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. 2017 Sep;9(9):1279-1293.
doi: 10.15252/emmm.201707565.

The Cardiac Microenvironment Uses Non-Canonical WNT Signaling to Activate Monocytes After Myocardial Infarction

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Free PMC article

The Cardiac Microenvironment Uses Non-Canonical WNT Signaling to Activate Monocytes After Myocardial Infarction

Ingmar Sören Meyer et al. EMBO Mol Med. .
Free PMC article

Abstract

A disturbed inflammatory response following myocardial infarction (MI) is associated with poor prognosis and increased tissue damage. Monocytes are key players in healing after MI, but little is known about the role of the cardiac niche in monocyte activation. This study investigated microenvironment-dependent changes in inflammatory monocytes after MI RNA sequencing analysis of murine Ly6Chigh monocytes on day 3 after MI revealed differential regulation depending on location. Notably, the local environment strongly impacted components of the WNT signaling cascade. Analysis of WNT modulators revealed a strong upregulation of WNT Inhibitory Factor 1 (WIF1) in cardiomyocytes-but not fibroblasts or endothelial cells-upon hypoxia. Compared to wild-type (WT) littermates, WIF1 knockout mice showed severe adverse remodeling marked by increased scar size and reduced ejection fraction 4 weeks after MI While FACS analysis on day 1 after MI revealed no differences in neutrophil numbers, the hearts of WIF1 knockouts contained significantly more inflammatory monocytes than hearts from WT animals. Next, we induced AAV-mediated cardiomyocyte-specific WIF1 overexpression, which attenuated the monocyte response and improved cardiac function after MI, as compared to control-AAV-treated animals. Finally, WIF1 overexpression in isolated cardiomyocytes limited the activation of non-canonical WNT signaling and led to reduced IL-1β and IL-6 expression in monocytes/macrophages. Taken together, we investigated the cardiac microenvironment's interaction with recruited monocytes after MI and identified a novel mechanism of monocyte activation. The local initiation of non-canonical WNT signaling shifts the accumulating myeloid cells toward a pro-inflammatory state and impacts healing after myocardial infarction.

Keywords: inflammation; monocytes; myocardial infarction.

Figures

Figure 1
Figure 1. Differential gene expression profiles in inflammatory monocytes sorted from the bone marrow, blood, and heart were found 3 days after MI

RNA‐seq analyses revealed differential expression of 1,482 genes in monocytes sorted from different bodily regions.

PANTHER pathway analysis of genes found in the transcriptomes.

Differential gene expression of WNT‐associated genes in monocytes.

Log2(x‐fold) of canonical WNT pathway inhibitors in Ly6Chi monocytes sorted from the heart compared to Ly6Chi monocytes in the bone marrow. Data are represented as mean ± SD (N = 3).

Log2(x‐fold) of non‐canonical WNT/PCP pathway mediators in Ly6Chi monocytes sorted from the heart compared to Ly6Chi monocytes in the bone marrow. Data are represented as mean ± SD (N = 3).

Figure 2
Figure 2. Non‐canonical WNT increases following MI

Representative Western blots of pJNK expression in the border zone of mouse hearts following MI.

Quantification of pJNK expression (mean ± SD, N = 5, *P = 8.6E‐05, unpaired two‐sided Student's t‐test).

(C) Representative Western blots and (D) quantification of pJNK (*P = 0.0001) and active β‐catenin (ABC) (*P = 0.001) expression in macrophages stimulated with supernatant of cardiomyocytes cultured under hypoxic conditions (unpaired two‐sided Student's t‐test, mean ± SD, N = 4).

Source data are available online for this figure.
Figure 3
Figure 3. WIF1 expression during MI

WNT inhibitor mRNA levels in isolated neonatal rat cardiomyocytes cultured under hypoxic conditions. Results from three independent experiments performed in triplicate (mean ± SD, WIF1: *P = 0.011, sFRP5: *P = 0.021; unpaired two‐sided Student's t‐test with Sidak correction).

WIF1 mRNA levels in isolated neonatal rat cardiac fibroblasts under hypoxic conditions. Results originate from three independent experiments performed in triplicate. Data are represented as mean ± SD.

Representative Western blots of WIF1 in hearts from sham‐operated (control) or LAD‐ligated (MI) mice.

Quantification of WIF1 protein expression in sham‐operated and LAD‐ligated animals (mean ± SD, N = 4, Day 1: *P = 0.0001, Day 3: *P = 0.043; unpaired two‐sided Student's t‐test).

(E) Representative immunofluorescence staining of WIF1 in murine heart tissue sections from control (top), infarcted animals (middle), and (F) heart tissue sections from deceased human patients free from cardiovascular disease (top) or following acute MI (bottom) (red: WIF1, blue: DAPI). WIF1 expression in mice was visualized 4 days after LAD ligation. Scale bar = 25 μm. Yellow dotted line indicates the transition between scar and border zone (E).

Source data are available online for this figure.
Figure 4
Figure 4. Global WIF1 knockout worsens MI outcome

Representative Masson trichrome staining of WIF1KO (right) and their WT littermates (left) 4 weeks after induced MI (scale bar: 1,500 μm).

Quantification of relative scar size in WIF1KO and their WT littermates 4 weeks after MI (*P = 0.0001).

Heart weight/tibia length ratios 4 weeks after induced MI (*P = 0.0008).

Representative B‐mode (left) and M‐mode (right) echocardiographic images of WIF1KO (bottom) and their WT littermates (top) 4 weeks after MI.

Echocardiographic results from WIF1KO and their WT littermates 4 weeks after MI (N = 11, FS: *P = 0.0192, EF: *P = 0.0173).

Representative images of flow cytometric analysis of heart tissue cell suspension following MI in WIF1KO and WT mice gated on neutrophils 1 day post‐MI.

Quantification of total neutrophils following MI (P = 0.91).

cTNT levels 1 day after LAD ligation (N = 11, P = 0.65).

Representative images of flow cytometric analysis of heart tissue cell suspension 4 days post‐MI in WIF1KO (bottom) and WT (top) mice gated on monocytes/macrophages.

Quantification of total cell numbers per mg heart tissue of inflammatory (Ly6Chi) monocytes (top left, *P = 0.0327); Ly6Chi monocytes per μl blood (top right, P = 0.1946); reparative (Ly6Clo) macrophages per mg heart tissue (bottom left, *P ≤ 0.0439); and Ly6Chi monocytes per femur (bottom right, P = 0.3026).

Data information: Results are represented as mean ± SD and analyzed using unpaired two‐sided Student's t‐test (Sidak corrected). In panels (A–D, F, G, I, and J) N = 8 (WT) and N = 11 (WIF1‐KO).
Figure 5
Figure 5. Cardiac‐specific AAV‐9‐mediated WIF1 overexpression improves heart function following MI

Timeline of AAV‐mediated WIF1 overexpression experiments.

cTNT levels 1 day after LAD ligation (N = 6, P = 0.19).

Heart weight/body weight ratios 4 weeks after induced MI (N = 6, *P = 0.0476).

Representative Masson trichrome staining of AAV‐WIF1 (bottom) and AAV‐LUC control animals (top) 4 weeks after induced MI (scale bar: 1,000 μm).

Quantification of relative scar size in AAV‐WIF1 and AAV‐LUC control animals 4 weeks after MI (N = 5, *P = 0.0264).

Representative B‐mode (left) and M‐mode (right) echocardiographic image of AAV9‐LUC‐injected control (top) and AAV9‐WIF1‐injected animals (bottom) 4 weeks after MI.

Echocardiographic results from AAV9‐LUC‐injected and AAV9‐WIF1‐injected mice 4 weeks after MI (N = 6, FS: *P = 0.0047, EF: *P = 0.038).

Representative images of flow cytometric analysis of heart tissue cell suspension following myocardial infarction of AAV‐LUC‐injected control (top) and AAV‐WIF1 (bottom) mice.

Quantification of inflammatory (Ly6Chi) monocytes (top, *P ≤ 0.04) and reparative (Ly6Clo) macrophages (bottom, P = 0.58) per mg heart tissue (N = 12 per group).

Data information: Results are represented as mean ± SD and were analyzed by unpaired two‐sided Student's t‐test with Sidak correction.
Figure 6
Figure 6. WIF1 inhibits non‐canonical WNT signaling

Scheme of in vitro experiments.

mRNA levels of inflammatory markers in macrophages stimulated with supernatant of control or WIF1 overexpressing cardiomyocytes cultured under hypoxic conditions (mean ± SD, N = 3, IL‐1β: AdCtrl (normoxia) versus AdWIF1 (normoxia) P = 0.96 (n.s.), AdCtrl (normoxia) versus AdCtrl (hypoxia) *P = 0.000577, AdCtrl (hypoxia) versus AdWIF1 (hypoxia) *P = 0.004577. IL‐6: AdCtrl (normoxia) versus AdWIF1 (normoxia) P = 0.99 (n.s.), AdCtrl (normoxia) versus AdCtrl (hypoxia) *P = 0.0156, AdCtrl (hypoxia) versus AdWIF1 (hypoxia) *P = 0.0141, one‐way ANOVA with Holm‐Sidak's multiple comparisons test).

(C) Representative Western blots of JNK and ATF2 expression in macrophages stimulated with supernatant of AdControl‐ or AdWIF1‐transfected hypoxic cardiomyocytes and (D) Quantification of pJNK and pATF2 expression in macrophages stimulated with supernatant of AdWIF1‐transfected hypoxic cardiomyocytes (mean ± SD, N = 4, JNK: AdCtrl (normoxia) versus AdWIF1 (normoxia) P = 0.99 (n.s.); AdCtrl (normoxia) versus AdCtrl (hypoxia) *P = 0.0001, AdCtrl (hypoxia) versus AdWIF1 (hypoxia) *P = 0.0001, ATF2: AdCtrl (normoxia) versus AdWIF1 (normoxia) P = 0.99 (n.s.); AdCtrl (normoxia) versus AdCtrl (hypoxia) *P = 0.0012, AdCtrl (hypoxia) versus AdWIF1 (hypoxia) *P = 0.0256, one‐way ANOVA with Holm‐Sidak's multiple comparisons test).

Source data are available online for this figure.
Figure 7
Figure 7. Schematic overview of the identified mechanism of local monocyte activation
Schematic representation of proposed non‐canonical WNT signaling activation in infiltrating monocytes by the cardiac microenvironment and inhibition by cardiomyocyte‐derived WIF1 that limits inflammatory processes.

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