Abstract
Adult mammalian ventricular cardiomyocytes are terminally differentiated cells that enlarge adaptively by hypertrophy. In this situation, genes normally expressed in the fetal ventricular cardiomyocyte (e.g. atrial natriuretic factor (ANF), beta-myosin heavy chain (beta-MHC), and skeletal muscle (SkM) alpha-actin) are re-expressed, and there is transient expression of immediate early genes (e.g. c-fos). Using appropriate reporter plasmids, we studied the effects of transfection of the constitutively active or dominant negative mitogen-activated protein kinase kinase MEK1 on ANF, beta-MHC, and SkM alpha-actin promoter activities in cultured ventricular cardiomyocytes. ANF expression was stimulated (maximally 75-fold) by the hypertrophic agonist phenylephrine in a dose-dependent manner (EC50, 10 microM), and this stimulation was inhibited by dominant negative MEK1. Cotransfection of dominant negative MEK1 with a dominant negative mitogen-activated protein kinase (extracellular signal-regulated protein kinase (ERK2)) increased this inhibition. Transfection with constitutively active MEK1 constructs doubled ANF promoter activity. The additional cotransfection of wild-type ERK2 stimulated ANF promoter activity by about 5-fold. Expression of beta-MHC and SkM alpha-actin was also stimulated. Promoter activity regulated by activator protein-1 or c-fos serum response element consensus sequences was also increased. We conclude that the MEK1/ERK2 cascade may play a role in regulating gene expression during hypertrophy.
Highlights
§ British Heart Foundation Lecturer in Basic Science. ʈ To whom correspondence should be addressed: Dept. of Cardiac Medicine, National Heart and Lung Inst., Dovehouse St., London SW3 6LY, UK
After exposure to 0.1 mM phenylephrine for 48 h, the area of the cardiomyocytes transfected with pON249 was 1433 Ϯ 122 m2 versus 987 Ϯ 123 m2 for transfected control cells cultured in serum-free medium
Transfection of cardiomyocytes with MEK1(E217/E221) ϩ ERK2(wt) (5 g of each) in addition to pON249 did not significantly increase cell area (1087 Ϯ 107 m2 versus 872 Ϯ 59 m2 for cells transfected with 10 g of vector, mean Ϯ S.E., p ϭ 0.078 by an unpaired two-tailed t test)
Summary
G. Rosenfeld (Howard Hughes Medical Institute, University of California San Diego). Transient Transfection of Ventricular Cardiomyocytes in Culture— Myocytes were isolated and cultured by a method based on that of Iwaki et al [24] They were dissociated from the ventricles of 1–2-day-old rat hearts using 0.4 mg/ml collagenase and 0.6 mg/ml pancreatin in 116 mM NaCl, 20 mM Hepes, 0.8 mM Na2HPO4, 5.6 mM glucose, 5.4 mM KCl, 0.8 mM MgSO4 (pH 7.35). Myocytes were standardly transfected with 15 g of LUX reporter plasmid, 4 g of pON249, and a total of 10 g of test plasmid(s). After transfection for 16 –20 h, cells were washed in maintenance medium containing 10% horse serum and twice with maintenance medium. Statistics—Statistical significance was assessed as appropriate by a two-tailed paired or unpaired Student’s t test with a significant difference taken as being established at p Ͻ 0.05
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