Massive alterations of sarcoplasmic reticulum free calcium in skeletal muscle fibers lacking calsequestrin revealed by a genetically encoded probe

Abstract

The cytosolic free Ca(2+) transients elicited by muscle fiber excitation are well characterized, but little is known about the free [Ca(2+)] dynamics within the sarcoplasmic reticulum (SR). A targetable ratiometric FRET-based calcium indicator (D1ER Cameleon) allowed us to investigate SR Ca(2+) dynamics and analyze the impact of calsequestrin (CSQ) on SR [Ca(2+)] in enzymatically dissociated flexor digitorum brevis muscle fibers from WT and CSQ-KO mice lacking isoform 1 (CSQ-KO) or both isoforms [CSQ-double KO (DKO)]. At rest, free SR [Ca(2+)] did not differ between WT, CSQ-KO, and CSQ-DKO fibers. During sustained contractions, changes were rather small in WT, reflecting powerful buffering of CSQ, whereas in CSQ-KO fibers, significant drops in SR [Ca(2+)] occurred. Their amplitude increased with stimulation frequency between 1 and 60 Hz. At 60 Hz, the SR became virtually depleted of Ca(2+), both in CSQ-KO and CSQ-DKO fibers. In CSQ-KO fibers, cytosolic free calcium detected with Fura-2 declined during repetitive stimulation, indicating that SR calcium content was insufficient for sustained contractile activity. SR Ca(2+) reuptake during and after stimulation trains appeared to be governed by three temporally distinct processes with rate constants of 50, 1-5, and 0.3 s(-1) (at 26 °C), reflecting activity of the SR Ca(2+) pump and interplay of luminal and cytosolic Ca(2+) buffers and pointing to store-operated calcium entry (SOCE). SOCE might play an essential role during muscle contractures responsible for the malignant hyperthermia-like syndrome in mice lacking CSQ.

Fig. 1.

(A) Localization of D1ER on both sides of Z lines stained with α-actinin antibody (D1ER, green; α-actinin antibody + rhodamine, red). Two different magnifications are shown. The sarcomere length is 2.04 μm. (B) Western blot showing the differences in expression of the two CSQ isoforms in WT, CSQ-KO, and CSQ-DKO fibers. Note the reduced expression of CSQ2 in the CSQ-KO preparation. (C) Typical recording of the SR D1ER FRET response to repetitive stimulation (20 s at 1 Hz) of a CSQ-KO muscle fiber. Stimulation started 5 s after the beginning of data acquisition (t = 5 s) and ended at t = 25 s. YFP and CFP signals (two lower traces, left ordinate) and their R (upper trace, right ordinate), measures of the free SR Ca²⁺ concentration, are shown. The YFP/CFP R values during the initial and final intervals of 5 s were used to calculate the baseline, which is indicated by the interrupted line. The ΔR (double-sided arrow) reflects the decrease in the SR Ca²⁺ concentration during the train of stimuli.

Fig. 2.

Typical recordings of changes in intraluminal SR Ca²⁺ concentrations during repetitive stimulation at different stimulation rates. The D1ER responses (YFP, red; CFP, blue; YFP/CFP R, green) are shown in a WT fiber (A) and a CSQ-KO fiber (B) at 1, 5, 20, and 60 Hz. The R signal has been corrected for the alterations in baseline as described in the text. Note the striking difference between WT and CSQ-KO in the amplitude of the changes in the R related to contractile activity. Resting R values obtained for the fibers shown in A are 1.83 (1 Hz), 1.81 (5 Hz), 1.78 (20 Hz), and 1.70 (60 Hz), and those for the fibers shown in B are 2.00 (1 Hz), 1.95 (5 Hz), 1.88 (20 Hz), and 1.81 (60 Hz).

Fig. 3.

Average amplitudes of the decline in the YFP/CFP R (ΔR) during contractile activity with increasing stimulation frequency. The decrease in the R in fibers from WT, CSQ-KO, and CSQ-DKO reflects the decrease in the SR Ca²⁺ concentration at the end of the trains of stimuli. The decrease is small and remains rather constant in WT fibers at the stimulation frequencies used but increases markedly in both KO groups.

Fig. 4.

Time course of the change in the YFP/CFP R during the SR refilling. (A) Time course of the increase in the YFP/CFP R after a train of stimuli at 60 Hz in a CSQ-KO fiber. (B) Time course of the variations in the YFP/CFP R during a train of stimuli at 1 Hz in a CSQ-KO fiber. YFP signal is shown as a surrogate marker of mechanical activity. In A, the rise in YFP intensity marks the onset of relaxation. In B, the YFP intensity reflects the twitch time course of the fiber. In B, the time-averaged response of the final 10 responses of the 15-s train of stimuli is shown. In A and B, a double exponential was fitted to the YFP/CFP data points. The parameter values in A are a_f = 0.21, k_f = 4.20 s⁻¹; a_s = 0.18, and k_s = 0.26 s⁻¹, and those in B are b_f = 0.021, m_f = 44.1 s⁻¹, b_s = 0.025, and m_s = 1.36 s⁻¹.

Fig. 5.

Fibers lacking CSQ are not able to maintain a high cytosolic calcium concentration during repetitive stimulations. Typical recordings of cytosolic Ca²⁺ concentrations visualized by the Fura-2 fluorescence R in a WT fiber (A) and CSQ-DKO fiber (B) during repetitive stimulation at 20 Hz are shown. Note the striking difference between WT and CSQ-DKO in the ability to maintain a high cytosolic calcium concentration during high-frequency repetitive stimulation.