In vivo visualization and molecular targeting of the cardiac conduction system
William R. Goodyer, … , Eben L. Rosenthal, Sean M. Wu
William R. Goodyer, … , Eben L. Rosenthal, Sean M. Wu
Published August 11, 2022
Citation Information: J Clin Invest. 2022;132(20):e156955. https://doi.org/10.1172/JCI156955.
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Research Article Cardiology

In vivo visualization and molecular targeting of the cardiac conduction system

Abstract

Accidental injury to the cardiac conduction system (CCS), a network of specialized cells embedded within the heart and indistinguishable from the surrounding heart muscle tissue, is a major complication in cardiac surgeries. Here, we addressed this unmet need by engineering targeted antibody-dye conjugates directed against the CCS, allowing for the visualization of the CCS in vivo following a single intravenous injection in mice. These optical imaging tools showed high sensitivity, specificity, and resolution, with no adverse effects on CCS function. Further, with the goal of creating a viable prototype for human use, we generated a fully human monoclonal Fab that similarly targets the CCS with high specificity. We demonstrate that, when conjugated to an alternative cargo, this Fab can also be used to modulate CCS biology in vivo, providing a proof of principle for targeted cardiac therapeutics. Finally, in performing differential gene expression analyses of the entire murine CCS at single-cell resolution, we uncovered and validated a suite of additional cell surface markers that can be used to molecularly target the distinct subcomponents of the CCS, each prone to distinct life-threatening arrhythmias. These findings lay the foundation for translational approaches targeting the CCS for visualization and therapy in cardiothoracic surgery, cardiac imaging, and arrhythmia management.

Authors

William R. Goodyer, Benjamin M. Beyersdorf, Lauren Duan, Nynke S. van den Berg, Sruthi Mantri, Francisco X. Galdos, Nazan Puluca, Jan W. Buikema, Soah Lee, Darren Salmi, Elise R. Robinson, Stephan Rogalla, Dillon P. Cogan, Chaitan Khosla, Eben L. Rosenthal, Sean M. Wu

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Figure 7

Systemic injection of mNptn-800 in mice safely labels the CCS in vivo.

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Systemic injection of mNptn-800 in mice safely labels the CCS in vivo. (...
(A) Experimental work flow. (B) Whole hearts from a wild-type (WT) mouse injected 1 day prior with mNptn-800 (n = 3) or IgG-800 (n = 3). Heart shown in posterior-anterior (PA) and right lateral (RL) views. Atria are outlined in white and cardiac chambers are listed. LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. Top: Brightfield. Bottom: Near infrared (NIR) signal demonstrating labeling of the CCS (blue→red = lowest→highest signal). Mean signal to background ratio (SBR) is indicated. Bar graph showing mean SBR in hearts exposed to 150 μg of either mNptn-800 or IgG-800 (control) (n = 3 each). Mean ± SD shown. Statistical analyses using 2-tailed, unpaired Student’s t test. (C and D) Sedated surface electrocardiograms (ECGs) with measured intervals (in ms) including PR and QRS in WT mice prior to (day 0 = baseline) and daily (day 1, day 2, day 3 = after injection) following a single tail vein injection of mNptn-800 (150 μg). n = 3 for all time points. Intervals on a given day after injection were compared to each mouse’s preinjection control baseline (day 0) using 1-way ANOVA with Tukey’s post hoc test. (E) Whole-body biodistribution of other tissue types, showing expected clearance within the liver and kidneys. (F–I) Heart sections from adult mice (n = 3) injected 1 day prior with mNptn-800. (F) Compact SAN (cSAN) labeled by mNptn-800 signal (red) and absence of Cx40 (green) as opposed to Cx40+ right atrial myocardium (RA). (G) AVN labeled with mNptn-800 (red) and consistently lacking Cx40 (green) expression. (H) His bundle (His) and proximal bundle branch (Prox BB) and (I) Purkinje fibers (PF) costained for Cx40 (green) and mNptn-800 signal (red). mNptn-800 signal was amplified using an anti-sheep 555 nm secondary antibody following tissue fixation. DAPI (blue, nuclei). IVS, interventricular septum; VM, ventricular myocardium. Scale bars: 5 mm (B), 100 μm (F and G), and 50 μm (H and I).