Expression and characterization of the Na+/H+ exchanger in the mammalian myocardium.
We examined two expression systems for studying the Na(+)/H(+) exchanger in the mammalian myocardium. Mammalian NHE1 with a hemagglutinin (HA) tag and was cloned behind the alpha myosin heavy chain promoter. Transgenic mice were made with wild type NHE1 protein or with a hyperactive NHE1 protein mutated at the calmodulin-binding domain. Three lines of transgenic mice were made of each cDNA with expression levels of each type varying from high to low. Higher levels and activity of the Na(+)/H(+) exchanger were associated with decreased long-term survival of mice, and with dilated or hypertrophic cardiomyopathy. The exogenous NHE1 protein was present in freshly made cardiomyocytes from transgenic mice, however, expression from the alpha myosin heavy chain promoter declined rapidly and little exogenous NHE1 was apparent on the fourth day after cardiomyocyte isolation. To express NHE1 protein in isolated cardiomyocytes, we transferred a mutated form of the protein into an adenoviral expression system. Infection of neonatal rat cardiomyocytes resulted in robust expression of the exogenous NHE1 protein. The mutant form of the NHE1 protein could be distinguished from the endogenous Na(+)/H(+) exchanger by its resistance to inhibition by amiloride analogs. Our results suggest that for in vivo studies on intact hearts and animals, expression in transgenic mice is an appropriate system, however for long-term studies on cardiomyocytes, this model is inappropriate due to waning expression from the alpha myosin heavy chain promoter. Therefore, infection by adenovirus is a superior system for long-term studies on cardiomyocytes in culture.
Coccaro
Ersilia
E
Department of Biochemistry, University of Alberta, 347 Medical Sciences Building, T6G 2H7, Edmonton, AB, Canada.
Mraiche
Fatima
F
Malo
Mackenzie
M
Vandertol-Vanier
Heather
H
Bullis
Bonnie
B
Robertson
Murray
M
Fliegel
Larry
L
D000256:Adenoviridae; D000818:Animals; D015153:Blotting, Western; D027682:Cation Transport Proteins / Q000378:metabolism*; D002469:Cell Separation; D002478:Cells, Cultured; D004452:Echocardiography; D053208:Kaplan-Meier Estimate; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D050505:Mutant Proteins / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism; D010641:Phenotype; D051381:Rats; D017207:Rats, Sprague-Dawley; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D014162:Transfection
D027682:Cation Transport Proteins; D008565:Membrane Proteins; D050505:Mutant Proteins; C490997:Slc9a1 protein, mouse; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers