Arrested Neutrophils in Capillaries is a Novel Mechanism of Myocardial Hypoperfusion in Heart Failure with Preserved Ejection Fraction

Abstract

Background Heart failure with preserved ejection fraction (HFpEF) is a common and serious condition that lacks evidence‐based therapies due to an incomplete understanding of its pathogenesis, although cardiac hypoperfusion is clearly implicated. Our aim was to characterize leukocyte interactions in the coronary microvasculature during HFpEF using the novel imaging technique of intravital cardiac multiphoton microscopy (MPM), with the goal of identifying cellular mechanisms of hypoperfusion. Methods Male and female, 8–12‐week‐old C57BL/6 mice were fed a high fat diet and the nitric oxide synthase inhibitor, L‐NAME, in their drinking water (HFpEF) or a normal chow diet (Chow) for 15‐weeks. Mice were assessed for systolic and diastolic function (echocardiography and Doppler imaging), blood pressure (tail‐cuff), hypertrophy (wheat‐germ‐agglutin), hypoxia (Hypoxyprobe™), exercise intolerance (treadmill exhaustion), and heart RNA sequencing. Intravital cardiac MPM was performed in the anesthetized mechanically ventilated mouse following a left thoracotomy and placement of a customized imaging window on the left ventricle. Transgenic‐reporter mice were used to visualize neutrophil (Rosa26‐Cre‐Ly6G+/TdTom) and macrophage/monocyte (Cx3Cr1+/GFP‐CCR2+/RFP) populations. Intravenous injection of fluorescent dyes labeled the vasculature. Results HFpEF mice developed systemic and clinical features of HFpEF compared to Chow mice; obesity, hypertension, left ventricular hypertrophy, diastolic dysfunction, preserved systolic function, pulmonary remodeling, myocardial hypoxia, and exercise intolerance (p<0.05 for all). Intravital MPM visualized Ly6G+ neutrophil motion in capillaries, revealing an increased frequency of slowed and arrested neutrophils in capillaries in HFpEF hearts compared to Chow (p<0.0001; Kolmogorov‐Smirnov test). Immunohistology for tissue infiltrated (non‐vascular) neutrophils revealed no increase in HFpEF. The number and frequency of slowed and arrested Cx3Cr1+ and CCR2+ capillary monocytes were similar between HFpEF and Chow mice, while tissue resident Cx3Cr1+ macrophages increased in HFpEF (p<0.05). Gene sets associated with inflammatory signaling (TNF‐α and TGF‐β) and cell junctions (epithelial‐mesenchymal transition and apical junctions) were upregulated in HFpEF. Acute neutrophil depletion with anti‐Ly6G antibody (αLy6G) administration (4mg/kg body weight, 24h) reduced the incidence of slowed and arrested neutrophils (p<0.05) and improved exercise tolerance in HFpEF. Chronic αLy6G (2mg/kg every 3 days for 4 weeks) improved myocardial hypoxia and measures of diastolic function (mitral e/e’) (p<0.05). Conclusion We have used the novel imaging technique of intravital cardiac MPM to discover a new behavior of neutrophils within myocardial capillaries during HFpEF. We demonstrate that arrested capillary neutrophils impair myocardial perfusion to promote HFpEF. Targeting arrested neutrophil behavior offers potential therapeutic benefits.

Imaging Targets to Visualize the Cardiac Immune Landscape in Heart Failure

“…72,73 Cardiac macrophage abundance is increased in HFpEF models. 74 These hearts display increased populations of infiltrating monocytes and macrophages, specifically CXCR4 + monocytes and CCR2 + macrophages, and decreased abundance of cardiac tissue-resident macrophages. 75,76 Inhibition of monocyte infiltration is sufficient to improve outcomes.…”

“…Neutrophil depletion by anti-Ly6G antibody administration improved HFpEF disease characteristics in the mouse model. 74 In summary, neutrophils might represent potential therapeutic targets in HFpEF to modulate their role in fibrosis, inflammation, and microvascular dysfunction.…”