Deficiency in the anti‐aging gene Klotho promotes aortic valve fibrosis through AMPK α‐mediated activation of RUNX 2

Summary Fibrotic aortic valve disease ( FAVD ) is an important cause of aortic stenosis, yet currently there is no effective treatment for FAVD due to its unknown etiology. The purpose of this study was to investigate whether deficiency in the anti‐aging Klotho gene ( KL ) promotes high‐fat‐diet‐induced FAVD and to explore the underlying molecular mechanism. Heterozygous Klotho‐deficient ( KL +/− ) mice and WT littermates were fed with a high‐fat diet ( HFD ) or normal diet for 13 weeks, followed by treatment with the AMPK α activator ( AICAR ) for an additional 2 weeks. A HFD caused a greater increase in collagen levels in the aortic valves of KL +/− mice than of WT mice, indicating that Klotho deficiency promotes HFD ‐induced aortic valve fibrosis ( AVF ). AMPK α activity (p AMPK α) was decreased, while protein expression of collagen I and RUNX 2 was increased in the aortic valves of KL +/− mice fed with a HFD . Treatment with AICAR markedly attenuated HFD ‐induced AVF in KL +/− mice. AICAR not only abolished the downregulation of p AMPK α but also eliminated the upregulation of collagen I and RUNX 2 in the aortic valves of KL +/− mice fed with HFD . In cultured porcine aortic valve interstitial cells, Klotho‐deficient serum plus cholesterol increased RUNX 2 and collagen I protein expression, which were attenuated by activation of AMPK α by AICAR . Interestingly, silencing of RUNX 2 abolished the stimulatory effect of Klotho deficiency on cholesterol‐induced upregulation of matrix proteins, including collagen I and osteocalcin. In conclusion, Klotho gene deficiency promotes HFD ‐induced fibrosis in aortic valves, likely through the AMPK α– RUNX 2 pathway.