Abstract 411: Interruption of Specific Domain-Domain Interaction within Ryanodine Receptor Mediates Stabilization of Ryanodine Receptor in Failing Hearts
Interaction between N-terminal and central domains of ryanodine receptor (RyR1/RyR2), where many mutations have been found in patients with polymorphic VT or malignant hyperthermia (MH) was recently found to play a critical role in channel regulation. Here, we report that DP2114–2149, a synthetic cardiac domain peptide corresponding to Val-Met (mutable domain in MH) of RyR1, shows a significant RyR2 stabilizing effect in failing hearts.
Method and Results: Cardiomyocytes and sarcoplasmic reticulum (SR) vesicles were isolated from dog LV muscles (normal: n=6, 4-weeks pacing-induced heart failure: n=6). Either normal SR vesicles or human recombinant RyR2 fragments (1– 610, 741–1260, 1245–1768, 1741–2270, 2234–2750) were fluorescently labeled with methylcoumarin acetate (MCA) using DP2114–2149 as a site directing carrier; the carrier was removed after labeling. RyR2 in SR was specifically MCA-labeled. After tryptic digestion, the major MCA-labeled fragment of RyR2 (145 kD) was detected by an antibody (Ab) against central region (Ab2132). Moreover, of several recombinant RyR2 fragments, only RyR2 fragment2234–2750 was specifically MCA-labeled. Addition of DP2114–2149 to MCA-labeled SR competitively induced domain unzipping between native domain2114–2149 and its counter domain (EC50=0.3μM), as confirmed by a quenching of the MCA fluorescence by a large-size fluorescence quencher. The DP2114–2149 prevented spontaneous Ca2+ leak from failing SR (IC50=0.2μM). In failing cardiomyocytes, diastolic Ca2+ spark frequency (s−1 100ìm−1) was markedly increased (4.2±1.6, p<0.01) compared with normal (1.4±0.5), and delayed afterdepolaryzation-mediated Ca2+ transient (DAD-CaT) were frequently observed. Incorporation of DPc2114–2149 (by protein delivery kit) markedly decreased the frequency of Ca2+ spark (1.6±0.7) and DAD-CaT. A single Val-to-Met mutation made in DPc2114–2149, mimicking MH mutation, abolished all of these effects that would have been produced by DP2114–2149.
Conclusions: Interruption of the inter-domain interaction between 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 lethal arrhythmia.