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, 96 (3), 433-43

Enhanced Binding of Calmodulin to the Ryanodine Receptor Corrects Contractile Dysfunction in Failing Hearts

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Enhanced Binding of Calmodulin to the Ryanodine Receptor Corrects Contractile Dysfunction in Failing Hearts

Akihiro Hino et al. Cardiovasc Res.

Abstract

Aims: The channel function of the cardiac ryanodine receptor (RyR2) is modulated by calmodulin (CaM). However, the involvement of CaM in aberrant Ca(2+) release in diseased hearts remains unclear. Here, we investigated the pathogenic role of defective CaM binding to the RyR2 in the channel dysfunction associated with heart failure.

Methods and results: The involvement of CaM in aberrant Ca(2+) release was assessed in normal and pacing-induced failing canine hearts. The apparent affinity of CaM for RyR2 was considerably lower in failing sarcoplasmic reticulum (SR) compared with normal SR. Thus, the amount of CaM bound to RyR2 was markedly decreased in failing myocytes. Expression of the CaM isoform Gly-Ser-His-CaM (GSH-CaM), which has much higher binding affinity than wild-type CaM for RyR1, restored normal CaM binding to RyR2 in both SR and myocytes of failing hearts. The Ca(2+) spark frequency (SpF) was markedly higher and the SR Ca(2+) content was lower in failing myocytes compared with normal myocytes. The incorporation of GSH-CaM into the failing myocytes corrected the aberrant SpF and SR Ca(2+) content to normal levels.

Conclusion: Reduced CaM binding to RyR2 seems to play a critical role in the pathogenesis of aberrant Ca(2+) release in failing hearts. Correction of the reduced CaM binding to RyR2 stabilizes the RyR2 channel function and thereby restores normal Ca(2+) handling and contractile function to failing hearts.

Figures

Figure 1
Figure 1
Cross-linking of CaM or GSH-CaM to the ryanodine receptor (RyR2). (A) Representative immunoblot of CaM or GSH-CaM cross-linked to RyR2. Note that the anti-CaM (or GSH-CaM) antibody detected only RyR2 among the many proteins in the SR. (B) Effect of the antibody against the CaM-binding domain (3583–3603; anti-CaMBD antibody) of RyR2 on CaM (or GSH-CaM) binding to RyR2.
Figure 2
Figure 2
(A) CaM (or GSH-CaM) concentration dependence of the binding of CaM to normal RyR2 (top) and the summarized data (bottom). The immunoblot density of the CaM cross-linked to RyR2 was determined at various concentrations of CaM (or GSH-CaM)-SANPAH as indicated and expressed as the ratio to the maximum value obtained at 1 µmol/L CaM (or GSH-CaM). Each datum point per concentration represents mean ± SD of four SR preparations from four hearts, and the sigmoid concentration-dependent relationships for CaM binding was fitted by an equation: y = aKnxn/(1 + Knxn), and the EC50 values were calculated as 1/K. (Inset) Comparison of EC50. Paired t-test was employed to determine the statistical significance of EC50. The numerical value in the parenthesis means the number of concentration-dependent relationships for CaM binding. (B) Cross-linking of CaM or GSH-CaM (128 nmol/L) to RyR2 in DPc10 (30 µmol/L)- or DP4090–4123 (30 µmol/L)-treated SR vesicles from normal and failing hearts. Representative immunoblots of CaM bound to RyR2 (top) and summarized data (bottom) are shown. The immunoblot density of CaM cross-linked to RyR2 was measured and expressed as the ratio to the control. Data represent the means ± SD obtained from four SR preparations.
Figure 3
Figure 3
The binding characteristics of exogenously introduced CaM in saponin-permeabilized cardiomyocytes. Delivery of various concentrations of CaM-Alexa or GSH-CaM-Alexa (left panel) and the summarized data (right panel) in normal cardiomyocytes and failing cardiomyocytes treated with RyR2 domain peptides (DPc10, 30 µmol/L, or DP4090–4123, 30 µmol/L). Either WT-CaM-Alexa or GSH-CaM-Alexa fluorescence was measured and expressed as the ratio to its maximum value. Each datum point per concentration represents mean ± SD of 8–10 cells from three to four hearts, and the sigmoid concentration-dependent relationships for CaM binding was fitted by an equation: y = aKnxn/(1 + Knxn), and the EC50 values were calculated as 1/K. (Inset) Comparison of EC50. Paired t-test was employed to determine the statistical significance of EC50. The numerical value in the parenthesis means the number of concentration-dependent relationships for CaM binding.
Figure 4
Figure 4
Effect of WT-CAM (or GSH-CaM) (200 nmol/L) on Ca2+ SpF and SR Ca2+ content in (saponin-permeabilized) normal cardiomyocytes and failing cardiomyocytes treated with RyR2 domain peptides (DPc10, 30 µmol/L, or DP4090–4123, 30 µmol/L). SR Ca2+ content was measured by adding 10 mmol/L caffeine. Ca2+ spark images were obtained in the presence of the CaMKII inhibitor KN-93 (1 µmol/L). (A) Representative images of Ca2+ sparks. (B) Summarized data of the FDHM, FWHM, Ca2+ spark amplitude, Ca2+ SpF, and SR Ca2+ content. Data represent the means ± SD of 8–32 cells from each of three to four hearts.
Figure 5
Figure 5
Effect of incorporation of WT-CaM or GSH-CaM on the binding of CaM to RyR2. Representative images of the binding of CaM to RyR2 (top) and the summarized data (bottom) are shown. Data represent means ± SD of 5–11 cells from each of two to three hearts.
Figure 6
Figure 6
Effect of CaM or GSH-CaM on Ca2+ transient and cell shortening in normal and failing cardiomyocytes. (A) Representative tracings of Ca2+ transient and cell shortening in normal and failing cardiomyocytes. (B) The characteristics of cell shortening and Ca2+ transient in normal and failing cardiomyocytes treated with RyR2 domain peptides (DPc10, 30 µmol/L, or DP4090–4123, 30 µmol/L).

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