Sirt1 in focus: unveiling molecular insights and therapeutic prospects in calcific aortic stenosis with sglt2i inhibitors
Authors: Valerio V , Massaiu I , Franchi M , Bonomi A , Pellegrini G , Moschetta D , Trombara F , Rusconi V , Bertolini F , Myasoedova V A , Marenzi G , Corrao G , Genovese S , Poggio P 
Published: May 2024

Abstract

Funding Acknowledgements
Type of funding sources: Foundation. Main funding source(s): Fondazione Gigi & Pupa Ferrari ONLUS

Background
Calcific aortic stenosis (AS) affects 3% of older adults and lacks medical treatment. The well-known deacetylase Sirtuin1 (SIRT1) could be involved in many pathways linked to AS progression. Sodium-glucose co-transporter 2 inhibitors (SGLT2i), a class of antihyperglycemic agents, have been shown to reduce cardiovascular events and hospitalization for heart failure (possibly via SIRT1), but their possible benefits in AS are not yet known.

Purpose
Our study aims to uncover if SIRT1 plays a key role in AS progression and if SGLT2i can potentially slow the valve fibro-calcification processes, potentially via SIRT1 modulation.

Methods
RNA sequencing of control (n=27) and AS (n=39) aortic valves was performed. SIRT1 knockdown (SIRT1-) and overexpressing (SIRT1+) valve interstitial cells (VIC) were generated by CRISPR/Cas9. A calcification assay with or without dapagliflozin (dapa 1uM as SGLT2i) was employed to evaluate valve endothelial cells (VEC) and VIC crass-talk on calcification potential. Patient data were collected from the Lombardy regional healthcare utilization database and diabetic patients who were taking sulphonylureas (sul; not affecting SIRT1) and SGLT2i (acting on SIRT1) were selected. Patients were matched 1:1 by age, sex, and multisource comorbidity score. Kaplan-Maier analysis was used to generate time-to-event curves for aortic valve replacement (AVR) cumulative incidence at 2 years and Cox regression analysis was used to account for confounding effects.

Results
Whole tissue RNA-seq showed that SIRT1 could be a master regulator of multiple pathways linked to AS. Functional analysis and validations on mutant VIC revealed that inflammation, extracellular-matrix remodeling, and calcification mechanisms are directly regulated by SIRT1. Calcium assays unveiled an increment of calcification in SIRT1- VICs (+50.8%, p&lt;0.01), while SIRT1+ VICs deposited less calcium (-23.6%; p&lt;0.01) compared to wild-type ones. VIC exposed to dapa-treated VEC medium showed reduced calcification (-35.2%, p&lt;0.01). In the dapa-treated VEC medium, we found an increase in NO levels (+19.9%, p&lt;0.01) and VIC treated with an NO donor (detanonate 50uM) showed reduced calcification (-23.2%, p&lt;0.05), while VIC treated with a SIRT1 inhibitor (EX527 25uM) showed an increased calcification (+68.1%, p&lt;0.01). To corroborate these findings, we retrospectively enrolled 233,546 diabetic patients, and after matching, we obtained two groups (sul and SGLT2i) with 37,081 patients each. Adjusted Cox regression analysis revealed that patients taking SGLT2i had a lower AVR incidence than patients taking sulp with a hazard ratio of 0.65 (95%CI 0.47-0.89) resulting in a 35% risk reduction.

Conclusion
Our data highlights SIRT1 as a key regulator of fibro-calcific processes involved in AS and SGLT2i exhibit potential in slowing AS progression, via SIRT1 modulation. This study unveils promising molecular insights and suggests SGLT2i as a potential therapeutic avenue for AS.