Intact Heart Loose Patch Photolysis Reveals Ionic Current Kinetics during Ventricular Action Potentials

Abstract

Rationale: Assessing the underlying ionic currents during a triggered action potential (AP) in intact perfused hearts offers the opportunity to link molecular mechanisms with pathophysiological problems in cardiovascular research. The developed loose patch photolysis technique can provide striking new insights into cardiac function at the whole heart level during health and disease. Objective: To measure transmembrane ionic currents during an AP to determine how and when surface Ca2+ influx that triggers Ca2+-induced Ca2+ release occurs and how Ca2+-activated conductances can contribute to the genesis of AP phase 2. Methods and Results: Loose patch photolysis allows the measurement of transmembrane ionic currents in intact hearts. During a triggered AP, a voltage-dependent Ca2+ conductance was fractionally activated (dis-inhibited) by rapidly photo-degrading nifedipine, the Ca2+ channel blocker. The ionic currents during a mouse ventricular AP showed a fast early component and a slower late component. Pharmacological studies established that the molecular basis underlying the early component was driven by an influx of Ca2+ through the L-type channel, CaV 1.2. The late component was identified as an Na+-Ca2+ exchanger current mediated by Ca2+ released from the sarcoplasmic reticulum. Conclusions: The novel loose patch photolysis technique allowed the dissection of transmembrane ionic currents in the intact heart. We were able to determine that during an AP, L-type Ca2+ current contributes to phase 1, whereas Na+-Ca2+ exchanger contributes to phase 2. In addition, loose patch photolysis revealed that the influx of Ca2+ through L-type Ca2+ channels terminates because of voltage-dependent deactivation and not by Ca2+-dependent inactivation, as commonly believed.