SIRT1 directly regulates myofibroblastic activation, extracellular-matrix remodeling, and calcification in aortic valve interstitial cells

Background and Aims: Calcific aortic valve stenosis (CAVS) is one of the most common valve disorders. The well-known deacetylase SIRT1 could be involved in many pathways linked to CAVS development and progression. We aim to unveil if SIRT1 could represent a novel important player in the homeostasis of aortic valve leaflets. Methods: RNA-sequencing of whole aortic valves or valve interstitial cells were used to identify the main regulated pathways linked to CAVS. SIRT1 knockdown (SIRT1-) and overexpressing (SIRT1+) immortalized valve interstitial cells (iVIC) were generated by CRISPR/Cas9 technology. Fibrosis was evaluated by quantitative PCR and immunofluorescence of well-known genes. A calcification assay was employed to evaluate the capability of mutant iVIC to produce calcium deposits. Results: Whole tissue RNA-sequencing showed that many pathways which are linked to SIRT1 are modulated in CAVS specimens. Functional analysis of SIRT1 mutant iVICs revealed that inflammation, extracellular-matrix remodeling, and calcification mechanisms are significantly regulated. Validations showed that fibrosis-associated genes were down-regulated in SIRT1+ iVICs compared to wild-type ones (COL1A1 log2FC=-1.0±0.4; TGFB2 log2FC=-0.9±0.1; FN1 log2FC=-0.4±0.1; ACTA2 log2FC=-1.0±0.1; BGN log2FC=-1.0±0.08; TAGLN log2FC=-0.4±0.04; all p<0.001) as well as αSMA protein (-52%, p<0.0001), while SIRT1- iVICs showed a significant up-regulation of COL1A1 protein (+50.5% p<0.0001). Calcium assays unveiled a significant increment of calcification in SIRT1- iVICs (+50.8%, p<0.01), while SIRT1+ iVICs deposited less calcium (-23.6%; p<0.01) compared to wild-type ones. Conclusions: Our data show that SIRT1 could represent a novel master regulator of fibro-calcific processes involved in CAVS progression.