Ca²⁺-Driven Selectivity of the Effect of the Cardiotonic Steroid Marinobufagenin on Rabbit Sinoatrial Node Function

The synergy between Na⁺-K⁺ pumps, Na⁺-Ca²⁺ exchangers, membrane currents and the sarcoplasmic reticulum (SR) generates the coupled-clock system, which governs the spontaneous electrical activity of heart sinoatrial node cells (SANCs). Ca²⁺ mediates the degree of clock coupling via local Ca²⁺ release (LCR) from the SR and activation of cAMP/PKA signaling. Marinobufagenin (MBG) is a natural Na⁺-K⁺ pump inhibitor whose effect on SANCs has not been measured before. The following two hypotheses were tested to determine if and how MBG mediates between the Na⁺-K⁺ pump and spontaneous SAN activity: (i) MBG has a distinct effect on beat interval (BI) due to variable effects on LCR characteristics, and (ii) Ca²⁺ is an important mediator between MBG and SANC activity. Ca²⁺ transients were measured by confocal microscopy during application of increasing concentrations of MBG. To further support the hypothesis that Ca²⁺ mediates between MBG and SANC activity, Ca²⁺ was chelated by the addition of BAPTA. Dose response tests found that 100 nM MBG led to no change in BI in 6 SANCs (no BI change group), and to BI prolongation in 10 SANCs (BI change group). At the same concentration, the LCR period was prolonged in both groups, but more significantly in the BI change group. BAPTA-AM prolonged the BI in 12 SANCs. In the presence of BAPTA, 100 nM MBG had no effect on SANC BI or on the LCR period. In conclusion, the MBG effects on SANC function are mediated by the coupled clock system, and Ca²⁺ is an important regulator of these effects.