Ischemia reperfusion injury (IRI) is one of the main cause of organ dysfunction after heart attack and solid organ transplantation, and occurs frequently when the new organ comes from a deceased individual, a major source of transplanted organs. Indeed, once organ perfusion has ceased, hypoxia increases with time and the reperfusion results in severe tissue damage. The molecular mechanisms underlying this process are poorly understood.
Dr. Paul Lampe’s lab (Public Health Sciences and Human Biology divisions) has a long-standing interest in gap junction biology. Gap junctions are an assembly of intercellular channels that allow cell-to-cell communication, provide a signaling scaffold and are involved in cellular responses to injury. Connexin-43 (Cx43) is the most prevalent gap junction protein and gap junction formation and stability are regulated through interaction of Cx43 with many kinases. For instance, Cx43 can be phosphorylated by Casein Kinase 1 (CK1) to increase the efficiency of gap junction assembly, while phosphorylation by ERK or PKC can destabilize gap junctions. Dr. Joell Solan and colleagues from the Lampe lab recently unraveled a mechanism involving Cx43 in myocardium IRI and published their results in the Journal of Biology and Chemistry.
To understand the involvement of CK1 and Cx43 in IRI, the authors used a mouse model in which all serines usually phosphorylated by CK1 were mutated to alanine (Cx43CK1) and compared the hearts response to ischemia and reperfusion in Cx43CK1 compared to wild-type (WT) animals. As aging also influences IRI, both young (3-6 months) and old (more than 12 months) animals were investigated. To simulate IRI, the researchers excised mouse hearts, cannulated the aorta and perfused the organ in a Langendorff System at constant pressure (80 mm Hg) with an oxygenated solution. After letting the heart stabilize for 30 minutes, the oxygenated flow was stopped for 30 minutes followed by reperfusion for two hours. The extent of tissue damage or myocardial infarct (MI) was assessed by staining heart slices with the vital dye, triphenyl tetrazolium chloride (TTC), and quantifying the area of staining to determine the area of live tissue compared to total tissue.
While old WT hearts show a decreased amount of healthy, live tissue after ischemia compared to the young WT hearts, old Cx43CK1 hearts had a comparable amount of live tissue relative to young hearts (either WT or Cx43CK1). The authors hypothesized that this effect could involve the ERK and Akt pathways, kinases that interact with Cx43 and are known to be involved in survival during IRI. Immunoblotting of protein extracts from hearts of young and old WT and Cx43CK1 animals revealed that in the absence of CK1-mediated phosphorylation of Cx43, phosphorylation of Cx43 by ERK is increased and ERK itself showed higher levels of activation. In vitro experiments of single amino acid mutagenesis and GST-pulldown assay demonstrated that phosphorylation of Cx43 at Serine 330 by CK1 prevents the interaction with ERK. They also found that Akt downstream signaling was downregulated in old WT hearts compared to their young counterparts, whereas young and old Cx43CK1 hearts maintained a high level of Akt signaling. Further, in the absence of CK1-mediated Cx43 phosphorylation, Cx43 interacted with NDRG1, a stress-inducible protein downstream of Akt that promotes survival. This interaction stabilized NDRG1 from proteosomal degradation, consistent with the protection from IRI observed ex vivo and the biological function of NDRG1. Thus the interaction of Cx43 with ERK and NDRG1 in absence of CK1-mediated phosphorylation is critical for the cells to survive ischemia/reperfusion.
The authors continue teasing apart the precise molecular mechanism to understand the importance of gap junction equilibrium in response to cellular stress. Dr. Joell Solan, the first author of the study, emphasizes the dynamic nature of gap junction biology in relation with their results: “ There is a clear connection between gap junction stability and many cell processes including cell survival, proliferation and migration. Factors that activate these cellular behaviors lead to a predictable sequence of events that result in a rapid pulse of gap junction growth followed by disassembly of the gap junction. This requires coordinated interaction of Akt, ERK and PKC with the gap junction protein Cx43. In this work, we showed that altering the events in this sequence can alter cell behavior. Indeed, we found that the very signaling pathways that control gap junction stability are directly controlled by changes in gap junction organization and stability (i.e. ERK and Akt). In particular, our discovery that Cx43 regulates the stability of the stress responsive protein, NDRG1, is exciting since we believe this interaction may represent a new and novel signaling node that may bridge ERK and Akt signaling.” This mechanism seems also to play a role in other pathological conditions, as explains Dr. Solan: “In addition to effects on cell survival, as we showed in this paper, we find these changes alter the quality and rate of epidermal wound healing and can increase lifespan by over 30% in a mouse model of pancreas cancer.”
This work was supported by the National Institute of Health.
Fred Hutch/UW Cancer Consortium member Dr. Lampe contributed to this research.
Solan JL., Márquez-Rosado L., Lampe PD. Cx43 phosphorylation mediated effects on ERK and Akt protect against ischemia reperfusion injury and alter stability of stress-inducible protein NDRG1. JBC. 2019. doi: 10.1074/jbc.RA119.009162