Inositol-1,4,5-trisphosphate-mediated spontaneous activity in mouse embryonic stem cell-derived cardiomyocytes.
Embryonic stem cell-derived cardiomyocytes (ESdCs) have been proposed as a source for cardiac cell-replacement therapy. The aim of this study was to determine the Ca2+-handling mechanisms that determine the frequency and duration of spontaneous Ca2+ transients in single ESdCs. With laser scanning confocal microscopy using the Ca2+-sensitive dye Fluo-4/AM, we determined that spontaneous Ca2+ transients in ESdCs at the onset of beating (day 9) depend on Ca2+ entry across the plasma membrane (50%) whereas Ca2+-induced Ca2+ release is the major contributor to Ca2+ transients in ESdCs after 16 days (72%). Likewise, Ca2+ extrusion in 9-day-old ESdCs depends on Na+-Ca2+ exchange (50.0+/-8%) whereas Ca2+ reuptake by the sarco(endo)plasmic Ca2+ ATPase (72+/-5%) dominates in further differentiated cells. Spontaneous Ca2+ transients were suppressed by the inositol-1,4,5-trisphosphate (IP3) receptor (IP3R) blocker 2-aminoethoxydiphenyl borate (2-APB) and the phospholipase C blocker U73122 but continued in the presence of caffeine. Stimulation of IP3 production by phenylephrine or endothelin-1 had a positive chronotropic effect that could be reversed by U73122 and 2-APB. The presence of Ca2+-free solution and block of L-type Ca2+ channels by nifedipine also resulted in a cessation of spontaneous activity. Overall, IP3R-mediated Ca2+ release in ESdCs is translated into a depolarization of the plasma membrane and a whole-cell Ca2+ transient is subsequently induced by voltage-dependent Ca2+ influx. Although ryanodine receptor-mediated Ca2+ release amplifies the IP3R-induced trigger for the Ca2+ transients and modulates its frequencies, it is not a prerequisite for spontaneous activity. The results of this study offer important insight into the role of IP3R-mediated Ca2+ release for pacemaker activity in differentiating cardiomyocytes.
Kapur
Nidhi
N
Department of Physiology, Stritch School of Medicine, Loyola University Chicago, 2160 South First Avenue, Maywood, IL 60153, USA.
Banach
Kathrin
K
D000200:Action Potentials; D000814:Aniline Compounds; D000818:Animals; D001896:Boron Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002454:Cell Differentiation; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D019332:Endothelin-1 / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005456:Fluorescent Dyes; D006339:Heart Rate / Q000187:drug effects / Q000502:physiology*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D009543:Nifedipine / Q000494:pharmacology; D010656:Phenylephrine / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D013997:Time Factors; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism; D014966:Xanthenes
D000814:Aniline Compounds; D001896:Boron Compounds; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D019332:Endothelin-1; D004791:Enzyme Inhibitors; D004963:Estrenes; C409648:Fluo 4; D005456:Fluorescent Dyes; D053496:Inositol 1,4,5-Trisphosphate Receptors; D011760:Pyrrolidinones; D019837:Ryanodine Receptor Calcium Release Channel; D019831:Sodium-Calcium Exchanger; D014966:Xanthenes; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D010656:Phenylephrine; D015544:Inositol 1,4,5-Trisphosphate; C109986:2-aminoethoxydiphenyl borate; D010738:Type C Phospholipases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D009543:Nifedipine; D002118:Calcium