cGAS/STING/NLRP3 Signaling Pathway-Mediated Pyroptosis in Hypertrophic Cardiomyopathy Radiotherapy.

Abstract

Background: Radiotherapy is a commonly employed treatment modality for cancer; however, its radiobiological effects in hypertrophic cardiomyopathy (HCM) remain unclear. Radiation exposure activates the cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway, which is functionally associated with the activation of NOD-like Receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasomes, known mediators of pyroptotic cell death. Nonetheless, the underlying mechanism requires further investigation. Therefore, the objective of this study is to elucidate the role of the cGAS/STING/NLRP3 pathway in the process of cardiomyocyte pyroptosis during radiotherapy for HCM.

Methods: Transverse aortic constriction surgery was conducted to establish a mouse model of pressure overload-induced HCM, followed by the administration of 30 Gray (Gy) radiation one-week post-surgery. Cardiac morphology and function were evaluated through echocardiographic techniques. Hematoxylin & Eosin staining, along with Wheat Germ Agglutinin (WGA) staining, were utilized to quantify the cross-sectional area of cardiomyocytes and the degree of left ventricular hypertrophy. The HL-1 mouse cardiac muscle cell line was subjected to 40 Gy of radiation using an X-ray irradiator to establish an in vitro model of HCM, with or without the application of the NLRP3 inhibitor MCC950 and cGAS overexpression. Various assays, including the Cell Counting Kit-8 (CCK8), enzyme-linked immunosorbent assay (ELISA), and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimi- dazolylcarbocyanine iodide (JC-1) probe assays, were performed to assess cell viability, the concentrations of Interleukin (IL)-1β, IL-18, and cGAMP, as well as mitochondrial membrane potential. The morphology of cell membranes and mitochondria was analyzed using scanning electron microscopy (SEM) and fluorescence in situ hybridization (FISH) dual labelling techniques. The expression levels of cGAS, STING, and NLRP3 were evaluated through by western blot analysis.

Results: Radiotherapy reduced cardiac hypertrophy, improved cardiac function, and decreased fibrotic changes in HCM mice when compared to control groups. The application of radiation resulted in pyroptosis in HL-1 cells and a reduction in cell viability; this effect that was alleviated by the inhibition of NLRP3, while overexpression of cGAS exacerbated the situation. Furthermore, radiation led to a decline in mitochondrial membrane potential and the leakage of mitochondrial DNA into the cytoplasm, which activated the cGAS-STING signaling pathway, thereby initiating pyroptosis. This activation was corroborated by elevated levels of pyroptosis-associated proteins, including cGAS, STING, NLRP3, caspase-1, Gasdermin D (GSDMD), cGAMP, IL-18, and IL-1β. Notably, the inhibition of NLRP3 effectively abolished the upregulation of IL-18, and IL-1β levels.

Conclusion: Radiation can improve cardiac function, decrease hypertrophy of myocardial cells, and induce oxidative stress. This oxidative stress results in the leakage of mitochondrial DNA (mtDNA), which subsequently activates the cGAS/STING/NLRP3 signalling pathway, culminating in pyroptosis.