Inducing Energetic Switching Using Klotho Improves Vascular Smooth Muscle Cell Phenotype

Abstract

The cardiovascular disease of atherosclerosis is characterised by aged vascular smooth muscle cells and compromised cell survival. Analysis of human and murine plaques highlights markers of DNA damage such as p53, Ataxia telangiectasia mutated (ATM), and defects in mitochondrial oxidative metabolism as significant observations. The antiageing protein Klotho could prolong VSMC survival in the atherosclerotic plaque and delay the consequences of plaque rupture by improving VSMC phenotype to delay heart attacks and stroke. Comparing wild-type VSMCs from an ApoE model of atherosclerosis with a flox'd Pink1 knockout of inducible mitochondrial dysfunction we show WT Pink1 is essential for normal cell viability, while Klotho mediates energetic switching which may preserve cell survival.

Methods: Wild-type ApoE VSMCs were screened to identify potential drug candidates that could improve longevity without inducing cytotoxicity. The central regulator of cell metabolism AMP Kinase was used as a readout of energy homeostasis. Functional energetic switching between oxidative and glycolytic metabolism was assessed using XF24 technology. Live cell imaging was then used as a functional readout for the WT drug response, compared with Pink1 (phosphatase-and-tensin-homolog (PTEN)-induced kinase-1) knockout cells.

Results: Candidate drugs were assessed to induce pACC, pAMPK, and pLKB1 before selecting Klotho for its improved ability to perform energetic switching. Klotho mediated an inverse dose-dependent effect and was able to switch between oxidative and glycolytic metabolism. Klotho mediated improved glycolytic energetics in wild-type cells which were not present in Pink1 knockout cells that model mitochondrial dysfunction. Klotho improved WT cell survival and migration, increasing proliferation and decreasing necrosis independent of effects on apoptosis.

Conclusions: Klotho plays an important role in VSMC energetics which requires Pink1 to mediate energetic switching between oxidative and glycolytic metabolism. Klotho improved VSMC phenotype and, if targeted to the plaque early in the disease, could be a useful strategy to delay the effects of plaque ageing and improve VSMC survival.

Keywords: glycolysis; mitochondrial ATP generation; mitochondrial dysfunction; oxidative metabolism; vascular smooth muscle cells.

Figure 1

Cytotoxicity to screen acute and chronic dose-response. A screen of potential energetic switching compounds was performed. Both chronic (24 h) and acute (2 h) dosing regimens were tested in wild-type cells and rate of XTT conversion was used as an index of both mitochondrial health and viability. Survival was compared with no drug carrier controls: (A) A769662; (B) berberine; (C) troglitazone; (D) etoposide; (E) losartan; (F) AICAR; (G) resveratrol; (H) salicylate; (I) metformin; (J) trimetazidine; (K) Klotho; (L) confocal immunofluorescent imaging of the AMPK downstream marker acetyl–CoA carboxylase in explant VSMCs that are α-SMA positive (inset secondary antibody control) was used as a recognised surrogate marker of AMPK activity. (Scale 100 µM) and confirm VSMC status One-way ANOVA with Dunnett’s post hoc test (n = 4), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 2

(A–D) Representative Western blot and quantification of pACC ser79, pAMPKα, pAMPKβ1, and LKB1 normalised to β-tubulin after a selection of drug treatments: protein ladder (L), no treatment control (C), blank X, metformin (M), A769662 (A7), resveratrol (R); (E) Western blots were quantified using LICOR technology (one-way ANOVA with Dunnett’s post hoc test (n = 4) ** p ≤ 0.01 vs. control. ** p ≤ 0.001 vs. control.

Figure 3

Plaque cell and tissue energetic analysis: (A) wild-type (Pink+/+) VSMC show decreased oxygen consumption rate (OCR) in response to Klotho and (B) concomitant energetic switching by upregulation of glycolysis (ECAR) in presence of Klotho; (C) no significant difference in survival between WT and Klotho-treated VSMCs; (D) rates of proliferation by completed mitosis events; (E) apoptosis rate over 24 h; (F) necrosis rate over 24 h; two-way ANOVA with Bonferroni’s post hoc test (n = 3–8), * p ≤ 0.05, *** p ≤0.001.

Figure 4

Live-cell comparison of wild-type (WT) and Pink1-KO VSMC: (A) wild-type survival, compared with Pink1-KO VSMC; (B) comparison of cell proliferation; (C) rates of apoptosis over 24 h; (D) rates of necrosis over 24 h. Two-way ANOVA with Bonferroni’s post hoc test (n = 3). * p ≤ 0.05, *** p ≤ 0.001.

Figure 5

Pink-1 KO and Klotho energetic performance with live-cell analysis: (A) oxygen metabolism in Pink1-KO with Klotho; (B) total change in OCR; (C) Pink1-KO glycolytic metabolism with Klotho; (D) total change in ECAR; (E) Pink1-KO cell survival over 24 h in presence of Klotho; (F) Klotho-inhibited proliferation of Pink1-KO cells; (G) reduced apoptosis; (H) enhanced necrosis over 24 h. Two-way ANOVA with Bonferroni’s post hoc test (n = 3). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.