Key points• Abnormal oscillations of calcium (Ca 2+ ) concentration in cardiac Purkinje cells (P-cells) have been associated with life-threatening arrhythmias, but the mechanism by which these cells control their Ca 2+ level in normal conditions remains unknown.• We modelled our previous hypothesis that the principal intracellular Ca 2+ compartment (endoplasmic reticulum; ER) which governs intracellular Ca 2+ concentration, formed, in P-cells, three concentric and adjacent layers, each including a distinct Ca 2+ release channel. We then tested the model against typical Ca 2+ variations observed in stimulated P-cells.• We found in swine P-cells, as in the rabbit and dog, that stimulation evokes an elevation of Ca 2+ concentration first under the membrane , which then propagates to the interior of the cell.• Our mathematical model could reproduce accurately this typical 'centripetal' Ca 2+ spread, hence supporting (1) the existence of the '3 layered' Ca 2+ compartment, and (2) its central role in the regulation of Ca 2+ concentration in P-cells.• To model the 'centripetal' Ca 2+ spread, local variations of Ca 2+ concentration were calculated for a virtual cell environment encompassing three different regions that mimicked the three layers of ER in P-cells. Various tests of the model revealed that the second intermediate layer was essential for 'forwarding' the Ca 2+ elevation from the periphery to the cell centre.• This novel finding suggests that a thin intermediate layer of specific ER Ca 2+ channels controls the entire Ca 2+ signalling of P-cells. Because Ca 2+ plays a role in the electric properties of P-cells, any abnormality affecting this intermediate region is likely to be pro-arrhythmic and could explain the origin of serious cardiac arrhythmias known to start in the Purkinje fibres.Abstract Despite strong suspicion that abnormal Ca 2+ handling in Purkinje cells (P-cells) is implicated in life-threatening forms of ventricular tachycardias, the mechanism underlying the Ca 2+ cycling of these cells under normal conditions is still unclear. There is mounting evidence that P-cells have a unique Ca 2+ handling system. Notably complex spontaneous Ca 2+ activity was previously recorded in canine P-cells and was explained by a mechanistic hypothesis involving a triple layered system of Ca 2+ release channels. Here we examined the validity of this hypothesis for the electrically evoked Ca 2+ transient which was shown, in the dog and rabbit, to occur progressively from the periphery to the interior of the cell. To do so, the hypothesis was incorporated in a model of intracellular Ca 2+ dynamics which was then used to reproduce numerically the Ca 2+ activity of P-cells under stimulated conditions. The modelling was thus performed through a 2D computational array that encompassed three distinct Ca 2+ release nodes arranged, respectively,

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sarcolemma or nuclear membranes), cytosolic (minimal distance to membranes >5mm) or nuclear. Nuclear events were considered membrane associated when occurring at a distance <1mm from the nuclear envelope or membranes of the nucleoplasmic reticulum. With tetracaine the Ca spark frequency decreased to 0.750.3 (from 8.851.2 in control), whereas the frequency of events detected after subsequent addition of IP3 increased to 2.951.3 (events/100mm/s). Subsequent exposure to 2-APB reduced the frequency to 1.850.8. Compared to Ca sparks, local Ca release events in the presence of IP3 were lower in amplitude, prolonged in duration and had a slower rise time. The increase in frequency of local Ca release events was particularly pronounced in the perinucler regions compared to the cytosol or the subsarcolemmal space. Furthermore, IP3-mediated Ca release events could also be detected within the nucleus. These nuclear events occurred at a higher incidence at locations that were closely associated with membrane structures of the nuclear envelope or the nucleoplasmic reticulum. In conclusion, in atrial myocytes IP3Rmediated Ca release is spatially inhomogeneous and preferentially occurs in the nuclear and perinuclear regions. Background: In cardiac cells, sarcoplasmic reticulum (SR) Ca 2þ -channels (RyRs) are regrouped into clusters, so-called Ca 2þ -release units (CRUs). Dynamic regulation of the individual RyRs in the cluster determines the characteristics of the SR-Ca 2þ -efflux during cell activation. The Ca 2þ -sensitivity of the channel plays a central role in this regulation. However, technical limitations make it difficult to directly assess the mechanism underlying the Ca 2þ -sensitivity of SR-Ca 2þ -release in the micro-domain of a CRU. Thus, mathematical models simulating intracellular Ca 2þ activities, such as Ca 2þ sparks and waves, incorporate a Ca 2þ -release function which approximates or, frequently, omits the regulation in the cluster micro-domain. Moreover, using nano-scale components (such as realistic spacing between RyRs in a CRU) in a micro-scale computational domain intending to reproduce the cell geometry requires massive computational resources. Method: To overcome both experimental and computational limitations, we developed a time-dependent Ca 2þ -release function that addresses activation and termination of Ca 2þ -release, mediated by dynamic regulatory mechanisms. This function has been used in systems of ODEs and PDEs applied to cardiac Purkinje cells (Pcells), which included other intracellular modulators of Ca 2þ transients, such as Ca 2þ -buffers and Ca 2þ -pumps. Results : Compared to experimental data from Pcells, the Ca 2þ -release function could predict the instantaneous variation of Ca 2þ -efflux from the CRU. Predicted values for the activation threshold of [Ca 2þ ] at the cytosolic side for RyR2 and RyR3 in Pcells were found to be 160 nM-190 nM and 105 nM-110 nM, respectively. These results matched the experimental findings. Conclusion: This function has been used successfully in 1D and 2...

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Familial AD (FAD) mutations in presenilins have been linked to Ca 2þ signaling abnormalities. Presenilins (PS) are 50 kDa proteins in the endoplasmic reticulum (ER) membrane. The cleaved presenilins are well known as catalytic components of a gamma-secretase, which cleaves the amyloid precursor protein (APP) and releases the amyloid beta-peptide. In addition uncleaved presenilins function as passive ER Ca 2þ leak channels which control steadystate ER Ca 2þ levels. It was found that many FAD mutations in presenilins result in loss of ER Ca 2þ leak function, leading to ER Ca 2þ overload and supranormal Ca2þ release from the ER. The ER Ca 2þ leak function of presenilins is independent of their gamma-secretase activity. We suggested that presenilins affect store-operated calcium influx (SOC) by controlling the filling state of ER Ca 2þ stores. To determinate the influence of FAD presenilins mutations on SOC we performed a series of patch-clamp experiments in whole-cell mode. PS1-M146V and PS1-DE9 mutants have been shown to have loss and gain of ER Ca 2þ leak channel function respectively. A decrease in maximum amplitude and speed of SOC current activation was observed in SK-N-SH neuroblastoma cells and primary culture of rat hippocampal neurons transfected with PS1-M146V mutant comparing to wild type PS1 transfected cells. An increase in maximum amplitude and speed of SOC current activation was observed in cells transfected by PS1-DE9 mutant comparing to wild type PS1. In experiments with triple transgenic AD mice hippocampal neurons (3XTg mice; KI-PS1M146V, Thy1-APPKM670/671NL, Thy1-tauP301L) the maximum amplitude of SOC were decreased comparing to WT, but the speed of activation was the same for 3XTg and WT hippocampal neurons. Electrophysiological properties of all impaired SOCs suggest that TRPC1 is the main target of presenilins FAD mutations affect.