Question:

Spontaneous diastolic Ca²⁺ efflux from the SR contributes to the generation of spontaneous activity in cardiac pacemaker cells. We asked, whether a SR Ca²⁺ efflux can be identified to characterize cardiac pacemaker-like active cells derived from differentiated mouse embryonic stem cells (PM-ESC).

Methods:

PM-ESC were loaded with Fluo-4/AM and analyzed by imaging.

Results:

PM-ESC generated spontaneous Ca-transients with variable frequencies (0.2 to 5.4 Hz) and amplitudes. Application of 10 mM caffeine induced a Ca²⁺ peak with an equivalent amplitude to the spontaneous Ca-transients (n=13). In presence of caffeine, spontaneous Ca-transients with decreased frequency and amplitude could be still detected in 69 % of PM-ESC. During SERCA inhibition by thapsigargin (2 µM), the amplitude (35±5%) and the frequency (45±15%) of Ca-transients decreased in 92 % of PM-ESC (n=24). Blockade of Na-Ca-exchanger by removal of extracellular Na⁺ and Ca²⁺ suppressed normal Ca-transients, and revealed irregular Ca²⁺ oscillations in 42 % of PM-ESC (n=23). Application of thapsigargin under this condition enhanced the rate of intracellular Ca²⁺ accumulation by four-fold within 4-5 min, and depleted SR Ca²⁺ content as shown by a reduced response to caffeine. When PM-ESC's were returned to culture medium, spontaneous Ca-transients recovered in 79 % of PM-ESC.

Conclusions:

Ca²⁺ release from the SR occurs in PM-ESC and augments the intrinsic frequency of Ca-transients, but is not essential for the basal activity. Intracellular Ca²⁺ accumulation during SERCA inhibition indicates Ca²⁺ leakage from the SR. A leaky SR and the tolerance to Ca²⁺ depletion characterize PM-ESC as cardiac pacemaker-like active cells.