Establishment of molecular targeting therapy for duonic heart failure bystabilizmgryanodine receptor

点击翻译按钮获取中文摘要

Two domains within the ryanodine receptor (RyR2) of sarcoplasmic reticulum (SR) {N-terminal (0-600) and central (2000-2500) domains} was found to interact with each other as a regulatory switch for channel gating. We previously reported that K201 (JTV519) inhibits Ca^<2+> leak by correcting the defective inter-domain interaction between the two domains in failing hearts. Here, we identified the K201-binding domain and diaracterizedtherole ofthis novel domain on RyR2 channel gating.An assay using a quartz-crystal microbalance revealed that K201 specifically bound to recornbinant RyR2 fragment: 1741. 2270 in the 1-2750 region. By further analysis of the fragnent1741-2270, 201 was found to specifically bind to its sub-fragment2114-2149. Using the peptide matching this sub-fragment (DP2114-2149)as a carrier, the RyR2 was specifically labeled with methylcoumarin acetate(MCA). Moreover, of several recombinant RyR2 fragments, only fragment 2234-2750 was specifically MCA-labeled; this suggests that the K201 binding domain2114-2149 binds with domain 2234-2750. Addition of DP2114-2149 to the MCA-labeled SR interfered with the interaction between domain2114-2149 and domain2234-2750 causing domain unzipping, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. In failing cardiomyocytes, the frequency of spontaneous Ca^<2+> spark (CaSF) was much higher than normal cardiomyocytes (p<0.01), whereas incorporation of DP2114-2149 markedly decreased CaSF to normal level; the same effect as that produced by K201.In conclusion, we first identified the K201-binding site as domain2114-2149 of RyR2 Interruption of the inter-domain interaction between the domain2114-2149 and central domain2234-2750 seems to mediate stabilization of RyR2 in failing hearts, which may lead to a novel therapeutic strategy against heart failure and perhaps lethal arrhythmia.