Early activation of the cardiac CX3CL1/CX3CR1 axis delays β-adrenergic-induced heart failure

Abstract

We recently highlighted a novel potential protective paracrine role of cardiac myeloid CD11b/c cells improving resistance of adult hypertrophied cardiomyocytes to oxidative stress and potentially delaying evolution towards heart failure (HF) in response to early β-adrenergic stimulation. Here we characterized macrophages (Mφ) in hearts early infused with isoproterenol as compared to control and failing hearts and evaluated the role of upregulated CX3CL1 in cardiac remodeling. Flow cytometry, immunohistology and Mφ-depletion experiments evidenced a transient increase in Mφ number in isoproterenol-infused hearts, proportional to early concentric hypertrophy (ECH) remodeling and limiting HF. Combining transcriptomic and secretomic approaches we characterized Mφ-enriched CD45⁺ cells from ECH hearts as CX3CL1- and TNFα-secreting cells. In-vivo experiments, using intramyocardial injection in ECH hearts of either Cx3cl1 or Cx3cr1 siRNA, or Cx3cr1^-/- knockout mice, identified the CX3CL1/CX3CR1 axis as a protective pathway delaying transition to HF. In-vitro results showed that CX3CL1 not only enhanced ECH Mφ proliferation and expansion but also supported adult cardiomyocyte hypertrophy via a synergistic action with TNFα. Our data underscore the in-vivo transient protective role of the CX3CL1/CX3CR1 axis in ECH remodeling and suggest the participation of CX3CL1-secreting Mφ and their crosstalk with CX3CR1-expressing cardiomyocytes to delay HF.

Figure 1

An increase in CD64⁺ Mφ is correlated with concentric hypertrophy in iso-induced ECH hearts. Mice implanted or not with an iso-pump for 14 or 28 days were subjected to echocardiographic analyses. (A) Schematic representation of the protocol. (B) Heart weight/tibia length; n = 8 (Ct), 11 (ECH) and 7 (HF) mice, Kruskal–Wallis followed by Dunn’s post-hoc test; *** p < 0.001. (C) Echocardiographic parameters; n = 8 (Ct), 11 (ECH) and 7 (HF) mice, Kruskal–Wallis followed by Dunn’s post-hoc test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (D) From same mice, evolution of the geometric parameter h/r (diastolic wall thickness to radius ratio) and heart weight. (E) Mice implanted with an iso-pump for 28 days: Spearman correlation coefficient between h/r at day 14 and FS at day 28; n = 13 from 2 different protocols. (F) Cardiac cells isolated from mice implanted or not with an iso-pump for 14 or 28 days were analyzed by flow cytometry. Cells were isolated from collagenase digested hearts, stained with the indicated antibodies and submitted to typical flow cytometry gating strategy to identify cardiac CD64⁺ Mφ. CD45⁺ leukocytes were identified, doublets excluded (by FSC-W vs. SSCA). Live CD45⁺ cells (after PI exclusion) were gated on CD64⁺ Mφ. Quantification of immune cells; n = 8 (Ct), 7 (ECH) and 13 (HF) cell isolation, Kruskal–Wallis followed by Dunn’s post-hoc test; ** p < 0.01. (G) Spearman correlation coefficient between cardiac CD64⁺ (flow cytometry analysis) cell number/mg and h/r; n = 6 mice (Ct), n = 5 mice (ECH).

Figure 2

Mφ-enriched adherent CD45⁺ cells from ECH hearts selectively trigger adult WT cardiomyocyte hypertrophy in-vitro. Mφ depletion during ECH hampers early hypertrophy in-vivo. (A) Experimental procedure. Cardiac immune cells were isolated from collagenase digested Ct, ECH and HF hearts. Following CD45 positive enrichment using mouse CD45 microbeads, adherent (Mφ-enriched) cells were kept in culture for 18 h before conditioned media (Cmed) recovery. Cmed were applied on cardiomyocytes from WT mice. Cell hypertrophy was analyzed 18 h later. (B) Cmed from adherent CD45⁺ isolated from iso-infused ECH WT mice selectively enhance hypertrophy of control WT cardiomyocytes. Mean ± SEM of 5 experiments performed in triplicate. Cardiomyocytes from 5 mice (225–750 cell area quantified per condition per experiment), Cmed from 4–10 mice, Kruskal–Wallis followed by Dunn post-hoc test; * p < 0.05, **** p < 0.0001. (C) Experimental procedure of clodronate or control liposomes treatment in mice implanted with an iso-pump. (D) Echocardiographic parameters measured at d0, d9, d14, d21 and d25; n = 4–9 mice/group, two-way ANOVA followed by Sidak’s post-tests; * p < 0.05 vs control at the same time point. (E) Survival of control or clodronate liposomes treated mice; n = 9 mice/group at d0, Gehan-Breslow-Wilcoxon Test.

Figure 3

Cardiac ECH CD64⁺ Mφ are characterized by a typical induction of Cx3cl1 mRNA and a co-secretion of CX3CL1 and TNFα, as compared to Ct and HF counterparts. (A) RNAseq transcriptomic analysis: three-way Venn diagrams. (B) Volcano Plots showing the number of genes differentially expressed between ECH and Ct or ECH and HF CD64⁺ Mφ. (C) Analysis of Cx3cl1 mRNA levels in isolated cardiac CD64⁺ cells by RNAseq (n = 4 mice/group, normalization and differential analysis were performed with the glm edgeR package) and qPCR (n = 6 (Ct), 5 (ECH) and 5 (HF) mice, Kruskal–Wallis followed by Dunn post-hoc test; * p < 0.05, ** p < 0.01). (D) Typical immuno-fluorescent stainings of cardiac sections from iso-infused ECH mice with anti-TNFα, CX3CL1, CD68 or CX3CR1 Abs. (E) Multiplex CX3CL1 and TNFα analysis in Cmed from adherent Mφ-enriched CD45⁺ cells prepared as described in Fig. 2A; n = 5 (Ct), 8 (ECH) and 5 (HF) mice, Kruskal–Wallis followed by Dunn’s post-hoc test; * p < 0.05, ** p < 0.01.

Figure 4

Determinant role of the CX3Cl1/CX3CR1 axis in the pro-hypertrophic effect of ECH Mφ-enriched CD45⁺ cells in adult WT cardiomyocytes: synergism between CX3CL1 and TNFα. (A) CX3CR1 protein expression in WT heart tissue (15 µg) or isolated adult WT cardiomyocytes (50 µg). Full unedited gels are provided in the supplementary material as well as detailed procedure (Figure S10). (B) Cmed from adherent CD45⁺ cells isolated from ECH WT mice enhances hypertrophy of WT cardiomyocytes in a CX3CL1-dependent manner. Experimental procedure described in Fig. 2A. (C) CX3CL1 requires the synergistic action of TNFα to enhance hypertrophy in control WT cardiomyocytes. (D) The pro-hypertrophic impact of CX3CL1 + TNFα relies on TNFR1 binding of TNFα. (E) Either alone or in combination, CX3CL1 and TNFα are without hypertrophic effect on adult cardiomyocytes from control Cx3cr1^-/- mice. Mean ± SEM of 2–6 experiments performed in triplicate. Cardiomyocytes from 2–6 mice (225–750 cell area quantified per condition per experiment), Cmed from 4–10 mice, Kruskal–Wallis followed by Dunn post-hoc test; * p < 0.05, ** p < 0.01 vs Ct medium.

Figure 5

Cx3cr1 knockout hampers the transient selective increase in CD64⁺ CCR2^- Ly6C^low MHCII^low Mφ detected in WT mice during ECH, suppresses iso-induced concentric hypertrophy and accelerates dilation and alteration of function. (A) Experimental procedure (B) Typical flow cytometry gating strategy to identify cardiac Mφ subpopulations. Cells were isolated from collagenase digested hearts and stained with the indicated antibodies before analysis. CD45⁺ leukocytes were identified, doublets excluded (by FSC-W vs. SSCA). Live CD45⁺ cells (after PI exclusion) were gated on CD11b⁺ CD64⁺ Mφ. CD11b⁺ CD64⁺ Mφ were further analyzed on their pattern of expression of CCR2 vs Ly6C and CCR2^- Ly6C^low cells were gated on MHCII^high and MHCII^low cells. Quantification of immune cells n = 4–7 cell isolation, two-way ANOVA followed by Sidak’s post-hoc tests. * p < 0.05 vs WT at the same time point. (C) Kinetics of echocardiography parameters in WT or Cx3cr1^-/- mice implanted with Iso pump at day 0. n = 14–38 mice, Two-way ANOVA followed by Sidak’s post-hoc tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs WT at the same time point. (D) Geometric parameter h/r versus HW n = 7–26 mice/group. (E) Cardiomyocyte cross-sectional area estimated in cardiac sections stained with WGA (typical image). Mean ± SEM from 6 mice/group, sacrificed at day14. Mann–Whitney U test. ** p < 0.001.

Figure 6

Cardiac CX3CL1 or CX3CR1 knockdown at the onset of ECH, via intramyocardial siRNA injection, reverses iso-induced concentric hypertrophy and favors alteration of function and dilation. (A,D) Schematic representation of the protocol where mice implanted with an iso-pump at day 0 were subjected to a unique ultrasound-guided intramyocardial transthoracic injection of Scramble, Cx3cl1 or Cx3cr1 siRNAs at day 7. (B,E) Echocardiographic parameters measured at d0, d7, d12, d15, d22 and d28 concerning siScramble or siCx3cl1 injections; n = 5–13 mice/group. Measurement at d0, d7, d12 and d15 for siScramble or siCx3cr1 injections; n = 10–14 mice/group, two-way ANOVA followed by Sidak’s post-hoc tests; * p < 0.05, ** p < 0.01 vs scramble at the same time point. (C,F) Efficient knockdown of CX3CL1 or CX3CR1 protein levels in cardiac homogenates at d12, d15 and d28 following siRNA injection. Full unedited gels are in the supplementary material as well as detailed procedure (Figure S12 and S13). Mean ± SEM of mice, n = 4–6 mice/group, Kruskal–Wallis followed by Dunn’s post-hoc tests; * p < 0.05, ** p < 0.01.

Figure 7

Protective role of the cardiac CX3CL1/CX3CR1 axis during β-adrenergic-induced early concentric hypertrophy remodeling: participation of a novel crosstalk between cardiac Mφ and cardiomyocytes. Early β-adrenergic stimulation activates the cardiac CX3CL1/CX3CR1 axis that supports early transient concentric remodeling and delays evolution towards heart failure: this is associated with a CX3CL1/CX3CR1-dependent expansion of cardiac Mφ. CX3CL1 and TNF-α secreted by cardiac Mφ synergistically trigger cardiomyocyte hypertrophy.