Looking at the Trigger for CICR During Rat Cardiac Action Potentials

Abstract

It is generally accepted that cardiac CICR is triggered by L-type Ca2+ channel activation during the action potential. At the microscopic level, CICR is revealed by calcium sparks whose activation by single L-type Ca2+ channels has been demonstrated. In this study, we have tried to visualize the Ca2+ influx (that triggers CICR) during normal action potentials and when the L-type Ca2+ channel is partially blocked and/or during the application of an L-type Ca2+ channel gating modifier FPL64176. Using Fluo-4 and high speed confocal line scanning we have detected the rise in Ca2+ that precedes SR Ca2+ release. When ∼90% of L-type Ca2+ channels are blocked with 10 uM nifedipine, L-type Ca2+ influx is seen as an increase in fluorescence of ∼2 %/ms, which is 8% of the rate of rise of Ca2+ associated with Ca2+ sparks. This is associated with a latency for Ca2+ spark activation of typically 9 ms; assuming a Kd for fluo-4 of 800 and a resting Ca2+ of 65nM the trigger in these conditions equates to a current of ∼1 nA for a 30 pL cell. An unexpected finding was that FPL64176 did not dramatically decrease the latency for Ca2+ spark activation, as might be expected if many short L-type Ca2+ channel openings are required to activate CICR. In addition, no ‘sparklets’ were observed as might be expected if L-type Ca2+ channels are located almost exclusively in the junctional space and Ca2+ sparks activate with minimal delay.