Hypoosmotic stress as a modulator of the NLRP3 inflammasome in adult cardiomyocytes

PEREYRA, ERICA; RACIOPPI, MARÍA FLORENCIA; RANDO, MONICA; GONANO, LUIS ALBERTO; PETROFF, MARTÍN VILA

Congreso; Reunión de International Society for Heart Research (ISHR) Latin America; 2022

In most cells, hypoosmotic-induced swelling is followed by a process called “regulatory volume decrease” (RVD) that depends on chloride (Cl-) efflux (ICl swell). Although it is not completely clear if adult cardiomyocytes can regulate their volume by RVD, this response does appear when Cl- efflux is increased experimentally with low Cl- hypotonic solutions. The NLRP3 inflammasome is responsible for inflammatory responses by releasing IL-1ß and IL-18 that can damage the heart. Osmotic stress and Cl- efflux are known NLRP3 activators in non-cardiac cells. However, if swelling activates NLRP3 in cardiac myocytes has not been studied.We aimed to determine if hypoosmotic stress can activate NLRP3 in adult cardiomyocytes and if this activation is associated with the RVD process, in particular with the activation of ICl swell.Ventricular cardiomyocytes were obtained from 3 months old male Wistar rats by enzymatic digestion. To assess swelling and RVD, cells were perfused with isotonic (300 mOsm) or either normal or low Cl- hypotonic solutions (HS; 217 mOsm). Cell width was determined by video edge-detection. RVD was absent in normal Cl- HS but detected in myocytes perfused with a low Cl- HS solution. Cardiomyocytes were incubated for 2 hours with 1µg/ml bacterial LPS to induce NLRP3 priming and then exposed to either isotonic, normal or low Cl- HS for 2 hours. As a marker of NLRP3 activation, IL-1ß concentrations in supernatants were quantified by ELISA. No significant difference was detected in IL-1ß levels when cells were exposed to isosmotic or normal Cl- HS, but a significant increase was found when cells were exposed to low Cl- HS (p=0,028, n=5 per group). This increase was significantly attenuated by treating myocytes with the anion channel inhibitor SITS (100 µM), which inhibits Cl- efflux. Statistical analysis was performed by ANOVA or T Test followed by post hoc-test. Data are expressed as means ± SEM. Differences were considered significant at p ≤ 0.05. Our results suggest that a Cl- mediated RVD response is necessary for NLRP3 inflammasome activation in adult cardiomyocytes under hypoosmotic stress.