Selective expansion of cardiac macrophage subtypes distinguishes their functional roles in disease and homeostasis
Rajesh K. Kasam, Ronald J. Vagnozzi, Yasuhide Kuwabara, Anne Katrine Z. Johansen, N. Scott Blair, Vikram Prasad, Suh-Chin J. Lin, Akanksha Rajput, Michelle Nieman, Jeffery D. Molkentin
Rajesh K. Kasam, Ronald J. Vagnozzi, Yasuhide Kuwabara, Anne Katrine Z. Johansen, N. Scott Blair, Vikram Prasad, Suh-Chin J. Lin, Akanksha Rajput, Michelle Nieman, Jeffery D. Molkentin
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Research Article Cardiology Immunology

Selective expansion of cardiac macrophage subtypes distinguishes their functional roles in disease and homeostasis

Abstract

Cardiac macrophages are broadly studied as 2 subtypes, tissue-resident CX3C chemokine motif receptor 1 positive (CX3CR1+) that are also CC motif chemokine receptor 2 negative (CCR2–) and monocyte-derived CCR2+. Previous systemic loss-of-function approaches suggested unique roles for each subtype in the heart, with CCR2+ being inflammatory and CX3CR1+ being prohealing. Here, we employed a cardiac-specific gain-of-function approach to selectively enhance either macrophage subtype. A robust increase in basal CCR2+ macrophages in the heart by targeted CC chemokine ligand 2 (Ccl2) expression did not induce inflammation, cause fibroblast activation, or impair cardiac function. However, increased CCR2+ macrophages reciprocally diminished self-renewing tissue-resident macrophages and worsened cardiac fibrosis due to pressure overload stimulation. Conversely, augmented expression of colony-stimulating factor-1 (Csf1) in the heart promoted selective expansion of resident CX3CR1+ macrophages, which exerted no pathophysiological consequences at steady state. However, pressure overload in these mice with expanded CX3CR1+ macrophages showed a CCR2+ macrophage–dependent inflammation leading to exacerbated cardiac dysfunction, simultaneously protecting from adverse remodeling and cardiac fibrosis. In conclusion, cardiac-specific selective enrichment of macrophage subtypes shows their intricate interplay and unique functional roles in regulating myocardial inflammation and fibrosis during hypertrophy and at homeostasis.

Authors

Rajesh K. Kasam, Ronald J. Vagnozzi, Yasuhide Kuwabara, Anne Katrine Z. Johansen, N. Scott Blair, Vikram Prasad, Suh-Chin J. Lin, Akanksha Rajput, Michelle Nieman, Jeffery D. Molkentin

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Figure 6

Augmented CX3CR1+ macrophages exacerbate cardiac dysfunction without fibrosis after pressure overload.

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Augmented CX3CR1+ macrophages exacerbate cardiac dysfunction without fib...
(A) Experimental scheme showing MyoAAV-Csf1 vector with injection in adult reporter mice at 8 weeks of age followed by TAC or sham surgery 3 weeks later and then harvesting 8 weeks later. (B–E) Flow cytometry quantification of macrophage subtypes in hearts of these 2 groups of mice as (B) total CX3CR1+, (C) CX3CR1+MHC-IIlo, (D) CX3CR1+MHC-IIhi, and (E) CCR2+ macrophages. n = 4–5 mice per group, and error bars denote ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by 1-way ANOVA with Tukey’s multiple-comparison test. (F and G) Echocardiography in the indicated groups of mice 8 weeks after TAC to assess (F) fractional shortening percentage (FS%) and (G) left ventricular dimension in diastole. n = 11–13 mice per group, and error bars denote ± SEM. *P < 0.05, ****P < 0.0001, by 1-way ANOVA with Tukey’s multiple-comparison test. The control data without sham or TAC are from Figure 4, L and M. (H) Ventricular weight to body weight (VW/BW) ratio in the indicated 2 groups of mice 8 weeks after sham or TAC stimulation. n = 7–15 mice per group, and error bars denote ± SEM. ***P < 0.001, ****P < 0.0001, by 1-way ANOVA with Tukey’s multiple-comparison test. (I) Fibroblast quantification by flow cytometry from hearts of the 2 indicated groups of mice 8 weeks after sham and TAC. n = 4–5 mice per group. Error bars denote ± SEM. *P < 0.05, by 1-way ANOVA with Tukey’s multiple-comparison test. (J) Representative cardiac histology images with Masson’s trichrome staining for fibrosis (blue) in the 2 groups of mice 8 weeks post-TAC. Scale bar = 100 μm. (K) Fibrosis quantitation from Masson’s trichrome–stained cardiac histological sections in the 2 groups of mice 8 weeks after TAC stimulation. n = 9–10 mice per group, and error bars denote ± SEM. Two-tailed unpaired Student’s t test. (L–S) qRT-PCR analysis of the indicated genes from the hearts of MyoAAV-Empty– and MyoAAV-Csf1–injected mice 8 weeks after sham or TAC. n = 4–7 mice per group, and error bars denote ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by 1-way ANOVA with Tukey’s multiple-comparison test.