Vasodilator therapy with hydralazine induces angiotensin AT receptor-mediated cardiomyocyte growth in mice lacking guanylyl cyclase-A

Abstract

Background and purpose: Recent clinical guidelines advocate the use of the isosorbide dinitrate/hydralazine combination in treatment for heart failure. However, clinical and laboratory evidence suggest that some vasodilators may induce cardiac hypertrophy under uncertain conditions. This study investigated the effects and underlying mechanism of action of the vasodilator hydralazine on cardiac growth.

Experimental approach: Wild-type mice and animals deficient in guanylyl cyclase-A (GCA) and/or angiotensin receptors (AT(1) and AT(2) subtypes) were treated with hydralazine ( approximately 24 mg.kg(-1).day(-1) in drinking water) for 5 weeks. Cardiac mass and/or cardiomyocyte cross-sectional area, fibrosis (van Giessen-staining) and cardiac gene expression (real-time RT-PCR) were measured.

Key results: Hydralazine lowered blood pressure in mice of all genotypes. However, this treatment increased the heart and left ventricular to body weight ratios, as well as cardiomyocyte cross-sectional area, and cardiac expression of atrial natriuretic peptide mRNA in mice lacking GCA. Hydralazine did not affect cardiac hypertrophy in wild-type mice and mice lacking either AT(1) or AT(2) receptors alone. However, the pro-hypertrophic effect of hydralazine was prevented in mice lacking both GCA and AT(2), but not GCA and AT(1) receptors. However, hydralazine did decrease cardiac collagen deposition and collagen I mRNA (signs of cardiac fibrosis) in mice that were deficient in GCA, or both GCA and AT(2) receptors.

Conclusions and implications: The vasodilator hydralazine induced AT(2) receptor-mediated cardiomyocyte growth under conditions of GCA deficiency. However, attenuation of cardiac fibrosis by hydralazine could be beneficial in the management of cardiac diseases.

Figure 1

Systolic blood pressure (SBP, A) and the ratios of heart weight to body weight (HW/BW, B) and left ventricular weight to body weight (LVW/BW, C) in wild-type (WT) and guanylyl cyclase-A-deficient [GCA knockout (KO) mice] mice. Hydralazine (HYD) was administered in drinking water (≈24 mg·kg⁻¹·day⁻¹) for 5 weeks, while the mice in control groups received drinking water only. SBP was measured in conscious mice prior to, and at weekly intervals after, the commencement of treatment using a computerized tail-cuff method. Hearts and left ventricles were weighed at the conclusion of the experiments (week 5), and HW/BW and LVW/BW were calculated. Values are means ± SEM (n = 7−14). *P < 0.05; ^$different from WT control (HYD −) or ^#different from GCA KO (HYD −) at corresponding time-points (P < 0.05). HYD −: control, without HYD; HYD +, with HYD.

Figure 2

Cardiomyocyte cross-sectional areas in wild-type (WT) and guanylyl cyclase-A (GCA) knockout (KO) mice. Hydralazine (HYD) was administered in drinking water (≈24 mg·kg⁻¹·day⁻¹) for 5 weeks, while the mice in control groups received drinking water only. Morphometry of left ventricular myocytes was performed to measure the myocyte cross-sectional area. Values are means ± SEM (n = 7−14). *P < 0.05. (A) Results of cardiomyocyte cross-sectional areas with or without HYD treatment. (B) Representative histological findings of cardiomyocytes in different experimental groups. Original magnification: ×400. HYD −: control, without HYD; HYD +, with HYD.

Figure 3

Cardiac fibrosis in wild-type (WT) and guanylyl cyclase-A (GCA) knockout (KO) mice. Hydralazine (HYD) was administered in drinking water (≈24 mg·kg⁻¹·day⁻¹) for 5 weeks, while the mice in control groups received drinking water only. After van Giessen-staining the area of collagen deposition in the interstitial region and the total left ventricular area were quantified using an image analysing system. (A) Interstitial fibrosis (%, the ratio of the area of interstitial collagen accumulation to the total left ventricular area); (B) Representative examples of interstitial fibrosis (red) (original magnification: ×200). Values are means ± SEM (n = 7–14). *P < 0.05. HYD −: control, without HYD; HYD +, with HYD.

Figure 4

Left ventricular expression of mRNA for atrial natriuretic peptide (ANP, A) or collagen I (B) in wild-type (WT) and guanylyl cyclase-A (GCA) knockout (KO) mice. Hydralazine (HYD) was administered in drinking water (≈24 mg·kg⁻¹·day⁻¹) for 5 weeks, while the mice in control groups received drinking water only. Total RNA was extracted from the left ventricular tissues using TRIzol. The relative levels of specific mRNAs were assessed by real-time RT-PCR. Results were normalized to GAPDH. Levels in WT group were arbitrarily assigned a value of 1. Values are means ± SEM (n = 7). *P < 0.05. HYD −: control, without HYD; HYD +, with HYD.

Figure 5

Systolic blood pressure (SBP, A), the ratios of heart weight to body weight (HW/BW, B) and left ventricular weight to body weight (LVW/BW, C) and cardiac interstitial fibrosis (D) in angiotensin II type 1 (AT₁) receptor knockout (KO) and guanylyl cyclase-A (GCA)/AT₁ double KO (DKO) mice. Hydralazine (HYD) was administered in drinking water (≈24 mg·kg⁻¹·day⁻¹) for 5 weeks, while the mice in control groups received drinking water only. SBP was measured in conscious mice prior to, and at weekly intervals after, the commencement of treatment using a computerized tail-cuff method. Hearts and left ventricles were weighed at the conclusion of the experiments (week 5), and HW/BW and LVW/BW were calculated. After van Giessen-staining the area of collagen deposition on the interstitial region and the total left ventricular area were quantified using an image analysing system. Values are means ± SEM (n = 6−7). *P < 0.05; ^$different from AT₁ KO (HYD −), or ^#different from GCA/AT₁ DKO (HYD −) at corresponding time-points (P < 0.05). HYD −: control, without HYD; HYD +, with HYD.

Figure 6

Left ventricular expression of mRNA for atrial natriuretic peptide (ANP, A) and collagen I (B) in angiotensin II type 1 (AT₁) receptor knockout (KO) and guanylyl cyclase-A (GCA)/AT₁ double KO (DKO) mice. Hydralazine (HYD) was administered in drinking water (≈24 mg·kg⁻¹·day⁻¹) for 5 weeks, while the mice in control groups received drinking water only. Total RNA was extracted from the left ventricular tissues using TRIzol. The relative levels of specific mRNAs were assessed by real-time RT-PCR. Results were normalized to GAPDH. Levels in WT group were arbitrarily assigned a value of 1. Values are means ± SEM (n = 6−7). *P < 0.05. HYD −: control, without HYD; HYD +, with HYD.

Figure 8

Left ventricular expression of mRNA for atrial natriuretic peptide (ANP, A) and collagen I (B) in angiotensin II type 2 (AT₂) receptor and in guanylyl cyclase-A (GCA) and AT₂ double knockout (DKO) mice. Hydralazine (HYD) was administered in drinking water (≈24 mg·kg⁻¹·day⁻¹) for 5 weeks, while the mice in control groups received drinking water only. Total RNA was extracted from the left ventricular tissues using TRIzol. The relative levels of specific mRNAs were assessed by real-time RT-PCR. Results were normalized to GAPDH. Levels in wild-type group were arbitrarily assigned a value of 1. Values are means ± SEM (n = 7–8). *P < 0.05. HYD −: control, without HYD; HYD +, with HYD.

Figure 7

Systolic blood pressure (SBP, A), the ratios of heart weight to body weight (HW/BW, B) and left ventricular weight to body weight (LVW/BW, C) and cardiac interstitial fibrosis (D) in angiotensin II type 2 (AT₂) receptor knockout (KO) and guanylyl cyclase-A (GCA)/AT₂ double KO (DKO) mice. Hydralazine (HYD) was administered in drinking water (≈24 mg·kg⁻¹·day⁻¹) for 5 weeks, while the mice in control groups received drinking water only. SBP was measured in conscious mice prior to, and at weekly intervals after, the commencement of treatment using a computerized tail-cuff method. Hearts and left ventricles were weighed at the conclusion of the experiments (week 5), and HW/BW and LVW/BW were calculated. After van Giessen-staining the area of collagen deposition on the interstitial region and the total left ventricular area were quantified using an image analysing system. Values are means ± SEM (n = 7–8). *P < 0.05; ^$different from AT₂ KO (HYD −), or ^#different from GCA/AT₂ DKO (HYD −) at corresponding time-points (P < 0.05). HYD −: control, without HYD; HYD +, with HYD.