Abstract 109: Regulation of TNF-α Signaling in Cardiomyocytes via a Novel Interaction Between TNF-α Receptor and RASSF1A

Abstract

Tumour necrosis factor-α (TNF-α) is a pro-inflammatory cytokine that plays key roles in the pathogenesis of heart failure. Cardiomyocytes express the TNF-α receptor (TNFR), however, the mechanism of TNF-α signal transmission in cardiomyocytes is not completely understood. Recent studies showed that in cancer cells TNFR is regulated by Ras-association domain family 1 isoformA (RASSF1A). Therefore, we investigated whether RASSF1A modulates TNF-α signalling in cardiomyocytes. We used RASSF1A knockout (KO) mice and wild type (WT) controls and stimulated them with TNF-α (10µg/kg i.v.). In WT mice acute treatment with low dose of TNF-α increased cardiac contractility as indicated by the change in end systolic elastance (Ees) (baseline Ees (mmHg/μl), 3.3±0.5; Ees after TNF-α stimulation, 5.7±0.8, P<0.05) which is consistent with previously published data (Circulation 2004; 109:406–411). However, KO mice showed a blunted contractile response following acute TNF-α treatment (baseline Ees: 3.05±0.4 vs Ees after TNF-α: 2.54±0.3). Consistently, isolated cardiomyocytes from WT mice showed 40% increase in calcium transient amplitude in response to TNF-α (10 ng/ml) stimulation (n=24 cells, p<0.05). However, KO cardiomyocytes showed no significant increase in calcium transient amplitude following the same stimulation (n=24 cells). We also found that RASSF1A formed a molecular complex with TNF-α receptor in cardiomyocytes and this interaction was essential in the recruitment of TRADD and TRAF2, the major downstream effectors of TNF-α signalling. By mapping the interaction domain we found that the C-terminal region of RASSF1A was responsible for the formation of TNF-α receptor complex. Furthermore, using an adenoviral-mediated shRNA construct we found that cardiomyocytes lacking RASSF1A exhibited reduced activation of NFkB, a downstream target of TNF-α. Overall, our data indicate an essential role of RASSF1A in regulating TNF-α signalling in cardiomyocytes, with RASSF1A being key in the formation of TNF receptor complex and in the signal transmission to the downstream targets.