In cardiac tissue, Cx43 forms GJs primarily at the ID. A prominent feature of structural heart disease is a redistribution of Cx43 to the lateral cell borders and a decrease in Cx43 expression levels6, 7, 29
. Despite the consistency of altered Cx43 expression and distribution in structural heart disease, the functional status and structural nature of lateralized Cx43 is incompletely understood. In this study we demonstrate that GJs are formed between the lateral membranes of cardiomyocytes with increased frequency in a canine model of pacing induced HF. However, the lack of co-localization with ZO-1 and cadherin, and the heterogeneous structure of lateralized GJs demonstrate that they are structurally and likely functionally distinct from those formed at the ID.
Cx43 has been shown to be directly bound by ZO-135
and it is thought that ZO-1 in part regulates the assembly of Cx43 into the periphery of GJ plaques36
. Changes in the relationship between ZO-1 and Cx43 have been reported in cardiac disease37, 38
and is emerging as a potentially critical interaction for the maintenance of proper GJ mediated intercellular communication. Furthermore, Cx43-containing vesicles have been shown to be directly delivered to cadherin-containing adherens junctions via microtubules39
. The formation of apparently atypical GJs at cardiomyocyte lateral membranes, devoid of ZO-1 and cadherin co-localization, suggests impairment in the mechanisms responsible for direct delivery of Cx43 to sites of mechanical junction formation.
Annular GJs have been described in a number of cells and tissues14, 17, 18
including isolated cardiomyocytes15, 40
and cardiac tissue subjected to stress29, 41
. Annular GJs have been suggested to be endocytosed GJ plaques destined for lysosomal or proteasomal degradation14, 15, 19, 21, 23
. Direct evidence for the origin of annular GJs comes from the work of Jordan et al17
using time lapse studies of fluorescently tagged Cx43, and Piehl et al18
using dye injection studies to demonstrate internalization of intercellular GJ plaques into one of two coupled cells. We suggest that intracellular uni- and multi-lamellar GJ-containing membrane rings observed in cardiac tissue represent annular GJs in progressively advanced stages of processing for degradation.
The incorporation of internalized GJs into multi-lamellar membranes, their close association with cellular debris, and the co-localization of Cx43 with LC3 is suggestive of autophagy playing a role in GJ clearance. The association of internalized GJs with autophagosomes has been suggested in the literature15, 42
but is not an accepted mechanism of GJ degradation. Autophagosomes are dynamic organelles which sequester cell contents for delivery to lysosomes43
. In post-mitotic cells, such as cardiomyocytes, autophagy is constitutively active to help maintain cell size and integrity. Under stressful conditions the rate of autophagy is increased, where it serves an adaptive function by providing a source of nutrients through catabolism of “unnecessary” cellular contents, as well as clearing damaged organelles44
. Autophagy has been implicated in the pathogenesis of many diseases, including ischemia/reperfusion injury and heart failure45, 46
, as a mechanism of cell survival. Internalized GJs are quite large and structurally distinct relative to typical endocytic organelles, and therefore connexins are relatively non-standard endocytic cargo. The cellular machinery involved in GJ internalization and degradation is therefore likely to be distinct from that involved in more conventional endoctyic processes. Thus, it is plausible that GJs would be sequestered by autophagic machinery for delivery to lysosomes.
The signaling mechanisms regulating the internalization and degradation of Cx43 GJs are poorly understood, but likely involve post-translational modification of Cx43. Cx43 is phosphorylated extensively on its carboxyl-terminus, regulating both the trafficking and permeability of Cx43 GJ channels47, 48
. By SDS-PAGE the three major bands observed for Cx43 are typically referred to as P0 (fastest migrating, least phosphorylated), P1 and P2 (slowest migrating, most phosphorylated), and the P2 form only appears after Cx43 has reached the plasma membrane and formed gap junctions34
. Changes in the phosphorylation state of cardiac Cx43 have been reported in heart disease7, 29
; however, the precise changes that occur and the functional consequences of these changes are unclear. In cell-based models, pharmacological interventions which promote the phosphorylation of Cx43 to the P2 state have been shown to enhance internalization and degradation of Cx43 GJs, suggesting that hyperphosphorylation of Cx43 may be involved in GJ degradation24, 49-52
. Other studies have reported enhanced expression and GJ formation upon pharmacological treatments also likely to result in Cx43 phosphorylation26, 28
. It is likely that the specific amino acids which are phosphorylated dictate the effects that Cx43 phosphorylation has on GJs. Thus, increased Cx43 phosphorylation, or perhaps differences in the pattern of phosphorylation, may result in more rapid kinetics at multiple stages of the GJ life cycle enhancing the formation of GJs as well as their internalization and degradation, increasing the overall rate of GJ assembly and turnover. Further studies will be required to determine the effect that specific phosphorylation events have on GJ function, and to determine which of these events are altered in disease.
Cx43 has been reported to target to buoyant, cholesterol and sphingolipid-rich membrane domains termed lipid rafts (LRs)31-33
, and there is evidence that the phosphorylation state may differ between LR and non-LR targeted populations32-34, 53
. Our observation that the P2 form of Cx43 is uniquely targeted to LRs suggests that LR targeting of Cx43 is a late event in the GJ life cycle, and may be involved in GJ degradation. Consistent with this hypothesis are studies which demonstrate that connexins can target to LRs, but that intercellular GJs themselves are not LRs31
. Musil et al34
have described the relationship between Cx43 Triton solubility, phosphorylation state, and assembly into GJs in vitro
. The authors suggest that Cx43 begins as a Triton soluble, hypo-phosphorylated form during its progression towards the plasma membrane. Upon arrival at the plasma membrane Cx43 acquires Triton resistance and matures into a hyper-phosphorylated form. The authors further suggest that the acquisition of Triton resistance and maturation to the P2 phosphorylated form corresponds to the formation of mature GJ plaques. Based on our ultrastructural characterization of fraction 5 (), which contains P2 phosphorylated Cx43, we do not believe these buoyant pentalaminar membranes represent GJ plaques coupling two cells at intercalated disks, but rather internalized GJs incorporated into multi-lamellar membranes. We therefore hypothesize that maturation to the P2 form in fact corresponds to the degradation of GJs through autophagic processing, although further experiments will be required to confirm this hypothesis.
Our data demonstrate that GJs with highly variable morphology are formed between cardiomyocyte lateral membranes with increased frequency in HF, and that GJ turnover likely occurs via an autophagic pathway. The mechanisms by which Cx43 GJs are targeted for internalization and degradation by autophagy appears to involve changes in phosphorylation, as well as processing through membranes with physicochemical properties of lipid rafts. The regulated process of GJ turnover via autophagy suggests a novel pathway for the regulation of cardiac GJs, and therefore conduction in the mammalian ventricle. These findings offer insight into the mechanisms regulating electrical conduction in mammalian myocardium, and may in part contribute to the arrhythmogenic electrical remodeling associated with heart failure.
There are limitations to the present study. We did not perform serial sectioning and/or 3D reconstruction of TEM sections. It is therefore possible that apparently internalized GJs are in reality extensively convoluted GJs still attached to the plasma membrane. Regardless of this possibility, the presence of GJs at lateral membranes is nonetheless more frequent in failing hearts, and lateral GJs are more convoluted, if not more extensively internalized, than GJs at the ID.
These data were obtained in a canine model of heart failure that in many respects mimics human heart failure, but is clearly different from the most common forms of heart failure occurring in humans. The detailed regulation of Cx43 and GJ localization may not be identical with human disease. Reassuringly, many of the features of GJ remodeling characterized in this model are similar to those previously observed in diseased human hearts5, 9
Although we have provided ultrastructural, microscopic and biochemical evidence in support of GJ association with autophagosomes, we have not demonstrated connexin degradation via autophagy per se. The precise contribution of autophagy to connexin turnover will require a more quantitative examination.