Abstract 13909: Interruption of Novel Inter-domain Interaction Within RyR2 as a Common Therapeutic Target by Channel Stabilization in Heart Failure and Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT)
We recently reported that K201(JTV519) specifically binds to domain2114–2149 of the ryanodine receptor (RyR2) and stabilizes the channel. Here, we further characterized the role of this novel domain on the channel gating in tachycardia induced canine heart failure model and human CPVT-associated RyR2 R2474S/+ knock-in (KI) mice model.
Methods and results: Using a synthetic peptide matching domain2114–2149 of RyR2 (DP2114–2149) as a site-directing carrier, either recombinant fragments (1–610, 741–1260, 1245–1768, 1741–2270, 2234–2750) of RyR2, or canine RyR2 was fluorescently labeled with methylcoumarin acetate (MCA). Of these recombinant RyR2 fragments, only fragment2234–2750 was specifically MCA-labeled. After tryptic digestion of this MCA-labeled fragment, domain2234–2370 (14.8 kDa) was further identified as the shortest MCA-labeled fragment. The MCA-labeling of the 155kDa tryptic fragment (harboring domain2234–2370) in canine RyR2 was also confirmed. Addition of DP2114–2149 to the MCA-labeled canine RyR2 interfered with the interaction between domain2114–2149 and domain2234–2370 (viz. domain unzipping by competing with native domain), as evidenced by an increased accessibility of a large-size fluorescence quencher to the bound MCA. Interestingly, in failing canine SR, (abnormal) domain zipping between the aforementioned two domains has already taken place. In failing (F) canine cardiomyocytes, the frequency of spontaneous Ca2+ spark (CaSF:s-1 100µm-1) was markedly increased compared with normal (N) canine cardiomyocytes (F:2.8±0.6, n=12, N:0.9±0.4, n=12; p<0.01 vs N), whereas incorporation of DP2114–2149 markedly decreased CaSF (1.2±0.5, p<0.01 vs F). In saponin-permeabilized KI cardiomyocytes, CaSF was much more increased in response to cAMP (0.1–1 µM) than in wild type (WT) cardiomyocytes (KI:18.2±0.4, n=14 vs WT:12.9±0.4, n=14; at [cAMP]=0.3 µM , p<0.01). DP2114–2149 abolished the cAMP-induced increase in CaSF in KI.
Conclusions: The abnormal interaction between domain2114–2149 and domain2234–2370 within RyR2 plays a critical role in channel destabilization. Interruption of this specific domain-domain interaction could be a novel common therapeutic strategy against heart failure and lethal arrhythmia.
- © 2010 by American Heart Association, Inc.