Exercise-Induced Cardiac Lymphatic Remodeling Mitigates Inflammation in the Aging Heart

Abstract

The lymphatic vasculature plays essential roles in fluid balance, immunity, and lipid transport. Chronic, low-grade inflammation in peripheral tissues develops when lymphatic structure or function is impaired, as observed during aging. While aging has been associated with a broad range of heart pathophysiology, its effect on cardiac lymphatic vasculature has not been characterized. Here, we analyzed cardiac lymphatics in aged 20-month-old mice versus young 2-month-old mice. Aged hearts showed reduced lymphatic vascular density, more dilated vessels, and increased inflammation and fibrosis in peri-lymphatic zones. As exercise has shown benefits in several different models of age-related heart disease, we further investigated the effects of aerobic training on cardiac lymphatics. Eight weeks of voluntary wheel running attenuated age-associated adverse remodeling of the cardiac lymphatics, including reversing their dilation, increasing lymph vessel density and branching, and reducing perilymphatic inflammation and fibrosis. Intravital lymphangiography demonstrated improved cardiac lymphatic flow after exercise training. Our findings illustrate that aging leads to cardiac lymphatic dysfunction, and that exercise can improve lymphatic health in aged animals.

Keywords: VEGF‐C; exercise; heart; lymphangiogenesis; lymphatic endothelial cells; lymphatic vessels; ventricular remodeling.

FIGURE 1

Exercise training increased ejection fraction of collecting popliteal lymphatic vessels (PLV) and enhanced lymph flow. (A) Representative intravital microscopy images of PLVs perfused with FITC‐dextran from sedentary (Sed) and exercise‐trained (ExTr) 2‐month‐old mice. (B) Representative lymphatic contraction curves in sedentary (Sed) control and exercise‐trained (ExTr) mice. (C) Quantification of lymphatic diameter and pumping (n = 5 per group). (D) Schematic diagram of lymphatic pumping and flow after FITC dextran injection into the foot pad. (E) Representative images of transport of FITC‐dextran through draining popliteal, inguinal, and axillary lymph nodes with (F) quantification of FITC in proximal to distal lymph nodes (n = 3 per group). *p < 0.05; **p < 0.01; ***p < 0.001; ns: Not significant.

FIGURE 2

Exercise training induced cardiac lymphangiogenesis in young mice after 8 weeks of voluntary wheel running. (A) LYVE‐1 immunohistochemical staining of the lymphatic network in hearts from sedentary (Sed) control and exercise‐trained (ExTr) mice. (B) Confocal imaging for LYVE‐1 and VEGFR‐3 in heart tissues from sedentary control and exercised mice with quantification of cardiac lymphatic density (n = 5 per group). (C) Representative whole mount staining of LYVE‐1+ epicardial lymphatics from sedentary control and exercised mice with quantification of lymphatic diameter and branching points (white arrows) (n = 5 per group). (D) mRNA levels of CD31 and lymphatic markers LYVE‐1, PDPN, and VEGFR‐3 in sedentary control and exercised mice. *p < 0.05; **p < 0.01; ns: Not significant.

FIGURE 3

Aging caused dilation of cardiac lymphatics and impaired integrity of lymphatic vessels. (A) Confocal imaging and quantification of vessel diameter of LYVE‐1+ lymphatic vessels in hearts from young 2‐month‐old and aged 20‐month‐old mice. (B) Representative whole mount staining of CD31 + LYVE‐1+ initial lymphatics in the hearts from young and aged mice with quantification of lymphatic diameter. (C) Representative whole mount staining of CD31 + LYVE1‐ collecting lymphatics from young and aged mice with quantification of lymphatic diameter. (D) Confocal imaging of VE cadherin‐positive initial lymphatics and (E) RNA quantification of VE‐cadherin in hearts from young and aged mice. (F) Confocal imaging and quantification of lymphatic vessel density in the epicardium of young and aged mice. Magnified insets show LYVE1+/VEGFR‐3+ lymphatic endothelial cells (yellow upon merge). (n = 3 for all groups) *p < 0.05; **p < 0.01.

FIGURE 4

Eight weeks of exercise training enhanced lymphangiogenesis and lymphatic remodeling in the hearts of 20‐month‐old mice. (A) Confocal imaging and quantification of lymphatic vessels costained with LYVE‐1 and the proliferative marker Ki67 in the hearts of aged sedentary (aSed) control and aged exercise‐trained (aExTr) mice (n = 3 per group). (B) Whole mount staining for LYVE‐1+ epicardial lymphatics from aged sedentary and aged exercised mice with quantification of diameter and branching points (white arrows) (n = 5 per group). (C) Flow plots and quantification of lymphatic endothelial cells (CD45‐/CD31+/LYVE‐1+/VEGFR‐3+) in hearts from aged sedentary and aged exercised mice (n = 3 per group). (D) mRNA levels of lymphangiogenic markers in aged hearts following exercise training (n = 4 per group). (E) Whole mount staining of peripheral lymphatics under the ear skin in aged sedentary and aged exercised mice with quantification of diameter and branch points (n = 4 per group). *p < 0.05; **p < 0.01.

FIGURE 5

Exercise remodeled the perilymphatic microenvironment in aged hearts. (A) Confocal imaging and quantification of CD3+ T cells in peri‐lymphatic regions in the hearts of aged sedentary (aSed) and aged exercise‐trained (aExTr) mice (n = 5 per group). (B) Confocal imaging and quantification of CD68+/CD206+ macrophages in peri‐lymphatic regions in the hearts of aged sedentary and aged exercised mice (n = 5 per group). (C) Confocal imaging of collagen1, alpha‐actinin‐2, and LYVE‐1 with quantification of perilymphatic collagen (n = 3 per group). (D) Representative images and quantification of perilipin1+ cardiac fat in aged sedentary and aged exercised mice (n = 4 per group). (E) Confocal imaging of WGA‐stained cardiomyocytes and quantification of their size after exercise training in regions either adjacent to or remote from lymphatic vessels (n = 3–4 per group). *p < 0.05; ***p < 0.001; ns: Not significant.

FIGURE 6

Exercise training enhanced cardiac lymphatic flow. (A) Schematic diagram of injection of fluorescent tracer (FITC‐dextran) into the apex of the beating heart. (B) Representative intravital microscopy images of epicardial lymphatic vessels perfused with FITC‐dextran in young sedentary (ySed), aged sedentary (aSed), and aged exercise‐trained (aExTr) mice. (C) Quantification of FITC fluorescence in the upper half of the heart (dotted box, panel B) and the (D) maximal distance traveled from the apex of the heart (white arrows, panel B) (n = 3–4 per group). (E) Representative echocardiographic waveforms showing peak velocities through the mitral valve during early (E) and late (A) diastole. (F) Mitral valve E/A ratio in aged sedentary and aged exercised mice (n = 3 per group). *p < 0.05; **p < 0.01.