title: Adenovirally delivered shRNA strongly inhibits Na+-Ca2+ exchanger expression but does not prevent contraction of neonatal cardiomyocytes.
abstract: The cardiac Na(+)-Ca(2+) exchanger (NCX1) is the main mechanism for Ca(2+) efflux in the heart and is thought to serve an essential role in cardiac excitation-contraction (E-C) coupling. The demonstration that an NCX1 gene knock-out is embryonic lethal provides further support for this essential role. However, a recent report employing the Cre/loxP technique for cardiac specific knock-out of NCX1 has revealed that cardiac function is remarkably preserved in these mice, which survived to adulthood. This controversy highlights the necessity for further investigation of NCX1 function in the heart. In this study, we report on a novel approach for depletion of NCX1 in postnatal rat myocytes that utilizes RNA interference (RNAi), administered with high efficiency via adenoviral transfection. Depletion of NCX1 was confirmed by immunocytochemical detection, Western blots and radioisotopic assays of Na(+)-Ca(2+) exchange activity. Exchanger expression was inhibited by up to approximately 94%. Surprisingly, spontaneous beating of these cardiomyocytes was still maintained, although at a lower frequency. Electrical stimulation could elicit a normal beating rhythm, although NCX depleted cells exhibited a depressed Ca(2+) transient amplitude, a depressed rate of Ca(2+) rise and decline, elevated diastolic [Ca(2+)], and shorter action potentials. We also observed a compensatory increase in sarcolemmal Ca(2+) pump expression. Our data support an important, though non-essential, role for the NCX1 in E-C coupling in these neonatal heart cells. Furthermore, this approach provides a valuable means for assessing the role of NCX1 and could be utilized to examine other cardiac proteins in physiological and pathological studies.
authors.affiliation: Division of Stroke and Vascular Disease, St. Boniface General Hospital Research Center, 351 Tache Avenue, Winnipeg, MB, Canada R2H 2A6.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Hurtado
authors.forename: Bradley P
authors.initials: BP
authors.lastname: Ander
authors.forename: Thane G
authors.initials: TG
authors.lastname: Maddaford
authors.forename: Anton
authors.initials: A
authors.lastname: Lukas
authors.forename: Larry V
authors.initials: LV
authors.lastname: Hryshko
authors.forename: Grant N
authors.initials: GN
authors.lastname: Pierce
mesh_terms: D000200:Action Potentials / Q000502:physiology; D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism; D015536:Down-Regulation; D009200:Myocardial Contraction / Q000502:physiology*; D032383:Myocytes, Cardiac / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D012313:RNA / Q000235:genetics; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
chemical_list: D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D012313:RNA; D012964:Sodium; D002118:Calcium