Overexpression of angiotensin AT1 receptor transgene in the mouse myocardium produces a lethal phenotype associated with myocyte hyperplasia and heart block - PubMed (original) (raw)

Overexpression of angiotensin AT1 receptor transgene in the mouse myocardium produces a lethal phenotype associated with myocyte hyperplasia and heart block

L Hein et al. Proc Natl Acad Sci U S A. 1997.

Abstract

Previous studies have suggested that angiotensin II (Ang II) modulates cardiac contractility, rhythm, metabolism, and structure. However, it is unclear whether the cardiac effects are due to direct actions of Ang II on the myocardium or if they are due to secondary effects mediated through the hemodynamic actions of Ang II. In this study, we used the alpha-myosin heavy chain (alphaMHC) promoter to generate transgenic mice overexpressing angiotensin II type 1 (AT1a) receptor selectively in cardiac myocytes. The specificity of transgene expression in the transgenic offspring was confirmed by radioligand binding studies and reverse transcription-PCR. The offspring displayed massive atrial enlargement with myocyte hyperplasia at birth, developed significant bradycardia with heart block, and died within the first weeks after birth. Thus, direct activation of AT1 receptor signaling in cardiac myocytes in vivo is sufficient to induce cardiac myocyte growth and alter electrical conduction.

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Figures

Figure 1

Figure 1

Transmission rate and survival of αMHC-AT1 transgenic mice. (A) Transgenic vector. The complete intergenic region between the β- and α-myosin heavy chain genes (αMHC promoter) was used as a cardiac-specific promoter to control expression of the mouse AT1a angiotensin receptor. At the 3′ end of the AT1a receptor cDNA, the intron and polyadenylylation signal of the SV40 T antigen was added (SV40 t intron+polyA). β, α1,2,3 exons of the βMHC and αMHC locus; Agtr1a, angiotensin AT1 receptor gene; A, T, M, primers for reverse transcriptase–PCR. (B and C) Transmission frequency of the αMHC-AT1 transgene. Transgenic founder mice were mated with wild-type FVB/N mice, and the frequency of transmission of the transgene to their offspring was determined by Southern blot analysis using part of the coding region of the AT1a receptor cDNA. The endogenous AT1a gene can be detected as a 9-kb _Bam_HI fragment (shaded arrowhead), the AT1b gene appears as a faint band at 10.6 kb size (open arrowhead), and the αMHC-AT1 transgene is detectable as a 8-kb fragment (solid arrowhead). For lines F2/9 and F2/12, blots from the F1 generation represent nontransgenic offspring, as no transgenic mice were born from these lines. Fifteen micrograms of genomic DNA was loaded per lane. Transmission rates are given in absolute numbers (number of transgenic offspring/total mice screened) and in percent values for the following age groups: at embryonic days 16–19 (E16–19), at 1 week after birth (P1w), at weaning age (3 weeks, P3w), and at 6 weeks of age (P6w). (C) Effect of ACE inhibitor treatment or AT1 receptor blockade on the survival of transgenic offspring of the M4/7 line. Pregnant mice were treated from day 12 after conception with captopril, losartan, or saline throughout pregnancy and during the lactation period. With both captopril and losartan, survival of the transgenic mice at 1 week of age was significantly improved as compared with untreated mice (∗, P < 0.05).

Figure 2

Figure 2

Cardiac-specific expression of the αMHC-AT1 transgene. (A) Detection of the expression of the αMHC-AT1 transgene by reverse transcriptase–PCR in 3-day-old transgenic offspring of line M2/5. Messenger RNA for the endogenous AT1a receptor gene could be detected in heart, lung, and liver, whereas mRNA for the αMHC-AT1 transgene could only be detected in the heart. (B) AT1 receptor saturation binding isotherms in heart membranes derived from transgenic lines M2/5 (Left) and M4/7 (Right). Hearts were obtained from transgenic mice and wild-type littermates at day 3 (line M2/5) or days 5–7 (line M4/7) after birth. Data shown represent means ± SEM for duplicate determinations of three hearts in each group. Transgenic offspring express significantly more AT1 receptors in the heart than their nontransgenic littermates.

Figure 3

Figure 3

Cardiac phenotype of the αMHC-AT1 transgenic mice. (A) Heart and lung preparation of transgenic (Left) and nontransgenic (Right) littermates from line M4/7 at day 4 after birth. The mother of these mice was treated with captopril from day 12 of pregnancy until day 4 after delivery (1 mg/ml drinking water). Compared with the control specimen, the transgenic heart displays a massive enlargement of left and right atria. Arrowheads mark the borders of the atria. RA, right atria; LA, left atria; V, ventricle. Bar = 2 mm. (B and C) Frontal section through the same transgenic (B) and nontransgenic (C) hearts as shown in Fig. 3_A_. The atrial cavity is greatly increased in transgenic atria compared with nontransgenic atria. RA, right atria; LA, left atria; LV, left ventricle; RV, right ventricle. Bar = 1.5 mm. (D and E) Horizontal cross-sections through the ventricles of transgenic (D) and nontransgenic (E) offspring from line M4/7 do not reveal any significant morphological differences between transgenic and nontransgenic ventricles. Bar = 1.2 mm.

Figure 4

Figure 4

Electrophysiological alterations in the αMHC-AT1 transgenic mice. ECG of αMHC-AT1 transgenic and nontransgenic littermate mice (offspring of line M4/7, captopril-treatment of the mothers) during the first 2 days after birth. Transgenic mice display severe bradycardia (A and B) with evidence of atrioventricular block (increased P–R interval) as well as a broadening of the QRS complex (A). Heart rate (B) was measured in 4 transgenic mice and 10 nontransgenic littermates, which were derived from a total of 3 litters. The mothers of these mice had received captopril through the drinking water from day 12 of pregnancy, as described.

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