Intravital Microscopy of the Beating Murine Heart to Understand Cardiac Leukocyte Dynamics

Allan-Rahill, Nathaniel H.; Lamont, Michael R. E.; Chilian, William M.; Nishimura, Nozomi; Small, David M.

doi:10.3389/fimmu.2020.00092

Cited by 11 publications

(8 citation statements)

References 107 publications

(126 reference statements)

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“…A left thoracotomy was performed as above and a 3D-printed titanium stabilization probe with imaging window ( Allan-Rahill et al, 2020 ; Jones et al, 2018 ) attached to the left ventricle free-wall using tissue adhesive (Vetbond). Texas Red-conjugated 70 kDa dextran (3% in saline; Thermo Fisher Scientific #D1830) was injected retro-orbitally to identify the vasculature.…”

Section: Methodsmentioning

confidence: 99%

Genetically engineered mice for combinatorial cardiovascular optobiology

Lee

Shui

et al. 2021

eLife

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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.

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Section: Methodsmentioning

confidence: 99%

Genetically engineered mice for combinatorial cardiovascular optobiology

Lee

Shui

et al. 2021

eLife

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“…A left thoracotomy was performed as above and a 3D-printed titanium stabilization probe with imaging window (Allan-Rahill, Lamont, Chilian, Nishimura, & Small, 2020; Jones, Small, & Nishimura, 2018) attached to the left ventricle free-wall using tissue adhesive (Vetbond). Texas-Red conjugated 70-kDa dextran (3% in saline; Thermo Fischer Scientific #D1830) was injected retro-orbitally to identify the vasculature.…”

Section: Methodsmentioning

confidence: 99%

Genetically Engineered Mice for Combinatorial Cardiovascular Optobiology

Lee

Shui

et al. 2021

Preprint

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RationaleOptogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. The effective combination of these optical tools in mice requires designed genetic models that are optically compatible and efficiently assembled. We designed strains for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology.ObjectiveTo address this lack of optogenetic resources by combining lineage-specific expression of optogenetic effectors and sensors in single mouse lines and demonstrating their utility.Methods and ResultsTwenty-two lines of mice were created in which optogenetic effectors (11 lines) or Ca2+ sensors (11 lines) were expressed in a lineage-specific manner in cardiac pacemaker cells (SAN), cardiomyocytes, vascular endothelial and smooth muscle cells, as well as other cell types. We show functional responses associated with optical formation of the second messengers InsP3 (optoα1AR) and cAMP (optoβ2AR), or Ca2+-permeant membrane channels (CatCh2), in targeted cells in these mice. Green (GCaMP5, 8 and 8.1) and red (RCaMP 1.07) Ca2+ sensors were chosen to enable simultaneous fluorescent detection of cellular responses. Biallelic crosses were created with robust expression of sensor/effector pairs. We present examples of novel in vivo and ex vivo experiments in selected strains including cardiac pacing with stimulation of HCN4 conduction cells, intravital cardiac imaging of GCaMP8 with two-photon microscopy, optical vascular dilation/constriction by smooth muscle (Acta2) and vasodilation by light activation of endothelium (Cdh5). These experiments highlight the potential of these mice to uncover new insight into mechanisms regulating cardiovascular function.ConclusionsThese new mouse lines efficiently express optical effectors and sensors in heart, blood vessels, and other tissues and can be efficiently combined enable novel in vivo and ex-vivo studies and can be combined or crossed with relevant cardiovascular disease models, constituting a new toolbox for cardiovascular biology.

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“…MPM-based intravital imaging of the ventricular wall has been performed (15,16,99,100); however, in vivo imaging of valves faces challenges due to accessibility, tissue movement, and blood flow (28,97,(101)(102)(103)(104). Limitations of tissue accessibility are being addressed by enhancing flexibility and miniaturization of microendoscopy tools, which will help facilitate preclinical and clinical translation of MPM (28,(101)(102)(103)105).…”

Section: Challenges Associated With Clinical Translation and Future Directionsmentioning

confidence: 99%

Label-Free Multiphoton Microscopy for the Detection and Monitoring of Calcific Aortic Valve Disease

Tandon

Quinn

Balachandran

2021

Front. Cardiovasc. Med.

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Calcific aortic valve disease (CAVD) is the most common valvular heart disease. CAVD results in a considerable socio-economic burden, especially considering the aging population in Europe and North America. The only treatment standard is surgical valve replacement as early diagnostic, mitigation, and drug strategies remain underdeveloped. Novel diagnostic techniques and biomarkers for early detection and monitoring of CAVD progression are thus a pressing need. Additionally, non-destructive tools are required for longitudinal in vitro and in vivo assessment of CAVD initiation and progression that can be translated into clinical practice in the future. Multiphoton microscopy (MPM) facilitates label-free and non-destructive imaging to obtain quantitative, optical biomarkers that have been shown to correlate with key events during CAVD progression. MPM can also be used to obtain spatiotemporal readouts of metabolic changes that occur in the cells. While cellular metabolism has been extensively explored for various cardiovascular disorders like atherosclerosis, hypertension, and heart failure, and has shown potential in elucidating key pathophysiological processes in heart valve diseases, it has yet to gain traction in the study of CAVD. Furthermore, MPM also provides structural, functional, and metabolic readouts that have the potential to correlate with key pathophysiological events in CAVD progression. This review outlines the applicability of MPM and its derived quantitative metrics for the detection and monitoring of early CAVD progression. The review will further focus on the MPM-detectable metabolic biomarkers that correlate with key biological events during valve pathogenesis and their potential role in assessing CAVD pathophysiology.

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Intravital Microscopy of the Beating Murine Heart to Understand Cardiac Leukocyte Dynamics

Cited by 11 publications

References 107 publications

Genetically engineered mice for combinatorial cardiovascular optobiology

Genetically engineered mice for combinatorial cardiovascular optobiology

Genetically Engineered Mice for Combinatorial Cardiovascular Optobiology

Label-Free Multiphoton Microscopy for the Detection and Monitoring of Calcific Aortic Valve Disease

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