Sarcoplasmic reticulum Ca2+ permeation explored from the lumen side in mdx muscle fibers under voltage control

Abstract

Under resting conditions, external Ca(2+) is known to enter skeletal muscle cells, whereas Ca(2+) stored in the sarcoplasmic reticulum (SR) leaks into the cytosol. The nature of the pathways involved in the sarcolemmal Ca(2+) entry and in the SR Ca(2+) leak is still a matter of debate, but several lines of evidence suggest that these Ca(2+) fluxes are up-regulated in Duchenne muscular dystrophy. We investigated here SR calcium permeation at resting potential and in response to depolarization in voltage-controlled skeletal muscle fibers from control and mdx mice, the mouse model of Duchenne muscular dystrophy. Using the cytosolic Ca(2+) dye Fura2, we first demonstrated that the rate of Ca(2+) increase in response to cyclopiazonic acid (CPA)-induced inhibition of SR Ca(2+)-ATPases at resting potential was significantly higher in mdx fibers, which suggests an elevated SR Ca(2+) leak. However, removal of external Ca(2+) reduced the rate of CPA-induced Ca(2+) increase in mdx and increased it in control fibers, which indicates an up-regulation of sarcolemmal Ca(2+) influx in mdx fibers. Fibers were then loaded with the low-affinity Ca(2+) dye Fluo5N-AM to measure intraluminal SR Ca(2+) changes. Trains of action potentials, chloro-m-cresol, and depolarization pulses evoked transient Fluo5N fluorescence decreases, and recovery of voltage-induced Fluo5N fluorescence changes were inhibited by CPA, demonstrating that Fluo5N actually reports intraluminal SR Ca(2+) changes. Voltage dependence and magnitude of depolarization-induced SR Ca(2+) depletion were found to be unchanged in mdx fibers, but the rate of the recovery phase that followed depletion was found to be faster, indicating a higher SR Ca(2+) reuptake activity in mdx fibers. Overall, CPA-induced SR Ca(2+) leak at -80 mV was found to be significantly higher in mdx fibers and was potentiated by removal of external Ca(2+) in control fibers. The elevated passive SR Ca(2+) leak may contribute to alteration of Ca(2+) homeostasis in mdx muscle.

Figure 1.

Changes in cytosolic Ca²⁺ induced by SR Ca²⁺-ATPase inhibition in control and in mdx fibers. (A) Changes in intracellular Ca²⁺ monitored by Fura-2 and induced by 50 µM CPA in a control (C57BL6 mouse) and in an mdx fiber at a holding potential of −80 mV (the fluorescence baselines before addition of CPA were superimposed to make easier the comparison of the fluorescence changes induced by CPA in the two fiber types). (B) Mean rates of CPA-induced Ca²⁺ changes in control (C57BL6 mice) and in mdx fibers in the presence of 2.5 mM Ca²⁺ or in the absence of Ca²⁺ in the external solution. The numbers above each bar indicate the number of cells tested. Mean values in control and in mdx fibers and mean values in the presence of Ca²⁺ and in its absence have been compared using a Student’s unpaired t test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005. Error bars indicate mean ± SEM.

Figure 2.

Changes in Fluo-5N fluorescence induced by a train of action potentials, CmC, and depolarizing pulses. (A) Simultaneous recordings of a train of action potentials (bottom left trace) induced by a burst of supraliminal current pulses, 0.5 ms in duration at a frequency of 50 Hz during 1 s in current clamp conditions, and of the associated changes in Fluo-5N fluorescence (top trace) in the presence of an external Tyrode solution in a control fiber (C57BL6 mouse). The first action potential of the train is shown on an expanded time scale next to the train. (B) Change in Fluo-5N fluorescence induced by exposure of the same fiber as in A 2 min later to 1 mM CmC at a holding potential of −80 mV in current clamp conditions. Fluorescence images were captured at a frequency of 15 Hz in A and 1 Hz in B.

Figure 3.

Changes in Fluo-5N fluorescence induced by depolarizing pulses of increasing amplitude. (A) Simultaneous recordings of membrane currents (top trace) and changes in Fluo-5N fluorescence (middle traces) in the same control fiber (OF1 mouse) in response to depolarizing pulses of 1 s duration and increasing amplitudes delivered every 2 min (bottom traces). Images recorded for fluorescence measurements were captured at a frequency of 25 Hz. (B) Relationship between the mean changes in Fluo-5N fluorescence and membrane potential. The change in Fluo-5N fluorescence induced by depolarization corresponds to the measured difference between the minimal value of fluorescence recorded during the voltage pulse and the baseline value before the pulse. The numbers above each data point indicate the number of cells tested. Error bars indicate mean ± SEM.

Figure 4.

Voltage-induced changes in Fluo-5N fluorescence in response to a single or to successive depolarizing pulses in the presence or absence of CPA. (A) On this representative recording, a muscle fiber was submitted to a 50-s depolarizing pulse to 0 mV in the presence of 50 µM CPA. (B) Three consecutive pulses to +30 mV of 1, 5, and 20 s in duration and separated by 2-min intervals were applied on the same fiber. Fluo-5N fluorescence images were collected at a frequency of 0.2 Hz in A and 15 Hz in B in two control fibers (OF1 mice).

Figure 5.

Changes in Fluo-5N fluorescence induced by depolarization in control and in mdx fibers. (A) Means and SEM of Fluo-5N fluorescence changes in response to depolarizing pulses to −10, +10, and +30 mV from a holding potential of −80 mV in 12 control (C57BL6 mice; left) and 11 mdx (right) fibers. Images were collected at a frequency of 12.5 Hz. (B) Relationship between the mean changes in Fluo-5N fluorescence and membrane potential in control and mdx fibers. The changes in Fluo-5N fluorescence were measured as explained in the legend of Fig. 4. (C) Mean percentages of fluorescence recovery 1 s after the end of the depolarizing pulses at +10, +20, +30, and +40 mV in the 12 control and 11 mdx fibers. Mean values have been compared using a Student’s unpaired t test. Error bars indicate mean ± SEM.

Figure 6.

Decrease in Fluo-5N fluorescence induced by CPA in control and in mdx fibers. (A) Effect of removal of external Ca²⁺ on the changes in Fluo-5N fluorescence induced by addition of 50 µM CPA in a control (C57BL6 mouse) fiber. (B) Mean rates of CPA-induced fluorescence decrease in the absence and in the presence of 2.5 mM external Ca²⁺. Mean values have been compared using a Student’s paired t test. (C) Changes in Fluo-5N fluorescence in response to the addition of 50 µM CPA in a control (C57BL6 mouse) and in an mdx fiber. (D) Mean rates of fluorescence decrease in control and in mdx fibers. Mean values have been compared using a Student’s unpaired t test. Images were collected at a frequency of 0.5 Hz in A and C. ***, P < 0.0005. Error bars indicate mean ± SEM.

Figure 7.

Model showing the Ca²⁺ fluxes in control and mdx fibers and the possible consequences of the changing of these fluxes on Ca²⁺ homeostasis in mdx fibers. Gray arrows correspond to Ca²⁺ fluxes. Partial filling with gray color of the SR compartment in mdx fibers indicates possible reduced SR Ca²⁺ content. Broken black arrows indicate that SR depletion and/or SR leak influence sarcolemmal Ca²⁺ influx and provoke in mdx fibers a higher sarcolemmal Ca²⁺ influx that contributes to higher cytosolic [Ca²⁺]. See text for details.