Cardiomyopathy in Irx4-deficient mice is preceded by abnormal ventricular gene expression

Abstract

To define the role of Irx4, a member of the Iroquois family of homeobox transcription factors in mammalian heart development and function, we disrupted the murine Irx4 gene. Cardiac morphology in Irx4-deficient mice (designated Irx4(Delta ex2/Delta ex2)) was normal during embryogenesis and in early postnatal life. Adult Irx4(Delta ex2/Delta ex2) mice developed a cardiomyopathy characterized by cardiac hypertrophy and impaired contractile function. Prior to the development of cardiomyopathy, Irx4(Delta ex2/Delta ex2) hearts had abnormal ventricular gene expression: Irx4-deficient embryos exhibited reduced ventricular expression of the basic helix-loop-helix transcription factor eHand (Hand1), increased Irx2 expression, and ventricular induction of an atrial chamber-specific transgene. In neonatal hearts, ventricular expression of atrial natriuretic factor and alpha-skeletal actin was markedly increased. Several weeks subsequent to these changes in embryonic and neonatal gene expression, increased expression of hypertrophic markers BNP and beta-myosin heavy chain accompanied adult-onset cardiac hypertrophy. Cardiac expression of Irx1, Irx2, and Irx5 may partially compensate for loss of Irx4 function. We conclude that Irx4 is not sufficient for ventricular chamber formation but is required for the establishment of some components of a ventricle-specific gene expression program. In the absence of genes under the control of Irx4, ventricular function deteriorates and cardiomyopathy ensues.

FIG. 1

Targeted disruption of Irx4. (A) Diagram of the Irx4 genomic locus, targeting construct, and targeted locus. Open boxes, untranslated sequences; solid boxes, coding sequences; hatched boxes, homeodomain-coding sequences. Only relevant restriction enzyme sites are shown. (B) Southern blot of XbaI-digested embryonic stem cell DNA using a probe external to the targeting construct, showing a targeting event (lane 2) as evidenced by two bands representing the endogenous 9-kb allele and the 6-kb targeted allele. (C) PCR identification of wild-type and targeted alleles in Irx4^+/+ (+/+), Irx4^Δex2/+ (+/−) and Irx4^Δex2/Δex2 (−/−) mice. (D) RT-PCR of mouse heart RNA for all three genotypes using primers internal to the deletion (ii and iii in panel A). (E) RT-PCR of mouse heart RNA for all three genotypes using primers external to the deletion (i and iv in panel A). Xb, XbaI; B, BamHI; N, NotI.

FIG. 2

Gene expression in wild-type (+/+) and Irx4^Δex2/Δex2 (−/−) mice. (A) Northern blot analysis of gene expression of ventricular RNA for wild-type, heterozygous (+/−), and Irx4^Δex2/Δex2 mice aged 10 days, 6 weeks, or 6 months. Representative signals for ANF, α-skeletal actin, βMHC, BNP, and GAPDH (as a loading control) are shown. (B and C) Expression of ANF by in situ hybridization on longitudinal sections of wild-type (B) and Irx4^Δex2/Δex2 (C) hearts at 6 months of age showing increased ANF transcript levels in Irx4^Δex2/Δex2 ventricles. (D to F) eHand expression in E10.5 embryos viewed ventrally (D) or in hearts dissected from E10.5 embryos viewed from the back (E) or the front (F). The arrow indicates lower eHand mRNA levels in Irx4^Δex2/Δex2 embryonic hearts. Dashed lines provide a comparison of the domains of eHand expression.

FIG. 3

SMyHC3-HAP transgene expression in wild-type (+/+) and Irx4^Δex2/Δex2 (−/−) embryos at E9, E10.5, and E12.5. Hearts were removed from E12.5 embryos for better visualization. a, atrium; v, ventricle; lv, LV; rv, RV.

FIG. 4

Cardiac expression of Irx family genes. Irx5 is robustly expressed in both atria and ventricles of E9.5 (A) and E10.5 (B and E) embryos. Expression of Irx1 was detected in a subset of ventricular cardiocytes at E10.5 (arrow in panel C, bracket in panel D). Irx2 is also expressed in a pattern that overlaps Irx1 (F and G; red arrows in panel F); Irx2 expression is increased in E10.5 Irx4^Δex2/Δex2 embryonic hearts (F and G) a, atrium; v, ventricle; rv, RV; lv, LV.

FIG. 5

Altered ventricular dimensions and function in 6-month-old wild-type and Irx4^Δex2/Δex2 mice. M-mode echocardiography shows increased LV wall thickness (LVWT) and LV end-systolic diameter (LVESD) but normal LV end-diastolic diameter (LVEDD) in Irx4^Δex2/Δex2 mice (right) compared to those for wild-type animals (left).