Journal: The Journal of Clinical Investigation

Article Title: In vivo visualization and molecular targeting of the cardiac conduction system

doi: 10.1172/JCI156955

Figure Lengend Snippet: ( A ) Antibody-dye conjugate (mCntn2-800) consists of a near-infrared (NIR) dye conjugated to an antibody against the CCS-specific surface marker Cntn2 (contactin 2). ( B ) Experimental work flow. ( C ) Whole-body biodistribution of other tissue types, showing expected clearance within the liver, bladder, and kidneys and notable absence from the brain. ( D ) Whole mouse heart ( n = 3) from a wild-type (WT) mouse injected 3 days prior with mCntn2-800, 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: NIR signal demonstrating labeling of the CCS (blue→red = lowest→highest signal). Mean signal to background ratio (SBR) is indicated. ( E ) Measured intervals (in ms) from sedated surface electrocardiograms (ECGs) including PR, QRS, QTc, and RR in WT mice prior to (day 0 = baseline, n = 12) and daily (day 1 n = 3, day 2 n = 9; after injection) following a single tail vein injection of mCntn2-800. Intervals (mean ± SD) 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. ( F – H ) Heart sections from a WT mouse injected 2 days prior with mCntn2-800. CCS components labeled with mCntn2-800 (purple) and costained with antibodies targeting known markers of the CCS, including anti-Hcn4 (red, SAN and His) or anti-Cx40 (green, PF). DAPI (blue, nuclei). LBB/RBB, left and right bundle branches; His, His bundle; PF, Purkinje fibers; SAN, sinoatrial node; VM, ventricular myocardium. Scale bars: 10 mm ( C ), 5 mm ( D ), and 100 μm ( F – H ).

Article Snippet: A phage display system was used (Promab Biotech, Inc) to screen for monoclonal Fab antibodies targeting the human CNTN2-His recombinant protein (Acro Biosystems, CN2-H5226).

Techniques: Marker, Injection, Labeling

Journal: The Journal of Clinical Investigation

Article Title: In vivo visualization and molecular targeting of the cardiac conduction system

doi: 10.1172/JCI156955

Figure Lengend Snippet: ( A ) Experimental workflow: wild-type mice ( n = 3) received a tail vein injection of mCntn2-800 and after 48 hours were sedated and received a sternotomy and cardiac incisions including a right atriotomy and right ventriculotomy to simulate a surgical scenario. Live imaging of the heart with a FLARE Intraoperative Near-Infrared (NIR) Fluorescence Imaging System. ( B and C ) Heart with visible sinoatrial node (SAN), atrioventricular node/His bundle (AVN/His), and Purkinje fiber (PF) network. Left: Color image of ex vivo heart. Right: mCntn2-800 NIR signal (green). Middle: Merged image of color image and NIR (green) signal. ( D ) Heart sections from the same heart, demonstrating mCntn2-800 signal (cyan) labeling the SAN, as costained with anti-Cntn2 (red) immunostaining. ( E ) Magnified region in SAN indicated by the white box in D . Right atrial (RA) tissue labeled with anti-Cx43 (purple). DAPI (blue, nuclei). IVS, interventricular septum; RV, right ventricle; SAN, sinoatrial node; SVC, superior vena cava. Scale bars: 5 mm ( B and C ), 300 μm ( D ), and 100 μm ( E ).

Article Snippet: A phage display system was used (Promab Biotech, Inc) to screen for monoclonal Fab antibodies targeting the human CNTN2-His recombinant protein (Acro Biosystems, CN2-H5226).

Techniques: Injection, Imaging, Fluorescence, Ex Vivo, Labeling, Immunostaining

Journal: The Journal of Clinical Investigation

Article Title: In vivo visualization and molecular targeting of the cardiac conduction system

doi: 10.1172/JCI156955

Figure Lengend Snippet: ( A ) Human anti-CNTN2 Fab antibody was biotinylated and conjugated to streptavidin-linked saporin, a cell toxin (hCNTN2-Sap). ( B ) Wild-type mice received a single tail vein injection of either hCNTN2-Sap (100 μg) ( n = 6) or Control-Sap (100 μg nonspecific human IgG similarly conjugated to saporin) ( n = 6). Mice received electrocardiograms (ECGs) on day 0 (baseline) and daily following injection with control or hCNTN2-Sap. On day 2, hearts were harvested, fixed, and immunostained. ( C ) Representative ECG tracings ( n = 6 per condition). ( D ) By day 2, mice injected with hCNTN2-Sap demonstrated marked conduction abnormalities, including prolonged PR, QRS, and RR intervals as compared with mice injected with Control-Sap (mean intervals in ms ± SD). Gray bar, QTc interval corrected for QRS intervals. Statistical analyses using 2-way ANOVA with Tukey’s post hoc test. ( E ) Consistent with targeted cell death, immunofluorescence of the CCS (red) showed subtotal loss of CCS cells as shown within the His bundle (His), right and left bundle branches (RBB/LBB) as stained by anti-Cntn2. His, His bundle; IVS, interventricular septum; LBB, left bundle branch; RBB, right bundle branch. Scale bar: 50 μm.

Article Snippet: A phage display system was used (Promab Biotech, Inc) to screen for monoclonal Fab antibodies targeting the human CNTN2-His recombinant protein (Acro Biosystems, CN2-H5226).

Techniques: Injection, Immunofluorescence, Staining