Therapeutics That Promote Sympathetic Reinnervation Modulate the Inflammatory Response After Myocardial Infarction

Abstract

Myocardial infarction (MI) triggers an inflammatory response that transitions from pro-inflammatory to reparative over time. Restoring sympathetic nerves in the heart after MI prevents arrhythmias. This study investigated if reinnervation altered the immune response after MI. This study used quantitative multiplex immunohistochemistry to identify the immune cells present in the heart 2 weeks after ischemia-reperfusion. Two therapeutics stimulated reinnervation, preventing arrhythmias and shifting the immune response from inflammatory to reparative, with fewer pro-inflammatory macrophages and more regulatory T cells and reparative macrophages. Treatments did not alter macrophage phenotype in vitro, which suggested reinnervation contributed to the altered immune response.

Keywords: ACh, acetylcholine; IP, intraperitoneal; ISP, intracellular sigma peptide; MI, myocardial infarction; NE, norepinephrine; PBS, phosphate-buffered saline; TH, tyrosine hydroxylase; Tregs, regulatory T cells; VEH, vehicle; inflammation; mIHC, multiplex immunohistochemistry; macrophages; multiplex IHC; myocardial infarction; sympathetic nervous system; β1-AR, adrenergic receptor.

Graphical abstract

Figure 1

Multiplex Immunohistochemistry (mIHC) Overview The multiplex immunohistochemistry (mIHC) process begins with multiple cycles of applying specific antibodies of interest to tissue sections, and scanning tissue sections after each stain. After the staining and scanning process, image gating cytometry is used to identify and classify immune cells. Finally, immune cell types are quantified.

Figure 2

ISP, HJ-01 and HJ-02 Injections Promote Sympathetic Reinnervation of the Infarct (A-D) Representative images of infarcted left ventricles from mice treated with (A) VEH, (B) HJ-01, (C) HJ-02, and (D) ISP 14 days after MI. Sections were stained for tyrosine hydroxylase (TH) to identify sympathetic nerve fibers and fibrinogen to identify the infarct. HJ-01, HJ-02, and ISP treatment resulted in extensive sympathetic reinnervation of the infarct. (E) Quantification of TH+ fiber density within the infarct 14 day post-MI (mean ± SD; n = 5/group; ∗∗P < 0.01; ∗∗∗P < 0.001; 2-way ANOVA with Tukey's multiple comparisons post-test). Dotted line denotes innervation density in sham animals.

Figure 3

Reinnervation Reduces Arrhythmia Susceptibility After MI (A to C) Representative electrocardiographic traces recorded in conscious ambulatory animals following (A) sham or (B and C) MI then treated with either (B) VEH or (C) HJ-02. Arrhythmias were induced by isoproterenol, and observed premature ventricular complexes (PVCs) are noted with asterisks. (D) Quantification of arrhythmias during the 45-minute period following isoproterenol injection in all groups. Data are mean ± SD, n = 5/group; ∗∗∗P < 0.001; 1-way ANOVA with Dunnett's multiple comparisons post-test. ANOVA = analysis of variance; ISO = isoproterenol; VEH = vehicle.

Figure 4

Representative mIHC Staining Used for Immune Cell Quantification (A) A cardiac section with CD45⁺ cells shown in yellow. To be quantified as an immune cell, cells must have stained positively for CD45. After CD45⁺ cells were identified, sequential antibody staining was used to phenotype immune cells. CD4⁺ T cells were identified with CD3⁺ and CD4⁺ staining and are shown in green in B. Following additional sequential staining, CD4⁺ cells were further analyzed into subpopulations. T regulatory cells (Tregs) were identified with the cell marker expression of CD45⁺CD3⁺CD4⁺RORɣT⁻FOXP3⁺GATA3⁻, and can be seen in purple in C. In addition to lymphoid lineage cells, we also detected CD3⁻ myeloid lineage cells as shown in red in D. Notice that none of the cells in the myeloid lineage (red) were positive for CD4 (lymphoid lineage). Abbreviation as in Figure 1.

Figure 5

Select Immune Cell Populations Are Impacted by Treatments Restoring Innervation (A) Quantification of immune cell populations from the hearts of mice treated with VEH, ISP, or HJ-02 are shown in (B to E). Quantification of immune cell populations are expressed as a percentage of total CD45+ cells. There was no difference in the percentage of B cells, granulocytes, CD8+ T cells, or CD4+ T cells between any of the groups. (F) Hearts from ISP- and HJ-02−treated animals had significantly more dendritic cells compared with VEH. (G) Hearts from ISP and HJ-02 treated animals had significantly increased macrophages compared with VEH. Data are mean ± SD, ∗P < 0.05; ∗∗P < 0.01; ISP, n = 6; VEH and HJ-02, n = 4; 1-way ANOVA with Tukey's multiple comparisons post-test. Abbreviations as in Figures 1 and 3.

Figure 6

Macrophage and T Cell Subtypes Are Altered by Treatments Restoring Innervation (A and B) Quantification of M1-like and M2-like macrophages, expressed as a percentage of total macrophages. Hearts from ISP- and HJ-02−treated animals had significantly higher levels of M2-like macrophages compared with vehicle (VEH), and hearts from VEH-treated animals had significantly higher levels of M1-like macrophages compared with ISP or HJ-02 treated animals. (C) Ratio of M1-like macrophages to M2-like macrophages. (D) Quantification of regulatory T cell (Treg) cells, expressed as a percentage of CD4⁺ T cells. Hearts from ISP- and HJ-02−treated animals had significantly higher levels of Treg cells compared with VEH. Data are mean ± SD. ∗P < 0.05; ∗∗P < 0.01. ISP (n = 6); VEH and HJ-02 (n = 4); 1-way ANOVA with Tukey's multiple comparisons post-test. Other abbreviations as in Figure 1.

Figure 7

ISP and HJ-02 Have No Effect on Macrophage Expansion or Polarization (A) Gating scheme for identifying peritoneal macrophages. (B) Quantification of macrophage population levels as a percent of control, normalized to each replicate. Cells were treated with 10 µM ISP or 100 nM HJ02 for 24 hours, and additional cells were treated with 100 ng/mL lipopolysaccharide + 5.0 ng/mL interferon-γ for 6 hours (M1-like) or 10 ng/mL interferon-4 for 24 hours (M2-like). Representative graphs of (C) CD86 and (D) CD206 expression levels. Graphs of mean fluorescence intensity (MFI) for (E) CD86 and (F) CD206, quantified via FlowJo v10.8; marker expression in both ISP and HJ-02 treated conditions remains unchanged from control. All data are mean ± SD (n = 4-5 experiments); ∗P < 0.05; ∗∗P < 0.01 versus control, 1-way ANOVA with Dunnett's multiple comparisons post-test. Abbreviations as in Figure 1.

Figure 8

HJ-01 and HJ-02, But Not ISP, Reduce Infarct Size and Preserve Cardiac Function (A and B) Quantification of cardiac output and ejection fraction in mice following sham or myocardial infarction (MI) procedures, then treated with vehicle, HJ-01, or HJ-02. (C) Example image of a heart after MI depicting the loss of autofluorescence in the infarct. (D) Quantification of infarct size as a percentage of left ventricle in mice treated with HJ-01, HJ-02, or intracellular sigma peptide (ISP). Data are mean ± SD; n = 5/group except sham (n = 4); ∗P < 0.05; ∗∗P < 0.01; 1-way analysis of variance (ANOVA) with Dunnett's multiple comparisons post-test. (control group = sham mice in A and B.

Figure 9

HJ-02 and ISP Have No Effect on Vascularization Following MI Representative images of an HJ-02−treated mouse heart (A and B) stained for CD31 and (C and D) α-smooth muscle actin (SMA). Quantification of CD31⁺ and α-SMA⁺ cells in mice treated with VEH, HJ-02, or ISP. Data are mean ± SD n = 4 for vehicle, n = 5-6 for HJ-02 and ISP.