Remodeling of mechanical junctions and of microtubule-associated proteins accompany cardiac connexin43 lateralization

Abstract

Background: Desmosomes and adherens junctions provide mechanical continuity between cardiac cells, whereas gap junctions allow for cell-cell electrical/metabolic coupling. These structures reside at the cardiac intercalated disc (ID). Also at the ID is the voltage-gated sodium channel (VGSC) complex. Functional interactions between desmosomes, gap junctions, and VGSC have been demonstrated. Separate studies show, under various conditions, reduced presence of gap junctions at the ID and redistribution of connexin43 (Cx43) to plaques oriented parallel to fiber direction (gap junction "lateralization").

Objective: To determine the mechanisms of Cx43 lateralization, and the fate of desmosomal and sodium channel molecules in the setting of Cx43 remodeling.

Methods: Adult sheep were subjected to right ventricular pressure overload (pulmonary hypertension). Tissue was analyzed by quantitative confocal microscopy and by transmission electron microscopy. Ionic currents were measured using conventional patch clamp.

Result: Quantitative confocal microscopy demonstrated lateralization of immunoreactive junctional molecules. Desmosomes and gap junctions in lateral membranes were demonstrable by electron microscopy. Cx43/desmosomal remodeling was accompanied by lateralization of 2 microtubule-associated proteins relevant for Cx43 trafficking: EB1 and kinesin protein Kif5b. In contrast, molecules of the VGSC failed to reorganize in plaques discernable by confocal microscopy. Patch-clamp studies demonstrated change in amplitude and kinetics of sodium current and a small reduction in electrical coupling between cells.

Conclusions: Cx43 lateralization is part of a complex remodeling that includes mechanical and gap junctions but may exclude components of the VGSC. We speculate that lateralization results from redirectionality of microtubule-mediated forward trafficking. Remodeling of junctional complexes may preserve electrical synchrony under conditions that disrupt ID integrity.

Figure 1

Localization of connexin43 (Cx43) and plakoglobin (PG). Confocal microscopy images from right ventricular tissue of control sheep (CNTR) (A, E, G) or sheep with pulmonary hypertension (PH) (B, F, H). Immunoreactive Cx43 (red) and PG (green) are presented separately (A, B), and (E, F) in merged images (G, H). a, a′, a″ enlarged images of Cx43 and PG along the long axis. Bar = 20 μm. C and D: Quantification plots representing percentage of Cx43 plaques found in a given orientation with respect to fiber direction in tissue CNTR (C) or PH (D). Total number of plaques: 84 (CNTR) and 403 (PH). I: Colocalization (Pearson’s coefficient) of PG and Cx43 in plaques oriented perpendicular (intercalated disc [ID]) or parallel (lateral membrane [LM]) to fiber orientation. Statistical analysis: 1-way analysis of variance, Tukey’s multiple comparison test. Mean ± SEM. P: >.05 (ns); .001–.0001 (***). Numbers of regions of interest analyzed: 48, 60, and 60 for ID CNTR, ID PH, and LM PH, respectively. SEM, standard error of the mean.

Figure 2

Electron microscopy image obtained from the right ventricle of a sheep afflicted with pulmonary hypertension. Low-magnification image = 2600× (A), shows preservation of structures and orientation of cells. Area within the red square is shown in B, at higher magnification = 34,000×. Notice the presence of lateralized desmosomes and gap junctions oriented parallel to fiber direction.

Figure 3

Nav1.5 localization in PH hearts. A: Confocal microscopy images obtained from CNTR or PH sheep. Nav1.5 (red), Cx43 (green). a: Enlarged area of intercalated disc (ID) from CNTR animal. Notice colocalization of Nav1.5 and Cx43 highlighted by yellow in merged image. b: Enlarged area of Cx43 with Nav1.5 along the long axis in PH tissue. Notice absence of immunoreactive Nav1.5 where immunoreactive Cx43 is present. c: Enlarged images of Cx43 and Nav1.5 of 2 IDs. Notice presence of immunoreactive Nav1.5 (red) in “left” ID and its loss in “right” one. Scale bar = 20 μm. B: Quantification of colocalization (Pearson’s coefficient) of Nav1.5 and Cx43 signals at areas of ID in CNTR and PH animals and at the lateral membrane (LM) of PH animals. Statistical analysis: 1-way analysis of variance, Tukey’s multiple comparison test. Mean ± SEM. P: .001–.0001 (***). Numbers of regions of interest analyzed: N = 61, 108, and 50 for ID CNTR, ID PH, and LM PH, respectively. C: Comparison of Pearson coefficient values for colocalization of Cx43 with various molecules (noted in abscisae) at LM in PH hearts. Analysis of variance-Tukey’s multiple comparison test showed that the value obtained for Cx43–Nav1.5 colocalization was highly different from that obtained for all other junctional molecules (P <.0001). CNTR = control; DP = desmoplakin; DSC = desmocollin; DSG = desmoglein; Cx43 = connexin43; N-Cad = cadherin; PG = plakoglobin; PH = pulmonary hypertension; PKP2 = plakophilin-2; SEM, standard error of the mean.

Figure 4

Electrophysiological changes. Electrophysiological analysis of ventricular myocytes dissociated from PH-afflicted sheep hearts. A–D: Peak average sodium current density (A), steady-state activation (B), steady-state inactivation (C), and recovery from inactivation kinetics (D) in ventricular myocytes dissociated from right ventricle (PH RV, blue) or left ventricle (CTL LV, black) of PH-afflicted sheep hearts, as well as from the RV of a control animal (CT LRV, red). E: Junctional conductance measured from cell pairs obtained from either the LV or the RV of control (blue) or PH-afflicted animals (yellow). PH = pulmonary hypertension.

Figure 5

Localization of EB1. Confocal microscopy image obtained from right ventricular tissue of CNTR or PH sheep. EB1 (green), Cx43 (red), nuclei (TO-PRO-3, blue). a and b: Enlarged areas of intercalated disc in CNTR (a) and PH (b) tissue. Notice that the area is rotated 90° clockwise in (a). Scale bar = 20 μm. CNTR = control; Cx43 = connexin43; PH = pulmonary hypertension.

Figure 6

Redirection of Kinesin-1 to the lateral membrane. A: Confocal microscopy image obtained from right ventricular tissue of CNTR or PH sheep. Kif5b (green), Cx43 (red), nuclei (TO-PRO-3, blue). a: Enlarged image of Cx43 with Kif5b at the intercalated disc in CNTR. b: Enlarged image of Cx43 with Kif5b along the long axis. Scale bar = 20 μm. B: Localization of Kif5b. TIRF microscopy images obtained from freshly isolated mouse ventricular cardiomyocyte. Notice colocalization of immunoreactive Kif5b and Cx43 (merged panel at the bottom, from box labeled “c”). Scale bar = 5 μm. CNTR = control; Cx43 = connexin43; PH = pulmonary hypertension; TIRF = total internal reflection fluorescence.