Hypersensitivity of excitation-contraction coupling in dystrophic cardiomyocytes

Abstract

Duchenne muscular dystrophy represents a severe inherited disease of striated muscle. It is caused by a mutation of the dystrophin gene and characterized by a progressive loss of skeletal muscle function. Most patients also develop a dystrophic cardiomyopathy, resulting in dilated hypertrophy and heart failure, but the cellular mechanisms leading to the deterioration of cardiac function remain elusive. In the present study, we tested whether defective excitation-contraction (E-C) coupling contributes to impaired cardiac performance. "E-C coupling gain" was determined in cardiomyocytes from control and dystrophin-deficient mdx mice. To this end, L-type Ca2+ currents (ICaL) were measured with the whole cell patch-clamp technique, whereas Ca2+ transients were simultaneously recorded with confocal imaging of fluo-3. Initial findings indicated subtle changes of E-C coupling in mdx cells despite matched Ca2+ loading of the sarcoplasmic reticulum (SR). However, lowering the extracellular Ca2+ concentration, a maneuver used to unmask latent E-C coupling problems, was surprisingly much better tolerated by mdx myocytes, suggesting a hypersensitive E-C coupling mechanism. Challenging the SR Ca2+ release by slow elevations of the intracellular Ca2+ concentration resulted in Ca2+ oscillations after a much shorter delay in mdx cells. This is consistent with an enhanced Ca2+ sensitivity of the SR Ca2+-release channels [ryanodine receptors (RyRs)]. The hypersensitivity could be normalized by the introduction of reducing agents, indicating that the elevated cellular ROS generation in dystrophy underlies the abnormal RyR sensitivity and hypersensitive E-C coupling. Our data suggest that in dystrophin-deficient cardiomyocytes, E-C coupling is altered due to potentially arrhythmogenic changes in the Ca2+ sensitivity of redox-modified RyRs.

Fig. 1.

L-type Ca²⁺ currents (I_CaL), Ca²⁺ transients, and excitation-contraction (E-C) coupling gain under control conditions [1.2 mM extracellular Ca²⁺ concentration ([Ca²⁺]_o)]. A: voltage protocol used for the experiments. The eight prepulses were always applied in control solution, whereas for the test step [Ca²⁺]_o was briefly varied according to the experimental challenge. B and C: representative traces for Ca²⁺ currents (bottom), line-scan images of Ca²⁺-related changes in fluorescence (ΔF/F₀; middle), and normalized cytosolic Ca²⁺ transients (top) elicited by a 400-ms test pulse to 0 mV in control solution in a wild-type (WT) cell (B) and a mdx cell (C). D: voltage dependence of current activation [current-voltage (I–V) curve, bottom traces] and corresponding Ca²⁺ transients (Ca²⁺-V curve, top traces) in WT (black; n = 8–21 cells) and mdx (red; n = 15–30 cells) myocytes. E: E-C coupling gain calculated from the data shown in D in WT (black) and mdx (red) myocytes at different test voltages.

Fig. 2.

I_CaL, Ca²⁺ transients, and E-C coupling gain challenged by low (0.5 mM) [Ca²⁺]_o. Please note that the prepulses to load the sarcoplasmic reticulum (SR) were done in control solution (1.2 mM [Ca²⁺]_o). A: voltage dependence of current (top lines) and corresponding Ca²⁺ transients (bottom lines) in WT (black) and mdx (red) cells. B: voltage dependence of the E-C coupling gain from the data shown in A in WT (black; n = 4–17 cells) and mdx (red; n = 9–25 cells) myocytes. C: statistical comparison of the E-C coupling gain in 1.2 and 0.5 mM Ca²⁺ in WT (black; n = 15 cells) and mdx (red; n = 25 cells) cells.

Fig. 3.

I_CaL, Ca²⁺ transients, and E-C coupling gain in 0.25 mM [Ca²⁺]_o elicited with the same voltage protocol as shown in Fig. 1. A–D: representative traces of Ca²⁺ currents (bottom), line-scan images (middle), and normalized cytosolic Ca²⁺ transients (top) elicited by test pulses to 0 mV (A and B) and −25 mV (C and D) in WT (A and C) and mdx (B and D) myocytes. Insets in C and D show enlarged Ca²⁺ signals at −25 mV. E: statistical comparison of the E-C coupling gain in 1.2 mM (from Fig. 2) and 0.25 mM Ca²⁺ at −25 mV for WT (black; n = 7 cells) and mdx (red; n = 9 cells) cells. *P < 0.05.

Fig. 4.

Ca²⁺ oscillations in WT and mdx cells. A and B: representative cytosolic Ca²⁺ signals in a WT cardiomyocyte (A) and a mdx (B) cardiomyocyte upon rapid reduction to 70 mM Na⁺ or complete removal of Na⁺ from the external solution (0 mM Na⁺). C and D: averaged frequency of oscillations (C) and time delay to their initiation (D) in WT (solid bars; n = 19) and mdx (shaded bars; n = 8) cardiomyocytes. *P < 0.05; **P < 0.01.

Fig. 5.

Activity of the Na⁺/Ca²⁺ exchanger (NCX) in WT and mdx myocytes. A: Ca²⁺ influx into a WT cardiomyocyte upon rapid reduction to 70 mM Na⁺ or complete removal of Na⁺ from the external solution (0 mM Na⁺) with eliminated SR function (1 μM thapsigargin and 10 μM ryanodine). B: averaged intracellular Ca²⁺ responses to a stepwise reduction or removal of external Na⁺ in WT (solid bars; n = 11) and mdx (shaded bars; n = 5) cells.

Fig. 6.

Estimation of luminal SR Ca²⁺ content. A: caffeine-induced cytosolic Ca²⁺ transients in a WT myocyte (black) and a mdx myocyte (red). Confocal line-scan images and average fluorescence signals are shown. B: averaged amplitudes of caffeine-induced Ca²⁺ transients in both types of cells. C: NCX current (top) and integrated NCX current (bottom) activated by caffeine-induced Ca²⁺ transients in a WT myocyte (left) and a mdx myocyte (right). D: averaged integrated NCX currents in WT (n = 6) and mdx (n = 8) myocytes. E: representative traces of I_CaL during a test step from −40 mV to maximal current at 0 mV under control conditions in WT (black line) and mdx (red line) cells. F: fast (τ₁) and slow (τ₂) time constants of current inactivation at 0 mV at different [Ca²⁺]_o in WT (black) and mdx (red) cells. At 1.2 mM [Ca²⁺]_o, τ₁ and τ₂ were 14.5 ± 1.1 and 78.9 ± 6.1 ms (n = 42) in WT cells and 18.8 ± 2.0 and 87.7 ± 5.5 ms (n = 57) in mdx cells, respectively.

Fig. 7.

Reduction of oxidative stress prevents the hypersensitivity of E-C coupling gain in mdx cardiomyocytes. E-C coupling gain data for WT and mdx are replotted from Figs. 2C and 3E for better comparison. Preincubation of mdx cells with 800 μM mercaptopropionyl-glycine (MPG; 30 min, n = 3) or with 10 μM Mn-cpx3 (30 min, n = 7) reduced the E-C coupling gain to levels similar to those in WT cells kept in control conditions (dashed lines). WT myocytes pretreated with 800 μM MPG also led to the expected reduction of the E-C coupling gain. *P < 0.05 and **P < 0.01.