© 2001 American Heart Association, Inc.
Cellular Biology |
Overexpression of FK506-Binding Protein FKBP12.6 in Cardiomyocytes Reduces Ryanodine Receptor–Mediated Ca2+ Leak From the Sarcoplasmic Reticulum and Increases Contractility
From the Department of Cardiology and Pneumology (J.P., P.M.L.J., A.P.J., O.Z., S.E.L., G.H.), Georg-August-University Goettingen, Goettingen, Germany, and Institute of Biomedical and Life Sciences (L.B., G.H.), Division of Neurosciences and Biomedical Systems, University of Glasgow, Glasgow, UK.
Correspondence to Juergen Prestle, PhD, Georg-August-Universitaet Goettingen, Zentrum Innere Medizin, Abt. Kardiologie & Pneumologie, Robert-Koch-Strasse 40, 37075 Goettingen, Germany. E-mail prestle{at}med.uni-goettingen.de
Abstract—The FK506-binding protein FKBP12.6 is tightly associated with the cardiac sarcoplasmic reticulum (SR) Ca2+-release channel (ryanodine receptor type 2 [RyR2]), but the physiological function of FKBP12.6 is unclear. We used adenovirus (Ad)-mediated gene transfer to overexpress FKBP12.6 in adult rabbit cardiomyocytes. Western immunoblot and reverse transcriptase–polymerase chain reaction analysis revealed specific overexpression of FKBP12.6, with unchanged expression of endogenous FKBP12. FKBP12.6-transfected myocytes displayed a significantly higher (21%) fractional shortening (FS) at 48 hours after transfection compared with Ad-GFP–infected control cells (4.8±0.2% FS versus 4±0.2% FS, respectively; n=79 each; P=0.001). SR-Ca2+ uptake rates were monitored in ß-escin–permeabilized myocytes using Fura-2. Ad-FKBP12.6–infected cells showed a statistically significant higher rate of Ca2+ uptake of 0.8±0.09 nmol/s-1/106 cells (n=8, P<0.05) compared with 0.52±0.1 nmol/s-1/106 cells in sham-infected cells (n=8) at a [Ca2+] of 1 µmol/L. In the presence of 5 µmol/L ruthenium red to block Ca2+ efflux via RyR2, SR-Ca2+ uptake rates were not significantly different between groups. From these measurements, we calculate that SR-Ca2+ leak through RyR2 is reduced by 53% in FKBP12.6-overexpressing cells. Caffeine-induced contractures were significantly larger in Ad-FKBP12.6–infected myocytes compared with Ad-GFP–infected control cells, indicating a higher SR-Ca2+ load. Taken together, these data suggest that FKBP12.6 stabilizes the closed conformation state of RyR2. This may reduce diastolic SR-Ca2+ leak and consequently increase SR-Ca2+ release and myocyte shortening.
Key Words: cardiac myocytes • calcium • sarcoplasmic reticulum • adenovirus • gene transfer
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