Interleukin 11 therapy causes acute left ventricular dysfunction

Abstract

Aims: Interleukin 11 (IL11) was initially thought important for platelet production, which led to recombinant IL11 being developed as a drug to treat thrombocytopenia. IL11 was later found to be redundant for haematopoiesis, and its use in patients is associated with unexplained and severe cardiac side effects. Here, we aim to identify, for the first time, direct cardiomyocyte toxicities associated with IL11, which was previously believed cardioprotective.

Methods and results: We injected recombinant mouse lL11 (rmIL11) into mice and studied its molecular effects in the heart using immunoblotting, qRT-PCR, bulk RNA-seq, single nuclei RNA-seq (snRNA-seq), and assay for transposase-accessible chromatin with sequencing (ATAC-seq). The physiological impact of IL11 was assessed by echocardiography in vivo and using cardiomyocyte contractility assays in vitro. To determine the activity of IL11 specifically in cardiomyocytes, we made two cardiomyocyte-specific Il11ra1 knockout (CMKO) mouse models using either AAV9-mediated and Tnnt2-restricted (vCMKO) or Myh6 (m6CMKO) Cre expression and an Il11ra1 floxed mouse strain. In pharmacologic studies, we studied the effects of JAK/STAT inhibition on rmIL11-induced cardiac toxicities. Injection of rmIL11 caused acute and dose-dependent impairment of left ventricular ejection fraction (saline: 62.4% ± 1.9; rmIL11: 32.6% ± 2.9, P < 0.001, n = 5). Following rmIL11 injection, myocardial STAT3 and JNK phosphorylation were increased and bulk RNA-seq revealed up-regulation of pro-inflammatory pathways (TNFα, NFκB, and JAK/STAT) and perturbed calcium handling. snRNA-seq showed rmIL11-induced expression of stress factors (Ankrd1, Ankrd23, Xirp2), activator protein-1 (AP-1) transcription factor genes, and Nppb in the cardiomyocyte compartment. Following rmIL11 injection, ATAC-seq identified the Ankrd1 and Nppb genes and loci enriched for stress-responsive, AP-1 transcription factor binding sites. Cardiomyocyte-specific effects were examined in vCMKO and m6CMKO mice, which were both protected from rmIL11-induced left ventricular impairment and molecular pathobiologies. In mechanistic studies, inhibition of JAK/STAT signalling with either ruxolitinib or tofacitinib prevented rmIL11-induced cardiac dysfunction.

Conclusions: Injection of IL11 directly activates IL11RA/JAK/STAT3 in cardiomyocytes to cause acute heart failure. Our data overturn the earlier assumption that IL11 is cardioprotective and explain the serious cardiac side effects associated with IL11 therapy.

Keywords: Cardiotoxicity; Fibrosis; Heart failure; Inflammation; Interleukin 11; JAK/STAT.

Graphical Abstract

Figure 1

IL11 causes acute left ventricular dysfunction and impairs cardiomyocyte calcium handling. Male C57BL/6J mice were injected with rmIL11 (200 µg/kg) (▪), rmIL6 (200 µg/kg) (▴), or an equivalent volume of saline (2 µL/kg) (●). (A) Representative electrocardiogram traces were recorded under light anaesthesia, 2 h after intraperitoneal (ip) injection of saline, rmIL11, or rmIL6. (B) Quantification of heart rate (n = 5 per group). (C) Representative m-mode images from echocardiography performed 2 h after injection of saline, rmIL11, or rmIL6. (D) Quantification of left ventricular ejection fraction (LVEF), (E) global circumferential strain (GCS), and (F) velocity time integral at the aortic arch (VTI) in each group (n = 5 per group). (G) LVEF 2 h after ip injection of rmIL11 to male mice at 0, 5, 10, 25, 50, 100, and 200 µg/kg (n = 5 per dose). (H) LVEF at baseline, 1, 2, 4, 6, and 24 h and 7 days after ip injection of rmIL11 (200 µg/kg) (n = 4 per time point). (I) Western blot of myocardial lysates from C57BL/6J male mice 0.5, 3, 6, and 24 h after ip rmIL11 injection (200 µg/kg). Blots are probed for pSTAT3, total STAT3, pERK, total ERK, pJNK, total JNK, and GAPDH. CMs isolated from male C57BL/6J mice were treated in vitro for 2 h with media supplemented with rmIL11 (10 ng/mL) or non-supplemented media (Cntrl) (n = 3 mice, 20 cells per mouse) and assessed for (J) contractility (effective n = 9.7) and (K) the systolic change of intracellular calcium concentration (effective n = 12). Statistics: one-way ANOVA with Sidak’s multiple comparisons test. Significance denoted as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. CM data: two-level hierarchical clustering P-values denoted as ***<0.001.

Figure 2

Transcriptional changes in the myocardium following rmIL11 injection. Volcano plot of all detected genes (A) 1 h (n = 3) and (B) 3 h (n = 4) after intraperitoneal injection of rmIL11 at 200 µg/kg. Vertical red lines are drawn at Log2FC of 1 and −1 and horizontal red lines at FDR of 0.05. (C) Chart of most significantly enriched KEGG terms from at 1 h post-injection of rmIL11 ranked by FDR. (D) Gene set enrichment analysis of the most highly enriched Hallmark gene sets from RNA-seq data at 1 h after injection of rmIL11 ranked by normalized enrichment score.

Figure 3

Single nuclear RNA sequencing reveals an IL11-induced cardiomyocyte stress signature. (A) Uniform Manifold Approximation and Projection (UMAP) embedding of all cell types from the left ventricle of male C57BL/6J mice 3 h after intraperitoneal injection of rmIL11 (200 µg/kg) or an equivalent volume of saline (n = 5). (B) Comparison of cellular composition of the left ventricle in rmIL11-treated mice compared to saline-treated mice. (C) UMAP embedding of cardiomyocyte fraction. Four distinct clusters are identified based on gene expression. (D) UMAP embedding of cardiomyocytes annotated with the treatment group. (E) UMAP embedding of cardiomyocyte fraction of saline- or rmIL11-treated cardiomyocytes annotated with relative expression of Nppb and Ankrd1. EC, endothelial cells.

Figure 4

ATAC-Seq reveals a stress signature that occurs acutely in the myocardium after rmIL11 injection. (A) Number of positively and negatively enriched genomic regions identified by ATAC-Seq analysis of the myocardium 3 h after injection of rmIL11 (n = 4). (B) Top 20 most strongly enriched DNA regions in ATAC-seq analysis and adjacent genes, when present (gene–chromosome). (C) Top 20 most strongly negatively enriched DNA regions in ATAC-seq analysis and adjacent genes (gene–chromosome). (D) De novo Homer motif analysis of ATAC-seq data most highly enriched motifs in myocardial samples. (E) Heatmap of AP-1 transcription factor family members from bulk RNA sequencing data of myocardium at baseline, 1, 3, and 6 h after rmIL11 injection. Genes differentially expressed in cardiomyocytes in single nuclear RNA sequencing data are marked with * and highlighted in red.

Figure 5

Viral-mediated Il11ra1 deletion in adult cardiomyocytes protects against IL11-driven cardiac dysfunction. (A) Schematic of experimental design for AAV9 mediated delivery of Tnnt2 promoter driven Cre-recombinase to male Il11ra1^fl/fl or Il11ra1^+/+ mice. (B) qPCR of relative myocardial expression of Il11ra1 in Il11ra1^+/+ or Il11ra1^fl/fl injected with AAV9-Cre or vehicle. (C) Western blot from myocardial lysate following rmIL11 injection (200 µg/kg) in Il11ra1^+/+ or Il11ra1^fl/fl treated with either AAV9-Cre or saline (n = 3). The membrane was probed with primary antibodies against GFP, pSTAT3, STAT3, and GAPDH. (D) Quantification of relative pSTAT3/STAT3 from (C). Echocardiographic assessment of vCMKO mice (▴) injected with rmIL11 (200 µg/kg) or saline were compared to WT mice (●) injected with rmIL11 (200 µg/kg) or saline. (E) Left ventricular ejection fraction, (F) global circumferential strain, (G) velocity time integral at the aortic arch, and (H) heart rate were measured 2 h after treatment (n = 3–5). (I) Contractility and (J) peak calcium amplitude in CMs isolated from vCMKO mice and treated for 2 h in vitro with rmIL11 containing media (10 ng/mL) or normal media. Statistics: one-way ANOVA with Sidak’s multiple comparisons testing. Significance denoted as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. CM data: two-level hierarchical clustering.

Figure 6

Germline deletion of Il11ra1 in cardiomyocytes prevents IL11-induced cardiac toxicities. (A) Breeding strategy to generate m6CMKO mice and litter-mate Il11ra1^fl/fl controls. (B) qPCR of Il11ra1 gene expression in Il11ra1^fl/fl controls and m6CMKO mice compared to male wild type C57BL/6J controls. (n = 4) (C) Western blot of phospho-STAT3 and total STAT3 signalling in male and female Il11ra1^fl/fl controls and m6CMKO mice with and without rmIL11 treatment. (D) Quantification of relative pSTAT and STAT3 expression. Male and female m6CMKO mice (CM Il11ra−) (▪) were treated with saline or rmIL11 and compared to wild type mice (CM Il11ra1+) (●) treated with saline or rmIL11 (n = 4). (E) LVEF, (F) GCS, (G) VTI in the aortic arch, and (H) heart rate were measured 2 h after rmIL11 injection. (n = 4). qPCR analysis of relative expression of (I) Nppb and (J) Fosl2 in the myocardium following rmIL11 treatment of m6CMKO mice and Il11ra1^fl/fl control mice (n = 3–4). Statistics: comparison between groups by two-way ANOVA with Sidak’s multiple comparisons. P-values denoted as *<0.05, **<0.01, ***<0.001, ****<0.0001.

Figure 7

The acute toxic effects of rmIl11 are mediated via JAK/STAT signalling. (A) Schematic of the pre-treatment of wild type male C57BL/6J mice with JAKi or vehicle 30 min before administration of rmIL11 or saline. (B) Western blot of myocardial lysate from mice 1 h after injection with saline or rmIL11 following pre-treatment with either ruxolitinib (30 mg/kg) (Ruxo) or vehicle (Veh). Membranes have been probed for pSTAT3, STAT3, and GAPDH (n = 3). Two hours after treatment, mice had an echocardiogram performed under isoflurane anaesthesia that measured (C) left ventricular ejection fraction, (D) global circumferential strain, (E) VTI in the aortic arch, and (F) heart rate (n = 4) in mice treated with a combination of vehicle (Veh), ruxolitinib (30 mg/kg) (Ruxo), or tofacitinib (20 mg/kg) (Tofa) and rmIL11 or saline. (G) qPCR of Nppb and (H) Fosl2 expression in myocardial tissue from combinations of ruxolitinib and rmIL11 treatments (n = 3–6). Statistics: comparison between groups by one-way ANOVA with Sidak’s multiple comparisons test. Significance denoted as *P < 0.05, **P < 0.01, ****P < 0.0001.