Hypoxia causes connexin 43 internalization in neonatal rat ventricular myocytes.
Gap junctions produce low resistance pathways between cardiomyocytes and are major determinants of electrical conduction in the heart. Altered distribution and function of connexin 43 (Cx43), the major gap junctional protein in the ventricles, can slow conduction, and thus contribute to arrhythmogenesis in experimental models such as ischemic rat heart and pacing-induced atrial fibrillation. The mechanisms underlying reduced gap junctional density and conductance during ischemia may involve decreased Cx43 synthesis, increased degradation and/or Cx43 migration into areas which do not contribute to intercellular communication. To test more rigorously the hypothesis that hypoxia resulting from ischemia causes Cx43 internalization, we infected neonatal rat ventricular myocytes (NRVM) with a Cx43-GFP chimera, which enabled us to investigate by means of confocal microscopy the effect of hypoxia (1% O2 for 5 h) on Cx43 distribution in live cardiomyocytes. Importantly, this protocol permitted each culture to serve as its own control. To this end we used life confocal microscopy analysis to determine in the same pair of myocytes the effects of hypoxia on Cx43-GFP distribution at the gap junctional (GJ) and non-GJ areas. In support of this hypothesis, we found that compared to normoxia, 5 h of hypoxia reduced the Cx43-GFP signal at the GJ areas (defined as the border area) and caused a corresponding increase in the Cx43-GFP signal at the non-border areas, thus providing an additional explanation for the intercellular uncoupling during ischemic conditions.
Danon
Asaf
A
Department of Physiology, Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.
Zeevi-Levin
Naama
N
Pinkovich
Dani Y
DY
Michaeli
Tomer
T
Berkovich
Alexander
A
Flugelman
Moshe
M
Eldar
Yonina C
YC
Rosen
Michael R
MR
Binah
Ofer
O
D000818:Animals; D000831:Animals, Newborn; D001693:Biological Transport, Active; D015687:Cell Hypoxia / Q000502:physiology*; D002478:Cells, Cultured; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D032383:Myocytes, Cardiac / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D018031:Connexin 43; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins