Dissociation of calmodulin from cardiac ryanodine receptor causes aberrant Ca(2+) release in heart failure

Abstract

Aims: Calmodulin (CaM) is well known to modulate the channel function of the cardiac ryanodine receptor (RyR2). However, the possible role of CaM on the aberrant Ca(2+) release in diseased hearts remains unclear. In this study, we investigated the state of RyR2-bound CaM and channel dysfunctions in pacing-induced failing hearts.

Methods and results: The characteristics of CaM binding to RyR2 and the role of CaM on the aberrant Ca(2+) release were assessed in normal and failing canine hearts. The affinity of CaM binding to RyR2 was lower in failing sarcoplasmic reticulum (SR) than in normal SR. Addition of FK506, which dissociates FKBP12.6 from RyR2, to normal SR reduced the CaM-binding affinity. Dantrolene restored a normal level of the CaM-binding affinity in either FK506-treated (normal) SR or failing SR, suggesting that the defective inter-domain interaction between the N-terminal domain and the central domain of RyR2 (the therapeutic target of dantrolene) is involved in the reduction of the CaM-binding affinity in failing hearts. In saponin-permeabilized cardiomyocytes, the frequency of spontaneous Ca(2+) sparks was much more increased in failing cardiomyocytes than in normal cardiomyocytes, whereas the addition of a high concentration of CaM attenuated the aberrant increase of Ca(2+) sparks.

Conclusion: The defective inter-domain interaction between N-terminal and central domains within RyR2 reduces the binding affinity of CaM to RyR2, thereby causing the spontaneous Ca(2+) release events in failing hearts. Correction of the defective CaM binding may be a new strategy to protect against the aberrant Ca(2+) release in heart failure.

Figure 1

Cross-linking of CaM to the RyR2 in normal and failing SR vesicles. (A) Representative western blotting of CaM cross-linked to the RyR2 (top) and the summarized data (bottom). The immunoblot density of CaM cross-linked to the RyR2 was determined at various concentrations of CaM-SANPAH as indicated and expressed as the ratio to maximum value obtained at 1 µmol/L CaM. (B) Effects of the antibody against CaMBD (3583–3603; Anti-CaMBD Ab), FK506 (30 µmol/L), and/or dantrolene (1 µmol/L) on the cross-linking of CaM (128 nmol/L) to the RyR2 in normal and failing SR vesicles. Anti-CaMBD Ab: anti-CaMBD antibody. Representative western blots of CaM bound to the RyR2 (top) and the summarized data (bottom). The immunoblot density of CaM cross-linked to the RyR2 was measured and expressed as the ratio to control. Data represent means ± SD of 3–4 SR preparations.

Figure 2

Localization and the binding characteristics of exogenously introduced CaM in saponin-permeabilized cardiomyocytes. The CaM, fluorescently labelled with Alexa Fluor 488 (Molecular Probes, OR, USA), was delivered into the cardiomyocytes. The fluorescently labelled cardiomyocytes were laser-scanned with the confocal microscope system (LSM-510, Carl Zeiss). (A) (left) Representative images of exogenously introduced CaM co-localized with the RyR2 in normal cardiomyocytes. Left; CaM-Alexa (green); middle; RyR2 (red); right; merged image. (Right) Periodical increases in the Alexa fluorescence signals of either RyR2 (red) or CaM (green). (B) Delivery of various concentrations of the CaM-Alexa (left) and the summarized data (right). Data represent means ± SD of 22–26 cells from 3–4 hearts. The CaM-Alexa fluorescence was measured and expressed as the ratio to its maximum value. (C) Effect of FK506 (30 µmol/L) or dantrolene (1 µmol/L) on the CaM-Alexa (200 nmol/L) binding on the RyR2 in normal or failing cardiomyocytes. Representative images (top) and the summarized data (bottom). The CaM-Alexa fluorescence was measured and expressed as the ratio to the control value. Data represent means ± SD of 24–40 cells from 3–4 hearts.

Figure 3

Localization and the binding characteristics of endogenous CaM in normal and failing cardiomyocytes. (A) (left) Representative images of the endogenous CaM, detected by the anti-CaM antibody, co-localized with the RyR2 in normal cardiomyocytes. Left; CaM (red); middle; RyR2 (green); right; merged image. (Right) Periodical increases in the Alexa fluorescence signals of either CaM (red) or RyR2 (green). (B) Effect of FK506 (30 µmol/L) and/or dantrolene (1 µmol/L) on the endogenous CaM binding on the RyR2. Representative images of the endogenous CaM bound to the RyR2 (top) and the summarized data (bottom). The fluorescence signal of the endogenous CaM was measured and expressed as the ratio to control. Data represent means ± SD of 19–22 cells from 3–4 hearts.

Figure 4

Effect of FK506 (30 µmol/L), dantrolene (1 µmol/L), and/or CaM (1 µmol/L) on Ca²⁺ sparks in normal and failing saponin-permeabilized cardiomyocytes. (A) Representative images. (B) Relationship between the Ca²⁺ spark frequency and SR Ca²⁺ content. SR Ca²⁺ content was obtained by the application of 10 mmol/L caffeine. Ca²⁺ spark images were obtained in the presence of the CaMKII inhibitor KN-93 (1 µmol/L). Data represent means ± SD of 31–54 cells from 4–5 hearts.

Figure 5

Fluorescence quenching analysis of the domain unzipping. Evaluation of the domain unzipping between the N-terminal (1–600) and the central domains (2000–2500). The accessibility of the RyR2-bound MCA to a macromolecular fluorescence quencher BSA-QSY conjugate was measured and the Stern–Volmer fluorescence quenching constant (i.e. the slope of the Fo/F vs. [BSA-QSY] plot) was determined as a measure of the degree of domain unzipping. Statistical comparison of the slope of each plot, which is equivalent to the Stern–Volmer quenching constant (K_Q) (bottom figures). Data represent means ± SD of four SR preparations. Diagrams for data interpretation are added at the bottom of each figure.