Expression and regulation of connexins in cultured ventricular myocytes isolated from adult rat hearts.
Gap junctions were assayed during re-differentiation of adult rat cardiomyocytes in long-term culture to gain insight into the processes of remodeling. Double immunostaining allowed the localization of connexins Cx40, Cx43, and Cx45 between myocytes and demonstrated co-expression and co-localization in individual cells and gap junction plaques, respectively. Immunoblots showed differential time-dependent changes in connexin expression and phosphorylation. The total amount of connexins and the ratio of phosphorylated/non-phosphorylated isoforms gradually increased during the re-establishment of intercellular communication. Dual voltage-clamp studies showed the involvement of several types of gap junction channels. Multichannel currents yielded diverse spectra of g(j,inst)=f( V(j)) and g(j,ss)=f( V(j)) relationships ( g(j,inst): instantaneous gap junction conductance; g(j,ss): conductance at steady state; V(j): transjunctional voltage), indicative of homotypic and heterotypic channels. Single-channel currents revealed two prominent conductances reflecting gamma(j,main) and gamma(j,residual). The histograms of gamma(j,main) showed four discrete peaks (41-44, 59-61, 70-76, and 100-107 pS) attributable to a combination of Cx45-Cx45, Cx40-Cx45 and Cx43-Cx45 channels (1st peak), Cx43-Cx43 and Cx40-Cx43 channels (2nd peak), Cx43-Cx43 channels (3rd peak) and Cx40-Cx40 and Cx40-Cx43 channels (4th peak). However, the presence of heteromeric channels cannot be excluded. The data are consistent with an up-regulation of Cx45 and Cx43 during re-differentiation.
Polontchouk
Lioudmila O
LO
Institute for Cell Biology, ETH Hönggerberg, 8093 Zurich, Switzerland.
Valiunas
Virginijus
V
Haefliger
Jacques-Antoine
JA
Eppenberger
Hans M
HM
Weingart
Robert
R
D000367:Age Factors; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D002478:Cells, Cultured; D018031:Connexin 43 / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D017630:Connexins / Q000032:analysis / Q000096:biosynthesis* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D017629:Gap Junctions / Q000737:chemistry / Q000378:metabolism*; D006352:Heart Ventricles / Q000166:cytology; D007473:Ion Channels / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D009206:Myocardium / Q000166:cytology*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D051381:Rats; D000097003:Gap Junction alpha-5 Protein
D018031:Connexin 43; D017630:Connexins; D007473:Ion Channels; D000097003:Gap Junction alpha-5 Protein; C082920:connexin 45