Overexpression of FK506-binding protein FKBP12.6 in cardiomyocytes reduces ryanodine receptor-mediated Ca(2+) leak from the sarcoplasmic reticulum and increases contractility.
The FK506-binding protein FKBP12.6 is tightly associated with the cardiac sarcoplasmic reticulum (SR) Ca(2+)-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-Ca(2+) uptake rates were monitored in beta-escin-permeabilized myocytes using Fura-2. Ad-FKBP12.6-infected cells showed a statistically significant higher rate of Ca(2+) uptake of 0.8+/-0.09 nmol/s(-)(1)/10(6) cells (n=8, P:<0.05) compared with 0.52+/-0.1 nmol/s(-)(1)/10(6) cells in sham-infected cells (n=8) at a [Ca(2+)] of 1 micromol/L. In the presence of 5 micromol/L ruthenium red to block Ca(2+) efflux via RyR2, SR-Ca(2+) uptake rates were not significantly different between groups. From these measurements, we calculate that SR-Ca(2+) 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-Ca(2+) load. Taken together, these data suggest that FKBP12.6 stabilizes the closed conformation state of RyR2. This may reduce diastolic SR-Ca(2+) leak and consequently increase SR-Ca(2+) release and myocyte shortening.
Prestle
J
J
Department of Cardiology and Pneumology, Georg-August-University Goettingen, Goettingen, Germany. prestle@med.uni-goettingen.de
Janssen
P M
PM
Janssen
A P
AP
Zeitz
O
O
Lehnart
S E
SE
Bruce
L
L
Smith
G L
GL
Hasenfuss
G
G
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D009200:Myocardial Contraction / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D020033:Protein Isoforms; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012430:Ruthenium Red / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology; D016559:Tacrolimus / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012430:Ruthenium Red; D002110:Caffeine; D022021:Tacrolimus Binding Proteins; C403484:tacrolimus binding protein 1B; D002118:Calcium; D020123:Sirolimus; D016559:Tacrolimus