote littermate (K). Quantification of ventricular wall thickness in <i>Gata4 G295S^ki/wt</i> and <i>Gata4 G295S^ki/ki</i> embryos at E9.5 (N) and 10.5 (O). RV, right ventricle; LV, left ventricle; red arrow, heart; A, atria; V, ventricle; OFT, outflow tract. Scale bars indicate 200 µm.</p

Targeting strategy for generation of <i>Gata4 G295S</i> knock-in mice.

<p>(A) Single nucleotide change resulting in the glycine to serine mutation was introduced into the mouse <i>Gata4</i> locus. Partial restriction map of the murine <i>Gata4</i> wildtype allele (top), the <i>Gata4</i> targeting vector (middle), and successfully targeted allele (bottom) are shown. Homologous recombination results in replacement of wildtype <i>Gata4</i> with genomic DNA harboring a substitution of glycine to serine at position 295 into the mouse <i>Gata4</i> locus, as well as the incorporation of neomycin cassette surrounded by loxP sites. <i>Gata4</i> coding exons are shown as empty boxes, whereas the exon used as a probe used for Southern blot analysis is highlighted by a black bar. NZf, amino- terminal zinc finger (exon 2); CZf, carboxy- terminal zinc finger (exon 3); E4, exon 4; E5, exon 5; E6, exon 6; B, BglI; S, SacI; E, EcoRV; and N, NotI. (B) Germline transmission of mutant allele was confirmed by Southern blotting after digestion of genomic DNA from <i>Gata4 G295S^ki/wt</i> and wiltype mice with BglI. A 3.8 kb wildtype band and a 12.5 kb mutant band using 3′ external probe are shown (black bar in A). (C) Direct sequencing confirmed the presence of mutated residue that altered glycine (GGC) to serine (AGC) in DNA from <i>Gata4 G295S^ki/ki</i> embryos. (D) Western blotting demonstrates that levels of Gata4 protein are equivalent in <i>Gata4 G295S^ki/ki</i> hearts (from three different embryos) as compared to wildtype E9.5 hearts. Equal protein loading is shown by Western blotting to GAPDH.</p