Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection.
We have previously shown that connexin 43 (Cx43) is present in mitochondria, that its genetic depletion abolishes the protection of ischemia- and diazoxide-induced preconditioning, and that it is involved in reactive oxygen species (ROS) formation in response to diazoxide. Here we investigated the intramitochondrial localization of Cx43, the mechanism of Cx43 translocation to mitochondria and the effect of inhibiting translocation on the protection of preconditioning. Confocal microscopy of mitochondria devoid of the outer membrane and Western blotting on fractionated mitochondria showed that Cx43 is located at the inner mitochondrial membrane, and coimmunoprecipitation of Cx43 with Tom20 (Translocase of the outer membrane 20) and with heat shock protein 90 (Hsp90) indicated that it interacts with the regular mitochondrial protein import machinery. In isolated rat hearts, geldanamycin, a blocker of Hsp90-dependent translocation of proteins to the inner mitochondrial membrane through the TOM pathway, rapidly (15 minutes) reduced mitochondrial Cx43 content by approximately one-third in the absence or presence of diazoxide. Geldanamycin alone had no effect on infarct size, but it ablated the protection against infarction afforded by diazoxide. Geldanamycin abolished the 2-fold increase in mitochondrial Cx43 induced by 2 preconditioning cycles of ischemia/reperfusion, but this effect was not associated with reduced protection. These results demonstrate that Cx43 is transported to the inner mitochondrial membrane through translocation via the TOM complex and that a normal mitochondrial Cx43 content is important for the diazoxide-related pathway of preconditioning.
Rodriguez-Sinovas
Antonio
A
Servicio de Cardiología, Hospital Vall d'Hebron, Barcelona, Spain.
Boengler
Kerstin
K
Cabestrero
Alberto
A
Gres
Petra
P
Morente
Miriam
M
Ruiz-Meana
Marisol
M
Konietzka
Ina
I
Miró
Elisabet
E
Totzeck
Andreas
A
Heusch
Gerd
G
Schulz
Rainer
R
Garcia-Dorado
David
D
D000818:Animals; D016227:Benzoquinones; D001692:Biological Transport / Q000502:physiology; D002316:Cardiotonic Agents / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D018031:Connexin 43 / Q000378:metabolism*; D003981:Diazoxide / Q000037:antagonists & inhibitors / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000502:physiology*; D066298:In Vitro Techniques; D019157:Ischemic Preconditioning, Myocardial; D047029:Lactams, Macrocyclic; D008297:Male; D051336:Mitochondrial Membranes / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D015428:Myocardial Reperfusion Injury / Q000503:physiopathology; D032383:Myocytes, Cardiac / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013552:Swine; D014018:Tissue Distribution
D016227:Benzoquinones; D002316:Cardiotonic Agents; D002352:Carrier Proteins; D018031:Connexin 43; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011809:Quinones; D003981:Diazoxide; C001277:geldanamycin