In Failing Cardiomyocytes, CaM-RyR2 Dissociation Leads to Defective Domain Interaction and Channel Destabilization

Calmodulin (CaM) binding to RyR2 inhibits diastolic channel activity, indicating that CaM stabilizes RyR2 in the closed state. In conditions where CaM dissociates from RyR2, like heart failure (HF), RyR2 exhibits destabilized gating and increased activity. Those characteristics make CaM a critical regulator of RyR2 and potential therapeutic target for HF. Another leading mechanism for the RyR2 dysfunction in HF is defective domain interaction between N-terminal (N: 0-600) and central (C: 2000-2500) domains. However, the relationship between CaM-RyR2 binding and defective domain interaction in HF is unclear, especially in cardiomyocytes. Here, in cardiomyocytes from rat with HF induced by coronary ligation, we use FRET between fluorescent FKBP12.6 and CaM to specifically detect RyR2-bound CaM and measure the RyR2-CaM binding affinity. In steady-state binding affinity measurements, the Kd for CaM-RyR2 binding in HF myocytes is ∼51 nM, which is ∼3 fold increased vs. normal myocytes. By measuring the binding kinetics of fluorescent domain peptide DPc10 (F-DPc10), we can detect defective interaction between N-terminal and central domains in myocytes. In HF myocytes, the F-DPc10-RyR2 association rate was significantly accelerated vs. normal myocytes, indicating a destabilized domain interaction (unzipping). However, in HF myocytes, saturating RyR2 with high [CaM] dramatically reduced F-DPc10 binding to RyR2 (Bmax) and greatly slowed the association rate (kon). We conclude that in HF myocytes, reduced CaM-RyR2 binding affinity leads to defective domain interaction, shifting the channel to an “unzipped” state'. Promoting CaM-RyR2 reassociation can restore the defective domain interaction and stabilize the channel.