Aldosterone Impairs Mitochondrial Function in Human Cardiac Fibroblasts via A-Kinase Anchor Protein 12

Abstract

Aldosterone (Aldo) contributes to mitochondrial dysfunction and cardiac oxidative stress. Using a proteomic approach, A-kinase anchor protein (AKAP)-12 has been identified as a down-regulated protein by Aldo in human cardiac fibroblasts. We aim to characterize whether AKAP-12 down-regulation could be a deleterious mechanism which induces mitochondrial dysfunction and oxidative stress in cardiac cells. Aldo down-regulated AKAP-12 via its mineralocorticoid receptor, increased oxidative stress and induced mitochondrial dysfunction characterized by decreased mitochondrial-DNA and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expressions in human cardiac fibroblasts. CRISPR/Cas9-mediated knock-down of AKAP-12 produced similar deleterious effects in human cardiac fibroblasts. CRISPR/Cas9-mediated activation of AKAP-12 blunted Aldo effects on mitochondrial dysfunction and oxidative stress in human cardiac fibroblasts. In Aldo-salt-treated rats, cardiac AKAP-12, mitochondrial-DNA and PGC-1α expressions were decreased and paralleled increased oxidative stress. In myocardial biopsies from patients with aortic stenosis (AS, n = 26), AKAP-12, mitochondrial-DNA and PGC-1α expressions were decreased as compared to Controls (n = 13). Circulating Aldo levels inversely correlated with cardiac AKAP-12. PGC-1α positively associated with AKAP-12 and with mitochondrial-DNA. Aldo decreased AKAP-12 expression, impairing mitochondrial biogenesis and increasing cardiac oxidative stress. AKAP-12 down-regulation triggered by Aldo may represent an important event in the development of mitochondrial dysfunction and cardiac oxidative stress.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Aldo modulates AKAP-12 and regulates oxidative stress in adult human cardiac fibroblasts. Aldo effects on AKAP-12 protein expression in human cardiac fibroblasts (A,B). Total mtDNA expression was measured in human cardiac fibroblasts (C). Oxidative markers (D,E) and mitochondrial function markers (F) expressed as a fold change relative to controls in lysate cells from Aldo-treated human cardiac fibroblasts. All conditions were performed at least in triplicate. Histogram bars represent the mean ± SEM of 6 assays. For Western blot experiments, the blots were cropped, protein densitometry was expressed in arbitrary units (AU) once normalized to β-actin. *p < 0.05 vs Control. AKAP-12, A-kinase anchoring protein 12; Aldo, Aldosterone; Spiro, Spironolactone; Total mtDNA, Total mitochondrial DNA; TAC, Total antioxidant capacity; CML, carboxy-methyl-lysine; MDA, malondialdehyde; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator 1 alpha; PHB, prohibitin.

Figure 2

AKAP-12 inhibition modifies mitochondrial function and oxidant status in adult human cardiac fibroblasts. AKAP-12 protein (A) expression was measured in AKAP-12-knock-down human cardiac fibroblast. Total mtDNA expression was quantified by RT-PCR (B). Effects of AKAP-12 knock-down on oxidative markers (C,D). Effects of AKAP-12 knock-down on PGC-1α and PHB protein expressions (E). Histogram bars represent the mean ± SEM of 6 assays. For Western blot experiments, the blots were cropped, protein densitometry was expressed in arbitrary units (AU) once normalized to β-actin. For RT-PCR experiments, data was normalized to HPRT and β-actin for cDNA. *p < 0.05 vs Scramble. AKAP-12, A-kinase anchoring protein 12; Total mtDNA, Total mitochondrial DNA; TAC, Total antioxidant capacity; CML, carboxy-methyl-lysine; MDA, malondialdehyde; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator 1 alpha; PHB, prohibitin.

Figure 3

AKAP-12 activation prevented Aldo effects on mitochondrial function and oxidative stress parameters in adult human cardiac fibroblasts. AKAP-12 protein (A) expression was measured in human cardiac fibroblasts over-expressing AKAP-12. Total mtDNA expression was quantified by RT-PCR (B). Effects of AKAP-12 over-expression on peroxide production (C) and oxidative stress markers (D). Effects of AKAP-12 activation on PGC-1α and PHB protein expressions (E). Histogram bars represent the mean ± SEM of 6 assays. For Western blot experiments the blots were cropped, protein densitometry was expressed in arbitrary units (AU) once normalized to β-actin. For RT-PCR experiments, data was normalized to HPRT and β-actin for cDNA. *p < 0.05 vs Scramble. $p < 0.05 vs Aldo. AKAP-12, A-kinase anchoring protein 12; Total mtDNA, Total mitochondrial DNA; TAC, Total antioxidant capacity; CML, carboxy-methyl-lysine; MDA, malondialdehyde; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator 1 alpha; PHB, prohibitin.

Figure 4

AKAP-12, mitochondrial function and oxidative stress markers in hearts from Aldosterone-salt-treated rats. AKAP-12 immunostaining and protein expression (A,B) in myocardium from Controls, Aldo-salt-treated rats and Aldo-salt + Spiro-treated rats. Total mtDNA expression was measured (C). TAC and peroxide production (D). Oxidative stress markers (E). PGC-1α and PHB expressions (F). PGC-1α immunostaining (G). Magnification of the microphotographs 40x. Histogram bars represent the mean ± SEM of each group of subjects. For Western blot experiments, the blots were cropped, protein densitometry was expressed in arbitrary units (AU) once normalized to β-actin. *p < 0.05 vs Control; $p < 0.05 vs Aldo. AKAP-12, A-kinase anchoring protein 12; Aldo, Aldosterone; Spiro, Spironolactone; Total mtDNA, Total mitochondrial DNA; TAC, Total antioxidant capacity; CML, carboxy-methyl-lysine; MDA, malondialdehyde; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator 1 alpha; PHB, prohibitin.

Figure 5

AKAP-12, mitochondrial function and oxidative stress markers in myocardial biopsies from aortic stenosis patients. AKAP-12 immunostaining and AKAP-12 mRNA levels in myocardial biopsies from AS patients (A,B). Total mtDNA expression was measured (C). PGC-1α and PHB mRNA levels (D) and PGC-1α immunostaining (E) in myocardial biopsies. Immunostaining of Nitrotyrosine and CML (E). Magnification of the microphotographs 40x. Histogram bars represent the mean ± SEM of each group of subjects (Control n = 13 and patients with AS, n = 26) in arbitrary units (AU) normalized to HPRT and β-actin for cDNA. *p < 0.05 vs. control group. AS, aortic stenosis; AKAP-12, A-kinase anchoring protein 12; Total mtDNA, Total mitochondrial DNA; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator 1 alpha; PHB, prohibitin; CML, carboxy-methyl-lysine.

Figure 6

Correlation studies in aortic stenosis patients. AKAP-12 protein expression negatively correlated with serum Aldo (A) and positively correlated with PGC-1α mRNA levels (B) in AS patients. Total mtDNA levels positively correlated with PGC-1α mRNA levels in the whole population (C). AKAP-12, A-kinase anchoring protein 12; Aldo, Aldosterone; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator 1 alpha.