BS03 Interleukin-11: pro-inflammatory, profibrotic and cardiotoxic – a new therapeutic target in heart failure

Introduction Fibrosis and inflammation are key pathophysiological factors in heart failure (HF) however no specific therapies exist which target these pathways. The interleukin-11 (IL11) cytokine was initially described as anti-inflammatory, cardioprotective and important for platelet production. However, while recombinant IL11 is used to treat thrombocytopenia it causes serious but unexplained cardiac side-effects, including HF. Serum IL11 is elevated in HF patients and murine HF models however the effect of IL11 on myocardial inflammation and cardiomyocyte function has not been previously investigated. Method Two transgenic mouse lines with a C57BL/6J background were created using the Cre-LoxP system. A mouse line with tamoxifen-inducible expression of IL11 in CMs (CM-IL11-Tg) was generated using Myh6 driven Cre-recombinase. CM IL11 expression was induced at 8 weeks by tamoxifen injection. An adeno-associated-virus-9 virus containing Tnnt2 driven Cre-recombinase was injected to wildtype mice or mice with the IL11 receptor flanked by loxP sites to generate controls and CM-specific IL11 receptor knockout (CM-IL11R-KO) mice respectively. 8-week-old CM-IL11R-KO and control mice were injected intraperitoneally with IL11 (recombinant mouse IL11, 200 mcg/kg). Cardiac function was assessed by echocardiography and myocardial samples were taken at various timepoints for for western blot, immunohistochemistry, RNAseq, ATACseq and single-nuclei RNAseq. CMs were isolated from wildtype and CM-IL11R-KO animals using a retrograde perfusion method and incubated with IL11 (10ng/mL) or standard media for 2 hours. Cells were paced at 1Hz and analysis was performed using a high throughput, cytocypher microscope, and calcium-sensitive (Fura2a) dye. Results Chronic transgenic CM expression of IL11 induced proinflammatory transcriptional responses by RNAseq analysis including upregulation of NF-κB, TNF-α and interferon-γ pathways [figure 1A]. This was associated with extensive perivascular fibrosis, inflammatory cell infiltration [figure 1B] and LV dysfunction (Ejection fraction: Cntrl, 63%±1.3; CM IL11 expression, 49%±2.7; p=0.005). Acutely after IL11 injection, single-nuclei RNAseq revealed a CM stress signature characterised by Nppb and Ankrd1 expression. JNK phosphorylation and expression of AP-1 transcription factors increased leading to AP-1 becoming the dominant transcriptional response to IL11 in the myocardium in ATACseq analysis. The L-type calcium channel inhibitor, Rrad, is a transcriptional target of AP-1 and was upregulated in the heart within 1 hour of IL11 injection. Following incubation with IL11 (10ng/mL, 2hrs), isolated CMs showed reduced systolic calcium influx (Cntrl, 1.00±0.34; IL11, 0.78±0.30; p=0.0002) and impaired CM contractility (Cntrl, 1.00±0.071; IL11: 0.67±0.47; p=0.0003) [figure 2B]. Consistent with these findings, LV function was impaired in vivo 2 hours after IL11 injection (Ejection fraction: Saline, 60%±3.1; IL11, 32%±2.0; p=<0.0001). [figure 2A]. CM-IL11R-KO prevented IL11 induced CM stress phenotype and improved calcium influx and contractile function. Conclusion IL11 causes acute and direct CM stress and dysfunction and chronically induces cardiac inflammation, fibrosis and failure. These insights explain the side-effects seen following IL11 injection that were previously thought indirect and idiosyncratic. Considering the proinflammatory, pro-fibrotic and directly negatively inotropic effects of IL11, anti-IL11 therapy may represent a holistic treatment option in HF. Conflict of Interest None