Impaired excitation-contraction coupling in muscle fibres from the dynamin2R465W mouse model of centronuclear myopathy

Abstract

Key points: Dynamin 2 is a ubiquitously expressed protein involved in membrane trafficking processes. Mutations in the gene encoding dynamin 2 are responsible for a congenital myopathy associated with centrally located nuclei in the muscle fibres. Using muscle fibres from a mouse model of the most common mutation responsible for this disease in humans, we tested whether altered Ca²⁺ signalling and excitation-contraction coupling contribute to muscle weakness. The plasma membrane network that carries the electrical excitation is moderately perturbed in the diseased muscle fibres. The excitation-activated Ca²⁺ input fluxes across both the plasma membrane and the membrane of the sarcoplasmic reticulum are defective in the diseased fibres, which probably contributes to muscle weakness in patients.

Abstract: Mutations in the gene encoding dynamin 2 (DNM2) are responsible for autosomal dominant centronuclear myopathy (AD-CNM). We studied the functional properties of Ca²⁺ signalling and excitation-contraction (EC) coupling in muscle fibres isolated from a knock-in (KI) mouse model of the disease, using confocal imaging and the voltage clamp technique. The transverse-tubule network organization appeared to be unaltered in the diseased fibres, although its density was reduced by ∼10% compared to that in control fibres. The density of Ca²⁺ current through CaV1.1 channels and the rate of voltage-activated sarcoplasmic reticulum Ca²⁺ release were reduced by ∼60% and 30%, respectively, in KI vs. control fibres. In addition, Ca²⁺ release in the KI fibres reached its peak value 10-50 ms later than in control ones. Activation of Ca²⁺ transients along the longitudinal axis of the fibres was more heterogeneous in the KI than in the control fibres, with the difference being exacerbated at intermediate membrane voltages. KI fibres exhibited spontaneous Ca²⁺ release events that were almost absent from control fibres. Overall, the results of the present study demonstrate that Ca²⁺ signalling and EC coupling exhibit a number of dysfunctions likely contributing to muscle weakness in DNM2-related AD-CNM.

Keywords: dynamin 2; excitation-contraction coupling; ryanodine receptor; sarcoplasmic reticulum Ca2+ release; skeletal muscle.

Figure 1. T‐tubule network in WT and KI‐Dnm2 ^R465 fibres

A, x,y fluorescence images of di‐8‐anepps staining of the t‐tubule network in a WT (left) and in a KI (right) muscle fibre. The analysis shown in (B) and (C) was performed within the region of interest highlighted by a white box in each image. B, longitudinal profile of fluorescence along the corresponding region of interest shown in (A). C, outlined t‐tubule network within each region of interest; thresholding was used to create a binary image of the network. The graph in the middle shows values for the t‐tubule density index in the two groups of fibres, calculated as the percentage of above‐threshold pixels in the regions of interest.

Figure 2. CaV.1.1 Ca²⁺ current in WT and KI‐Dnm2 ^R465 fibres

A, representative Ca²⁺ current traces in a WT and in a KI fibre in response to 0.5 s long depolarizing pulses from −80 mV to the range of indicated values, by 10 mV steps. B, mean voltage‐dependence of the peak Ca²⁺ current in the two groups of fibres from measurements performed as shown in (A). Superimposed continuous lines show the result from fitting the mean WT and KI data points with eqn (1). C, mean values for the parameters obtained from fitting each current vs. voltage dataset with eqn (1) (see Methods). D, expression of CaV1.1 in tibialis anterior muscles: western‐blot using an antibody against the α1 subunit of the dihydropyridine receptor was performed on protein extracts from five WT and four KI mice at 5 months of age. An α‐tubulin antibody was used as loading control.

Figure 3. Voltage‐activated SR Ca²⁺ release in WT and KI‐Dnm2 ^R465 fibres

A, representative rhod‐2 Ca²⁺ transients in a WT and in a KI fibre in response to 0.5 s long depolarizing pulses from −80 mV to the range of indicated values, by 10 mV steps. B, corresponding Ca²⁺ release flux (d[Ca_Tot]/dt) traces calculated as described in the Methods. C, mean voltage‐dependence of the peak rate of SR Ca²⁺ release in WT and KI fibres. Superimposed continuous lines show the result from fitting the mean WT and KI data points with a Boltzmann equation. The inset shows the mean values for maximal rate of SR Ca²⁺ release in the two groups of fibres, as assessed from Boltzmann fits to data from each fibre. D, mean values for the total amount of released Ca²⁺ calculated from the time integral of the Ca²⁺ release traces, in WT and KI fibres. E, mean values for the time to peak Ca²⁺ release in WT and KI fibres.

Figure 4. Spatial heterogeneity of time to peak SR Ca²⁺ release in KI‐Dnm2 ^R465 fibres

A, F/F ₀ rhod‐2 line‐scan images taken from a WT and a KI fibre in response to a 0.5 s long depolarizing pulse from −80 to −10 mV. Traces below show the corresponding average F/F ₀ signal over the full line. B, corresponding images of the rate of change in F/F ₀ (dF/F ₀/dt); these images were resampled from 512 to 64 pixels (0.1–0.8 μm per pixel) after linear averaging in the space domain, and the time derivative was calculated with a dt of 4 pixels (4.6 ms). Traces below show the average rate of change in F/F ₀ over the full line. C, corresponding distribution of the time to peak rate of change in F/F ₀ at each position along the scanned line. D, mean ± SD of the time to peak rate of change in rhod‐2 F/F ₀ in WT (n = 21) and KI fibres (n = 25), in response to a pulse to −10 mV (left) and to +10 mV (right).

Figure 5. Examples of spatial variability of peak Ca²⁺ release rate in KI‐Dnm2 ^R465W fibres at −10 and + 10 mV

A–C, images of the rate of change in rhod‐2 fluorescence (dF/F ₀/dt) in three distinct KI fibres, respectively, stimulated by a voltage pulse from −80 to −10 mV (left) and by a voltage pulse from −80 to +10 mV (right). Traces underneath each image show the time course of the rate of change in F/F ₀ at the three positions indicated by arrows along the line. Scale bars = 0.4 ms⁻¹.

Figure 6. Ca²⁺ sparks in intact KI‐Dnm2 ^R465W fibres

A, sum of 10 x,y confocal images of fluo‐4 fluorescence from an isolated KI muscle fibre showing a localized increase in [Ca²⁺]. Colour bar is in arbitrary units. B, first image from the cumulated series shown in (A) after correction for baseline fluorescence (F ₀) and removal of structure elements (see Methods), expressed as F/F ₀. C, enlarged view of the individual spark highlighted by the square box in (B). Colour bar is the same for (B) and (C). D, Ca²⁺ release events amplitudes. E, FWHM of the Ca²⁺ release events, for profiles parallel (X) and perpendicular (Y) to the fibres axis. Arrows in (D) and (E) indicate the corresponding average values measured for the few events detected in WT fibres.

Figure 7. Ca²⁺ sparks in intact KI‐Dnm2 ^R465W fibres detected in line‐scan mode

A and B, normalized line‐scan images (F/F ₀) of fluo‐4 fluorescence from two KI fibres showing typical calcium release events corresponding to either lone Ca²⁺ sparks (A) or a series of such events (B, bottom of the image) or long embers (B, marked by arrow). C, enlarged portion of the image in (A) displaying an individual event at increased temporal and spatial resolution. Traces on the right and underneath show the corresponding spatial distribution and time course of the fluorescence transient, respectively. D and E, amplitude and FWHM histograms, respectively, of the lone calcium sparks from DNM2 KI fibres.