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Thrombin supports coagulation-independent inflammation via protease-activated receptors (PAR). PAR4 is specifically increased in obese human atria, correlating with NLRP3 inflammasome activation. PAR4-mediated NLRP3 inflammasome activation in atrial cardiomyocytes is not known, nor have signaling partners been identified. Thrombin transactivates the hepatocyte growth factor receptor in some cancer cells, so we examined PAR4/c-met cross-talk in atrial cardiomyocytes and its possible significance in obesity. Cardiomyocytes from right atrial appendages (RAA) of obese patients expressed more PAR1 and PAR4 compared to non-obese. In HL-1 atrial cardiomyocytes, thrombin induced caspase-1 auto-activation and IL-1β maturation; IL-1β secretion was evoked by PAR4-activating peptide (AP), but not PAR1-AP. PAR4-AP additionally increased phosphorylated CaMKII-Thr287, mTOR-Ser2481, and Akt-Ser473 while suppressing AMPK-Thr172 phosphorylation. Total kinase levels were largely unaltered. PAR4AP rapidly increased phosphorylated c-met in HL-1 cells and over time also transcriptionally upregulated c-met. The c-met inhibitor SGX-523 abrogated the effects of PAR4-AP on CaMKII/AKT/mTOR phosphorylation but did not affect PAR4-stimulated IL-1β production. Obese human RAA contained more IL-1β, phospho-c-met, and phospho-mTOR than non-obese RAA; CamKII phosphorylation was not modified. Atria from high-fat diet (HFD) versus chow-fed mice also contained more IL-1β, together with higher myeloperoxidase activity, Acta2 mRNA total and phosphorylated c-met; these increases were blunted in PAR4 -/- HFD-fed mice. Thrombin cross-activates c-met via PAR4 in atrial cardiomyocytes. Transactivated c-met contributes partially to PAR4-mediated signaling, but NLRP3 inflammasome activation appears to be largely independent of c-met. Abundance of PAR4 and activated c-met increases with obesity, providing therapeutic targets for management of adiposity-driven AF., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Trained immunity of monocytes, endothelial, and smooth muscle cells augments the cytokine response to secondary stimuli. Immune training is characterized by stabilization of hypoxia-inducible factor (HIF)-1α, mTOR activation, and aerobic glycolysis. Cardiac fibroblast (CF)-myofibroblast transition upon myocardial ischemia/reperfusion (I/R) features epigenetic and metabolic adaptations reminiscent of trained immunity. We assessed the impact of I/R on characteristics of immune training in human CF and mouse myocardium. I/R was simulated in vitro with transient metabolic inhibition. CF primed with simulated I/R or control buffer were 5 days later re-stimulated with Pam3CSK for 24 h. Mice underwent transient left anterior descending artery occlusion or sham operation with reperfusion for up to 5 days. HIF-regulated metabolic targets and cytokines were assessed by qPCR, immunoblot, and ELISA and glucose consumption, lactate release, and lactate dehydrogenase (LDH) by chromogenic assay. Simulated I/R increased HIF-1α stabilization, mTOR phosphorylation, glucose consumption, lactate production, and transcription of PFKB3 and F2RL3, a HIF-regulated target gene, in human CF. PGK1 and LDH mRNAs were suppressed. Intracellular LDH transiently increased after simulated I/R, and extracellular LDH showed sustained elevation. I/R priming increased abundance of pro-caspase-1, auto-cleaved active caspase-1, and the expression and secretion of interleukin (IL)-1β, but did not augment Pam3CSK-stimulated cytokine transcription or secretion. Myocardial I/R in vivo increased abundance of HIF-1 and the precursor and cleaved forms of caspase-1, caspase-11, and caspase-8, but not of LDH-A or phospho-mTOR. I/R partially reproduces features of immune training in human CF, specifically HIF-1α stabilization, aerobic glycolysis, mTOR phosphorylation, and PFKB3 transcription. I/R does not augment PGK1 or LDH expression or the cytokine response to Pam3CSK. Regulation of PAR4 and inflammasome caspases likely occurs independently of an immune training repertoire., (© 2024. The Author(s).)

Thrombin inhibition suppresses adiposity, WAT inflammation and metabolic dysfunction in mice. Protease-activated receptor (PAR)1 does not account for thrombin-driven obesity, so we explored the culprit role of PAR4 in this context. Male WT and PAR-4 -/- mice received a high fat diet (HFD) for 8 weeks, WT controls received standard chow. Body fat was quantified by NMR. Epididymal WAT was assessed by histology, immunohistochemistry, qPCR and lipase activity assay. 3T3-L1 preadipocytes were differentiated ± thrombin, acutely stimulated ± PAR4 activating peptide (AP) and assessed by immunoblot, qPCR and U937 monocyte adhesion. Epicardial adipose tissue (EAT) from obese and lean patients was assessed by immunoblot. PAR4 was upregulated in mouse WAT under HFD. PAR4 -/- mice developed less visceral adiposity and glucose intolerance under HFD, featuring smaller adipocytes, fewer macrophages and lower expression of adipogenic (leptin, PPARγ) and pro-inflammatory genes (CCL2, IL-1β) in WAT. HFD-modified activity and expression of lipases or perilipin were unaffected by PAR4 deletion. 3T3-L1 adipocytes differentiated with thrombin retained Ki67 expression, further upregulated IL-1β and CCL2 and were more adhesive for monocytes. In mature adipocytes, PAR4-AP increased phosphorylated ERK1/2 and AKT, upregulated Ki67, CCl2, IL-β and hyaluronan synthase 1 but not TNF-α mRNA, and augmented hyaluronidase-sensitive monocyte adhesion. Obese human EAT expressed more PAR4, CD68 and CD54 than lean EAT. PAR4 upregulated in obesity supports adipocyte hypertrophy, WAT expansion and thrombo-inflammation. The emerging PAR4 antagonists provide a therapeutic perspective in this context beyond their canonical antiplatelet action., (© 2024. The Author(s).)

Activators of protease-activated receptors PAR-1 and PAR-2 such as thrombin and synthetic hexapeptides promote hypertrophy of isolated neonatal cardiomyocytes at pathological concentrations. Since PAR-activating proteases often show dual actions at low vs. high concentrations, the potential hypertrophic effects of low-level PAR activation were examined. In H9c2 cardiomyoblasts, messenger RNA (mRNA) expression of the hypertrophic marker atrial natriuretic peptide (ANP) was significantly increased only by higher concentrations of thrombin, trypsin or the synthetic PAR-2 agonist SLIGRL. The dual PAR-1/PAR-2 agonist SFLLRN did not influence basal ANP mRNA expression in H9c2 cells. Low concentration of thrombin or trypsin (up to 0.1 U/mL) or of the synthetic ligands SFLLRN and SLIGRL (1 μM); however, all suppressed ANP mRNA expression stimulated by angiotensin II (Ang II). The PAR-1 selective ligand TFLLRN exerted a comparable effect as SFLLRN. In adult rat cardiomyocytes, protein synthesis determined by [(3)H]phenylalanine incorporation was not increased by various PAR agonists at concentrations tenfold lower than conventionally used to study PAR function in vitro (10 μM for SFLLRN or SLIGRL, 0.1 U/mL for thrombin or trypsin). The positive control endothelin-1 (ET-1, 60 nM) however significantly increased protein synthesis in adult rat cardiomyocytes. Addition of low concentrations of PAR agonists to cardiomyocytes treated with ET-1 or Ang II suppressed [(3)H]phenylalanine incorporation induced by the hypertrophic stimuli. The inhibitory effect of SFLLRN effect was partially reversed by the PAR-1 antagonist RWJ56110. These findings suggest that physiological concentrations of PAR activators may suppress hypertrophy, in contrast to the pro-hypertrophic effects evident at high concentrations. PAR-1 and PAR-2 may dynamically control cardiomyocyte growth, with the net effect critically dependent upon local agonist concentrations. The precise significance of proposed concept of bimodal PAR function in cardiomyocytes remains to be defined, particularly in vivo where hemodynamic and other regulatory factors may counteract or mask the direct cellular actions described here.