Cells expressing unique Na+/Ca2+ exchange (NCX1) splice variants exhibit different susceptibilities to Ca2+ overload

1 Division of Stroke and Vascular Disease and 3 Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, and 2 Departments of Physiology and Biochemistry and Medical Genetics, Faculties of Medicine and 4 Pharmacy, University of Manitoba, Winnipeg, Manitoba, Canada Submitted 8 September 2005 ; accepted in final form 20 December 2005 The Na + /Ca 2+ exchanger (NCX) NCX1 exhibits tissue-specific alternative splicing. Such NCX splice variants as NCX1.1 and NCX1.3 are also differentially regulated by Na + and Ca 2+ , although the physiological implications of these regulatory characteristics are unclear. On the basis of their distinct regulatory profiles, we hypothesized that cells expressing these different splice variants might exhibit unique responses to conditions promoting Ca 2+ overload, such as during exposure to cardiac glycosides or simulated ischemia. NCX1.1 or NCX1.3 was expressed in human embryonic kidney (HEK)-293 cells or rat neonatal ventricular cardiomyocytes (NVC), and expression was confirmed by Western blotting and immunocytochemical analyses. HEK-293 cells lacked NCX1 protein before transfection. With use of adenoviral vectors, neonatal cardiomyocytes were induced to overexpress the NCX1.1 splice variant by nearly twofold, whereas the NCX1.3 isoform was expressed on the endogenous NCX1.1 background. Total expression was comparable for NCX1.1 and NCX1.3. Exposure of NVC to ouabain induced a significant increase in cellular Ca 2+ , an effect that was exaggerated in cells overexpressing NCX1.1, but not NCX1.3. The increase in intracellular Ca 2+ was inhibited by 5 µM KB-R7943. Cardiomyocytes overexpressing NCX1.1 also exhibited a greater accumulation of intracellular Ca 2+ in response to simulated ischemia than did cells expressing NCX1.3. Similar responses were observed in HEK-293 cells where NCX1.1 was expressed. We conclude that expression of the NCX1.3 splice variant protects against severe Ca 2+ overload, whereas NCX1.1 promotes Ca 2+ overload in response to cardiac glycosides and ischemic challenges. These results highlight the importance of ionic regulation in controlling NCX1 activity under conditions that promote Ca 2+ overload. NCX1.1; NCX1.3; ouabain; ischemia Address for reprint requests and other correspondence: G. N. Pierce, Div. of Stroke & Vascular Disease, St. Boniface Hospital Research Centre, 351 Tache Ave., Winnipeg, MB, Canada R2H 2A6