Thrombin receptor PAR4 regulates the NLRP3 inflammasome/GRP78 signaling tandem in obese atria

Background:

Obese patients are highly prone to atrial cardiomyopathy, which provides the critical substrate for the development of atrial fibrillation (AF). We recently identified a functional connection between the NLRP3 inflammasome and the ER stress chaperone GRP78 in this context. Druggable upstream regulators of this signaling tandem are not known. One candidate is protease-activated receptor PAR4, which is upregulated in obesity and mediates coagulation-independent inflammatory signaling. Atrial fibroblasts are an emerging nexus for NLRP3 inflammasome activity in AF, an interaction with PAR4 and GRP78 especially in obesity is not known.

Methods:

Right atrial appendages (RAA) were collected from obese and non-obese patients undergoing cardiac surgery. Human primary atrial fibroblasts were stimulated with the PAR4 agonist peptide AYPGKF-NH2. Pooled left and right atria were isolated from wildtype and PAR4-/- mice fed chow or high fat diet (HFD, 16 weeks). Tissues, cell lysates and supernatants were assayed by qPCR, immunoblot and ELISA.

Results:

Obese human atria showed higher abundance of activated NLRP3 inflammasome-associated caspase-1, interleukin (IL)-1β, PAR4 and GRP78, compared to non-obese atria. Other markers of a triggered ER stress response (HSP70, ATG7, cleaved ATF6, spliced XBP1) did not significantly differ. Obese atria also displayed higher phosphorylation of the master regulator mTOR and more fibrotic markers. In human atrial fibroblasts, PAR4 activation increased mTOR phosphorylation and the transcriptional upregulation and secretion of GRP78 and IL-1β. Other ER stress mediators were not regulated. Atria of HFD vs. chow mice contained more GRP78, but not other ER stress markers, together with increased caspase-1, IL-1β, phospho-mTOR and Acta2. All of these signals, with the unexpected exception of GRP78, were abrogated in HFD-fed PAR4-/- mouse atria.

Conclusion:

Upregulated atrial abundance of the thrombin receptor PAR4 in obesity is causally linked with mTOR/NLRP3 inflammasome activation and an increased expression and mobilization of the ER stress chaperone GRP78. At least in part this convergence of multiple signaling platforms occurs at the level of atrial fibroblasts and likely supports fibrotic remodeling, independently of a full ER stress response. The emerging PAR4 antagonists provide new therapeutic perspective to modify obesity-associated atrial cardiomyopathy.