Modulation of Rxrα Expression in Mononuclear Phagocytes Impacts on Cardiac Remodeling after Ischemia-Reperfusion Injury

Figures

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  Figure 1. Cx3cr1Cre-ert2-YFPRxrαflox/flox mice show reduced expression of Rxrα in blood monocytes, which has no impact on PB parameters. (A) Study design (created with BioRender.com); (B) Rxrα wildtype allele and Rxrα L- allele expression in PB and different tissues of Cx3cr1ΔRxrα and control mice assessed by PCR and gel electrophoresis; (C) comparison of blood monocyte Rxrα expression between Cx3cr1ΔRxrα and control mice; (D) analysis of different blood parameters in Cx3cr1ΔRxrα and control mice. Arb: arbitrary; BM: bone marrow; CCR2: C-C chemokine receptor type 2; control (+/+): control mice [littermates with a floxed Rxrα allele but without Cre expression]; Cx3cr1ΔRxrα (Cre/+): mice with a floxed Rxrα allele and Cre expression; CyK: cytokine; ELP: electrophoresis; FACS: Fluorescence activated cell sorting; I/R injury: Ischemia/Reperfusion injury; mac: macrophage; mo: monocyte; PB: peripheral blood; PET: positron emission tomography; Rxr: Retinoid X receptor; qPCR: real-time polymerase chain reaction.

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  Figure 2. Reduced Rxrα expression in myeloid cells influences monocyte and macrophage subset composition. (A) FACS gating strategy to assess monocyte subsets in peripheral blood; (B) FACS gating strategy to analyze cardiac macrophage populations; (C) amount of Ly6chi monocytes in PB during steady state in Cx3cr1ΔRxrα compared to control mice; (D) proportions of Ly6chi monocytes in PB and MHCIIhi macrophages in the heart of Cx3cr1ΔRxrα in relation to control mice on d1 after I/R injury; (E) intrinsic YFP expression in Ly6chi versus Ly6clo monocytes of Cre/+ compared to control mice. Flowchart was created with BioRender.com. control (+/+): control mice [littermates with a floxed Rxrα allele but without Cre expression]; Cx3cr1ΔRxrα (Cre/+): mice with a floxed Rxrα allele and Cre expression; FACS: fluorescence activated cell sorting; FSC: forward scatter; hi: high; I/R injury: Ischemia/Reperfusion injury; lo: low; Lin: Lineage; macs: macrophages; Rxr: Retinoid X receptor; SS: Steady state; SSC: sideward scatter.

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  Figure 3. Reduction of myeloid Rxrα expression increases infarct size after I/R injury. (A) PET imaging to assess the cardiac defect on d6 and d30 after MI; (B) comparison of cardiac defect on d6 and d30 after MI between Cx3cr1ΔRxrα and control mice; (C) assessment of functional parameters (EDV and EF) on d6 and d30 after MI in Cx3cr1ΔRxrα and control mice. Flowchart was created with BioRender.com. control (+/+): control mice [littermates with a floxed Rxrα allele but without Cre expression]; Cx3cr1ΔRxrα (Cre/+): mice with a floxed Rxrα allele and Cre expression; EDV: end-diastolic volume; EF: ejection fraction; I/R injury: Ischemia/Reperfusion injury; PET: positron emission tomography; Rxr: Retinoid X receptor.

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  Figure 4. Reduction of Rxrα expression in myeloid cells impairs their phagocytic function and is associated with an increase in apoptotic cells after I/R injury. (A) FACS gating strategy to analyze the phagocytic function of PB monocytes; (B) comparison of the phagocytic activity of PB monocytes from Cx3cr1ΔRxrα and control mice; (C) co-staining of apoptotic cells using an ApopTag Plus Fluorescein In Situ Apoptosis Detection Kit and the nuclear counterstain Hoechst; (D) quantification of apoptotic cells per area in the heart of Cx3cr1ΔRxrα compared to control mice on d1 after MI. Flowchart was created with BioRender.com. control (+/+): control mice [littermates with a floxed Rxrα allele but without Cre expression]; Cx3cr1ΔRxrα (Cre/+): mice with a floxed Rxrα allele and Cre expression; FACS: fluorescence activated cell sorting; FSC: forward scatter; I/R injury: Ischemia/Reperfusion injury; mo: monocytes; phago.: phagocytic; Rxr: Retinoid X receptor; SSC: sideward scatter.

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  Figure 5. Decreased Rxrα expression is linked to a reduction in macrophage proliferation and vessel density in the heart after I/R injury. (A) IF staining to detect proliferating macrophages (F4/80: macrophage marker, Ki-67: proliferation marker, Hoechst: nuclear counterstain); (B) comparison of proliferating cardiac macrophages on d2 after MI between Cx3cr1ΔRxrα and control mice; (C) wheat Germ Agglutinin staining to detect the IZ using a WGA-Alexa Fluor™ 350 conjugated antibody; (D) visualization of cardiac vessel density using a CD31 antibody; (E) quantification of cardiac vessel density in the border zone on d30 after MI in Cx3cr1ΔRxrα compared to control mice. Flowchart was created with BioRender.com. BZ: border zone; control (+/+): control mice [littermates with a floxed Rxrα allele but without Cre expression]; Cx3cr1ΔRxrα (Cre/+): mice with a floxed Rxrα allele and Cre expression; IF: immunofluorescence microscopy; I/R injury: Ischemia/Reperfusion injury; IZ: infarct zone; mac: macrophages; MI: myocardial infarction; Rxr: Retinoid X receptor; WGA: Wheat Germ Agglutinin.

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  Figure 6. A Reduced expression of Rxrα in myeloid cells has no impact on cardiac cytokine and chemokine profiles on d1 after I/R injury. Cardiac cytokine and chemokine concentrations assessed by signal intensities using the Proteome ProfilerTM Mouse XL Cytokine Array Kit. Analytes are summarized in supplementary Table S2. Flowchart was created with BioRender.com. control (+/+): control mice [littermates with a floxed Rxrα allele but without Cre expression]; Cx3cr1ΔRxrα (Cre/+): mice with a floxed Rxrα allele and Cre expression; I/R injury: Ischemia/Reperfusion injury; Rxr: Retinoid X receptor.