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. 2003 May;111(9):1399-407.
doi: 10.1172/JCI17061.

Pressure-independent cardiac hypertrophy in mice with cardiomyocyte-restricted inactivation of the atrial natriuretic peptide receptor guanylyl cyclase-A

Rita Holtwick  1 , Martin van Eickels, Boris V Skryabin, Hideo A Baba, Alexander Bubikat, Frank Begrow, Michael D Schneider, David L Garbers, Michaela Kuhn
Affiliations

Pressure-independent cardiac hypertrophy in mice with cardiomyocyte-restricted inactivation of the atrial natriuretic peptide receptor guanylyl cyclase-A

Rita Holtwick et al. J Clin Invest. 2003 May.

Abstract

Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Atrial natriuretic peptide (ANP) has been postulated to exert local antihypertrophic effects in the heart. Thus, a loss of function of the ANP receptor guanylyl cyclase-A (GC-A) might contribute to the increased propensity to cardiac hypertrophy, although a causative role in vivo has not been definitively demonstrated. To test whether local ANP modulates cardiomyocyte growth, we inactivated the GC-A gene selectively in cardiomyocytes by homologous loxP/Cre-mediated recombination. Thereby we have circumvented the systemic, hypertensive phenotype associated with germline inactivation of GC-A. Mice with cardiomyocyte-restricted GC-A deletion exhibited mild cardiac hypertrophy, markedly increased mRNA expression of cardiac hypertrophy markers such as ANP (fivefold), alpha-skeletal actin (1.7-fold), and beta-myosin heavy chain (twofold), and increased systemic circulating ANP levels. Their blood pressure was 7-10 mmHg below normal, probably because of the elevated systemic levels and endocrine actions of ANP. Furthermore, cardiac hypertrophic responses to aortic constriction were enhanced and accompanied by marked deterioration of cardiac function. This phenotype is consistent with a local function of the ANP/GC-A system to moderate the molecular program of cardiac hypertrophy.

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Figures

Figure 1
Figure 1
Generation and Southern blot characterization of “flox/flox GC-A” and “flox/flox GC-A × αMHC-Cretg” mice. (a) Restriction map of the targeting construct and the αMHC-Cre-generated deletion allele. B, BamHI site. (b) Southern blot analyses of genomic DNA obtained from tails, left ventricles, and enriched ventricular cardiomyocytes of “flox/flox GC-A” and “flox/flox GC-A × αMHC-Cretg” mice, demonstrating the deletion event that occurs in cardiomyocytes from mice harboring the αMHC-Cre transgene (CM GC-A KO mice). The Cre-mediated deletion event abolishes the additional BamHI site inserted in intron 1 by the recombination event. BamHI-digested DNA was hybridized with a specific cDNA probe (spanning E2–E6); 11-kb and 7.5-kb bands represent the deletion allele and targeted allele, respectively. (c) Efficiency of Cre-mediated GC-A deletion (n = 8).
Figure 2
Figure 2
Demonstration of the GC-A deletion at the mRNA and protein level. (a) RT-PCR analyses of GC-A and GAPDH mRNA in preparations of isolated adult ventricular cardiomyocytes from floxed GC-A and CM GC-A KO mice (20–30 single cells were collected from each heart; five hearts were studied per genotype). Left: Ethidium bromide visualization of RT-PCR products. Left ventricle (LV) of a floxed GC-A mouse was used as positive control. Right: GC-A signal intensities were normalized to GAPDH. (b) Basal and ANP-stimulated guanylyl cyclase activity of plasma membranes isolated from whole hearts of floxed GC-A and CM GC-A KO mice. Enzymatic activity is expressed as picomoles of cGMP formed per milligram of protein over 10 minutes (n = 6). *P < 0.05 vs. floxed GC-A.
Figure 3
Figure 3
Effect of cardiomyocyte-restricted disruption of the GC-A gene on systolic blood pressure (SBP) (a), heart weight/body weight (HW/BW) ratios (b), and the relationship between left ventricular weight/body weight (LVW/BW) ratios and SBP (c) (n = 14 per sex and genotype, n = 28 per group, *P < 0.05 vs. floxed GC-A).
Figure 4
Figure 4
Histological analysis of floxed GC-A and CM GC-A KO hearts. Cardiomyocyte diameters and interstitial collagen fractions were estimated by quantitative morphometry of PAS-stained (a) or Sirius red–stained (b) ventricular sections, respectively. Black lines (a) indicate cardiomyocyte diameters in the region of the cell nucleus, which were significantly increased in CM GC-A KO hearts (c), without changes in interstitial collagen (d) (n = 16, *P < 0.05 vs. floxed GC-A).
Figure 5
Figure 5
Representative Northern blots. Messenger RNA expression levels of ANP, α-sk-actin, αMHC, and βMHC, as well as GAPDH, in cardiac ventricles of floxed GC-A and CM GC-A KO mice under baseline conditions (a) and after TAC (b).
Figure 6
Figure 6
Plasma concentrations of ANP in CM GC-A KO mice compared with their floxed GC-A littermates (n = 18, *P < 0.05 vs. floxed GC-A).
Figure 7
Figure 7
In vivo hemodynamic measurements of cardiac function by left ventricular catheterization. Heart rate (a), peak left ventricular systolic pressure (LVSP) (b), and the maximal rates of contraction (dP/dtmax) (c) and relaxation (dP/dtmin) (d) are shown at baseline (0) and during infusion of dobutamine in CM GC-A KO and floxed GC-A mice (n = 6, *P < 0.05 vs. floxed GC-A).
Figure 8
Figure 8
(a) TAC led to a substantial increase in systolic blood pressure (SBP) proximal to the stenosis. SBP was significantly lower in the CM GC-A KO than in the floxed GC-A mice. For reference, pressures in sham-operated mice averaged 100 ± 6 mmHg (floxed GC-A) and 93 ± 10 mmHg (CM GC-A KO), indicating that a substantial pressure load was induced by TAC in both genotypes. (b) Despite the lower SBP, the CM GC-A KO mice had an increased LVW/BW index compared with the floxed GC-A mice. (c) The linear regression of SBP and LVW/BW index revealed that in the CM GC-A KO mice (filled circles and solid line), a stronger hypertrophic response in the heart correlated with lower blood pressures, while in the floxed GC-A mice (open circles and dashed line), blood pressure and LVW/BW index were positively correlated. (d) To compare the hypertrophic response independently of the differences in blood pressure in this figure, we divided the LVW/BW index of each animal by the individual SBP and normalized this value to the mean SBP of 145 mmHg (n = 18 floxed GC-A and 27 CM GC-A KO mice, *P < 0.05 vs. floxed GC-A).
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