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

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel the endoplasmic (ER)/sarcoplasmic reticulum that plays a cen-tral role in the excitation-contraction coupling in the heart. peractivity of RyR2 has been linked to ventricular arrhythmias patients with catecholaminergic polymorphic ventricular tachy-cardia and heart failure, where spontaneous Ca2+ release 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 com-pound library using a recently developed ER Ca2+-based say, where the inhibition of RyR2 activity was detected by increase in ER Ca2+ signals from R-CEPIA1er, a genetically coded ER Ca2+ indicator, in RyR2-expressing HEK293 cells. 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-mu- tant 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.