Screening for Novel Type 2 Ryanodine Receptor Inhibitors by Endoplasmic Reticulum Ca2+ Monitoring.

Type 2 ryanodine receptor (RyR2) is a Ca²⁺ release channel on the endoplasmic (ER)/sarcoplasmic reticulum that plays a central role in the excitation-contraction coupling in the heart. Hyperactivity of RyR2 has been linked to ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia and heart failure, where spontaneous Ca²⁺ release via hyperactivated RyR2 depolarizes diastolic membrane potential to induce triggered activity. In such cases, drugs that suppress RyR2 activity are expected to prevent the arrhythmias, but there is no clinically available RyR2 inhibitors at present. In this study, we searched for RyR2 inhibitors from a well-characterized compound library using a recently developed ER Ca²⁺-based assay, where the inhibition of RyR2 activity was detected by the increase in ER Ca²⁺ signals from R-CEPIA1er, a genetically encoded ER Ca²⁺ indicator, in RyR2-expressing HEK293 cells. By screening 1535 compounds in the library, we identified three compounds (chloroxylenol, methyl orsellinate, and riluzole) that greatly increased the ER Ca²⁺ signal. All of the three compounds suppressed spontaneous Ca²⁺ oscillations in RyR2-expressing HEK293 cells and correspondingly reduced the Ca²⁺-dependent [³H]ryanodine binding activity. In cardiomyocytes from RyR2-mutant mice, the three compounds effectively suppressed abnormal Ca²⁺ waves without substantial effects on the action-potential-induced Ca²⁺ transients. These results confirm that ER Ca²⁺-based screening is useful for identifying modulators of ER Ca²⁺ release channels and suggest that RyR2 inhibitors have potential to be developed as a new category of antiarrhythmic drugs. SIGNIFICANCE STATEMENT: We successfully identified three compounds having RyR2 inhibitory action from a well-characterized compound library using an endoplasmic reticulum Ca²⁺-based assay, and demonstrated that these compounds suppressed arrhythmogenic Ca²⁺ wave generation without substantially affecting physiological action-potential induced Ca²⁺ transients in cardiomyocytes. This study will facilitate the development of RyR2-specific inhibitors as a potential new class of drugs for life-threatening arrhythmias induced by hyperactivation of RyR2.