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.