Metformin ameliorates valve interstitial cell calcification by promoting autophagic flux

Abstract

Calcific aortic valve disease (CAVD) is the most common heart disease of the developed world. It has previously been established that metformin administration reduces arterial calcification via autophagy; however, whether metformin directly regulates CAVD has yet to be elucidated. In the present study we investigated whether metformin alleviates valvular calcification through the autophagy-mediated recycling of Runx2. Calcification was reduced in rat valve interstitial cells (RVICs) by metformin treatment (0.5-1.5 mM) (P < 0.01), with a marked decrease in Runx2 protein expression compared to control cells (P < 0.05). Additionally, upregulated expression of Atg3 and Atg7 (key proteins required for autophagosome formation), was observed following metformin treatment (1 mM). Blocking autophagic flux using Bafilomycin-A1 revealed colocalisation of Runx2 with LC3 puncta in metformin treated RVICs (P < 0.001). Comparable Runx2 accumulation was seen in LC3 positive autolysosomes present within cells that had been treated with both metformin and hydroxychloroquine in combination (P < 0.001). Mechanistic studies employing three-way co-immunoprecipitation with Runx2, p62 and LC3 suggested that Runx2 binds to LC3-II upon metformin treatment in VICs. Together these studies suggest that the utilisation of metformin may represent a novel strategy for the treatment of CAVD.

Figure 1

Metformin inhibits calcification in RVICs and reduces the induction of RUNX-2 and bone sialoprotein expression. (A) Calcium content (mg/mg protein) of cells treated with metformin (Met) (0.5–1.0 mM), (n = 3). (B) Alizarin red staining and its quantification in cells treated with metformin (1 mM), (n = 3). (C) mRNA expression of Runx2 in calcified RVICs cultured in presence/absence of metformin (1 mM) for 48 and 72 h. (n = 3). (D) Representative western blots and (E, F) western blot quantification showing the effect of metformin treatment (1 mM) on the protein levels of Runx2 and bone sialoprotein compared to β-actin (n = 4). Data shown as mean + / − S.E.M *P < 0.05; **P < 0.01; ***P < 0.001 compared to control. Full-length blots are presented in Supplementary Fig. 2.

Figure 2

Metformin induces autophagy in calcified RVICs. RVICs were cultured in the presence/absence of calcium (2.5 mM) and phosphate (2.7 mM Pi) with 0.3% DMSO for 72 h in the presence/absence of metformin (Met) (1mM) and/or 5nM Bafilomycin-A1 (Baf-A) or 10 μM Hydroxychloroquine (HCQ). (A) Representative western blots and (B–E) western blot quantification showing the effect of metformin (1 mM) on Atg7, Beclin-1, Atg3 and p62 expression compared with β-actin (n = 4). (F, G) Representative confocal images showing the effect of Bafilomycin-A (Baf-A; 5 nM) and Hydroxychloroquine (10 μM) and/or metformin treatment on LC3 expression (n = 3; scale bar = 10 μm) with (H, I) quantification of LC3 puncta. Data shown as mean + / − S.E.M *P < 0.05, ***P < 0.001, ****P < 0.0001 compared to control. Full-length blots are presented in Supplementary Fig. 2.

Figure 3

Treatment of calcified RVICs with metformin leads to colocalisation of Runx2 with LC3 in autophagosomes. RVICs were cultured with calcium (2.5 mM) and phosphate (2.7mM Pi) with 0.3% DMSO for 72 h in the presence/absence of 1mM metformin (Met) and/or 5nM Bafilomycin-A1 (Baf-A) or 10μM Hydroxychloroquine (HCQ). (A) Representative confocal images showing Runx2 and LC3 staining in Baf-A cells (B) Representative confocal images showing Runx2 and LC3 staining in HCQ treated cells. (C) Reduced expression of Runx2 in the nucleus of the calcified RVICs treated with metformin (quantified on nuclei from lanes 1 and 2). (D) Colocalisation of Runx2 with LC3-II puncta in both Baf-A and HCQ treated cells. Data shown as mean + / − S.E.M ***P < 0.001 compared to control.

Figure 4

Treatment of calcified VICs with metformin leads to Runx2 degradation via autophagy and the ubiquitin proteasome pathway. RVICs were cultured with calcium (2.5mM) and phosphate (2.7 mM Pi) with 0.3% DMSO for 72 h in the presence/absence of 1mM metformin (Met) and/or 5nM Bafilomycin-A1 (Baf-A) or 50nM MG132. (A) Representative western blots and (B) western blot quantification showing LC3-II expression compared with β-actin (n = 3), MG132 was used as a negative control (C) Representative western blots and (D) western blot quantification showing Runx2 expression compared with β-actin (n = 3), MG132 was used to block the ubiquitin proteasome pathway. Data shown as mean + / − S.E.M *P < 0.05; ***P < 0.001; ****P < 0.0001 compared to control. Full-length blots are presented in Supplementary Fig. 2.

Figure 5

Metformin induces LC3-II mediated sequestering of Runx2 in calcified RVICs autophagosomes/autolysosomes. RVICs were cultured with calcium (2.5 mM) and phosphate (2.7 mM Pi) with 0.3% DMSO for 72 h in the presence/absence of 1 mM metformin (Met) and/or 5nM Bafilomycin-A1 (Baf-A). Representative western blots for Runx2, p62 and LC3 expression with (A) Runx2 co-immuno precipitate (IP) lysate (B) p62 co-IP lysate and (C) LC3 co-IP lysate. Beads alone were used as a negative control (n = 2). (D) Input blots for the IP. (E–H) Quantification of ratios of Co-IP Runx2: IP p62, Co-IP Runx2: IP LC3-II, Co-IP p62: IP Runx2 and Co-IP p62: IP LC3-II. Data shown as mean + / − S.E.M *P < 0.05. Full-length blots are presented in Supplementary Fig. 2.

Figure 6

Proposed mechanism of action for metformin in CAVD. Metformin is proposed to decrease calcification in RVICs through the selective binding of Runx2 with mature autophagosome marker LC3-II and the autophagy adaptor p62.