Amitriptyline Activates Cardiac Ryanodine Channels and Causes Spontaneous Sarcoplasmic Reticulum Calcium Release

Patients taking amitriptyline (AMT) have an increased risk of sudden cardiac death, yet the mechanism for AMT's proar-rhythmic effects remains incompletely understood. Here, we hypothesize that AMT activates cardiac ryanodine channels (RyR2), causing premature Ca2+ release from the sarcoplasmic reticulum (SR), a mechanism identified by genetic studies as a cause of ventricular arrhythmias and sudden cardiac death. To test this hypothesis, we measured the effect of AMT on RyR2 channels from mice and sheep and on intact mouse cardio-myocytes loaded with the Ca2+ fluorescent indicator Fura-2 acetoxymethyl ester. AMT induced trains of long channel openings (bursts) with 60 to 90% of normal conductance in RyR2 channels incorporated in lipid bilayers. The [AMT], voltage, and open probability (P-o) dependencies of burst frequency and duration indicated that AMT binds primarily to open RyR2 channels. AMT also activated RyR2 channels isolated from transgenic mice lacking cardiac calsequestrin. Reducing RyR2 P-o by increasing cytoplasmic [Mg2+] significantly inhibited the AMT effect on RyR2 channels. Consistent with the single RyR2 channel data, AMT increased the rate of spontaneous Ca2+ releases and decreased the SR Ca2+ content in intact cardiomyocytes. Intracellular [AMT] were approximately 5-fold higher than extracellular [AMT], explaining AMT's higher potency in cardiomyocytes at clinically relevant concentrations (0.5-3 mu M) compared with its effect in lipid bilayers (5-10 mu M). Increasing extracellular [Mg2+] attenuated the effect of AMT in intact myocytes. We conclude that the heretofore unrecognized activation of RyR2 channels and increased SR Ca2+ leak may contribute to AMT's proarrhythmic and cardiotoxic effects, which may be counteracted by interventions that reduce RyR2 channel open probability.