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

Oates Institute for Experimental Therapeutics and Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee (N.C., S.S.D., B.C.K.); and Department of Biomedical Sciences, University of Newcastle and HMRI, Callaghan, Australia (D.L.).

Patients taking amitriptyline (AMT) have an increased risk of sudden cardiac death, yet the mechanism for AMT's proarrhythmic 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 cardiomyocytes 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 µM) compared with its effect in lipid bilayers (5-10 µM). 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.

Address correspondence to: Dr. Björn C. Knollmann, Oates Institute for Experimental Therapeutics, Division of Clinical Pharmacology, Vanderbilt University Medical Center, 1265 Medical Research Building IV, Nashville, TN 37232-0575. E-mail: bjorn.knollmann{at}vanderbilt.edu