Fig 1.
Effect of triadin ablation on the expression of couplon proteins.
A, Western blot of proteins of interest from WT and Tr-null total homogenates. Band signal mass was normalized to signal in the corresponding lane of the gel in B (boxed region a corrected for baseline level in b). Details in Methods and [48]. B.1, Ponceau-stained proteins separated by PAGE. B.2, uncropped blot showing immunoreactivity of triadin antibody. C, distribution of results in Tr-null samples as a ratio of the WT average. Different symbols identify values obtained from individual mice. Error bars represent SEM. Asterisks mark changes statistically significant at the 0.05 (*) level. Statistical parameters are listed in Table 1.
Table 1.
Levels of proteins expression.
Fig 2.
Resting calcium concentration in cytosol and sarcoplasmic reticulum.
A, B, distributions of [Ca2+]cyto and [Ca2+]SR, measured ratiometrically in WT and Tr-null myofibers. Multiple measurements are represented by the vertical histograms, while N symbols represent averages of measures in m myofibers of N mice and box plots summarize the distribution of individual measurements. A, average [Ca2+]SR: in Tr-null, 224 ± 9 μM (N = 6, m = 204); in WT, 343 ± 10 μM (N = 5, m = 162); p < 0.001 in three-level hierarchical analysis. B, averages of [Ca2+]cyto: in Tr-null, 167 ± 2 nM (N = 6, m = 291); in WT 139 ± 3 nM (N = 5, m = 195); p < 0.001.
Table 2.
Properties of Ca2+ release elicited by clamp depolarization.
Fig 3.
Voltage dependence of voltage sensor charge movement.
A, B, representative charge movement currents obtained in myofibers held at -90 mV and depolarized for 50 ms to voltage V, ranging between -60 and +50 mV. C, D, charge displaced Qon, vs. V in WT and Tr-null fibers. Thin lines (measured values joined by straight segments) trace results in individual myofibers. These individual Qon(V) sets were fitted with a “Boltzmann” function. The thick smooth curves trace Boltzmann functions with parameters equal to the averages of parameters fitted to individual sets. Both average curves are plotted in C and D for ease of comparison. Details are presented in Table 3. E, kinetics; off sections of IQ(t) after depolarization pulses, with exponential fit to decay in red traces. F, distribution of fitted time constants. As in the preceding figures, symbols represent averages of measurements in individual mice, with replicates in m myofibers represented as vertical histograms.
Table 3.
Parameters of charge distribution and total calcium release.
Fig 4.
Calcium release elicited by maximal clamp depolarization.
A, line-scan image F(x, t) of a WT myofiber of FDB muscle, upon application of the voltage pulse represented (+30 mV for 100 ms); the space average F(t) is overlaid. B, red: Ca2+ release flux , derived from the [Ca2+]cyto(t) elicited by this pulse and others. The peak flux value is represented as
. Black: quantity R(t) of Ca2+ released via voltage activation of RyR channels. Note definitions of the quantities RT, RP and RH. A dip in the slope of R(t) (corresponding to the local minimum in
) marks the end of the peak stage and allows the calculation of RP. C, distribution of
. The averages are nearly equal (~92 mM/s) in 8 Tr-null and 8 WT muscles. Details in Table 3: D, distribution of quantities R(t). Symbols represent values from line-scan images in separate myofibers; those from the same animal are represented by the same symbol. While the distribution of RP was similar in Tr-null and WT, RT was lower by ~16% in the null, with p = 0.011. As can be inferred from the difference in RT, the approximate constancy of RP and the definition of RH, nearly all the difference in RT was due to a smaller RH component (p = 0.001). Fluxes and quantities of Ca2+ released at specific times were calculated from these records as illustrated in Fig 4B–4D; these include RT, the total quantity released up to the end of the pulse, RP, the calcium released during the peak stage, from the beginning up to the dip that precedes the hump, and RH, the calcium released during the hump stage, defined as RT−RP.
Fig 5.
Voltage dependence of Ca2+ release.
A, B, Fluo-4 fluorescence transients (ΔF/F0), upon 50 ms depolarizations to the voltages indicated. C, D, voltage dependence of fluorescence increases at end of pulses. Thin lines plot ΔF/F0(V) for every myofiber studied in WT (C) and Tr-null (D). Smooth curves trace the Boltzmann functions obtained with the averages of the best fit parameters of the same function fitted to the individual myofiber data. Average Boltzmann curves for both WT and Tr-nulls are repeated in C and D for ease of comparison. E, F, representative records of quantity RT of Ca2+ released. G, H, voltage dependence of RT. Average Boltzmann curves for both WT and Tr-nulls are repeated in G and H. RT(max) was lower, by ~19%, in the Tr-nulls; the difference was statistically significant (p = 0.025). was shifted to more depolarized values by 2 mV and κ was 16% greater in Tr-null myofibers. Statistical details are provided in Table 3.
Fig 6.
Recovery of Ca2+ release flux.
A, fluorescence signals F(t) elicited by pairs of supramaximal pulses (+30 mV, 100 ms) separated by an interval ranging between 100 and 800 ms. The sets of paired pulses were separated by a resetting time of 3 minutes. B, Ca2+ release flux derived from the F(t) in A, plus flux elicited by 3 additional pairs of pulses at the greater intervals listed. C, peak flux during the 2nd pulse, represented as percent of
during the first one. D, quantity RH in the hump stage of Ca2+ release elicited by the second pulse, represented as percent of RH during the first one. The recovery of
is faster, while that of RH is slower in Tr-null fibers.
Table 4.
Ca2+ release elicited by two successive pulses.
Fig 7.
Recovery of the quantity of Ca2+ released.
A, R(t) elicited by a pair of pulses 800 ms apart. Two-head arrows represent maximum quantities (RT1 and RT2) released with the respective pulses. B, data from A as fractions of quantity RT1. C, RT2 as fraction of RT1. Normalized thus, the quantity of Ca2+ released is ~32% greater in Tr-null at the 800 ms interval. The superposition of WT and null traces marks clearly the end of the peak stage and allows an accurate measurement of the quantity of Ca2+ contributed by the hump, as the difference between the total, RT1, and the level at the start of the hump. The dependence of RT2 on pulse interval is represented and summarized in panel C. This difference is in the same direction as that noted for peak flux; both suggest faster recovery of SR luminal free [Ca2+] in the absence of triadin.
Fig 8.
Kinetics of decay of cytosolic Ca2+.
A, B, increase in [Ca2+]cyto(t) elicited by a supramaximal pulse. Traces from 8 WT animals are shown in A and 9 Tr-null in B; continuous smooth curves are exponential decay functions obtained with the average parameters of exponentials fitted individually to the decay of each myofiber (in A, solid black trace: WT and in B, solid red trace: Tr-null). WT and Tr-null curves are repeated in A and B. C, distribution of individual time constants in A, B; averages ± SEM (Tr-null: 434 ± 16. WT: 527 ± 45 ms. p = 0.04). D, Net quantity of Ca2+ released upon paired voltage-clamp pulses 800 ms apart (calculated from all records in A and B) shown normalized to the total quantity released by the first pulse, RT1. Consistent with the analysis illustrated with Fig 7, the 2nd pulse releases more Ca2+ (as a fraction of that released in the first pulse) in the Tr-null. E, distribution of individual time constants of decay. Their average is ~36% greater in the WT (3.79 ± 0.19 s in WT vs. 2.79 ± 0.04 in the null; p < 0.001). Similar numbers are found by analyzing the decay of the average time courses, as shown in panel D. The large difference between time constants of [Ca2+]cyto and Rnet is justified in Discussion.