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Effects of PP1/PP2A inhibitor calyculin A on the E-C coupling cascade in murine ventricular myocytes
William H duBell1, Marisa S Gigena, Silvia Guatimosim
1Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene St., Baltimore, MD 21201, USA.
Abstract:
Calyculin A was used to examine the importance of phosphatases in the modulation of cardiac contractile magnitude in the absence of any neural or humoral stimulation. Protein phosphatase (PP)1 and PP2A activity, twitch contractions, intracellular Ca(2+) concentration ([Ca(2+)](i)) transients, action potentials, membrane currents, and myofilament Ca(2+) sensitivity were measured in isolated mouse ventricular myocytes. Calyculin A (125 nM) inhibited PP1 and PP2A by 50% and 85%, respectively, whereas it doubled the twitch magnitude and increased twitch duration by 50% in field-stimulated cells. Calyculin A-evoked increases in L-type Ca(2+) current (70%) and the resulting [Ca(2+)](i) transient (83%) explain the positive inotropic response. However, increases in twitch and action potential durations did not result from increased myofilament Ca(2+) sensitivity or K(+) current inhibition, respectively. Comparison of the effects of calyculin A and isoproterenol on [Ca(2+)](i) transients and twitch contractions revealed that calyculin A had a much smaller lusitropic effect than the beta-agonist, indicating that calyculin A did not significantly increase sarcoplasmic reticulum Ca(2+) reuptake. Thus while cardiac contractile magnitude is controlled by a steady-state kinase/phosphatase balance, this regulation is not equally operative at all of the steps in the excitation-contraction coupling pathway and may in fact be most important to the regulation of the L-type Ca(2+) channel.
Insights
Calyculin A significantly increased cardiac twitch magnitude by inhibiting protein phosphatases (PP1 and PP2A), primarily by enhancing L-type Ca(2+) current. This highlights the critical role of phosphatases in regulating cardiac contraction.
Area of Science:
- Cardiology
- Molecular Biology
- Cell Physiology
Background:
- Protein phosphatases (PP1 and PP2A) play a role in cardiac function.
- Understanding their specific roles in excitation-contraction coupling is crucial.
Purpose of the Study:
- To investigate the role of phosphatases in cardiac contractile magnitude without neural or humoral stimulation.
- To determine the specific mechanisms by which phosphatases modulate cardiac contraction.
Main Methods:
- Isolated mouse ventricular myocytes were used.
- Measurements included protein phosphatase activity, twitch contractions, intracellular Ca(2+) transients, action potentials, membrane currents, and myofilament Ca(2+) sensitivity.
- Calyculin A was administered to inhibit PP1 and PP2A.
Main Results:
- Calyculin A (125 nM) inhibited PP1 (50%) and PP2A (85%).
- Twitch magnitude doubled and twitch duration increased by 50%.
- Increased L-type Ca(2+) current (70%) and intracellular Ca(2+) transients (83%) explained the positive inotropic effect.
Conclusions:
- Cardiac contractile magnitude is regulated by a kinase/phosphatase balance.
- This regulation is most critical for the L-type Ca(2+) channel in excitation-contraction coupling.
- Phosphatase inhibition primarily impacts L-type Ca(2+) current, not myofilament Ca(2+) sensitivity or sarcoplasmic reticulum Ca(2+) reuptake.