In this study we have identified Cx43 to be localized on mitochondria of RRECs, predominately in the inner mitochondrial membrane, and that it is downregulated by HG, which in turn, triggers cytochrome c release in the RRECs. Furthermore, we observed that mitochondrial Cx43 channel activity is necessary for maintenance of mitochondrial morphology, and importantly in isolated mitochondria inhibition of mitochondrial Cx43 channel activity–induced cytochrome c release. Results also indicate that HG-induced mitochondrial morphology changes in retinal vascular cells, which we previously reported,6
may be associated with dysfunction of mitochondrial Cx43 channel activity. While it is evident that HG-induced mitochondrial morphology changes and cytochrome c release leads to apoptosis,14
the underlying mechanisms of mitochondrial morphology disruption remain unclear. Our findings presented in this study suggest HG-induced mitochondrial Cx43 downregulation and reduced channel activity may contribute to cytochrome c release and promote apoptosis.
Several studies have shown that mitochondrial dysfunction plays a critical role in diabetes-induced apoptosis.1,3,24,25
Cx43 has been reported to be downregulated by HG and in diabetes where the canonical role of Cx43 as a gap junction channel is compromised, leading to reduced gap junction intercellular communication activity and triggering apoptosis.10–14
Here, we show that Cx43 located on the inner membrane is decreased by HG, which may result in overall decreased channel activity, provoking the release of cytochrome c. Presumably, these Cx43 on the mitochondrial inner membrane are functioning as hemi-channels (connexons); however, the limited studies on mitochondrial Cx43 have not ruled out other possible docked Cx43 channel activity in the mitochondrial membranes, or its functional role through interacting with key mitochondrial proteins.26
Cx43 hemi-channels on the plasma membrane of cells facilitate Na+ and Ca++ exchange between cells and the environment, H+ regulation and plasma membrane polarization, and other functions related to cell survival.27
Similarly, Cx43 hemi-channels in the mitochondrial inner membrane are involved in regulating mitochondrial ion exchange or mitochondrial volume and swelling.16
Other studies have shown mitochondrial Cx43 upregulation to promote ischaemic preconditioning, where mitochondrial ion regulation and swelling are established players in the preconditioning process.18
In addition, respiration defect was observed in isolated mitochondria from Cx43-deficient mice15
; thus, findings from these studies and our current study point to the important role of Cx43 in normal mitochondrial functioning.
Mechanisms for Cx43 intracellular trafficking is not completely understood at this present time. Our studies using plasmid–Cx43-GFP showed a majority of the Cx43 plaques would localize on the cell plasma membrane, whereas a small percentage (<5%) would localize on the mitochondria. This difference in the distribution of Cx43 localization on mitochondria has been verified by more quantitative studies in rat myocardium.26,28
In retinal endothelial cells, overall Cx43 protein level both in the mitochondria and cell surface is significantly reduced under HG condition10
; however, it is unclear whether HG alters this ratio. Presumably, an imbalance between the levels of Cx43 between the plasma membrane and the mitochondria may disrupt normal ion homeostasis. Cx43 is imported into the mitochondria by the transporter outer membrane (TOM) and transporter inner membrane (TIM) mitochondrial import machinery, and currently, it is believed that a certain percentage of the total Cx43 is targeted to the mitochondria.17
Further studies are needed to better understand Cx43 targeting to the mitochondria versus the cell membrane, and the consequences resulting from an imbalance between the two compartments.
Recent studies have stressed the importance of mitochondrial morphology changes and its association with disease processes. In diabetes, mitochondrial morphology changes have been shown to induce mitochondrial dysfunction through mitochondrial morphology changes in pancreatic islets,29
coronary endothelial cells,32
and retinal endothelial cells6
Maintenance of normal mitochondrial morphology has been shown to regulate several aspects of mitochondrial functioning, including mitochondrial metabolism, reactive oxygen species production, and induction of apoptosis.4
Importantly, our previous studies have shown how mitochondrial fragmentation under HG condition promotes mitochondrial oxygen consumption defects and increased apoptosis of both retinal endothelial cells6
two cell types that undergo cell death in the early stages of diabetic retinopathy.33
In a diabetic animal model we have observed Cx43 downregualtion in the retina and subsequent retinal pathology similar to those seen in human diabetic retinopathy.13
Here we present evidence that inhibition of normal mitochondrial Cx43 channel activity can lead to mitochondrial fragmentation, similar to the HG-induced mitochondrial morphology changes. This may point to an important role for HG-induced mitochondrial Cx43 inhibition in promoting mitochondrial fragmentation and subsequent apoptosis of retinal microvascular cells.
In this study we present data demonstrating the localization and important functional role of Cx43 on the mitochondria of retinal endothelial cells. As of yet, it is still unclear what is the functional role of Cx43 in the mitochondria, and whether it forms hemi-channels and what are the key molecules that are being exchanged through this channel. Only recently, Cx43 has been identified on the mitochondria of different cell types. It is clear that mitochondrial Cx43 plays an important role in mitochondrial function, specifically regulation of apoptosis.8,26
Our data demonstrates that HG is a critical player that drives mitochondrial Cx43-mediated cellular disturbances. Identifying means to improve proper functioning of Cx43 channel activity on mitochondria may hold therapeutic promise for treatment of diabetic retinopathy.