Replacement of K-Ras with H-Ras supports normal embryonic development despite inducing cardiovascular pathology in adult mice

Abstract

Ras proteins are highly related GTPases that have key roles in regulating growth, differentiation and tumorigenesis. Gene-targeting experiments have shown that, out of the three mammalian ras genes, only K-ras is essential for normal mouse embryogenesis, and that mice deprived of H-ras and/or N-ras show no major phenotype. We generated mice (HrasKI) in which the K-ras gene had been modified to encode H-Ras protein. HrasKI mice produce undetectable amounts of K-Ras but-in contrast to mice homozygous for a null K-ras allele-they are born at the expected mendelian frequency, indicating that H-Ras can be substituted for K-Ras in embryonic development. However, adult HrasKI mice show dilated cardiomyopathy associated with arterial hypertension. Our results show that K-Ras can be replaced by H-Ras in its essential function in embryogenesis, and indicate that K-Ras has a unique role in cardiovascular homeostasis.

Figure 1

Targeting strategy of knock-in of H-ras (or K-ras) cDNA in K-ras locus. (A) Wild-type K-ras targeted region with targeting vector and the predicted mutant locus. The coding exons are represented by open boxes. The HSV-TK (herpes simplex virus thymidine kinase) and neo cassettes are indicated. The cloned coding sequences for H- or K-ras are represented by dashed boxes. The 3′ untranslated region is represented by a grey box. The probes used for Southern hybridization are shown by black boxes and the expected sizes of the signals are indicated by dashed lines. EI, EcoRI; EV, EcoRV; S, SacI; X, XhoI. (B) Southern blot analysis of genomic DNA from ES clones using 3′ (left) and 5′ (right) probes. +/+, wild type; +/−, HrasKI heterozygous.

Figure 2

Expression of the Ras isoforms in different tissues from wild-type (WT), KrasKI and HrasKI adult mice. The protein extracts were probed with antibodies specific for each Ras protein. Pan-Ras antibody was used to detect the total expression levels of Ras proteins. Tubulin antibody was used to normalize the amount of loaded proteins.

Figure 3

Left ventricular remodelling. (A) Representative M-mode left ventricular echocardiographic recordings of wild-type (+/+), KrasKI and HrasKI mice. All measurements were determined in a short-axis view at the level of papillary muscles. (B) Collagen deposition (light blue), indicative of fibrosis, evaluated by Masson's trichrome staining in the left ventricle. (C) Left ventricles processed for histology to measure the cardiomyocyte crosssectional area from +/+, KrasKI and HrasKI mice. (D) Mean values from 200 cardiomyocyte area determinations per mouse. ^*P<0.05 versus +/+.

Figure 4

BP measurements. (A) Systolic BP evaluated non-invasively by tail-cuff measurements in conscious mice (n=6 for each group). ^*P<0.01 versus wild type (+/+) and KrasKI. Data are mean±s.e.m. (B) Systolic and diastolic BP evaluated invasively by radiotelemetry in conscious mice (n=3 for each group). ^*P<0.02 versus +/+ and KrasKI. Data are mean±s.e.m.