Functional coupling of calcineurin and protein kinase A in mouse ventricular myocytes

• Published in: The Journal of physiology 2002-10, Vol.544 (1), p.57-69
• Main Authors: Santana, L. F., Chase, E. G., Votaw, V. S., Nelson, Mark T., Greven, R.
• Format: Article
• Language: English
• Subjects: Action Potentials - drug effects; Animals; Calcineurin - metabolism; Calcineurin Inhibitors; Calcium - metabolism; Calcium Channels - physiology; Calcium Signaling - physiology; Cyclic AMP-Dependent Protein Kinases - metabolism; Cyclosporine - pharmacology; Cytoplasm - metabolism; Electric Conductivity; Enzyme Inhibitors - pharmacology; Heart Ventricles; Isoenzymes - metabolism; Mice; Mice, Inbred Strains; Myocardial Contraction - physiology; Myocytes, Cardiac - metabolism; Original; Osmolar Concentration; Potassium Channels - drug effects; Potassium Channels - physiology; Reference Values; Sarcoplasmic Reticulum - metabolism; Tissue Distribution
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/jphysiol.2002.020552

We examined the role of the Ca 2+ -regulated protein phosphatase calcineurin in controlling Ca 2+ signalling in mouse ventricular myocytes. Membrane currents and voltage were measured in single myocytes using the patch-clamp technique. Cytoplasmic Ca 2+ concentration ([Ca 2+ ] i ) was measured in cells loaded with the fluorescent Ca 2+ indicators fluo-4 or fura-2 using a confocal or epifluorescence microscope. Inhibition of calcineurin with cyclosporin A (CsA, 100 n m ) or the calcineurin auto-inhibitory peptide (CiP, 100 μM), increased the amplitude and rate of decay of the evoked [Ca 2+ ] i transient and also prolonged the action potential (AP) of ventricular myocytes to a similar extent. The effects of CsA (100 n m ) and 100 μM CiP on the [Ca 2+ ] i transient and AP were not additive. Calcineurin inhibition did not modify the K + currents responsible for repolarisation of the mouse ventricle. Instead, inhibition of calcineurin increased the amplitude of the Ca 2+ current ( I Ca ) and the evoked calcium transient normalized to the I Ca . Calcium sparks, which underlie the [Ca 2+ ] i transient, had a higher frequency and amplitude, suggesting an elevation of SR calcium load. Inhibition of protein kinase A (PKA) prevented the effects of calcineurin inhibition, indicating that calcineurin opposes the actions of PKA. Finally, immunofluorescence images suggest that calcineurin and PKA co-localize near the T-tubules of ventricular myocytes. We propose that calcineurin and PKA are co-localized to control Ca 2+ influx through calcium channels and calcium release through ryanodine receptors.