Regulation of Cardiac Gap Junctional Communication: Metabolic regulation of Cx40 and Cx43 via phosphorylation

• Author / Creator

  Iden, Jason, B.

• Alterations in myocardial metabolism and cardiac electrophysiology associated with structural heart disease generate lethal arrhythmias. Differential levels of energy (ATP) supply and demand are generated with structural heart disease that is expected to activate 5’-amp activated protein kinase (AMPK) and other pathways that may contribute to an arrhythmogenic substrate in the human heart. Cardiac gap junctions (GJ) determine the level of electrical and ‘macroscopic/metabolic’ communication between heart cells. This level of conductance is one factor responsible for generating an arrhythmogenic substrate in humans and experimental animal models. Regulation of cardiac connexins by phosphorylation has been demonstrated and the cardiac GJ proteins Cx40, Cx43, and Cx45 contain putative AMPK phosphorylation sites. Initially we tested the hypothesis that rapid pacing (which also simulates atrial ventricular tachycardia) would activate AMPK and result in reduced GJ conductance and arrhythmia. These effects were readily apparent in a porcine model treated with rapid (200bpm) pacing for 3h. AMPK activity levels were increased, as were levels of (activated) phospho-AMPK. These animals exhibited atrial and ventricular arrhythmia after pacing not found in control animals. Expression levels of Cx43 (LV), and Cx40 (LA) were not significantly decreased despite the reduction in LV GJ conductance. We also show that Cx43 may be phosphorylated in vitro by AMPK but the residues affected were not identified. Due to complicating factors, we attempted to monitor Cx conductance in a variety of ‘simpler’ models so as to allow us to use viral and pharmacological methods to activate or inhibit AMPK. The models included isolated Langendorf perfused rat hearts (epicardial electrical mapping, tissue resistivity, optical mapping), in isolated neonatal cardiac myocytes (dye injections, scrape loading of dye, and growth on multi-electrode arrays), and in communication deficient cell lines transfected with Cx40 and Cx43 (dual cell patch clamp, dye injection/ transfer, and fluorescence recovery after photo-bleaching GapFRAP). These techniques have been used in the GJ literature. Of the available techniques we chose to focus on the GapFRAP method as it was cost effective, most amenable to our manipulations, and had a high rate of success. Using the GapFRAP system, we examined the effects of pharmacological AMPK activation (using AICAR, phenformin, and infection with either constitutively active or dominant negative AMPK adenovirus) on the rate of dye transfer ‘macroscopic conductance’ between pairs of Hek293 cells. We were able to show that viral infection alone caused a significant decrease in junctional conductance, and that phenformin significantly reduced Cx40 and Cx43 conductance, which was associated with a decrease in plasmalemmal Cx protein expression. AICAR had no effect on conductance indicating that this decrease was likely independent of direct phosphorylation on Cx43 or Cx40. AICAR did appear to alter the distribution of Cx40 at the membrane including some evidence for a shift in the overall phosphorylation level, and internalization associated with increased AMPK activity. To follow up, we generated stable cell lines expressing mutated Cx40, to mimic or prevent phosphorylation by converting four putative Cx40 phosphorylation sites to alanine (a) or aspartic acid (d). Based on these data we show that phospho-mimetic mutation of Cx40 in the cytoplasmic loop (CL) significantly altered Cx40 permeability. These effects were opposite in the two nearby residues (120,122), and were associated with altered expression, and in the modeled 3D structure including surface charge distribution in the pore forming regions. The data indicates that phosphorylation at one site s122 was essential for normal function, and that phosphorylation of s120 was not likely responsible for previously observed conductance increase due to cAMP activation. We also show that mutation of a highly conserved n-terminal residue t19 significantly reduces its conductance and expression at the plasma membrane. In all, we have shown that atrial or ventricular tachycardia can degenerate quite rapidly into a highly arrhythmogenic substrate. That Cx40 and Cx43 can be affected by altered metabolic states which activate AMPK and other stress response pathways, that phosphorylation of Cx43 by AMPK may occur, and we have identified three novel Cx40 phosphorylation sites, including two in the CL that can affect its function and expression. These combined results highlight the current lack of knowledge related to Cx40 modulation by phosphorylation, and the importance of expanding that knowledge with respect to the generation of arrhythmogenic substrates in the heart.

• Subjects / Keywords

  • Electrophysiology
  • Connexin
  • AMPK
  • Cardiac Electrophysiology
  • Gap junction
  • Phosphorylation
  • Arrhythmia

• Graduation date

  Fall 2015

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3VD6PG31

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.