© 1998 by European Society of Cardiology
Copyright © 1998, European Society of Cardiology
Proteolysis of connexin43-containing gap junctions in normal and heat-stressed cardiac myocytes
aDepartment of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, USA
bDepartment of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA
cDepartment of Pathology, Washington University School of Medicine, St. Louis, MO 63110, USA
dDepartment of Pediatrics, University of Chicago, Chicago, IL 60637-1470, USA
* Corresponding author. Section of Pediatric Hematology/Oncology, University of Chicago Children's Hospital, MC 4060, 5841 S. Maryland Ave., Chicago, IL 60637-1470, USA. Tel.: +1 (773) 834-1498; Fax: +1 (773) 702-9881; E-mail: ebeyer@peds.bsd.uchicago.edu
Objective: The present studies were performed to examine the degradation of connexin43-containing gap junctions by the lysosome or the proteasome in normal and heat-stressed cultures of neonatal rat ventricular myocytes. Methods: Primary cultures were prepared from neonatal rat ventricular myocytes. Connexin43 was detected by immunoblotting, immunofluorescence, or immunoprecipitation. Gap junction profiles were detected by transmission electron microscopy. Results: Immunoblots of whole cell lysates demonstrated increased levels of connexin43 in cultures treated with lysosomal inhibitors (chloroquine, leupeptin, E-64, or ammonium chloride) or proteasomal inhibitors (lactacystin or ALLN). Pulse-chase experiments showed that the half-life of connexin43 was 1.4 h in control cultures, but was prolonged to 2.0 or 2.8 h in cultures treated with chloroquine or lactacystin, respectively. Immunofluorescence and electron microscopy showed a significant increase in the number of gap junction profiles in myocytes treated with either chloroquine or lactacystin. Heat treatment of cultures (43.5°C for 30 min) produced a rapid loss of connexin43 as detected by immunoblotting or immunofluorescence. Heat-induced connexin43 degradation was prevented by simultaneous treatment with lactacystin, ALLN, or chloroquine. Connexin43 levels and distribution returned to normal by 3 h following a heat shock and were resistant to a subsequent repeat heat stress. The heat shock also led to production of HSP70 as detected by immunoblotting. Conclusions: These data suggest that Cx43 gap junctions in myocytes are degraded by the proteasome and the lysosome, that this proteolysis can be augmented by heat stress, and that inducible factors such as HSP70 may protect against Cx43 degradation.
KEYWORDS Cx43, connexin43; ALLN, N-acetyl-L-leucyl-L-leucyl-norleucinal; NH4Cl, ammonium chloride; ALLM, N-acetyl-leucyl-leucyl-methioninal; PBS, phosphate-buffered saline
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