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Phospholamban mediates the beta-adrenergic-enhanced Ca2+ uptake in mammalian ventricular myocytes
1Department of Physiology, University of Pennsylvania, Philadelphia 19104-6085.
Insights
A new antibody targeting phospholamban enhances calcium uptake in heart cells. This suggests phospholamban phosphorylation is key to catecholamine-mediated relaxation in cardiac muscle.
Area of Science:
- Cardiac Physiology
- Molecular Cardiology
- Calcium Signaling
Background:
- Catecholamines induce cardiac muscle relaxation through complex molecular mechanisms.
- Phospholamban is a key regulator of sarcoplasmic reticulum calcium uptake in cardiomyocytes.
Purpose of the Study:
- To investigate the role of phospholamban in the relaxant effects of catecholamines.
- To elucidate the molecular mechanism underlying catecholamine-induced changes in cardiac calcium handling.
Main Methods:
- Utilized a monoclonal antibody (2D12) against phospholamban in guinea pig ventricular myocytes.
- Simultaneously measured intracellular Ca2+ transient and Ca2+ current.
- Assessed effects on sarcoplasmic reticular vesicles and whole cell calcium uptake/release.
Main Results:
- The anti-phospholamban antibody (2D12) stimulated Ca2+ uptake in sarcoplasmic reticulum vesicles.
- Antibody effects mimicked protein kinase A and were blocked by a phospholamban peptide.
- Intracellular dialysis with the antibody enhanced Ca2+ uptake and suppressed isoproterenol's effects.
Conclusions:
- Phosphorylation of phospholamban is critical for sarcoplasmic reticulum Ca2+ sequestration.
- This phospholamban-mediated process likely accounts for catecholamine-enhanced Ca2+ handling in heart muscle.
- Targeting phospholamban offers potential insights into cardiac function regulation.
Abstract:
To probe the molecular mechanism responsible for the relaxant effect of catecholamines in heart muscle, we studied the effect of a monoclonal antibody (2D12) against phospholamban in intact whole cell clamped guinea pig ventricular myocytes, in which intracellular Ca2+ transient and Ca2+ current were simultaneously measured. The antibody stimulated Ca2+ uptake in guinea pig ventricular sarcoplasmic reticular vesicles, shifting the apparent dissociation constant for activation by Ca2+ from 200 to 60 nM. The stimulatory effect of the antibody could be mimicked by the catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent kinase and could be blocked by phospholamban peptide 2-25. Dialysis of ventricular myocytes with the antibody enhanced the rate of uptake of Ca2+ and significantly suppressed the ability of isoproterenol to enhance the rate of uptake and release of Ca2+ by depolarizing pulses. These data suggest that not only is phosphorylation of phospholamban crucial in sequestration of Ca2+ by the sarcoplasmic reticulum, but that this process may account for the catecholamine-enhanced rate of Ca2+ uptake release in heart muscle.