Remodeling of the distribution of gap junctions is an important featur
e of anatomic substrates of arrhythmias in patients with healed myocar
dial infarcts. Mechanisms underlying this process are poorly understoo
d but probably involve changes in gap junction protein (connexin) synt
hesis, assembly into channels, and degradation. The half-life of the p
rincipal 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 c
onnections 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 pr
oteolytic pathways involved in Cx43 degradation in isolated perfused a
dult rat hearts. Hearts were labeled for 40 minutes with Krebs-Hensele
it buffer containing [S-35]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 hea
rts at each time point yielded a monoexponential decay curve indicatin
g 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 pr
oteolysis, Immunoblot analysis demonstrated significant increases (app
roximate to 30%) in Cx43 content in hearts perfused with either lysoso
mal or proteasomal pathway inhibitors. Most of the Cx43 in hearts perf
used with lysosomal inhibitors consisted of phosphorylated isoforms, w
hereas nonphosphorylated Cx43 accumulated selectively in hearts perfus
ed 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 im
portant role in gap junction remodeling in response to cardiac injury.