© 1998 American Heart Association, Inc.
Original Contributions |
Rapid Turnover of Connexin43 in the Adult Rat Heart
From the Departments of Medicine and Pathology (M.A.B., J.G.L., J.E.S.), Washington University School of Medicine, St Louis, Mo, and the Department of Pediatrics (E.C.B.), University of Chicago, Chicago, Ill.
Correspondence to Jeffrey E. Saffitz, MD, PhD, Department of Pathology, Box 8118, Washington University School of Medicine, 660 South Euclid Ave, St Louis, MO 63110. E-mail saffitz{at}pathology.wustl.edu
Abstract—Remodeling of the
distribution of gap junctions is an important feature of anatomic
substrates of arrhythmias in patients with healed myocardial
infarcts. Mechanisms underlying this process are poorly understood but
probably involve changes in gap junction protein (connexin) synthesis,
assembly into channels, and degradation. The half-life of the principal
cardiac gap junction protein, connexin43 (Cx43), is only 1.5 to 2 hours
in primary cultures of neonatal myocytes, but it is unknown whether
rapid turnover of Cx43 occurs in the adult heart or is unique to
disaggregated neonatal myocytes that are actively reestablishing
connections in vitro. To characterize connexin turnover dynamics in the
adult heart and to elucidate its potential role in remodeling of gap
junctions, we measured Cx43 turnover kinetics and characterized the
proteolytic pathways involved in Cx43 degradation in isolated perfused
adult rat hearts. Hearts were labeled for 40 minutes with
Krebs-Henseleit buffer containing [35S]methionine, and
then chase perfusions were performed with nonradioactive buffer for 0,
60, 120, and 240 minutes. Quantitative immunoprecipitation assays of
Cx43 radioactivity in 4 hearts at each time point yielded a
monoexponential decay curve indicating a Cx43 half-life
of 1.3 hours. Proteolytic pathways responsible for Cx43 degradation
were elucidated by perfusing isolated rat hearts for 4 hours with
specific inhibitors of either lysosomal or proteasomal
proteolysis. Immunoblot analysis demonstrated
significant increases (
30%) in Cx43 content in hearts perfused with
either lysosomal or proteasomal pathway inhibitors. Most of
the Cx43 in hearts perfused with lysosomal inhibitors
consisted of phosphorylated isoforms, whereas
nonphosphorylated Cx43 accumulated selectively in
hearts perfused with a specific proteasomal inhibitor.
These results indicate that Cx43 turns over rapidly in the adult heart
and is degraded by multiple proteolytic pathways. Regulation of Cx43
degradation could play an important role in gap junction remodeling in
response to cardiac injury.
Key Words: gap junction • connexin43 • proteolysis • anatomic substrate of arrhythmia
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