Ca(2+) channel modulation by recombinant auxiliary beta subunits expressed in young adult heart cells.
L-type Ca(2+) channels contribute importantly to the normal excitation-contraction coupling of physiological hearts, and to the functional derangement seen in heart failure. Although Ca(2+) channel auxiliary beta(1-4) subunits are among the strongest modulators of channel properties, little is known about their role in regulating channel behavior in actual heart cells. Current understanding draws almost exclusively from heterologous expression of recombinant subunits in model systems, which may differ from cardiocytes. To study beta-subunit effects in the cardiac setting, we here used an adenoviral-component gene-delivery strategy to express recombinant beta subunits in young adult ventricular myocytes cultured from 4- to 6-week-old rats. The main results were the following. (1) A component system of replication-deficient adenovirus, poly-L-lysine, and expression plasmids encoding beta subunits could be optimized to transfect young adult myocytes with 1% to 10% efficiency. (2) A reporter gene strategy based on green fluorescent protein (GFP) could be used to identify successfully transfected cells. Because fusion of GFP to beta subunits altered intrinsic beta-subunit properties, we favored the use of a bicistronic expression plasmid encoding both GFP and a beta subunit. (3) Despite the heteromultimeric composition of L-type channels (composed of alpha(1C), beta, and alpha(2)delta), expression of recombinant beta subunits alone enhanced Ca(2+) channel current density up to 3- to 4-fold, which argues that beta subunits are "rate limiting" for expression of current in heart. (4) Overexpression of the putative "cardiac" beta(2a) subunit more than halved the rate of voltage-dependent inactivation at +10 mV. This result demonstrates that beta subunits can tune inactivation in the myocardium and suggests that other beta subunits may be functionally dominant in the heart. Overall, this study points to the possible therapeutic potential of beta subunits to ameliorate contractile dysfunction and excitability in heart failure.
Wei
S K
SK
Program in Molecular and Cellular Systems Physiology, Departments of Biomedical Engineering and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Colecraft
H M
HM
DeMaria
C D
CD
Peterson
B Z
BZ
Zhang
R
R
Kohout
T A
TA
Rogers
T B
TB
Yue
D T
DT
D000256:Adenoviridae; D000367:Age Factors; D000818:Animals; D020746:Calcium Channels, L-Type / Q000235:genetics*; D002460:Cell Line; D015870:Gene Expression / Q000502:physiology; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006352:Heart Ventricles / Q000737:chemistry / Q000166:cytology; D007202:Indicators and Reagents / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics; D018485:Muscle Fibers, Skeletal / Q000737:chemistry* / Q000166:cytology / Q000502:physiology; D009200:Myocardial Contraction / Q000502:physiology; D009206:Myocardium / Q000737:chemistry* / Q000166:cytology; D018408:Patch-Clamp Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics
D020746:Calcium Channels, L-Type; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins