Decoy calcium channel beta subunits modulate contractile function in myocytes.
To test the hypothesis that mutated beta2-subunits of the L-type calcium channel could serve as a decoy and interdict calcium channel trafficking and function, we engineered a beta2 subunit that contained the beta interaction domain for alpha1c subunit interaction, but lacked N- and C-terminal domains that might be essential for sarcolemmal localization. An adenoviral vector was constructed containing the gene for the beta-interaction domain (BID) fused to green fluorescence protein (GFP), using a vector containing only GFP as control. Freshly plated, dissociated adult rat myocytes were infected and expression and function were assessed at 60 h. Fluorescence microscopy confirmed GFP expression; immunoblot analysis confirmed dose-dependent GFP-BID expression. Mechanical properties of adult rat ventricular myocytes were evaluated using a video edge-detection system. Contractility analysis (optical/video, field stimulation) demonstrated that contracting cells decreased from 60 to 2%. Contractile amplitude (percent shortening) decreases significantly from 5.6 vs. 2.4% with no change in time to peak twitch. Recombinant adenovirus overexpressing mutated beta2 subunits in adult mammalian myocytes can markedly alter excitation-contraction coupling. This paradigm may offer new approaches to understanding and modulating EC coupling.
Fan
Q Ivy
QI
Program in Molecular and Cellular Cardiology, Department of Internal Medicine, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Vanderpool
Kathleen M
KM
O'Connor
Jessica
J
Marsh
James D
JD
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008297:Male; D032342:Muscle Cells / Q000737:chemistry / Q000378:metabolism*; D009119:Muscle Contraction; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D015220:Calcium Channels; D008164:Luminescent Proteins; D021122:Protein Subunits; D049452:Green Fluorescent Proteins