Abstract 490: Amitriptyline Activates Cardiac Ryanodine Receptors Independently of its Action on Cardiac Calsequestrin - Role for Amitriptyline Linked Sudden Cardiac Death
Cardiac calsequestrin (Casq2), a junctional sarcoplasmic reticulum (SR) protein, regulates ryanodine Ca2+-release channels (RyR2). Casq2 mutations cause spontaneous SR Ca2+-releases (SCRs) and ventricular arrhythmia in mice and humans. Amitriptyline (AMT), linked to increased risk of ventricular arrhythmia and sudden cardiac death in epidemiologic studies, binds to Casq2 and decreases its Ca2+ buffering capacity in vitro. Hence, AMT binding to Casq2 may render RyR2 channels leaky and cause ventricular arrhythmias akin to Casq2 mutations. We tested this hypothesis using wild-type, heterozygous and Casq2 null mice and examined the effect of AMT in ventricular myocytes, lipid bilayers and in vivo.
Results: AMT caused SCRs in wild-type myocytes (peak effect 1 μ M, n=139) and decreased SR Ca2+ content in a concentration dependant fashion (EC50 6 μM, n=139). Surprisingly, AMT had similar effects in Casq2 null myocytes, suggesting that AMT disrupts SR Ca2+ handling independently of its binding to Casq2. Accordingly, AMT activated native RyR2 channels isolated from Casq2 null hearts in lipid bilayers (EC50 3 μM, n=5, p<0.05). To explore the consequence of AMT action in vivo, wild-type and Casq2 heterozygous mice with implanted ECG telemeters were challenged with AMT (20 mg/kg). Unexpectedly, AMT challenge did not cause ventricular arrhythmias in vivo, even though peak AMT plasma and cardiac concentrations reached values high enough to activate the RyR2 channels in bilayers (1.8±1.3 and 34.5±1.9 μM, n=3 respectively). Moreover, AMT significantly reduced the incidence of ventricular ectopy after isoproterenol challenge in heterozygous mice (6/6 vs 0/6, p<0.05). However, the QRS widening after AMT challenge suggested that significant Na+ channel inhibition occurred in vivo, consistent with the AMT IC50 of 450±80 nM in recombinant Na+ channels (n=26).
Conclusion: We show for the first time that AMT directly activates RyR2 channels causing SCRs and SR Ca2+ depletion. However, AMT failed to induce ventricular ectopy in vivo, likely due to its potent Na+ channel blocking effect. This previously unrecognized AMT action on RyR2 may contribute to the complex clinical presentations, notably arrhythmias and depressed contractile function, seen with AMT overdoses.