Mineralocorticoid receptor antagonism prevents coronary microvascular dysfunction in intermittent hypoxia independent of blood pressure

Abstract

Study objectives: Obstructive sleep apnea (OSA), is characterized by intermittent hypoxia (IH), and is associated with increased cardiovascular mortality that may not be reduced by standard therapies. Inappropriate activation of the renin-angiotensin-aldosterone system occurs in IH, and mineralocorticoid receptor (MR) blockade has been shown to improve vascular outcomes in cardiovascular disease. Thus, we hypothesized that MR inhibition prevents coronary and renal vascular dysfunction in mice exposed to chronic IH.

Methods: Human and mouse coronary vascular cells and male C57BL/6J mice were exposed to IH or room air (RA) for 12 hours/day for 3 days (in vitro) and 6 weeks with or without treatments with spironolactone (SPL) or hydrochlorothiazide (HTZ).

Results: In vitro studies demonstrated that IH increased MR gene expression in human and mouse coronary artery endothelial and smooth muscle cells. Exposure to IH in mice increased blood pressure, reduced coronary flow velocity reserve (CFVR), attenuated endothelium-dependent dilation, and enhanced vasoconstrictor responsiveness in coronary, but not renal arteries. Importantly, SPL treatment prevented altered coronary vascular function independent of blood pressure as normalization of BP with HTZ did not improve CFVR or coronary vasomotor function.

Conclusions: These data demonstrate that chronic IH, which mimics the hypoxia-reoxygenation cycles of moderate-to-severe OSA, increases coronary vascular MR expression in vitro. It also selectively promotes coronary vascular dysfunction in mice. Importantly, this dysfunction is sensitive to MR antagonism by SPL, independent of blood pressure. These findings suggest that MR blockade could serve as an adjuvant therapy to improve long-term cardiovascular outcomes in patients with OSA.

Keywords: blood pressure; coronary microvascular disease; intermittent hypoxia; mineralocorticoid receptor; obstructive sleep apnea; spironolactone.

Graphical Abstract

Figure 1.

Schematic of experimental treatment groups and measured outcomes.

Figure 2.

IH exposure in vitro increases MR gene expression in human and mouse coronary vascular cells. Fold change in MR gene expression in human and mouse endothelial cells (EC) and smooth muscle cells (SMC) after exposure to IH for 3 days (n = 5–6 wells) with 50k cells/well. Data represented as mean ± SD. Statistical analysis was done using the Student t-test. **p < .01, ***p < .001, ****p < .0001. IH, intermittent hypoxia; RA, room air; MR, mineralocorticoid receptor; TBP, TATA-box binding protein.

Figure 3.

SPL treatment normalizes blood pressure and ameliorates coronary, but not renal, artery vasomotor dysfunction in mice exposed to 6 weeks of IH. (A) Mean arterial blood pressure (MBP). Coronary artery cumulative concentration-response curves to the thromboxane A₂ analog U46619 (B), acetylcholine (ACh; C), and sodium nitroprusside (SNP; D). Renal artery cumulative dose–response curves to phenylephrine (PE; E), ACh (F), and SNP (G). Data are shown as mean ± S.D (n = 6–8). Statistical analysis was done using two-way ANOVA followed by Tukey post hoc test. ****p < .0001 for MAP. ^†p < .05 vs. all groups. IH, intermittent hypoxia; RA, room air; SPL, spironolactone.

Figure 4.

SPL treatment improves CFVR in mice exposed to 6 weeks of IH. (A) Blood flow velocity images of coronary arteries responses at baseline (1%) and hyperemic inducing (2.5%) concentrations of isoflurane. (B) Coronary flow velocity reserve (CFVR) values are calculated as a ratio of hyperemic to baseline responses (H/B) to isoflurane. Data represented as mean ± S.D (n = 6) mice. Statistical analysis was done using two-way ANOVA followed by Tukey post hoc test. *** p < .001. IH, intermittent hypoxia; RA, room air; SPL, spironolactone.

Figure 5.

HTZ treatment normalizes blood pressure but does not attenuate coronary artery vasomotor dysfunction in mice exposed to 6 weeks of IH. (A) Mean arterial blood pressure (MBP). Coronary artery cumulative concentration-response curves to thromboxane A₂ analog U46619 (B), acetylcholine (ACh; C), and sodium nitroprusside (SNP; D). Data are shown as mean ± S.D (n = 5). Statistical analysis was done using two-way ANOVA followed by Tukey post hoc test. ^†p < .05 vs. RA, ^‡p < .05 vs. RA-HTZ. IH, intermittent hypoxia; RA, room air; HTZ, hydrochlorothiazide.

Figure 6.

HTZ treatment does not improve CFVR in mice exposed to 6 weeks of IH. (A) Blood flow velocity images of coronary arteries responses at baseline (1%) and hyperemic (2.5%) concentrations of isoflurane. (B) Coronary flow velocity reserve (CFVR) values are calculated as a ratio of hyperemic to baseline responses (H/B) to isoflurane. Data represented as mean ± S.D (n = 5). Statistical analysis was done using two-way ANOVA followed by Tukey post hoc test. **p < .01, ***p < .001, ****p < .0001. IH, intermittent hypoxia; RA, room air; HTZ, hydrochlorothiazide.