Redox modification of ryanodine receptors contributes to sarcoplasmic reticulum Ca2+ leak in chronic heart failure

Abstract

Abnormal cardiac ryanodine receptor (RyR2) function is recognized as an important factor in the pathogenesis of heart failure (HF). However, the specific molecular causes underlying RyR2 defects in HF remain poorly understood. In the present study, we used a canine model of chronic HF to test the hypothesis that the HF-related alterations in RyR2 function are caused by posttranslational modification by reactive oxygen species generated in the failing heart. Experimental approaches included imaging of cytosolic ([Ca(2+)](c)) and sarcoplasmic reticulum (SR) luminal Ca(2+) ([Ca(2+)]SR) in isolated intact and permeabilized ventricular myocytes and single RyR2 channel recording using the planar lipid bilayer technique. The ratio of reduced to oxidized glutathione, as well as the level of free thiols on RyR2 decreased markedly in failing versus control hearts consistent with increased oxidative stress in HF. RyR2-mediated SR Ca(2+) leak was significantly enhanced in permeabilized myocytes, resulting in reduced [Ca(2+)](SR) in HF compared to control cells. Both SR Ca(2+) leak and [Ca(2+)](SR) were partially normalized by treating HF myocytes with reducing agents. Conversely, oxidizing agents accelerated SR Ca(2+) leak and decreased [Ca(2+)](SR) in cells from normal hearts. Moreover, exposure to antioxidants significantly improved intracellular Ca(2+)-handling parameters in intact HF myocytes. Single RyR2 channel activity was significantly higher in HF versus control because of increased sensitivity to activation by luminal Ca(2+) and was partially normalized by reducing agents through restoring luminal Ca(2+) sensitivity oxidation of control RyR2s enhanced their luminal Ca(2+) sensitivity, thus reproducing the HF phenotype. These findings suggest that redox modification contributes to abnormal function of RyR2s in HF, presenting a potential therapeutic target for treating HF.

Figure 1

Increases in oxidative stress, ROS formation, and RyR2 oxidation in heart failure. A, Averages of the levels of reduced form of glutathione (GSH) measured in tissue homogenates from normal (38.9±14.6 nmol per mg tissue) and failing hearts (29.4±11.2 nmol per mg tissue). B, Averages of ratios of reduced to oxidized glutathione (GSH/GSSG) measured in tissue homogenates from normal (28.6±10.4) and failing hearts (17.7±5.1). *P<0.05 (n=9 and 8 for control and HF, respectively). C, Fluorescence of the ROS-sensitive indicator DCFDA loaded into myocytes from normal and failing hearts. Representative images of DCFDA-loaded cells from control (top) and failing hearts under basal conditions (middle) and after 30 minutes of incubation with 800 μmol/L MPG (bottom). D, Summary graph for averages of DCFDA fluorescence normalized to the signal in the presence of 10 mmol/L H₂O₂ (F_max) measured in myocytes from control hearts (2.47±0.09) and myocytes from failing hearts under basal conditions (10.77±1.01) and in the presence of MPG (2.48±0.28). *P<0.05 vs control (n=21 to 32). E, Representative images of Coomassie-stained gels and mBB fluorescence intensity of RyR2 from normal and failing hearts. F, Pooled data for RyR2 content in samples from normal vs failing hearts. G, Relative free thiol content (%) of RyR2s from control vs HF samples obtained by normalizing mBB fluorescence to RyR2 level. *P<0.05 vs control (n=10).

Figure 2

Effects of oxidizing and reducing reagents on Ca²⁺ transients in control and HF myocytes. A, Representative linescan images and corresponding time-dependent profiles of cytosolic Ca²⁺ transients recorded during field stimulation (0.3 Hz) of control (left) and HF (right) myocytes under baseline (BL) conditions and after treatment of the cells with DTDP (100 μmol/L) or reducing MPG (800 μmol/L for 30 minutes), respectively. B, Summary graph for averages of amplitudes of Ca²⁺ transient. Rhod-2 ΔF/F₀ values were 1.07±0.09 for control, 0.65±0.04 for control+DTDP, 0.53±0.12 for HF, and 0.94±0.05 for HF+MPG (n from 5 to 9). *P<0.05 vs baseline, **P<0.05 vs control.

Figure 3

Effects of oxidizing and reducing agents on intra-SR Ca²⁺ in saponin-permeabilized myocytes from normal and failing hearts. A, Representative traces of time-dependent changes of cell-averaged luminal Fluo-5N signals in control (left) and HF permeabilized (right) myocytes. The experimental protocol involved consecutive applications of DTDP (50 μmol/L), DTT (1 mmol/L) and RR (30 μmol/L) in control cells and consecutive applications of DTT (1 mmol/L) and RR (30 μmol/L) in HF cells, as indicated. The values of F_min and F_max in both control and HF myocytes after removal of RR were obtained on application of 10 mmol/L caffeine with low Ca²⁺ and high Ca²⁺ (10 mmol/L), respectively. B, Summary graph of averaged Fluo-5N signals. The values of normalized Fluo-5N signal were 0.812±0.016, 0.456±0.012, 0.688±0.019, and 0.895±0.028 for baseline and in the presence of DTDP, DTT, and RR, respectively, in myocytes from normal hearts. In myocytes from failing hearts, the values for baseline and in the presence of DTT and RR were 0.610±0.012, 0.712±0.021, and 0.860±0.029, respectively. *P<0.05 vs baseline (BL), **P<0.05 vs control (n=10 to 12 for control and HF, respectively). C, SR Ca²⁺ leak determined as the difference in [Ca²⁺]_SR measured at the baseline conditions and in the presence of the 30 μmol/L RR in myocytes from healthy (0.083±0.026) and diseased hearts (0.25±0.017). D and E, Dose dependencies of DTDP and DTT effects on intra-SR [Ca²⁺] in myocytes from healthy (control) and failing hearts, respectively (n was from 7 to 14 for each data point). Data sets were fitted by logistic functions (red lines) with IC₅₀ values of 26 μmol/L for DTDP and 28 μmol/L for DTT.

Figure 4

Effects of oxidizing and reducing reagents on SR Ca²⁺ leak in control and HF permeabilized myocytes. A, Representative recordings of intra-SR Fluo-5N fluorescence as measures of SR Ca²⁺ leak unmasked by inhibition of the SERCA pump with Tg (10 μmol/L) in control (left) and HF permeabilized myocytes (right) under baseline conditions and after treatment of the control and HF cells with DTDP (50 μmol/L) or DTT (1 mmol/L), respectively. The decline of Fluo-5N signal in the presence of Tg was fitted by exponential functions (red lines). B, Average time constants (from exponential fits) of SR Ca²⁺ leak for control cells at baseline (BL) conditions (856±191 s, n=12) or in the presence of DTDP (139±20 seconds; n=8) (black bars) and for myocytes from failing hearts at baseline conditions (337±52 seconds; n=10) or in the presence of DTT (625±140 seconds; n=6) (light bars). *P<0.05 vs baseline (BL), **P<0.05 vs control.

Figure 5

Effects of oxidizing and reducing reagents on activity of RyRs from control and failing hearts. A, Representative recordings of single RyR2 channels from control and HF samples (top and bottom recordings) at low and high trans [Ca²⁺] and in the presence of either DTDP (10 μmol/L) or DTT (5 mmol/L) as indicated. Single-channel activities were recorded in 350 mmol/L symmetrical CsCH₃SO₃ solutions at 40 mV. Channel openings are shown as upward deflections from the closed level. B, Summary data of relative values of P_o in control RyR2s before and after the addition (to the cis side) of 10 μmol/L DTDP at 20 μmol/L Ca²⁺ trans [Ca²⁺] and following elevation of trans [Ca²⁺] to 2 mmol/L. *P<0.05 vs baseline (n=6 channels). C, Summary data of relative values of P_o in HF RyR2s before and after the addition (to the cis side) of 5 mmol/L DTT with 20 μmol/L [Ca²⁺] trans and following elevation of trans [Ca²⁺]to 2 mmol/L. *P<0.05 vs baseline, **P<0.05 vs 20 μmol/L Ca²⁺ trans [Ca²⁺](n=7 channels). D, Summary data of relative values of P_o at low and high luminal Ca²⁺ as in B and C.