Aldosterone, through novel signaling proteins, is a fundamental molecular bridge between the genetic defect and the cardiac phenotype of hypertrophic cardiomyopathy

Abstract

Background: Human hypertrophic cardiomyopathy (HCM), the most common cause of sudden cardiac death in the young, is characterized by cardiac hypertrophy, myocyte disarray, and interstitial fibrosis. The genetic basis of HCM is largely known; however, the molecular mediators of cardiac phenotypes are unknown.

Methods and results: We show myocardial aldosterone and aldosterone synthase mRNA levels were elevated by 4- to 6-fold in humans with HCM, whereas cAMP levels were normal. Aldosterone provoked expression of hypertrophic markers (NPPA, NPPB, and ACTA1) in rat cardiac myocytes by phosphorylation of protein kinase D (PKD) and expression of collagens (COL1A1, COL1A2, and COL3A1) and transforming growth factor-beta1 in rat cardiac fibroblasts by upregulation of phosphoinositide 3-kinase (PI3K)-p100delta. Inhibition of PKD and PI3K-p110delta abrogated the hypertrophic and profibrotic effects, respectively, as did the mineralocorticoid receptor (MR) antagonist spironolactone. Spironolactone reversed interstitial fibrosis, attenuated myocyte disarray by 50%, and improved diastolic function in the cardiac troponin T (cTnT)-Q92 transgenic mouse model of human HCM. Myocyte disarray was associated with increased levels of phosphorylated beta-catenin (serine 38) and reduced beta-catenin-N-cadherin complexing in the heart of cTnT-Q92 mice. Concordantly, distribution of N-cadherin, predominantly localized to cell membrane in normal myocardium, was diffuse in disarrayed myocardium. Spironolactone restored beta-catenin-N-cadherin complexing and cellular distribution of N-cadherin and reduced myocyte disarray in 2 independent randomized studies.

Conclusions: The results implicate aldosterone as a major link between sarcomeric mutations and cardiac phenotype in HCM and, if confirmed in additional models, signal the need for clinical studies to determine the potential beneficial effects of MR blockade in human HCM.

Figures

Figure 1

Aldosterone levels in HCM. Serum (A) and myocardial (B) aldosterone levels in patient with HCM (n=8) and controls (n=11). C, Relative expression levels of CYP11B2 mRNA in HCM (n=8) compared with controls (n=4).

Figure 2

Hypertrophic effect of aldosterone. Relative expression levels of mRNAs for atrial natriuretic peptide (NPPA), brain natriuretic peptide (NPPB), and skeletal α-actin (ACTA1) at 24 and 48 hours after treatment of cardiac myocytes with 2 doses of aldosterone (12-hour data not shown). C indicates control myocytes; AT, angiotensin II; A25 and A50, myocytes treated with aldosterone 25 and 50 nmol/L, respectively; A+S, myocytes treated with aldosterone 50 nmol/L in presence of spironolactone 0.5 μmol/L. *P<0.05 vs C; ¶P<0.05 vs A50.

Figure 3

Profibrotic effect of aldosterone. Relative expression levels of mRNAs for 3 main cardiac collagen genes at 24 and 48 hours after treatment of cardiac fibroblasts with aldosterone are shown. Abbreviations and symbols as in Figure 2.

Figure 4

Induction of expression of TGF-β1 with aldosterone in cardiac fibroblasts. Top, Induction of expression of TGF-β1 after 30 and 60 minutes of treatment with aldosterone 50 nmol/L and its blockade with spironolactone. Bottom (blot), Expression levels of α-tubulin, control for protein loading conditions.

Figure 5

Molecular mediators of aldosterone signaling in cardiac myocytes and fibroblasts. A, Immunoblot showing expression levels of serine 916 and serine 744 to 748 phosphorylated (p) and total PKD in cardiac myocytes treated with aldosterone at 6 time points. B, Expression levels of phosphorylated (p) and total PKD proteins in cardiac fibroblasts treated with aldosterone. C, Expression levels of phosphorylated (p) and total PKD proteins in mice hearts treated with aldosterone. D, Expression levels of PI3K signaling molecules and PTEN (phosphatase and tensin homolog deleted on chromosome 10) in cultured cardiac fibroblasts treated with aldosterone at 6 different time points. E, Expression of PI3K-p110δ in cardiac myocytes (CM) and fibroblasts (CF) isolated from hearts of mice treated with aldosterone.

Figure 6

Inhibition of cardiac hypertrophic and profibrotic responses of aldosterone. Top panels represent relative expression levels of 3 cardiac hypertrophic markers, as described in Figure 2. C indicates control myocytes; AT, angiotensin II; A, aldosterone; and A+G100 and A+G500, myocytes treated with aldosterone and Go6976 100 or 500 nmol/L, respectively. Bottom panels show inhibition of induction of expression of 3 major cardiac collagen genes in cardiac fibroblasts. Abbreviations are as above, except CFs were used and W50 and W100 denote fibroblasts treated with aldosterone and wortmannin 50 and 100 nmol/L, respectively. *P<0.05 vs C; ¶P<0.05 vs aldosterone.

Figure 7

Molecular basis for myocyte disarray and its attenuation with spironolactone. A, Confocal micrographs showing distribution of N-cadherin in hearts of nontransgenic (NTG) mice and cTnT-Q92 mice treated either with placebo or spironolactone. Lower panels show hematoxylin-and-eosin–stained thin sections of myocardium in experimental groups. B, Immunoblots depicting expression levels of serine 37-phosphorylated β-catenin in NTG, placebo, and spironolactone groups (upper panel); middle panel depicts expression levels of N-cadherin in experimental groups; and lower panel represents expression levels of sarcomeric actin, control for protein loading control. C, Representative immunoblot showing expression levels of β-catenin after immunoprecipitation with anti–N-cadherin antibody. IP indicates intraperitoneal.