Histone demethylase LSD1 deficiency during high-salt diet is associated with enhanced vascular contraction, altered NO-cGMP relaxation pathway, and hypertension

Abstract

Histone methylation, a determinant of chromatin structure and gene transcription, was thought to be irreversible, but recent evidence suggests that lysine-specific demethylase-1 (LSD1, Kdm1a) induces demethylation of histone H3 lysine 4 (H3K4) or H3K9 and thereby alters gene transcription. We previously demonstrated a human LSD1 phenotype associated with salt-sensitive hypertension. To test the hypothesis that LSD1 plays a role in the regulation of blood pressure (BP) via vascular mechanisms and gene transcription, we measured BP and examined vascular function and endothelial nitric oxide (NO) synthase (eNOS) expression in thoracic aorta of male wild-type (WT) and heterozygous LSD1 knockout mice (LSD1(+/-)) fed either a liberal salt (HS; 4% NaCl) or restricted salt diet (LS; 0.08% NaCl). BP was higher in LSD1(+/-) than WT mice on the HS diet but not different between LSD1(+/-) and WT mice on the LS diet. Further examination of the mechanisms of this salt-sensitive hypertension in LSD1(+/-) mice on the HS diet demonstrated that plasma renin activity and plasma levels and urinary excretion of aldosterone were less in LSD1(+/-) than WT, suggesting suppressed renin-angiotensin-aldosterone system. In contrast, phenylephrine (Phe)-induced aortic contraction was greater in LSD1(+/-) than WT mice on the HS diet. Treatment of aortic rings with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; a blocker of guanylate cyclase) enhanced Phe contraction in LSD1(+/-) compared with WT mice on the HS diet. Acetylcholine (Ach)-induced relaxation was less in LSD1(+/-) than WT mice on the HS diet. Endothelium removal or pretreatment with N(ω)-nitro-L-arginine methyl ester (blocker of NOS) or ODQ abolished Ach-induced relaxation in aorta of WT but had minimal effect in LSD1(+/-). Vascular relaxation to sodium nitroprusside, an exogenous NO donor and guanylate cyclase activator, was decreased in LSD1(+/-) vs. WT mice on the HS diet. RT-PCR and Western blots revealed decreased eNOS mRNA expression and eNOS and guanylate cyclase protein in the heart and aorta of LSD1(+/-) compared with WT mice on HS diet. Thus, during the HS diet, LSD1 deficiency is associated with hypertension, enhanced vascular contraction, and reduced relaxation via NO-cGMP pathway. The data support a role for LSD1-mediated histone demethylation in the regulation of NOS/guanylate cyclase gene expression, vascular function, and BP during the HS diet.

Fig. 1.

RT-PCR of lysine-specific demethylase-1 (LSD1) mRNA expression in the heart (A) and Western blot analysis of LSD1 protein amount in the heart (B) and aorta (C) of WT and LSD1^+/− mice. Data represent means ± SE (n = 4). Two representative gels for LSD1 and actin immunoreactive bands are presented for each genotype. *Measurements in LSD1^+/− mice are significantly different (P < 0.05) from corresponding measurements in wild-type (WT) mice.

Fig. 2.

Systolic blood pressure (BP) in WT and LSD1^+/− mice on restricted salt (LS) and liberal salt (HS) diets. Data represent means ± SE (n = 6). *Measurements in LSD1^+/− mice are significantly different (P < 0.05) from corresponding measurements in WT. §Measurements in LSD1^+/− mice on HS diet are significantly different (P < 0.05) from corresponding measurements in LSD1^+/− mice on LS diet.

Fig. 3.

Plasma renin activity (PRA; A) and plasma levels (B) and 24-h urinary excretion of aldosterone (ALSO; C) in WT and LSD1^+/− mice on HS diet. Data represent means ± SE (n = 4). *Measurements in LSD1^+/− mice are significantly different (P < 0.05) from corresponding measurements in WT.

Fig. 4.

Phe- and KCl-induced contraction in aortic rings of WT and LSD1^+/− mice on LS and HS diets. Aortic rings from each genotype and treatment group were stimulated with increasing concentrations of Phe, the contractile response was measured and presented in grams (A) or as %maximum Phe contraction in mice on LS (B) and HS diet (C). In other experiments, aortic rings were stimulated with high 96 mM KCl depolarizing solution and the contractile response was measured and presented in grams (D). Data represent means ± SE (n = 6 to 8).*Measurements in LSD1^+/− mice on HS intake are significantly different (P < 0.05) from corresponding measurements in WT mice on HS intake. §Measurements in LSD1^+/− mice on HS intake diet are significantly different (P < 0.05) from corresponding measurements in LSD1^+/− mice on LS diet.

Fig. 5.

Effect of nitric oxide synthase (NOS) and guanylate cyclase blockade on Phe-induced contraction in aortic rings of WT and LSD1^+/− mice on HS intake. Aortic rings of WT (A) and LSD1^+/− mice (B) were either nontreated (circles) or pretreated with the NOS inhibitor N^ω-nitro-l-arginine methyl ester (l-NAME; 3 × 10⁻⁴ M; ▴) or the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10⁻⁵M) for 10 min (■). Tissues were stimulated with increasing concentrations of Phe, and the contractile response was measured and presented in grams. Data represent means ± SE (n = 4 to 8). *Measurements in ODQ-treated aortic segments are significantly different (P < 0.05) from corresponding measurements in nontreated segments.

Fig. 6.

Ach-induced relaxation in aortic rings of WT and LSD1^+/− mice on HS intake. Aortic rings of WT and LSD1^+/− mice were precontracted with Phe (10⁻⁵ M), increasing concentrations of Ach were added and the %relaxation of Phe contraction was measured (A). In other experiments, Ach-induced relaxation of aortic segments of WT (B) and LSD1^+/− mice (C) was compared in endothelium-intact aortic segments (○), endothelium-denuded segments (●), and aortic segments pretreated with l-NAME (3 × 10⁻⁴M; ▵) or ODQ (10⁻⁵M; ◊). Data represent means ± SE (n = 4 to 8). *Measurements in LSD1^+/− mice are significantly different (P < 0.05) from corresponding measurements in WT mice. ^#Measurements in endothelium-denuded and l-NAME- or ODQ-treated aortic segments are significantly different (P < 0.05) from corresponding measurements in endothelium-intact nontreated segments.

Fig. 7.

RT-PCR of endothelial (e)NOS mRNA expression in the heart (A) and Western blot analysis of eNOS protein amount in the heart (B) and aorta (C) of WT and LSD1^+/− mice on HS intake. Data represent means ± SE (n = 4). Two representative gels for eNOS and actin immunoreactive bands are presented for each genotype. *Measurements in LSD1^+/− mice are significantly different (P < 0.05) from corresponding measurements in WT mice.

Fig. 8.

A: sodium nitroprusside (SNP)-induced relaxation in aortic rings of WT and LSD1^+/− mice on HS intake. Aortic rings of WT and LSD1^+/− mice were precontracted with Phe (10⁻⁵ M), increasing concentrations of SNP were added, and the %relaxation of Phe contraction was measured. Data represent means ± SE (n = 6 to 8). B: Western blot analysis of guanylate cyclase protein levels in aortic tissues from WT and LSD1^+/− mice on HS intake. Data represent means ± SE (n = 4). Two representative gels for guanylate cyclase and actin immunoreactive bands are presented for each genotype. *Measurements in LSD1^+/− mice are significantly different (P < 0.05) from corresponding measurements in WT mice.