Abstract
In many non-excitable and excitable cells, store-operated calcium entry (SOCE) represents an additional pathway for calcium entry upon Ca2+ store depletion. In a previous study, we demonstrated SOCE activity in intact mouse cardiac pacemaker tissue, specifically from sinoatrial node (SAN) tissue. However, store content as a key determinant of SOCE activity is difficult to measure in intact SAN tissue. Therefore, to investigate the interaction between SOCE and store content and its role in cardiac pacemaking, it is necessary to investigate SOCE activity in single cardiac pacemaker cells. Furthermore, recent studies in other tissues have identified two new proteins involved in SOCE, stromal interacting molecule (STIM), which is an ER Ca2+ sensor, and the surface membrane channel Orai, a prototypic gene encoding for SOCE. However, whether STIM and Orai are expressed in native pacemaker cells is still unknown. In this current study, we examined SOCE activity in single firing pacemaker cells isolated from mouse sinoatrial node tissue. We found a significant rise in Ca2+ entry in response to Ca2+ store depletion. SOCE blockers reduced the amplitude and frequency of spontaneous Ca2+ transients and reduced Ca2+ store content. We demonstrated for the first time that STIM and Orai are expressed in pacemaker cells. After store depletion, STIM1 redistributed to the cell periphery and showed increased co-localization with surface membrane located Orai1, indicating a possible involvement of these proteins in SOCE activity in native cardiac pacemaker cells. These results suggest the novel concept that SOCE plays a functional role in regulating intracellular Ca2+ of cardiac pacemaker cells.
Highlights
Cardiac contraction originates in the spontaneous firing of pacemaker cells in the sinoatrial node (SAN) of the heart
Given that pacemaker cells generate robust spontaneous firing by a combination of voltage-dependent channels and Ca2+ cycling, one might question what functional role SOCE can play in pacemaker cells
Application of the blockers to the normally firing pacemaker cells caused only modest reductions of the firing rate, though the fact that they were accompanied by a reduction in store level strengthens the case for the effect arising from blocking SOCE
Summary
Cardiac contraction originates in the spontaneous firing of pacemaker cells in the sinoatrial node (SAN) of the heart. It was thought that the spontaneous firing of pacemaker cells was driven purely by voltage-dependent membrane currents (Noble, 1960) but subsequently it has been found that intracellular Ca2+ cycling plays an important role (Rigg and Terrar, 1996; Ju and Allen, 1998, 1999; Rigg et al, 2000; Lakatta et al, 2010). Ca2+ extrusion through the Na+/Ca2+ exchanger generates an inward current that contributes to pacemaker diastolic depolarization.(Rigg and Terrar, 1996; Ju and Allen, 1998, 2000; Rigg et al, 2000; Vinogradova et al, 2000, 2006). Controversies still exist about the relative importance of intracellular Ca2+ cycling over membrane currents (Lakatta and DiFrancesco, 2009; Himeno et al, 2011), it is generally accepted that pacemaker activity is orchestrated by the coupled system of membrane ionic currents (the “membrane clock”) and intracellular SR calcium cycling (the “calcium clock”) (for review see, Lakatta et al, 2010)
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