Mineralocorticoid Receptor in Smooth Muscle Contributes to Pressure Overload-Induced Heart Failure

Abstract

Background: Mineralocorticoid receptor (MR) antagonists decrease heart failure (HF) hospitalization and mortality, but the mechanisms are unknown. Preclinical studies reveal that the benefits on cardiac remodeling and dysfunction are not completely explained by inhibition of MR in cardiomyocytes, fibroblasts, or endothelial cells. The role of MR in smooth muscle cells (SMCs) in HF has never been explored.

Methods: Male mice with inducible deletion of MR from SMCs (SMC-MR-knockout) and their MR-intact littermates were exposed to HF induced by 27-gauge transverse aortic constriction versus sham surgery. HF phenotypes and mechanisms were measured 4 weeks later using cardiac ultrasound, intracardiac pressure measurements, exercise testing, histology, cardiac gene expression, and leukocyte flow cytometry.

Results: Deletion of MR from SMC attenuated transverse aortic constriction-induced HF with statistically significant improvements in ejection fraction, cardiac stiffness, chamber dimensions, intracardiac pressure, pulmonary edema, and exercise capacity. Mechanistically, SMC-MR-knockout protected from adverse cardiac remodeling as evidenced by decreased cardiomyocyte hypertrophy and fetal gene expression, interstitial and perivascular fibrosis, and inflammatory and fibrotic gene expression. Exposure to pressure overload resulted in a statistically significant decline in cardiac capillary density and coronary flow reserve in MR-intact mice. These vascular parameters were improved in SMC-MR-knockout mice compared with MR-intact littermates exposed to transverse aortic constriction.

Conclusions: These results provide a novel paradigm by which MR inhibition may be beneficial in HF by blocking MR in SMC, thereby improving cardiac blood supply in the setting of pressure overload-induced hypertrophy, which in turn mitigates the adverse cardiac remodeling that contributes to HF progression and symptoms.

Keywords: constriction; endothelial cell; hospitalization; hypertrophy; phenotype.

Figure 1:. Deletion of mineralocorticoid receptor in smooth muscle cell (SMC-MR-KO) attenuates cardiac dysfunction induced by pressure overload

(A) LV ejection fraction (EF) and (B) fractional shortening (FS) measured by echocardiography. (C) Representative images of M-mode echocardiography. LV function indices assessed by invasive LV pressure-volume loop analysis to quantify (D) maximal rate of rise in LV pressure (dP/dt max), (E) minimal rate of rise in LV pressure (dP/dt min), (F) end-systolic pressure-volume relationship (ESPVR), a measure of systolic cardiac stiffness and (G) end-diastolic pressure-volume relationship (EDPVR), a measure of diastolic cardiac stiffness. A, B: MR-intact Sham n=9, MR-intact TAC n=27, SMC-MR-KO Sham n=10, SMC-MR-KO TAC n=17. D-G: MR-intact Sham n=7, MR-intact TAC n=23, SMC-MR-KO Sham n=9, SMC-MR-KO TAC n=14. Two-way ANOVA with Tukey post-hoc test.

Figure 2:. Deletion of mineralocorticoid receptor in smooth muscle cell (SMC-MR-KO) ameliorates pressure overload-induced cardiac dilation and heart failure.

(A) End-systolic diameter (ESD) and (B) end-diastolic diameter (EDD) of the left ventricle (LV) were measured by echocardiography; (C) LV end-diastolic pressure (EDP) as assessed by invasive left ventricular pressure-volume loop analysis; (D) Pulmonary edema as measured by lung wet mass minus dried mass; (E) Circulating aldosterone levels measured in serum; (F) Whole body endurance exercise capacity determined by measuring run to exhaustion time normalized to body mass and vertical distance on the rodent treadmill. A-D: MR-intact Sham n=9, MR-intact TAC n=27, SMC-MR-KO Sham n=10, SMC-MR-KO TAC n=17. E: MR-intact Sham n=5, MR-intact TAC n=10, SMC-MR-KO Sham n=7, SMC-MR-KO TAC n=6. F: MR-intact Sham n=5, MR-intact TAC n=11, SMC-MR-KO Sham n=6, SMC-MR-KO TAC n=8. Two-way ANOVA with Tukey post-hoc test.

Figure 3:. Mineralocorticoid receptor in smooth muscle cell (SMC-MR) contributes to pathological cardiac hypertrophy induced by pressure overload.

(A) LV hypertrophy measured by LV weight to tibia length ratio; (B) Left: Representative echocardiographic images used to quantify posterior wall thickness (PWT, right). (C) Left: Representative LV sections stained with H&E for assessment of cardiomyocyte area (red outline shows an example of a representative cardiomyocyte that was quantified) and quantification of cardiomyocyte area (right). Scale bar=100 μm. Results of quantitative RT-PCR for (D) atrial natriuretic peptide (Nppa), (E) brain natriuretic peptide (Nppb) and (F) the ratio of myosin heavy chain-β (Myh7) to myosin heavy chain-α (Myh6) in LV tissue. A, B: MR-intact Sham n=9, MR-intact TAC n=27, SMC-MR-KO Sham n=10, SMC-MR-KO TAC n=17. C: MR-intact Sham n=3, MR-intact TAC n=12, SMC-MR-KO Sham n=3, SMC-MR-KO TAC n=6. D-F: MR-intact Sham n=3, MR-intact TAC n=5, SMC-MR-KO Sham n=3, SMC-MR-KO TAC n=6. Two-way ANOVA with Tukey post-hoc test.

Figure 4:. Deletion of mineralocorticoid receptor in smooth muscle cell (SMC-MR-KO) reduces pressure overload-induced cardiac fibrosis.

Representative LV sections stained with picrosirius red for assessment of perivascular (A) or interstitial (B) collagen deposition. Scale bar=50 μm. Results of quantitative RT-PCR with primers specific for: (C) connective tissue growth factor (CTGF); (D) collagen type 1 alpha (Col1α); (E) collagen type 3 alpha (Col3α); (F) alpha smooth muscle actin (alpha-SMA); (G) matrix metalloproteinase-2 (MMP2); and (H) transforming growth factor beta 1 (TGFβ1). A, B: MR-intact Sham n=3, MR-intact TAC n=12, SMC-MR-KO Sham n=3, SMC-MR-KO TAC n=6. C-H: MR-intact Sham n=3, MR-intact TAC n=6, SMC-MR-KO Sham n=3, SMC-MR-KO TAC n=6. Two-way ANOVA with Tukey post-hoc test.

Figure 5:. Deletion of mineralocorticoid receptor in smooth muscle cells (SMC-MR-KO) reduces pressure overload-induced cardiac inflammation.

Leukocytes were quantified from LV tissue 4 weeks after TAC by flow cytometry. (A) Total leukocytes (CD45+), (B) T cells (CD45+/CD11b−/CD3+) and (C) myeloid cells (CD45+/CD3−/CD11b+) were compared between Shams, MR-intact TAC or SMC-MR-KO TAC mice. Results of quantitative RT-PCR for inflammatory gene expression in the LV: (D) monocyte chemoattractant protein-1 (MCP-1), (E) regulated on activation, normal T cell expressed and secreted (RANTES), (F) interleukin 1 beta (IL1-B), (G) interleukin 6 (IL-6), and (H) tumor necrosis factor alpha (TNFα). Results of quantitative RT-PCR for adhesion molecules (I) intercellular adhesion molecule 1 (ICAM1), (J) vascular cell adhesion protein 1 (VCAM1), and (K) endothelial-leukocyte adhesion molecule (E-selectin). A-C: Sham n=9: (MR-intact Sham n=5, SMC-MR-KO Sham n=4), MR-intact TAC n=8, SMC-MR-KO TAC n=6. D-K: MR-intact Sham n=3, MR-intact TAC n=6, SMC-MR-KO Sham n=3, SMC-MR-KO TAC n=6. A-C: One-way ANOVA with Tukey post-hoc test. D-K: Two-way ANOVA with Tukey post-hoc test.

Figure 6:. Deletion of mineralocorticoid receptor in smooth muscle cell (SMC-MR-KO) improves capillary density and coronary flow reserve in pressure overload-induced heart failure.

Results of quantitative RT-PCR for angiogenesis markers: (A) basic fibroblast growth factor (FGF2), (B) placental growth factor (PGF), and (C) vascular endothelial growth factor receptor 1 (FLT1). (D) Representative LV sections stained with CD31 to mark endothelial cells. Scale bar=50 μm. (E) Quantification of capillary density using CD31 stained area normalized to cardiomyocyte area. (F) Coronary flow reserve assessed by echocardiography before and after a hyperemic stimulus. A-C: MR-intact Sham n=3, MR-intact TAC n=6, SMC-MR-KO Sham n=3, SMC-MR-KO TAC n=6. E: MR-intact Sham n=3, MR-intact TAC n=11, SMC-MR-KO Sham n=3, SMC-MR-KO TAC n=5. F: MR-intact Sham n=6, MR-intact TAC n=16, SMC-MR-KO Sham n=7, SMC-MR-KO TAC n=13. Two-way ANOVA with Tukey post-hoc test.