D. Jaishankar1, M. Taylor1, Y. Lee1, K. Madavaraju1, A.P. Strouse1, D.B. Srivastava1, C. Atkinson1, S.N. Nadig1 1Northwestern University, Surgery, Chicago, ILLINOIS, USA
Introduction: The phases of heart transplantation (HTx), from procurement to preservation and implantation into recipients, inevitably result in ischemia-reperfusion injury (IRI). The first target of IRI is the endothelial cell (EC) barrier lining the blood vessels of the donor allograft. The EC barrier is damaged, allowing extravasation of immune cells into the allograft, triggering early alloimmune injury and exacerbating IRI. Therefore, protecting the EC barrier can mitigate IRI and acute alloimmune rejection. Gap junctions are membrane proteins that facilitate cell-to-cell communication. In the heart, connexin 43 (Cx43) is the predominant gap junction protein. The role of Cx43 in cardiac donor allografts during IRI in HTx is poorly understood.
Methods: We hypothesize that EC Cx43 gap junctions are reduced during IRI disrupting the EC barrier and exacerbating IRI-associated sequelae. We further hypothesize that preventing the reduction of EC Cx43 gap junction in donor allografts confers allograft protection after implantation. Firstly, to determine the protein expression of Cx43 gap junctions and evaluate the EC barrier permeability, murine microvascular cardiac EC (MCEC) were subjected to an in vitro model of IRI wherein cells undergo cold storage (CS) in a hypoxic chamber in University of Wisconsin (UW) organ preservation solution at 4oC followed by reperfusion in warm culture medium at 37oC (CS-WR). MCEC under normothermic (NT) conditions (at 37oC) served as a control. Secondly, to improve Cx43 gap junction expression and EC barrier in MCEC, we used the therapeutic peptide alpha-connexin carboxyl-terminus 1 (aCT1), which improves the stability and enhances Cx43 gap junction expression. Lastly, to demonstrate translational relevance in improving the EC barrier and mitigating IRI, donor cardiac allografts were preconditioned with aCT1 during CS and implanted into recipients using an in vivo murine heterotopic HTx model.
Results: We observed that MCEC subjected to the CS condition had significantly reduced protein expression of Cx43 at the gap junction compared to the NT condition, and the expression continued to remain reduced during CS-WR. We also observed significantly increased barrier permeability during CS-WR compared to NT, suggesting a compromised EC barrier. Since Cx43 expression was reduced during CS, we observed significantly enhanced expression of Cx43 gap junctions and improved MCEC health and barrier when aCT1 therapy was administered during CS. Similar results were recapitulated in vivo, where we observed an improved EC barrier, reduced EC death, and mitigated inflammation in donor hearts preconditioned with aCT1 during CS before HTx.
Conclusion: Collectively, our results demonstrate that Cx43 gap junction expression is reduced in EC of donor cardiac allografts, and preconditioning donor cardiac allografts with aCT1 therapy to bolster Cx43 gap junction expression can mitigate IRI after implantation and improve allograft outcomes.