Elevated Ca2+ at the triad junction underlies dysregulation of Ca2+ signaling in dysferlin-null skeletal muscle

Abstract

Dysferlin-null A/J myofibers generate abnormal Ca²⁺ transients that are slightly reduced in amplitude compared to controls. These are further reduced in amplitude by hypoosmotic shock and often appear as Ca²⁺ waves (Lukyanenko et al., J. Physiol., 2017). Ca²⁺ waves are typically associated with Ca²⁺-induced Ca²⁺ release, or CICR, which can be myopathic. We tested the ability of a permeable Ca²⁺ chelator, BAPTA-AM, to inhibit CICR in injured dysferlin-null fibers and found that 10-50 nM BAPTA-AM suppressed all Ca²⁺ waves. The same concentrations of BAPTA-AM increased the amplitude of the Ca²⁺ transient in A/J fibers to wild type levels and protected transients against the loss of amplitude after hypoosmotic shock, as also seen in wild type fibers. Incubation with 10 nM BAPTA-AM led to intracellular BAPTA concentrations of ∼60 nM, as estimated with its fluorescent analog, Fluo-4AM. This should be sufficient to restore intracellular Ca²⁺ to levels seen in wild type muscle. Fluo-4AM was ∼10-fold less effective than BAPTA-AM, however, consistent with its lower affinity for Ca²⁺. EGTA, which has an affinity for Ca²⁺ similar to BAPTA, but with much slower kinetics of binding, was even less potent when introduced as the -AM derivative. By contrast, a dysferlin variant with GCaMP6f_u in place of its C2A domain accumulated at triad junctions, like wild type dysferlin, and suppressed all abnormal Ca²⁺ signaling. GCaMP6f_u introduced as a Venus chimera did not accumulate at junctions and failed to suppress abnormal Ca²⁺ signaling. Our results suggest that leak of Ca²⁺ into the triad junctional cleft underlies dysregulation of Ca²⁺ signaling in dysferlin-null myofibers, and that dysferlin's C2A domain suppresses abnormal Ca²⁺ signaling and protects muscle against injury by binding Ca²⁺ in the cleft.

Keywords: BAPTA; CICR; Ca-induced Ca release; GCaMP; dysferlinopathy; injury; osmotic shock.

FIGURE 1

Effect of osmotic shock injury on Ca²⁺ transients in FDB fibers preloaded with different concentrations of BAPTA-AM. A/J or A/JCr myofibers were loaded with Rhod-2AM, with or without additional BAPTA-AM, subjected to 1 Hz stimulation, and imaged in line-scan mode under confocal optics (see Methods). Representative line-scan confocal images of voltage-induced Ca²⁺ transients in A/JCr (A) and sham (vehicle only) A/J [(B): A/J] fibers and in A/J fibers preloaded with BAPTA-AM at 5, 10, 50 and 100 nM (C). All examples in (B,C) were exposed to equal amounts of DMSO (1.5% by volume). All examples are shown before osmotic shock injury (OSI) and 5 min after OSI. Bars, 100 µm (vertical) and 250 ms (horizontal).

FIGURE 2

Effect of osmotic shock injury on Ca²⁺ transients in FDB fibers preloaded with different concentrations of BAPTA-AM. Data from experiments like those shown in Figure 1 were analyzed. (A), averaged amplitudes of Ca²⁺ transients before OSI presented as (F_max-F₀)/F₀. (B), averaged data for recovery of Ca²⁺ transients at 5 min after OSI. (C), averaged data for frequency of Ca²⁺ waves at 5 min after OSI. (D), % fibers that produced Ca²⁺ waves at 5 min after OSI. N is indicated in each bar. *. p < 0.05 compared to A/J (sham). **, p < 0.05 compared to A/JCr control. Student’s t test was used for A-C; Χ ² was used for (D). VICT = Voltage-induced Ca²⁺ transient.

FIGURE 3

Effect of osmotic shock injury on Ca²⁺ transients in A/J FDB fibers preloaded with different concentrations of Fluo-4AM. As in Figure 2, but with myofibers loaded with Fluo-4AM. (A), averaged amplitudes of Ca²⁺ release before OSI presented as (F_max-F₀)/F₀. (B), averaged data for recovery of Ca²⁺ transients at 5 min after OSI. (C), averaged data for frequency of Ca²⁺ waves at 5 min after OSI. (D), % fibers that produced Ca²⁺ waves at 5 min after OSI. Dashed lines represent values obtained with A/JCr fibers. *, p < 0.05 compared to A/J. N is indicated in each bar. Student’s t test was used for (A–C); Χ ² was used for (D). VICT = Voltage-induced Ca²⁺ transient.

FIGURE 4

Effect of osmotic shock injury on Ca²⁺ transients in A/J FDB fibers preloaded with different concentrations of EGTA-AM. As in Figure 2, but with myofibers loaded with EGTA-AM. (A), averaged amplitudes of Ca²⁺ release before OSI presented as (F_max-F₀)/F₀. (B), averaged data for recovery of Ca²⁺ transients at 5 min after OSI. (C), averaged data for frequency of Ca²⁺ waves at 5 min after OSI. (D), % fibers that produced Ca²⁺ waves at 5 min after OSI. Dashed lines represent values obtained with A/JCr fibers. *, p < 0.05 compared to A/J. N is indicated in each bar. Student’s t test was used for (A–C); Χ ² was used for (D)

FIGURE 5

Distributions of GCaMP6f_u and GCaMP6f_u-DYSF-ΔC2A in sarcoplasm. (A). Cartoon diagrams of chimeric structures, which include the CMV promoter, the GCaMP6f_u reporter, and, for GCaMP6f_u-DYSF-ΔC2A, the dysferlin ORF excluding most of the C2A domain (i.e., residues 108–2080) but including the remaining C2 domains B thought G (green hexagons), the Fer and DysF domains in the middle of the molecule (blue and pink outlined rectangles) and the transmembrane domain (blue rectangle near the C terminus; see Methods). (B). Subcellular distribution of Venus-GCaMP6f_u and GCaMP6f_u-DYSF-ΔC2A in transfected A/J myofibers. Plasmids were electroporated into A/J myofibers and imaged under confocal optics 2 weeks later. Double arrows: transverse tubules at level of triad junctions, as reported (Kerr et al., 2013; Muriel et al., 2022); single arrows: Z-disks, as reported (Muriel et al., 2022). Bars, 10 µm.

FIGURE 6

Effect of osmotic shock injury on Ca²⁺ transients in A/J FDB fibers transfected with GCaMP6f_u-Dysf-ΔC2A. Myofibers were transfected by electroporation. Two weeks later, they were loaded with Rhod-2AM and assayed as in Figure 2. (A), representative line-scan images of Ca²⁺ transients before and 5 min after OSI, as in Figure 1. (B), averaged amplitudes of Ca²⁺ release before OSI for A/J fibers transfected with Venus or with GCaMP6fu-Dysf-ΔC2A. (C), averaged data for recovery of Ca²⁺ transients from OSI for A/J fibers transfected with Venus or with GCaMP6fu-Dysf-ΔC2A at 5 min after OSI. Dashed line represents recovery in A/J fibers transfected with WT dysferlin. (D), averaged data for frequency of Ca²⁺ waves at 5 min after OSI. N is indicated in each bar. Student’s t test was used for (A–C); Χ ² was used for (D) *, p < 0.05 compared to A/J fibers transfected with Venus. (E). Recovery from OSI and frequency of Ca²⁺ waves as a function of GCaMP6f_u-DYSF-ΔC2A expression. GCaMP6f_u-DYSF-ΔC2A levels were determined in AU by measuring the intensity of the GCaMP6f_u fluorescence, after setting the background autofluorescence to 180 AU. For earlier data for A/J fibers expressing Venus- Dysf-ΔC2A, see Muriel et al., 2022.

FIGURE 7

Effect of OSI on Ca²⁺ transients in A/J FDB fibers transfected with Venus-GCaMP6f_u. Myofibers were transfected by electroporation. Two weeks later, they were loaded with Rhod-2AM and assayed as in Figure 2. (A), representative line-scan images of Ca²⁺ transients before and 5 min after OSI. (B), averaged amplitudes of Ca²⁺ release before OSI for A/J fibers transfected with Venus or with Venus-GCaMP6f_u. (C), averaged data for recovery of Ca²⁺ transients from OSI for A/J fibers transfected with Venus or with Venus-GCaMP6f_u at 5 min after OSI. The dashed line represents recovery in A/J fibers transfected with WT dysferlin. (D), averaged data for frequency of Ca²⁺ waves at 5 min after OSI. N is indicated in each bar. Student’s t test was used for (A–C); Χ ² was used for (D) *, p < 0.05 compared to A/J fibers transfected with Venus. (E). Recovery from OSI and frequency of Ca²⁺ waves as a function of Venus-GCaMP6f_u expression. Panels B–D show results obtained by imaging either Rhod-2 or GCaMP6f_u fluorescence. Panel E shows results obtained only with Rhod-2. Venus-GCaMP6f_u levels were determined in AU by measuring the intensity of the Venus fluorescence, after setting the background autofluorescence to 200 AU. For earlier data with A/J fibers expressing Venus alone, see Lukyanenko et al., 2017).

FIGURE 8

RyR1 but not RyR2 or RyR3 are expressed in fast twitch skeletal muscle. Immunoblots of extracts of Tibialis anterior muscles with antibodies to RyR1, RyR2, and RyR3. Only anti-RyR1 shows a strong band at ∼550 kDa.