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Ca(2+) channel modulation by recombinant auxiliary beta subunits expressed in young adult heart cells
S K Wei1, H M Colecraft, C D DeMaria
1Program in Molecular and Cellular Systems Physiology, Departments of Biomedical Engineering and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Abstract:
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.
Insights
This study shows that beta subunits are key regulators of L-type Ca(2+) channels in heart cells, influencing current density and inactivation. This finding suggests potential therapeutic strategies for heart failure by targeting these beta subunits.
Area of Science:
- Cardiovascular Physiology
- Molecular Cardiology
- Ion Channel Biology
Background:
- L-type Ca(2+) channels are crucial for heart contraction and are implicated in heart failure.
- Beta subunits significantly modulate Ca(2+) channel function, but their role in native cardiac cells is poorly understood.
- Existing research relies heavily on heterologous expression systems, which may not accurately reflect cardiomyocyte behavior.
Purpose of the Study:
- To investigate the role of L-type Ca(2+) channel beta subunits in native rat ventricular myocytes.
- To develop and optimize a gene delivery method for expressing beta subunits in adult cardiomyocytes.
- To determine the impact of beta subunit overexpression on Ca(2+) channel current and inactivation properties.
Main Methods:
- Utilized an adenoviral-component gene delivery system to express recombinant beta subunits in cultured adult rat ventricular myocytes.
- Employed a bicistronic expression plasmid encoding both green fluorescent protein (GFP) and beta subunits to identify transfected cells and avoid altering subunit function.
- Measured Ca(2+) channel current density and voltage-dependent inactivation using electrophysiological techniques.
Main Results:
- Optimized adenoviral gene delivery achieved 1-10% transfection efficiency in adult myocytes.
- Overexpression of recombinant beta subunits alone increased L-type Ca(2+) channel current density by 3- to 4-fold, indicating they are rate-limiting.
- Overexpression of the beta(2a) subunit significantly slowed voltage-dependent inactivation of Ca(2+) channels at +10 mV.
Conclusions:
- Beta subunits play a critical role in regulating L-type Ca(2+) channel expression and function in cardiomyocytes.
- The beta(2a) subunit can modulate channel inactivation kinetics in the myocardium.
- Targeting beta subunits may offer a novel therapeutic approach to address contractile dysfunction and excitability issues in heart failure.