Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage-specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca2+ sensors (10 lines) were selectively expressed in cardiac pacemaker cells, cardiomyocytes, vascular endothelial and smooth muscle cells, alveolar epithelial cells, lymphocytes, glia, and other cell types. Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP3 (optoα1AR) and cAMP (optoß2AR), or Ca2+-permeant membrane channels (CatCh2) in smooth muscle (Acta2) and endothelium (Cdh5). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca2+ imaging in Hcn4BAC-CatCh2/Myh6-GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.

Cardiovascular disease is the leading cause of worldwide mortality. Intravital microscopy has provided unprecedented insight into leukocyte biology by enabling the visualization of dynamic responses within living organ systems at the cell-scale. The heart presents a uniquely dynamic microenvironment driven by periodic, synchronous electrical conduction leading to rhythmic contractions of cardiomyocytes, and phasic coronary blood flow. In addition to functions shared throughout the body, immune cells have specific functions in the heart including tissue-resident macrophage-facilitated electrical conduction and rapid monocyte infiltration upon injury. Leukocyte responses to cardiac pathologies, including myocardial infarction and heart failure, have been well-studied using standard techniques, however, certain questions related to spatiotemporal relationships remain unanswered. Intravital imaging techniques could greatly benefit our understanding of the complexities of in vivo leukocyte behavior within cardiac tissue, but these techniques have been challenging to apply. Different approaches have been developed including high frame rate imaging of the beating heart, explantation models, micro-endoscopy, and mechanical stabilization coupled with various acquisition schemes to overcome challenges specific to the heart. The field of cardiac science has only begun to benefit from intravital microscopy techniques. The current focused review presents an overview of leukocyte responses in the heart, recent developments in intravital microscopy for the murine heart, and a discussion of future developments and applications for cardiovascular immunology.

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.

Background The 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. Methods ApoE−/− 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). Results ApoE−/−‐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. Conclusion Intravital 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 Information AHA17POST33680127, NSFDBI1707312, NIH5R21EB02469403

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