Functional coupling of calcineurin and protein kinase A in mouse ventricular myocytes.
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 nM) or the calcineurin auto-inhibitory peptide (CiP, 100 microM), 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 nM) and 100 microM 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.
D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D019703:Calcineurin / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000502:physiology; D020013:Calcium Signaling / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D016572:Cyclosporine / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004553:Electric Conductivity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006352:Heart Ventricles; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D009200:Myocardial Contraction / Q000502:physiology; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009994:Osmolar Concentration; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology; D012016:Reference Values; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D014018:Tissue Distribution
D065095:Calcineurin Inhibitors; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D015221:Potassium Channels; D016572:Cyclosporine; D017868:Cyclic AMP-Dependent Protein Kinases; D019703:Calcineurin; D002118:Calcium