Contactin-2 expression in the cardiac Purkinje fiber network

Abstract

Background: Purkinje cells (PCs) comprise the most distal component of the cardiac conduction system, and their unique electrophysiological properties and the anatomic complexity of the Purkinje fiber network may account for the prominent role these cells play in the genesis of various arrhythmic syndromes.

Methods and results: Differential transcriptional profiling of murine Purkinje fibers and working ventricular myocytes was performed to identify novel genes expressed in PCs. The most highly enriched transcript in Purkinje fibers encoded Contactin-2 (Cntn2), a cell adhesion molecule critical for neuronal patterning and ion channel clustering. Endogenous expression of Cntn2 in the murine ventricle was restricted to a subendocardial network of myocytes that also express beta-galactosidase in CCS-lacZ transgenic mice and the connexin40 gap junction protein. Both Cntn2-lacZ knockin mice and Cntn2-EGFP BAC transgenic reporter mice confirmed expression of Cntn2 in the Purkinje fiber network, as did immunohistochemical staining of single canine Purkinje fibers. Whole-cell patch-clamp recordings and measurements of Ca(2+) transients in Cntn2-EGFP(+) cells revealed electrophysiological properties indicative of PCs and distinctive from those of cardiac myocytes, including prolonged action potentials and frequent afterdepolarizations.

Conclusions: Cntn2 is a novel marker of the specialized cardiac conduction system. Endogenous expression of Cntn2 as well as Cntn2-dependent transcriptional reporters provides a new tool through which Purkinje cell biology and pathophysiology can now more readily be deciphered. Expression of a contactin family member within the CCS may provide a mechanistic basis for patterning of the conduction system network and the organization of ion channels within Purkinje cells.

Fig. 1. Identification of novel Purkinje fiber-specific transcripts by comparative microarray analysis

(A) PF⁺ and PF⁻ samples were micro dissected distal to the bundle branches (Top, arrow) from the subendocardial and subepicardial regions (Bottom) of CCS-lacZ hearts. (B) Enrichment of PF-specific transcripts in PF⁺ samples was confirmed by increased expression of the PF markers CCS-lacZ and Cx40 compared to PF⁻ samples, assessed by XGal staining (Top) and/or RT-PCR (Bottom). (C) QRT-PCR showing up-regulation of Cntn2 and the PF-specific markers CCS-lacZ (βGal) and Cx40 in micro dissected PFs. Values are expressed as Arbitrary Units (1AU= RV in PF⁻ sample). PFs, Purkinje fibers; RW, right ventricular wall; LW, left ventricular wall; RV, right ventricle; LV, left ventricle; e, endocardium; E, epicardium. Black outline, subendocardial region; Red outline, subepicardial region; IS, interventricular septum. Size bar: 200μM.

Fig. 2. Cntn2 is specifically expressed in Purkinje fibers

(A Left) Confocal image of mouse heart sections confirming that Cntn2⁺ cells (green) express the cardiac marker sarcomeric actinin (red, Left). (Inset) High power image showing an optical slice of a Cntn2⁺/sarcomeric actinin⁺ myocyte. (B Left) Epifluorescent images of CCS-lacZ hearts showing a Cntn2⁺/βGal⁺ (green/red) cell (arrowhead) under a layer of Cntn2⁻/βGal⁻ endocardial cells (arrows). (B Right) Images of CCS-lacZ heart serial sections stained for XGal (Upper) and Cntn2 (red, Lower) showing co-localization of Cntn2 and the PF-specific marker CCS-lacZ. (C) Epifluorescent image of E13.5 mouse brain used as positive control, showing Cntn2 expression (red) in the intermediate and marginal zones (arrowheads) and in the preplate region (arrows) of the ventral telenchephalon. LW, left ventricular wall; LV, left ventricle; RW, right ventricular wall; RV, right ventricle. Size bars: 20μM. A Left, Inset: 35μM.

Fig. 3. Cntn2 expression delineates the CCS in the adult heart

(A) Whole mounts (Top) and cryosections (Bottom) of adult Cntn2-lacZ (Left; XGal staining) and Cntn2-EGFP hearts (Right) showing expression of Cntn2 throughout the CCS. (B) Epifluorescent images of Cntn2-EGFP hearts confirming expression of Cntn2 (green) in the SAN (Top), AVN (Center) and PFs (Bottom), identified by anatomical landmarks and expression of the nodal marker Hcn4 (red) or the PF marker Cx40 (red). (Top) Cntn2-EGFP⁺ cells (green) were found in the SAN, localized at the junction of the VC with the right atrium and extending from the CT (0 μM), posteriorly, to the base (+40 μM), and the walls (+190 μM) of the VC, anteriorly. Cntn2 identifies a sub-population of elongated Hcn4⁺ cells in the SAN. Cntn2-EGFP⁺/Hcn4⁺ cells were also found in the AVN, 20–40 μM posteriorly to the His Bundle (Center). (Bottom) Cntn2-EGFP⁺ (green)/Cx40⁺ (red) PFs were observed in both ventricles. (C) Cntn2-EGFP transgene expression recapitulates Cntn2 protein expression as shown by co-localization of anti-Cntn2-TR antibody (red) and EGFP (green) signals in the AVN (Top) and the PFs (Bottom). (Bottom Right) Confocal image detail of PFs. His/Br, His bundle and bundle branches; AVN, atrioventricular node; SAN, sinoatrial node; lPFs, left ventricle Purkinje fibers; rPFs, right ventricle Purkinje Fibers; Ct, crista terminalis; VC, vena cava; A, aorta and pulmonary artery; arrow, SAN (in B); CCS, cardiac conduction system. Blue fluorescence: DAPI nuclear counter stain, in A (Bottom, Right,), B and C. Size bars: (A) 50μM, all except for Bottom, Left, 500μM; (B) 20μM, all except for Top, Left, 200μM, and Bottom, Left, 50μM; (C), 100μM.

Fig. 4. Cntn2 is expressed in canine Purkinje fibers

(A) Epifluorescent images (Top) of micro dissected canine Purkinje fiber (PF) bundles showing expression of Cntn2 (red, Left). In the negative control (Right) the primary antibody was omitted. (B) Confocal images of the same canine Purkinje Fiber bundles, showing expression of Cntn2 (red) on the membrane (arrowheads) of Cx40⁺ (green) canine Purkinje cells (PCs), sectioned either longitudinally (Left) or transversally (Right). Cx40⁺ intercalated disks (stars) are clearly visible at the junction of Cntn2⁺ adjacent PCs. (Inset) High power en face image of an entire intercalated disk showing Cntn2 signal at the periphery of Cx40⁺ domains with typical gap junction organization with larger fluorescent patches at the periphery and smaller areas in the center. Cntn2 (arrowhead) is also visible on the sarcolemma of two longitudinal PCs, separated by a Cx40⁺ intercalated disk (star). (C) High power confocal image of cell surface optical slice of a single canine PC confirming expression of Cntn2 on the sarcolemma, identified by expression of Cx40 (green) at the intercalated disk junctions (arrowheads). Cntn2⁺ cells co-express the cardiac marker Nav1.5 as visualized at the cell surface (D), or in a subsurface slice (E). (F) Negative control: no primary antibody. Size bars: 20μM (A, B); 10μM (C–F).

Fig. 5. Analysis of electrophysiological features of Cntn2-EGFP⁺ cells

(A) Representative phase contrast and corresponding epifluorescence micrographs of dissociated myocytes from Cntn2-EGFP transgenic hearts. Cntn2-EGFP⁺ cells can be differentiated from surrounding cardiac myocytes by their rod-shaped morphology (Left) and their Cx43⁻ (Center), Cx40⁺ (Right) phenotype. (B) Representative whole cell recordings of action potentials from Cntn2-EGFP⁻ and Cntn2-EGFP⁺ cells. (C) APs from Cntn2-EGFP⁺ myocytes demonstrated spontaneous electrical oscillations in phase 2 which resulted in EADs (Top, arrows), at lower pacing rates (1Hz), and DADs (Bottom, arrowheads), at higher pacing rates (5Hz). (D) Line-scan fluorescence images and corresponding fluorescence profiles generated by Ca²⁺ transients in Cntn2-EGFP⁺ (Right) and Cntn2-EGFP⁻ (Left) myocytes. (E) Comparison of kinetic parameters corresponding to Ca²⁺ transients in Cntn2-EGFP⁺ and Cntn2-EGFP⁻ myocytes. (F) Line plots (G) and graphs showing that Cntn2-EGFP⁺ myocytes displayed a higher frequency of unstimulated Ca²⁺ events arising from DADs compared to Cntn2-EGFP⁻ myocytes. EAD, early afterdepolarization; DAD, delayed afterdepolarization; AP, action potential. N=number of cells analyzed. VM, ventricular myocyte; PF, Purkinje Fiber; APs, action potentials; EADs, early afterdepolarizations; DADs, delayed afterdepolarizations.