Rhythmic beating of stem cell-derived cardiac cells requires dynamic coupling of electrophysiology and Ca cycling

Abstract There is an intense interest in differentiating embryonic stem cells to engineer biological pacemakers as an alternative to electronic pacemakers for patients with cardiac pacemaker function deficiency. Embryonic stem cell-derived cardiocytes (ESCs), however, often exhibit dysrhythmic excitations. Using Ca2+ imaging and patch-clamp techniques, we studied requirements for generation of spontaneous rhythmic action potentials (APs) in late-stage mouse ESCs. Sarcoplasmic reticulum (SR) of ESCs generates spontaneous, rhythmic, wavelet-like L ocal C a2+ R eleases (LCRs) (inhibited by ryanodine, tetracaine, or thapsigargin). L-type Ca2+ current ( ICaL ) induces a global Ca2+ release (CICR), depleting the Ca2+ content SR which resets the phases of LCR oscillators. Following a delay, SR then generates a highly synchronized spontaneous Ca2+ release of multiple LCRs throughout the cell. The LCRs generate an inward Na+ / Ca2+ exchanger (NCX) current (absent in Na+ -free solution) that ignites the next AP. Interfering with SR Ca2+ cycling (ryanodine, caffeine, thapsigargin, cyclopiazonic acid, BAPTA-AM), NCX (Na+ -free solution), or ICaL (nifedipine) results in dysrhythmic excitations or cessation of automaticity. Inhibition of cAMP/PKA signaling by a specific PKA inhibitor, PKI, decreases SR Ca2+ loading, substantially reducing both spontaneous LCRs (number, size, and amplitude) and rhythmic AP firing. In contrast, enhancing PKA signaling by cAMP increases the LCRs (number, size, duration) and converts irregularly beating ESCs to rhythmic “pacemaker-like” cells. SR Ca2+ loading and LCR activity could be also increased with a selective activation of SR Ca2+ pumping by a phospholamban antibody. We conclude that SR Ca2+ loading and spontaneous rhythmic LCRs are driven by inherent cAMP/PKA activity. ICaL synchronizes multiple LCR oscillators resulting in strong, partially synchronized diastolic Ca2+ release and NCX current. Rhythmic ESC automaticity can be achieved by boosting “coupling” factors, such as cAMP/PKA signaling, that enhance interactions between SR and sarcolemma.