Activation of cGMP-PKG signaling pathway attenuates aortic valve calcification through ULK1-mediated autophagy

Abstract Background and Purpose Calcific aortic valve disease (CAVD) is a prevalent cardiovascular disorder associated with high morbidity and mortality in developed countries. Currently, no pharmacotherapy has proven effective in halting disease progression, leaving aortic valve replacement as the only available treatment. A recent pilot study demonstrated the therapeutic potential of soluble guanylate cyclase (sGC) activation in patients with moderate aortic valve stenosis; however, the mechanisms by which sGC exerts its anti-calcific effects remain unclear. Given that the cyclic guanosine monophosphate (cGMP)-dependent protein kinase G (PKG) pathway is a classical downstream mediator of sGC, its role in CAVD is yet to be elucidated. This study focuses on the contribution of the cGMP-PKG signaling pathway to the osteogenic differentiation of human valvular interstitial cells (hVICs). Methods cGMP and PKG expression in aortic valve tissues and serum from CAVD patients were characterized using enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunofluorescence. PKG knockout mice were subjected to aortic wire valve injury (AVWI) to induce CAVD and subsequently treated with the sGC activator. In vitro, hVICs were cultured under osteogenic medium (OM) conditions and treated with the sGC activator in the presence of a PKG inhibitor or following PKG knockdown via small interfering RNA (siRNA). Osteogenic marker expression and calcium nodule formation were evaluated by Western blotting, Alizarin Red staining, and alkaline phosphatase (ALP) staining. Further exploration of the downstream mechanisms and regulators was performed using RNA sequencing, tandem mass tag (TMT)-based quantitative proteomics, and phosphoproteomics. Additionally, the interaction between PKG and downstream regulators was confirmed by co-immunoprecipitation followed by mass spectrometry analysis. Results Our results reveal that the cGMP-PKG pathway is deactivated in CAVD patients, as evidenced by decreased serum cGMP levels and reduced PKG expression in aortic valve tissues. Treatment with an sGC activator alleviated calcification both in vivo and ex vivo. Notably, inhibition or knockdown of PKG abrogated the anti-calcific effects of sGC activation in both the AVWI mouse model and in OM-induced calcification of hVICs. Mechanistically, PKG directly phosphorylates unc-51–like autophagy activating kinase 1 (ULK1) and regulates ULK1-mediated autophagy. Furthermore, inhibition of ULK1, either by siRNA or a selective inhibitor, abolished the anti-calcific effects conferred by PKG activation. Conclusion Our study demonstrates that activation of the cGMP-PKG signaling pathway is crucial for preventing aortic valve calcification. PKG promotes autophagy by phosphorylating ULK1, thereby mitigating the osteogenic phenotype of hVICs. These findings elucidate the molecular mechanisms underlying the anti-calcific effects of sGC agonists and suggest a potential therapeutic strategy for CAVD.