PROTEOLYSIS OF CONNEXIN43-CONTAINING GAP-JUNCTIONS IN NORMAL AND HEAT-STRESSED CARDIAC MYOCYTES

Objective: The present studies were performed to examine the degradati on of connexin43-containing gap junctions by the lysosome or the prote asome in normal and heat-stressed cultures of neonatal rat ventricular myocytes. Methods: Primary cultures were prepared from neonatal rat v entricular myocytes. Connexin43 was detected by immunoblotting, immuno fluorescence, or immunoprecipitation. Gap junction profiles were detec ted by transmission electron microscopy. Results: Immunoblots of whole cell lysates demonstrated increased levels of connexin43 in cultures heated with lysosomal inhibitors (chloroquine, leupeptin, E-64, or amm onium chloride) or proteasomal inhibitors (lactacystin or ALLN). Pulse -chase experiments showed that the half-life of connexin43 was 1.4 h i n control cultures, but was prolonged to 2.0 or 2.8 h in cultures trea ted 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 degrees C for 30 min) pr oduced a rapid loss of connexin43 as detected by immunoblotting or imm unofluorescence. Heat-induced connexin43 degradation was prevented by simultaneous treatment with lactacystin, ALLN, or chloroquine. Connexi n43 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 ar e degraded by the proteasome and the lysosome, that this proteolysis c an be augmented by heat stress, and that inducible factors such as HSP 70 may protect against Cx43 degradation. (C) 1998 Elsevier Science B.V . All rights reserved.