Abstract 408: Functional Separation of IP3R- and RyR- Controlled Ca Stores in Embryonic Stem Cell Derived Cardiomyocytes
Background: In adult cardiac tissue the role of IP3-mediated Ca release is thought to contribute to arrhythmic spontaneous activity and excitation-transcription coupling. However, the location of these stores and their functional interaction with RyR-controlled Ca stores are ill defined. Wetested the hypothesis that in embryonic stem cell-derived cardiomyocytes (ESdCs) IP3Rs and RyRs control functionally distinct but interconnected Ca stores.
Method: ESdCs were dissociated at different developmental stages starting at the onset of beating (day 8 - day 18). Single ESdCs were loaded with the Ca indicator Fluo-4/AM and spontaneous Ca transient were recorded in the line scan mode of the confocal microscope.
Results: ESdCs exhibit spontaneous Ca transients that increase in frequency and decrease in duration during developmental differentiation. ESdCs dissociated on day 9 already have RyR-controlled Ca stores as determined by caffeine (Caf: 10 mM) -induced Ca transients. Spontaneous Ca transients continued in the presence of Caf and were blocked by IP3R blocker 2-APB (1 μM) or PLC inhibitor U73122. The contribution of IP3R-mediated Ca release to spontaneous Ca transients was further supported by a phenylephrine (10 μM)-induced increase in frequency that was reversed by U73122. Spontaneous Ca transients continued up to 10 min with decreasing frequency in the presence of Caf and tetracaine (100 μM) indicating that IP3R Ca release is not immediately affected by inhibition of RyR controlled Ca stores. This was further supported by the observation that thapsigargin (1 μM) and CPA (20 μM), inhibitors of the SR Ca-ATPase, blocked spontaneous activity before depletion of Caf sensitive stores. However, the refilling of Caf depleted Ca-stores in Ca-free solution supports that IP3R- and RyR- controlled Ca stores are interconnected.
Conclusion: IP3R-mediated Ca release maintains the spontaneous activity in ESdCs. Although IP3R- and RyR- controlled Ca stores are functionally separated intra-SR Ca can be redistributed between them.