Intravital Multiphoton Microscopy of the Beating Mouse Heart Reveals Altered Cardiomyocyte Contraction Dynamics and Increased Microvascular Patrolling by Leukocytes during Cardiac Hypertrophy

Abstract

BackgroundThe study of functional cardiomyocyte adaptation and inflammatory cell behavior at the micro‐scale in vivo has been challenging due to limited imaging tools. We recently developed intravital multiphoton microscopy (MPM) methods that enable visualization and quantification of cardiac dynamics at the cell and micro‐vessel level throughout the cardiac cycle. We aimed to determine the dynamic cellular changes that occur due to high fat diet (HFD) induced hypertrophy using intravital cardiac MPM.MethodsApoE−/− C57Bl6 mice started a HFD at 6 weeks of age (ApoE−/−‐HFD, n=11), while age‐matched wild‐type mice were fed a normal chow diet (WT‐ND, n=10). At 26‐weeks, mice were assessed by cardiac echocardiography and intravital MPM in the intact beating heart. Intravenous injections of rhodamine‐6G (R6g) labeled cardiomyocytes and leukocytes, and Texas‐Red dextran labeled vasculature during intravital MPM. 3D volumes were reconstructed throughout the cardiac cycle to quantify cell motion using automated algorithms for cell displacement and regional deformation. Post‐mortem immuno‐histology were performed for myocardial macrophages (CD68), capillary density and cross‐sectional area (wheat‐germ‐agglutin).ResultsApoE−/−‐HFD hearts underwent hypertrophy compared to WT‐ND with increased heart weight‐to‐tibial length ratio (13±1.1 vs 10±0.8), left ventricle wall thickness (1.13±0.06 mm vs 1.07±0.03 mm) and myocyte cross‐sectional area (387±22.1 mm2 vs 278±16.6 mm2, p<0.05 for all), while ejection fraction remained preserved (59±3% vs 66±3%). Intravital MPM demonstrated that cardiomyocytes move a greater total distance during each cardiac cycle in ApoE−/−‐HFD vs WT‐ND. Maximum displacement in the apex‐base and anterior‐posterior directions increased by 56% in ApoE−/−‐HFD compared to WT‐ND (32±11 mm vs 18±6 mm), whereas regional absolute deformation remains similar between groups. R6g+ leukocytes were visible moving in capillaries. The incidence of patrolling behavior (defined as slow moving cells, visible for longer than one heartbeat) increased in capillaries of ApoE−/−‐HFD compared to WT‐ND (3.4±0.5/min vs 0.12±0.1/min, p<0.01), while the incidence of flowing (visible for less than one heartbeat) and non‐flowing (visible for 500 heartbeats) remained similar. Myocardial CD68+ macrophages increased (780±121/mm2 vs 89±20/mm2, p<0.0001) and capillary density decreased (3271±167/mm2 vs 3886±105/mm2, p=0.0067) in ApoE−/ −‐HFD hearts compared to WT‐ND in post‐mortem sections.ConclusionIntravital cardiac MPM provides a new perspective to study cardiac hypertrophy by capturing the simultaneous contributions of inflammatory cells and cardiomyocyte function in the beating heart. These results suggest that hypertrophied cardiomyocytes increase overall tissue motion to compensate for unchanged cardiomyocyte contraction to maintain a healthy ejection fraction. Increased capillary leukocyte patrolling behavior may promote myocardial hypertrophy.Support or Funding InformationAHA17POST33680127, NSFDBI1707312, NIH5R21EB02469403