Enhanced ryanodine receptor-mediated calcium leak determines reduced sarcoplasmic reticulum calcium content in chronic canine heart failure

Abstract

In this study, we investigated the role of elevated sarcoplasmic reticulum (SR) Ca(2+) leak through ryanodine receptors (RyR2s) in heart failure (HF)-related abnormalities of intracellular Ca(2+) handling, using a canine model of chronic HF. The cytosolic Ca(2+) transients were reduced in amplitude and slowed in duration in HF myocytes compared with control, changes paralleled by a dramatic reduction in the total SR Ca(2+) content. Direct measurements of [Ca(2+)](SR) in both intact and permeabilized cardiac myocytes demonstrated that SR luminal [Ca(2+)] is markedly lowered in HF, suggesting that alterations in Ca(2+) transport rather than fractional SR volume reduction accounts for the diminished Ca(2+) release capacity of SR in HF. SR Ca(2+) ATPase (SERCA2)-mediated SR Ca(2+) uptake rate was not significantly altered, and Na(+)/Ca(2+) exchange activity was accelerated in HF myocytes. At the same time, SR Ca(2+) leak, measured directly as a loss of [Ca(2+)](SR) after inhibition of SERCA2 by thapsigargin, was markedly enhanced in HF myocytes. Moreover, the reduced [Ca(2+)](SR) in HF myocytes could be nearly completely restored by the RyR2 channel blocker ruthenium red. The effects of HF on cytosolic and SR luminal Ca(2+) signals could be reasonably well mimicked by the RyR2 channel agonist caffeine. Taken together, these results suggest that RyR2-mediated SR Ca(2+) leak is a major factor in the abnormal intracellular Ca(2+) handling that critically contributes to the reduced SR Ca(2+) content of failing cardiomyocytes.

FIGURE 1

Simultaneous measurements of cytosolic and intra-SR [Ca²⁺] transients. (A) Representative line-scan images of Rhod-2 (upper) and Fluo-5N (lower) fluorescence in control (C) and heart failure (HF) myocytes. Ca²⁺ transients were evoked by 0.5-Hz electrical field stimulation. (B) Average amplitudes (F/F₀) of cytosolic Ca²⁺ transients were 2.21 ± 0.12 in control (C) and 1.44 ± 0.06 in HF myocytes. (C) Average amplitudes (ΔF/ΔF_MAX) of SR Ca²⁺ depletion were 0.14 ± 0.04 in control (C) and 0.04 ± 0.007 in HF myocytes. (D) The average levels of free diastolic SR Ca²⁺ (ΔF_CAF/ΔF_MAX) in control (C) and HF myocytes were 0.53 ± 0.07 and 0.39 ± 0.02, respectively (*p < 0.05; **p < 0.01 versus controls).

FIGURE 2

HF slows the kinetics of cytosolic and SR Ca²⁺ transients. (A) Representative time-dependent profiles of cytosolic (upper panel) and intra-SR (lower panel) Ca²⁺ transients induced by 0.5-Hz electrical field stimulation in control (C) and HF myocytes. The decay of the transients was fit by a single exponential function with specified time constants. Insets show Ca²⁺ transients with normalized amplitudes. (B) Average time constants of cytosolic Ca²⁺ transient decays were 295 ± 24 ms in control (C) and 447 ± 32 ms in HF myocytes. (C) Average time constants of the SR Ca²⁺ transient decays were 307 ± 26 ms in control (C) and 441 ± 26 ms in HF myocytes (*p < 0.05; **p < 0.01).

FIGURE 3

HF reduces SR Ca²⁺ content and moderately increases the rate of cytosolic Ca²⁺ removal by NCX. (A) Representative recordings of Ca²⁺ transients induced by rapid application of 10 mM caffeine in control (C) and HF myocytes. The inset shows caffeine-induced Ca²⁺ transients with normalized amplitude. (B) Average amplitudes (F/F₀) of caffeine-induced Ca²⁺ transients were 6.14 ± 0.47 in control and 4.69 ± 0.30 in HF myocytes, respectively. (C) The decay time constants of the caffeine-induced Ca²⁺ transients in control and HF myocytes were 2.8 ± 0.3 and 2.0 ± 0.1 s, respectively (*p < 0.05).

FIGURE 4

SERCA2-mediated SR Ca²⁺ uptake is not significantly altered in HF myocytes. (A) Time course of SR Ca²⁺ uptake in control and HF permeabilized myocytes, measured with Fluo-5N-loaded SR, in the presence of 10 μM ruthenium red (Rut Red). The SR [Ca²⁺] was depleted with 10 mM caffeine in Ca²⁺-free solution, and SR Ca²⁺ uptake was initiated by the addition of either 100 or 500 nM of Ca²⁺. (B) Average time constants (from exponential fit) of SR Ca²⁺ uptake were 45.5 ± 3.1 s in control (C) and 48.9 ± 2.4 s in HF myocytes, when measured in the presence of 100 nM Ca²⁺, and 12.0 ± 0.7 s in control and 10.5 ± 0.6 s in HF myocytes, when measured in the presence of 500 nM Ca²⁺.

FIGURE 5

HF increases SR Ca²⁺ leak. (A) Time-dependent profiles of intra-SR Ca²⁺ signals. Application of 10 μM of thapsigargin (Tg) evokes a steady loss of intra-SR Ca²⁺, visualized by the decline of the Fluo-5N signal, which was significantly faster in permeabilized myocytes from failing hearts. Then 10 mM caffeine (Caf) was applied to determine the Fluo-5N signal with depleted SR. Maximal Fluo-5N signal was determined by simultaneous application of 1 μM ionomycin with 10 mM Ca²⁺ and 10 mM BDM. (B) Time course of normalized Fluo-5N signals in control and HF myocytes measured in the presence of Tg, as demonstrated in panel A. Kinetic analysis was performed in both control and HF over the same range of Fluo-5N signal, as indicated in panel A by the wide black line. Dotted lines represent exponential fit to the data. (C) Time-dependent profiles of intra-SR Fluo-5N signals before and after application of 10 μM ruthenium red (Rut Red) in control and HF cells. (D) In control myocytes, normalized [Ca²⁺]_SR levels were 0.83 ± 0.02 in the absence of, and 0.92 ± 0.02 in the presence of, Rut Red, respectively. In HF myocytes, normalized [Ca²⁺]_SR levels were 0.61 ± 0.03 in the absence of, and 0.77 ± 0.04 in the presence of, Rut Red, respectively (*p < 0.05 versus control; **p < 0.01 versus control).

FIGURE 6

Caffeine mimics the properties of global Ca²⁺ release observed in HF. Representative simultaneous recordings of cytosolic (measured with Rhod-2) and intra-SR [Ca²⁺] (measured with Fluo-5N) signals in voltage-clamped myocytes before and after application of the specified concentrations of caffeine. Caffeine produced significant deceleration of relaxation and decreased amplitudes of the cytosolic and intra-SR [Ca²⁺] transients. Ca²⁺ transients were evoked by 300-ms depolarizing steps from a holding potential of −50 mV to 0 mV every 2 s.

FIGURE 7

Caffeine application to control myocytes mimics the properties of the SR Ca²⁺ leak observed in HF myocytes. (A) Time-dependent profiles of intra-SR Ca²⁺ signals. Application of 10 μM thapsigargin (Tg) evokes a steady loss of intra-SR Ca²⁺, visualized by the decline of Fluo-5N signal. Caffeine (1 mM) produces SR Ca²⁺ leak acceleration that is qualitatively similar to that occurring in HF. Dotted lines represent time-dependent profiles of intra-SR Ca²⁺ signals recorded in control (black) and HF (gray) myocytes. (B) Decline of Fluo-5N signal in the presence of thapsigargin was fit by an exponential function. The average time constants were 713 ± 160, 337 ± 53, and 201 ± 27 s in controls, HF myocytes, and control myocytes in the presence of 1 mM caffeine, respectively. (C) Time-dependent profiles of intra-SR Fluo-5N signals in control cells before and after application of 10 μM ruthenium red (Rut Red) in the absence and in the presence of specified concentrations of caffeine. Caffeine (Caf, 10 mM) was applied to determine Fluo-5N signal with Ca²⁺-depleted SR. The maximal Fluo-5N signal was determined by simultaneous application of ionomycin with 10 mM Ca²⁺ and 10 mM BDM. In the absence of caffeine, normalized [Ca²⁺]_SR levels were 0.73 ± 0.03 before and 0.92 ± 0.03 during application of Rut Red, respectively. In the presence of 2 mM caffeine, normalized [Ca²⁺]_SR levels were 0.28 ± 0.03 before and 0.76 ± 0.03 during the application of Rut Red, respectively (*p < 0.05 versus control; **p < 0.01 versus control).