Cardiac Lymphatic Insufficiency Leads to Diastolic Dysfunction Via Myocardial Morphologic Change

Figure 1. Establishment of a New Murine Cardiac Lymphatic Vessel Ablation Model (A and B) Both lymph trunks on the anterior and posterior side of the heart were ablated on the proximal side, indicated by red arrowheads, after the Evans blue dye injection. (C) Fluorescein isothiocyanate (FITC)–dextran was injected into the apex side of heart to detect the path of lymph drainage as recorded by an in vivo imaging system. (D) An electrocardiogram was obtained immediately after lymphatic ablation to determine if lymphatic ablation resulted in ST-segment elevation. (E) T2-weighted magnetic resonance imaging was used to assess tissue edema. (F) An assessment of the levels of circulating FITC-dextran. Data are presented as mean ± SD (n = 5). ∗∗∗P < 0.001 vs sham; unpaired Student’s t-test was used to evaluate statistical significance between 2 groups. (G) Representative images of the heart 6 weeks after lymph ablation.

Figure 2. Morphologic Changes of Myocardium Size, Cardiac Fibrosis, and Cardiac Hypertrophy Under Chronic Lymphatic Dysfunction (A and B) Heart weight to body weight (HW/BW) and heart weight to tibia length (HW/TL) ratios. (C) Frozen sections were immunostained with anti-F4/80 (red) to detect macrophages. Magnification ×20. Scale bar 100 μm. (D) Quantitative analysis of the number of macrophages per field. (E) Masson’s trichrome staining (blue) of heart paraffin sections. Magnification ×20. Scale bar 100 μm. (F) The percentage of fibrosis area per field. (H) Wheat germ agglutinin staining (red). 6-Diamidino-2-phenylindole–stained nuclei are shown in blue. Magnification ×20. Scale bar 100 μm. (I and G) Quantification of cardiomyocyte cross-sectional area and perimeter. Data are presented as mean ± SEM (n = 5). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 vs sham, ###P < 0.001 vs L+H; 1-way ANOVA and Tukey post hoc tests. HFD = high-fat diet; L+H = lymphatic ablation plus high-fat diet.

Figure 3. Cardiac Inflammation, Fibrosis, and Hypertrophic Signaling After Cardiac Lymphatic Ablation (A) The mRNA expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were assessed in the myocardium of sham-operated (sham), HFD, lymph ablation, and L+H mice 6 weeks after surgery. (B) The mRNA expression levels of fibrosis mediation factors collagen type I, alpha 1 (COL1A1), transforming growth factor (TGF) β, and periostin (Postn) increased after cardiac lymph ablation. (C) The mRNA expression levels of sensitive markers of pathologic hypertrophy β-myosin heavy chain (MHC), transient receptor potential cation channel subfamily C member 6 (TRPC6), regulator of calcineurin (RCAN) 1, and cardiotropin (CT) 1,were increased after cardiac lymph ablation. Data are presented as mean ± SEM (n = 5). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 vs sham; ###I < 0.001 vs L+H; 1-way ANOVA and Tukey post hoc tests. Abbreviations as in Figure 1.

Figure 4. Cardiac Lymphatic Ablation Induced Cardiac Diastolic Function Impairments in Murine Models (A and B) Six weeks after surgery, there were no significant differences in left ventricular ejection fraction (EF). (C and D) The diastolic interventricular septum thickness (IVS-D) and diastolic left ventricular posterior wall thickness (LVPW-D) were increased by cardiac lymph ablation. (E and G) Doppler (E/A ratio) and tissue Doppler (e′ velocity) profiles for the assessment of left ventricular diastolic function for the 4 groups 6 weeks after surgery (F) Ratio of flow Doppler E-wave to A-wave amplitude. (H) Ratio of flow Doppler E-wave amplitude to tissue Doppler E′-wave amplitude. Data are presented as mean ± SEM (n = 5). ∗∗P < 0.01; ∗∗∗P < 0.001 vs sham; #P < 0.05; ###P < 0.001 vs L+H; 1-way ANOVA and Tukey post hoc tests. Abbreviations as in Figure 1.

Figure 5. Cardiac Lymphangiogenesis Caused by ADRC Implantation Improved Cardiac Inflammation, Fibrosis, and Hypertrophy (A) Immunostaining with anti–lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) (green) and 4,6-diamidino-2-phenylindole (blue). Scale bar 100 μm. (B) Quantitative analysis of the LYVE1-positive cells. (C) Quantification of lymphatic area. (D) Representative images of the heart 6 weeks after ADRC implantation. (E and F) Heart weight to body weight (HW/BW) and heart weight to tibia length (HW/TL) ratios in the 3 groups. (G) Immunostaining with anti-F4/80 (red). (H) Quantitative analysis of the number of macrophages per field. (I) Masson’s trichrome staining (blue) of heart paraffin sections. (J) The percentage of fibrosis area per field. (K) Representative images of wheat germ agglutinin staining (red). (L) Quantification of cardiomyocyte cross-sectional area and perimeter. Data are presented as mean ± SEM (n = 5). ∗∗P < 0.01; ∗∗∗P < 0.001 vs sham; ###P < 0.001 vs L+H; 1-way ANOVA and Tukey post hoc tests. Abbreviations as Figure 1.

Figure 6. Expression of Cardiac Inflammatory, Fibrotic, and Hypertrophic Markers Was Decreased by ADRC-Induced Cardiac Lymphangiogenesis (A) The mRNA expression levels of TNF-α, IL-1β, and IL-6 were assessed in the myocardium of sham-operated, L+H, and L+H+ADRC implantation groups after 6 weeks (B) The mRNA expression levels of fibrosis mediation factors COL1A1, TGF-β, and Postn. (C) The mRNA expression levels of sensitive markers of pathologic hypertrophy, namely β-MHC, TRPC6, RCAN-1, and CT-1, were decreased in the ADRC implantation group. Data are presented as mean ± SEM (n = 5). ∗P <0.05; ∗∗P <0.01; ∗∗∗P <0.001 vs sham; ###P < 0.001 vs L+H; 1-way ANOVA and Tukey post hoc tests. Abbreviations as in Figures 1, 3, and 5.

Figure 7. ADRC Implantation Improved Cardiac Diastolic Function Through Cardiac Lymphangiogenesis (A and B) There were no significant differences in ejection fraction (EF). (C) The IVS-D and LVPW-D trends. (D) Circulating B-type natriuretic peptide (BNP) levels detected by enzyme-linked immunosorbent assay were improved after ADRC treatment. (E and G) Doppler (E/A ratio) and tissue Doppler (e′ velocity) profiles for the assessment of left ventricular diastolic function for the 4 groups 6 weeks after surgery (F) Ratio of flow Doppler E-wave to A-wave amplitude. (H) Ratio of flow Doppler E-wave amplitude to tissue Doppler E′-wave amplitude. Data are presented as mean ± SEM (n = 5). ∗∗∗P < 0.001 vs sham; ##P < 0.01; ###P < 0.001 vs L+H; 1-way ANOVA and Tukey post hoc tests. Abbreviations as in Figures 1, 4, and 5.