CX3CR1 is a prerequisite for the development of cardiac hypertrophy and left ventricular dysfunction in mice upon transverse aortic constriction

Abstract

The CX3CL1/CX3CR1 axis mediates recruitment and extravasation of CX3CR1-expressing subsets of leukocytes and plays a pivotal role in the inflammation-driven pathology of cardiovascular disease. The cardiac immune response differs depending on the underlying causes. This suggests that for the development of successful immunomodulatory therapy in heart failure due to chronic pressure overload induced left ventricular (LV) hypertrophy, the underlying immune patterns must be examined. Here, the authors demonstrate that Fraktalkine-receptor CX3CR1 is a prerequisite for the development of cardiac hypertrophy and left ventricular dysfunction in a mouse model of transverse aortic constriction (TAC). The comparison of C57BL/6 mice with CX3CR1 deficient mice displayed reduced LV hypertrophy and preserved cardiac function in response to pressure overload in mice lacking CX3CR1. Moreover, the normal immune response following TAC induced pressure overload which is dominated by Ly6Clow macrophages changed to an early pro-inflammatory immune response driven by neutrophils, Ly6Chigh macrophages and altered cytokine expression pattern in CX3CR1 deficient mice. In this early inflammatory phase of LV hypertrophy Ly6Chigh monocytes infiltrated the heart in response to a C-C chemokine ligand 2 burst. CX3CR1 expression impacts the immune response in the development of LV hypertrophy and its absence has clear cardioprotective effects. Hence, suppression of CX3CR1 may be an important immunomodulatory therapeutic target to ameliorate pressure-overload induced heart failure.

Fig 1. Cx3cr1^GFP/GFP mice exhibit a cardioprotective phenotype in response to chronic pressure overload.

Using pressure volume measurement, the cardiac function of Cx3cr1^GFP/GFP mice and Wt mice was evaluated 21 days after TAC or sham operation. (A, B) Ejection fraction and cardiac output are depicted as plots, more information is provided in Table 1. N = 6–8 mice/group; * above individual columns indicate significant differences between TAC and respective sham group; *P <0.05, **P<0.01, *** P<0.001.

Fig 2. CX3CR1 expression modulates the development of LV hypertrophy in response to pressure overload.

(A) Heart-Weight/Body-Weight-Index of Wt and Cx3cr1^GFP/GFP mice calculated 3, 6 and 21 days after transverse aortic constriction (TAC) and sham operation; n = 7–9 mice/group. (B) Aldolase mRNA expression pattern in LV tissue determined 3 and 6 days after TAC and sham operation in Wt and Cx3cr1^GFP/GFP mice; n = 6–14 mice/group. (C) BNP mRNA expression pattern in LV tissue determined 3 and 6 days after TAC and sham operation in Wt and Cx3cr1^GFP/GFP mice; n = 6–14 mice/group. (D) Representative sections of the left ventricle stained with sirious red of Wt and Cx3cr1^GFP/GFP mice 21 days after TAC or Sham operation to determine cardiomyocyte size. Dashed lines highlight exemplary individual cardiomyocytes; scale bar indicating 50μm. Quantification of cardiomyocyte size by area planimetry; n = 5–6 mice/group, 4 slides per mouse were randomly analyzed by manual cell count of at least 50 cells/slide and mean was used for statistical analysis. * above individual columns indicate significant differences between TAC and respective sham group; **P<0.01, *** P<0.001, ****p < 0.0001.

Fig 3. CX3CR1 deficiency drives the immune response to a pro-inflammatory phenotype during chronic pressure overload.

Quantification of Ly6C^low F4/80⁺ macrophages in the spleen and of (B) Ly6C^low and Ly6C^high CD115⁺ monocytes in the blood of Wt and Cx3cr1^GFP/GFP mice determined by flow cytometry 3 and 6 days after TAC and sham ioperation. (C-E) Number of neutrophils, Ly6C^low and Ly6C^high macrophages in the LV tissue was determined by flow cytometry analysis in Cx3cr1^GFP/GFP and Wt mice 3 and 6 days after TAC and sham operation. The particular cell subsets were defined as follows: neutrophils as CD45⁺ F4/80^- Ly6G^+, macrophages as CD45⁺ F4/80⁺ Ly6G^-; Ly6C^high and Ly6C^low macrophages were further discriminated according to the respective Ly6C surface expression; (F-I) Concentrations of the pro-inflammatory cytokines IL6 and IL-1β, the chemokine MCP1 and IL-10 were determined in LV tissue homogenates at day 3 and 6 after surgical intervention in Cx3cr1^GFP/GFP and Wt mice using ELISA assays. (J-M) BrdU content of cardiac Ly6C^high and Ly6C^low macrophages was determined by flow cytometry analysis at day 3 after TAC. BrdU was administered 18h before analysis. Histograms are based on concetenated plots of 4 mice. N = 6–8 mice/group; * above individual columns indicate significant differences between TAC and respective sham group; *P <0.05, **P<0.01, *** P<0.001, ****p < 0.0001.