Cardiomyocyte NLRP3 Contributes to the Cardiac Inflammatory Response

Abstract

Despite extensive clinical and basic science research, cardiovascular diseases continue to be the leading cause of death worldwide. In response to pathological insults, the heart attempts to adapt and normalize cardiac function by initiating a compensatory response that drives wound healing and remodeling of the myocardium. The immune system and its inflammatory mediators are currently recognized as active participants in cardiac disease progression and the development of heart failure. We have previously demonstrated that the cardiomyocyte is the initial site of proinflammatory gene transcription following transverse aortic constriction (TAC) and that this occurs through signal transduction by activated Ca2+/Calmodulin-Dependent Kinase II (CaMKIIδ). Activation of cardiomyocyte CaMKIIδ transduces cardiac cellular stress signals into inflammatory gene expression via canonical NFκβ signaling and the NLRP3 inflammasome. The NLRP3 inflammasome is rapidly primed and activated in cardiomyocytes following TAC as indicated by caspase-1 activation and generation of proinflammatory cytokines IL-1β and IL-18. Interleukin-1β is regarded as a potent mediator of sterile inflammation often associated with neutrophilic infiltration, and its biological activity is tightly controlled by multiple levels of regulation including the requirement for proteolytic cleavage of the full-length transcript to its biologically active form by active caspase-1 recruited to the NLRP3 inflammasome complex. To investigate the role of the NLRP3 inflammasome in cardiomyocytes, we have generated mice with constitutive NLRP3 (designated as FCAS after the human mutation in familial cold autoinflammatory syndrome) expression in cardiomyocytes. This was achieved by administration of cardiomyocyte-specific adeno-virus associated virus serotype 9 (AAV9) expressing cre recombinase into mice floxed with the mutant FCAS gene. At the conclusion of 3 weeks after virus administration, a low dose of 2mg/kg lipopolysaccharide (LPS) was administered as a priming event for the transcription of inflammasome signaling components. At the conclusion of 6 hours after LPS injection, blood was collected for measurements of systemic inflammation, and whole hearts were collected for analysis NLRP3 priming (gene expression) and NLRP3 activation using qPCR and measuring caspase1 enzymatic activity. Inflammasome priming, as measured by an increase in Il-18 and caspase-1, were increased in FCAS LPS animals compared to wild type control LPS animals, although we did not observe differences in Il-1β or NLRP3 gene expression. Whole ventricular tissue from these mice were probed for activated NLRP3 via the measurement of caspase-1 enzymatic activity AAV9-Cre administered mice with floxed FCAS gene increased NLRP3 inflammasome activation compared to virus injection into wild type mice, but there were no measured differences between FCAS LPS and wild type control LPS mice. Blood from FCAS LPS mice had significantly increased serum Il-1β levels compared to control LPS mice suggesting that cardiomyocyte-derived inflammasome products can contribute to appreciable levels of inflammation.