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. 2017 Jan;102:83-93.
doi: 10.1016/j.yjmcc.2016.12.002. Epub 2016 Dec 10.

Cardiac Inflammation in Genetic Dilated Cardiomyopathy Caused by MYBPC3 Mutation

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Free PMC article

Cardiac Inflammation in Genetic Dilated Cardiomyopathy Caused by MYBPC3 Mutation

Thomas L Lynch 4th et al. J Mol Cell Cardiol. .
Free PMC article

Abstract

Cardiomyopathies are a leading cause of heart failure and are often caused by mutations in sarcomeric genes, resulting in contractile dysfunction and cellular damage. This may stimulate the production of a robust proinflammatory response. To determine whether myocardial inflammation is associated with cardiac dysfunction in dilated cardiomyopathy (DCM) caused by MYBPC3 mutation, we used the well-characterized cMyBP-C(t/t) mouse model of DCM at 3months of age. Compared to wild type (WT) mice, DCM mice exhibited significantly decreased fractional shortening (36.4±2% vs. 15.5±1.0%, p<0.0001) and significantly increased spleen weight (5.3±0.3 vs. 7.2±0.4mg/mm, p=0.002). Intriguingly, flow cytometry analysis revealed a significant increase in total (CD45+CD11b+Ly6C-MHCII+F480+) macrophages (6.5±1.4% vs. 14.8±1.4%, p=0.002) and classically activated (CD45+CD11b+Ly6C-MHCII+F480+CD206-) proinflammatory (M1) macrophages (3.4±0.8% vs. 10.3±1.2%, p=0.0009) in DCM hearts as compared with WT hearts. These results were further confirmed by immunofluorescence analysis of heart tissue sections. Splenic red pulp (CD11b+Ly6C+MHCIIlowF480hi) macrophages were significantly elevated (1.3±0.1% vs. 2.4±0.1%, p=0.0001) in DCM compared to WT animals. Serum cytokine analysis in DCM animals exhibited a significant increase (0.65±0.2 vs. 2.175±0.5pg/mL, p=0.02) in interleukin (IL)-6 compared to WT animals. Furthermore, RNA-seq analysis revealed the upregulation of inflammatory pathways in the DCM hearts. Together, these data indicate a robust proinflammatory response in DCM hearts, likely in response to cellular damage triggered by MYBPC3 mutation and resultant contractile dysfunction.

Keywords: Dilated cardiomyopathy; Inflammation; MYBPC3; Mouse models; Sarcomere biology.

Figures

Fig. 1
Fig. 1. DCM mice exhibit cardiac dysfunction and splenomegaly
(A) Representative short-axis M-mode echocardiographic images of WT and DCM hearts. LVID at (B) peak diastole and (C) peak systole in WT (n = 7) and DCM (n = 10) hearts (****p < 0.0001). Functional measurements derived from LVID measurements showing (D) fractional shortening (FS) and global longitudinal strain (GLS) in WT (n = 7) and DCM (n = 10) hearts (**p = 0.05, ****p < 0.0001). Quantification of the ratio of (F) heart weight (HW) to tibia length (TL), (G) Lung weight (LW) to TL, (H) spleen weight (SW) to TL in WT (n = 7) and DCM (n = 10) animals (**p = 0.002, ****p<0.0001). Representative Masson’s trichrome-stained spleen sections from WT and DCM mice. The lower magnification (2X) of cross-sectioned spleens (top) shows a rounded edge in DCM spleens, which is a hallmark of splenomegaly. The high power magnification (20X) (bottom) shows less abundant splenic macrophage populations in the border zone area of DCM mouse spleens compared to WT mouse spleens. The dotted circle shows the border between red pulp (RP) and white pulp regions (WP) of the spleen. BZ= border zone.
Fig. 2
Fig. 2. Proinflammatory macrophages infiltrate DCM hearts
Cardiac mononuclear cells were isolated following tissue digestion and gradient centrifugation purification. (A) Representative gating strategy and scatter plots used to identify activated macrophages (CD45+CD11b+Ly6CMHCII+F480+), proinflammatory M1 macrophages (CD45+CD11b+Ly6CMHCII+F480+CD206), and anti-inflammatory M2 macrophages (CD45+CD11b+Ly6CMHCII+F480+CD206+) in WT and DCM hearts. Flow cytometry quantification of (B) activated macrophages, (C) M1 macrophages, and (D) M2 macrophages in WT (n = 6) and DCM (n = 7–8) hearts (**p = 0.002, ***p = 0.0009).
Fig 3
Fig 3. Elevation in CD68+ proinflammatory macrophages in DCM heart tissue
Representative confocal images and corresponding quantification of (A, B) CD68+ macrophages and (A, C) MRC1+ M2 anti-inflammatory macrophages (n = 58 sections from 5 WT hearts, 75 sections from 5 DCM hearts, *p = 0.016) and their merge, as well as (D, E) NIMP14+ (n = 40 sections from 4 WT hearts, 40 sections from 4 DCM hearts) and (F, G) Ly6G+ (n = 18 sections from 3 WT hearts, 29 sections from 1 DCM heart) neutrophils in WT and DCM heart tissue sections. DAPI (4′,6-diamidino-2-phenylindole).
Fig. 4
Fig. 4. Serum IL-6 levels are elevated in DCM animals
Serum cytokine levels of (A) IL-6, (B) MCP-1, (C) IFN-γ, (D) IL12p70, IL-10, and TNF-α in WT (n = 10) and DCM (n = 12–13) peripheral blood as determined by cytometric bead array immunoassay (*p = 0.02). IL (interleukin); tumor necrosis factor (TNF), interferon (IFN), monocyte chemoattractant protein (MCP).
Fig. 5
Fig. 5. Splenic red pulp macrophages (RPM) are augmented in DCM animals
(A) Representative scatter plots used to identify splenic monocytes (CD11b+Ly6C+ cells), monocytes within the red pulp region of the spleen (Ly6GCD11b+Ly6C+F480hi), and red pulp macrophages (CD11b+Ly6C+MHCIIlowF480hi cells) in WT and DCM spleens. Flow cytometry quantification of (B) splenic monocytes, (C) monocytes within the red pulp region of the spleen, and (D) red pulp macrophages in WT (n = 6–7) and DCM (n = 6–7) spleens (***p = 0.001).
Fig. 6
Fig. 6. Circulating mononuclear phagocytes in DCM and WT peripheral blood
(A) Representative gating strategy used to identify total monocytes (CD45+Gr1CD11b+Ly6C+), proinflammatory monocytes (CD45+Gr1CD11b+Ly6Chi), and anti-inflammatory monocytes (CD45+Gr1CD11b+Ly6Clow) in peripheral blood samples from WT and DCM mice. Flow cytometry quantification of (B) total monocytes, (C) proinflammatory monocytes, and (D) anti-inflammatory monocytes in WT (n = 4) and DCM (n = 5) peripheral blood samples.
Fig. 7
Fig. 7. Differentially regulated genes in DCM compared to WT hearts
(A) RNA-Seq heat maps depicting clusters of genes differentially regulated in DCM versus WT hearts. Shown is a subset of the most upregulated (red) and downregulated (green) genes from the total gene set. The fold change, up or down, is represented in the keys for the respective panels. n = 3 pooled samples per genotype. (B) Network of significantly enriched gene ontology terms for upregulated genes in DCM hearts. The red hexagons denote upregulated genes in DCM compared to WT hearts. Blue rectangles represent significantly enriched (p < 0.05; FDR) biological processes using the ToppFun application of ToppGene Suite.

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