Force dramatically drops after lengthening contractions in mdx but not C57 mice

By analyzing in situ TA isometric muscle force production in response to tetanic nerve
stimulation (125 Hz, 500 ms), we observed an immediate force drop following the third,
sixth, and ninth lengthening contractions in mdx mice (p??0.05) (Fig. 1a, b). There was no major force drop in C57 mice (Fig. 1b). In contrast, there was no force drop following isometric contractions in mdx mice
(p??0.05), indicating that the force drop was related to the muscle lengthening, but
not fatigue, and may be due to the higher force production which occurs during lengthening
versus isometric contractions (+65.9?±?3.6 % in mdx mice and +68.9?±?4.0 % in C57
mice). It should be noted that maximal force was not fully recovered even after 30 min
of recovery (Fig. 1c).

Fig. 1. Susceptibility to contraction-induced muscle injury, i.e., force drop following lengthening
contractions in TA muscle from mdx mice and neuromuscular transmission. a Example of force traces during lengthening contractions in mdx mice. The 10th contraction
was isometric. b Force drop following lengthening and isometric contractions in mdx and C57 mice.
c Force drop following nine lengthening contractions in mdx mice, recovery, and direct
muscle stimulation with high voltage. Mdx LC lengthening contractions in mdx mice, Mdx IC isometric contractions in mdx mice, C57 LC lengthening contractions in C57 mice, 0LC before lengthening contractions, 9LC after nine lengthening contractions, 9LC?+?muscle stimul stimulating electrodes were located on the muscle after the ninth contractions, 9LC?+?recovery 30 min force was measured 30 min after the ninth lengthening contractions, a significantly different from before lengthening contractions (p??0.05), b significant difference between strains (p??0.05), c significant difference from 9LC (p??0.05). ?=?8–21 per group

Is neuromuscular transmission impaired?

To determine whether neuromuscular transmission failure contributes to force drop,
we performed electrical TA muscle stimulation that can directly initiate muscle action
potentials, without the need of neuromuscular transmission 30], 31]. Stimulating electrodes were positioned on the midbelly of the muscle, and the muscle
was stimulated with a high strength voltage (80 V). Under basal conditions (before
lengthening contractions), nerve and muscle stimulations produced the same maximal
force (data not shown). We found that direct muscle stimulation with a high strength
voltage did not markedly improve maximal force production after the nine lengthening
contractions in mdx mice (Fig. 1b).

Moreover, we performed electromyographic measurements in order to measure CMAP decrement
following repetitive nerve stimulation (0.1 ms, 3 and 10 Hz, 20 s). CMAP (amplitude)
decrement following 3 or 10 Hz stimulation was higher in the case of neuromuscular
transmission failure 24]. We found no 3 and 10 Hz CMAP decrement differences between mdx and C57 mice before
and after the nine lengthening contractions (Fig. 2a, b).

Fig. 2. Neuromuscular transmission and neuromuscular junction morphology following lengthening
contractions in the TA muscle from mdx mice. a Compound muscle action potential (CMAP) (amplitude) in response to 3 Hz nerve stimulation.
b CMAP (amplitude) in response to 10 Hz nerve stimulation. cf representative images of neuromuscular junction morphology using ?-bungarotoxin staining.
Bar?=?20 ?m. CMAP compound muscle action potential, Mdx?+?9LC after nine lengthening contractions, Mdx LC lengthening contractions in mdx mice, Mdx and C57 before lengthening contractions. ?=?6–13 per group for a and b

To determine whether lengthening contractions cause immediate neuromuscular junction
structural changes in mdx mice as previously described 17], fibers from the TA muscle were stained with ?-BTX to label acetylcholine receptor
(AChR) clusters. Subsequently, z-serial images were collected with a confocal microscope
and collapsed into single images. Neuromuscular junctions of mdx mice were severely
fragmented before lengthening contractions (Fig. 2c). Indeed, postsynatic network was discontinuous and isolated AChR clusters were observed,
indicating a severe dismantlement of neuromuscular junctions. In contrast, AChR clusters
exhibited characteristic pretzel-like morphology, with complex continuous branched
network, in C57 mice (Fig. 2d). Importantly, the nine lengthening contractions did not appear to markedly affect
neuromuscular junction morphology, in both mdx and C57 (Fig. 2e, f). We measured the total neuromuscular junction area, defined by the area contained
within a boundary encompassing the outermost edges of the neuromuscular junction,
as revealed by ?-BTX label. We found a decrease in total neuromuscular junction area
in mdx mice following the nine lengthening contractions (p??0.05) but not in C57 mice (Table 1). Since the neuromuscular junction area contained within the outside boundary does
not really reflected the occupied area of AChR clusters, we also measured the AChR-rich
endplate area per neuromuscular junction corresponding to the ?-BTX-labeled area per
synapse. Accordingly, the occupied AChR cluster area was also reduced following the
nine lengthening contractions in mdx mice (p??0.05) (Table 1). However, the complexity within neuromuscular junction (calculated as (AChR-rich
endplate area/total neuromuscular junction area)*100) was unchanged following the
nine lengthening contractions in mdx mice (Table 1). Moreover, we found no effect of the nine lengthening contractions on the number
of fragments and the area of each AChR fragment in mdx mice (Table 1).

Table 1. Morphology parameters of neuromuscular junction following lengthening contractions
in mdx mice

Together, these results indicate that neuromuscular transmission failure was not a
major mechanism of the force drop following lengthening contractions in mdx mice.

Is muscle excitability depressed?

A decreased generation and propagation of muscle action potential, i.e., reduced muscle
excitability, could contribute to the force drop following lengthening contractions.
To test this hypothesis, we simultaneously measured maximal force and CMAP (RMS) during
lengthening contractions. We found that CMAP in response to nerve stimulation (0.1 ms,
500 ms, 125 Hz) decreased following lengthening contractions in mdx mice (p??0.05), similar to maximal force, but not in C57 mice (Fig. 3a). In contrast, CMAP did not decrease in mdx mice following isometric contractions
(Fig. 3b).

Fig. 3. Muscle excitability following lengthening contractions in the TA muscle from mdx mice.
Compound muscle action potential (CMAP) was measured in response to nerve (a, b, and d) or muscle stimulation (c). a CMAP (root mean square, RMS) in response to nerve stimulation recorded during lengthening
contractions in mdx mice. The force drop was also shown. b CMAP (RMS) in response to nerve stimulation recorded during lengthening or isometric
contractions in mdx mice. The force drop was also shown. c Voltage strength (V) needs to obtain 50 % of maximal force in response to muscle
stimulation when neuromuscular transmission was inhibited by tubocurarine in mdx mice.
d Force and CMAP (RMS) relationship obtained when CMAP in response to nerve stimulation
was experimentally reduced by tubocurarine, in the mdx muscle at the basal state (before
lengthening contractions). e CMAP (root mean square, RMS) in response to nerve stimulation recorded during lengthening
and isometric contractions in C57 mice. The force drop was also shown and was induced
by a severe lengthening contraction protocol (12?×?20 % L0). f Force drop following nine lengthening contractions in C57 mice and direct muscle
stimulation with high voltage (80 V). CMAP compound muscle action potential, Mdx?+?9LC after nine lengthening contractions in mdx mice, Mdx LC lengthening contractions in mdx mice, Mdx IC isometric contractions in mdx mice, 12LC (20 % L0) 12?×?20 % lengthening contractions were performed in C57 mice, 12LC(20 % L0)?+?muscle stimul stimulating electrodes were located on the muscle after 12?×?20 % L0 lengthening
contractions in C57 mice, C57 LC 12?×?20 % L0 lengthening contractions in C57 mice, C57 IC 12 isometric contractions in C57 mice, a significantly different from before lengthening contractions (p??0.05), b significant difference between strains (p??0.05), c significant difference from IC (p??0.05). ?=?6–13 per group

Next, we determined the necessary muscle stimulation strength (in V) for 50 % of maximal
force production when the muscle is directly activated, i.e., not excited via neuromuscular
transmission. TA muscles were injected with tubocurarine (15 ?l at 0.07 mg/ml), a
neuromuscular transmission blocker. The stimulating electrodes were positioned on
the muscle surface. We found that the voltage needed to elicit 50 % of maximal force
markedly increased following the nine lengthening contractions (Fig. 3c) (p??0.05), indicating an increased threshold for action potential generation, confirming
reduced excitability.

Then, we determined the relationship between CMAP (RMS) and force in intact mdx muscles
(that did not performed lengthening contractions) to determine whether an experimental
reduction in CMAP caused a proportional decreases in force. TA muscles from mdx mice
were injected with various doses of tubocurarine (15 ?l at 0.007–0.07 mg/ml) in order
to pharmacologically reduced CMAP, and force was measured 5 to 15 min after. We found
that absolute maximal force decreased proportionally with CMAP (Fig. 3d). In fact, linear regression analysis revealed a strong correlation between CMAP
and absolute maximal force (r2
?=?0.93) (p??0.0001). Since the slope of the regression line was ~1 (0.94?±?0.03), a given reduction
in CMAP caused a similar decrease in force. Therefore, the force drop following lengthening
contractions could be mimicked by an experimental reduction in CMAP, i.e., muscle
excitation. Together, these results indicate that the reduced muscle excitability
contributes to the force drop following lengthening contractions in mdx mice.

To determine whether reduced muscle excitability was also reduced in C57 mice when
the force drop is important, they performed 12?×?20 % L0 lengthening contractions,
a more severe lengthening contraction protocol than the protocol used for mdx mice
(9?×?10 % L0 lengthening contractions). We found that 12?×?20 % L0 lengthening contractions
induced a marked force drop (up to ?67 %) in C57 mice (Fig. 3e, f). Electromyography analysis indicated that CMAP was also decreased following 12?×?20 %
L0 lengthening contractions in C57 mice (p??0.05), but not isometric contractions (Fig. 3e). Moreover, muscle stimulation with high strength current (80 V) did not markedly
reduce the force drop following lengthening contractions in C57 mice (Fig. 3f), indicating that the reduced CMAP was independent from neuromuscular transmission
failure. Together, these results suggest that reduced muscle excitability is also
a mechanism of the force drop following lengthening contractions in C57 mice.

With the aim to increase muscle excitability, mdx mice were treated, before the lengthening
contractions, with salbutamol that activates the Na
+
,K
+
pump 32]. A recent study suggests that the Na
+
,K
+
pump is depressed in mdx mice at the basal state, i.e., before lengthening contractions
33]. We found that salbutamol administration (2 mg/kg, ip) did not reduce the force drop
following lengthening contractions (Fig. 4a), even though the initial maximal force (before lengthening contractions) was reduced
by salbutamol (Fig. 4b) (p??0.05). Mdx mice were then treated after the ninth lengthening contraction, with
anthracene-9-carboxylic acid (9AC) (30 mg/kg, ip) that increases muscle excitability
via an inhibition of the chloride channel 34]. Mdx mice were treated after (and not before) the lengthening contractions, to avoid
complications due to 9AC-induced slowing of the relaxation during lengthening contractions.
We found that 9AC treatment improved the maximal force 10 min after the last lengthening
contraction in mdx mice (Fig. 4c) (p??0.05). Finally, mdx mice were treated before the lengthening contractions with
mexiletine (40 mg/kg, ip), a sodium channel blocker, that reduced muscle excitability
34]. We confirmed that mexiletine decreased CMAP in mdx mice before lengthening contractions
(Fig. 4d) (p??0.05). Since mexiletine also induces a tetanic fade (decrease in force within a
train of stimulation) with the usual 125-Hz stimulation, we performed 75 Hz stimulation
that did not produce such a phenomenon. We found that mexiletine treatment had no
effect on either force or CMAP—during lengthening contractions (Fig. 4d, e), suggesting that alteration in sodium channels does not contribute to the reduced
excitability following lengthening contractions. It should be noted that mexiletine
did not alter initial maximal force (before lengthening contractions) (Fig. 4f). Together, these results may suggest that the reduced excitability following lengthening
contractions results, at least partly, from an alteration in chloride channels but
not in either the Na
+
,K
+
pump or sodium channels.

Fig. 4. Effect of pharmacological modulation of muscle excitability (salbutamol, 9AC, mexiletine)
in the TA muscle from mdx mice. a Force drop following lengthening contractions in mdx mice treated with salbutamol.
b Maximal force before lengthening contractions (initial) in mdx mice treated with
salbutamol. c Force drop following nine lengthening contractions in mdx mice treated with anthracene-9-carboxylic
acid (9AC). d CMAP (RMS) during 125 Hz stimulation in mdx mice treated with mexiletine, before
lengthening contractions. e Force and CMAP (RMS) drops following lengthening contractions in mdx mice treated
with mexiletine. f Maximal force before lengthening contractions in mdx mice treated with mexiletine.
CMAP compound muscle action potential, Mdx?+?Salbu mdx mice treated with salbutamol, Mdx?+?9AC mdx mice treated with 9AC, Mdx?+?Mexi mdx mice treated with mexiletine, Mdx LC lengthening contractions in mdx mice, a significantly different from before lengthening contractions (p??0.05), c significant difference with non-treated mice (p??0.05). ?=?6–15 per group

Is the excitation-contraction uncoupled?

We then tested the possibility that a defect in excitation-contraction coupling also
contributes to the force drop in mdx mice. A decrease in the ratio of the force at
low frequency (25 Hz) to the force at high-frequency (125 Hz) stimulation following
lengthening contractions was used as a marker of excitation-contraction uncoupling
35], 36]. At low frequency, insufficient calcium would be released (resulting in reduced submaximal
force) that could be overcome by high-frequency stimulation (resulting in near normal
maximal force). Figure 5a showed that the 25 Hz/125 Hz force ratio did not decrease following lengthening contractions
in mdx mice. These results suggest that excitation-contraction uncoupling did not
contribute to the force drop following lengthening contractions in mdx mice. However,
it remains to be confirmed if this ratio can be applicable to test excitation-contraction
coupling following lengthening contractions in mdx muscle.

Fig. 5. Excitation-contraction coupling (25 Hz/125 Hz force ratio) following lengthening contractions
and effect of pharmacological modulation of ryanodine receptor (caffeine, dantrolene)
in the TA muscle from mdx mice. a Ratio of 25 and 125 Hz forces following lengthening contractions in mdx mice, used
as an index of excitation-contraction coupling. b Force drop following lengthening contractions in the TA muscle from mdx mice treated
with caffeine or dantrolene, i.e., modulators of ryanodine receptor. c Maximal force before lengthening contractions (initial) in the TA muscle of mdx mice
treated with caffeine or dantrolene. Mdx?+?Caf mdx mice treated with caffeine, Mdx?+?Dant mdx mice treated with dantrolene, a significantly different from before lengthening contractions (p??0.05), c significant difference with non-treated mice (p??0.05). ?=?6–10 per group

Is the calcium release also limited because of RyR alteration?

To determine whether the force drop following lengthening contractions was also increased
by further ryanodide receptor (RyR) dysfunction 37], 38], we treated mdx mice with caffeine or dantrolene, two pharmacological agents known
to increase or reduce calcium release by modulating RyR functions 35], 39]. Our hypotheses were that caffeine or dantrolene would increase or decrease the calcium
leak related to RyR dysfunction, thus the force drop following lengthening contractions.
The results shown that caffeine (8 mg/kg, ip) increased the force drop following lengthening
contractions in mdx mice (Fig. 5b). However, dantrolene (15 mg/kg, ip) did not reduce it (Fig. 5b). It should be noted that dantrolene reduced maximal force before lengthening contraction
(Fig. 5c). These results suggest that a further worsening of RyR dysfunction following lengthening
contractions did not contribute to the force drop in mdx mice since dantrolene did
not improve it.

Is the contractile apparatus preserved?

We then tested the possibility that lengthening contractions altered myofibrillar
function. Immediately following in situ the nine lengthening contractions, skinned
fibers were prepared from the TA muscle and maximal activated calcium force was measured
in both mdx and C57 mice. We found no reduction in maximal activated calcium force
following the nine lengthening contractions in both mdx- and C57-skinned fibers expressing
the types IIx and IIb MHC isoforms (Fig. 6a) (p??0.05), indicating that myofibrillar dysfunction did not contribute to the force
drop following lengthening contractions in mdx mice.

Fig. 6. Myofribrillar function, muscle fiber ultrastructure, and optimal muscle length following
lengthening contractions in the TA muscle from mdx mice and effect of prior cardiotoxin
injection. a Calcium maximally activated force of skinned muscle fibers (myofibrillar function)
after nine lengthening contractions. b, c Representative images of electron microscopy from mdx mouse illustrating the absence
of morphological alterations following nine lengthening contractions (c) as compared to before lengthening contractions (b). d Force drop following nine lengthening contractions in mdx mice, muscles were stretched
to try to obtain a new optimal length (L0). e Force drop following lengthening contractions in the cardiotoxin-treated (freshly
regenerated) mdx muscle. C57 mice were also shown. f Maximal force before lengthening contractions (initial) in mdx muscle treated with
cardiotoxin. C57 mice were also shown. 0LC before lengthening contractions, 9LC after nine lengthening contractions, 9LC?+?newL0 muscles were stretched to try to obtain a new optimal length (L0) after nine lengthening
contractions, Cardio muscle injected with cardiotoxin 3 weeks before, a significantly different from before lengthening contractions (p??0.05), b significant difference between strains (p??0.05), c significantly different from 9LC (p??0.05), d significantly different from C57 (p??0.05). ???20 per group for a; ?=?7–10 per group for df

Accordingly, we observed that lengthening contractions induced no major change in
sarcomere ultrastructure, using electron microscopy (Fig. 6b, c). In line with previous studies 40], we did find morphological abnormalities in some TA muscle fibers of mdx mice before
lengthening contractions, such as enlarged SR cisternae, focal Z-line absence or streaming,
degenerating fibers, and central nuclei (data not shown). However, a thorough comparison
of the contralateral muscle fixed immediately after the nine lengthening contractions
did not reveal any specific additional lesions, such as sarcolemmal ruptures, sarcomere
tearing, thus arguing against any structural injuries directly linked to lengthening
contractions in mdx mice (Fig. 6c).

In agreement with these previous findings, we found no indication of reduced myofilament
overlapping, i.e., disrupted sarcomeres, which has been proposed to be responsible
for the force drop following lengthening contractions in healthy mice. The popping-sarcomere
hypothesis is based on the proposal that during muscle lengthening, the length change
will be taken up by the weakest sarcomeres, resulting in no myofilament overlap in
the latter ones 41]. At the end of the lengthening, these overstretched sarcomeres do not re-interdigitate,
and thus a shift in muscle optimal length for maximal force production (L0) would
occur. To test this mechanism, we determined whether muscle recovered maximal force
following the nine lengthening contractions when an attempt was made to reach a possible
new L0. As showed in Fig. 6d, maximal force was only slightly improved after this procedure, indicating that the
presence of overstretched sarcomeres/sarcomere disruption is not the major explanation
for the force drop in mdx mice.

Are freshly regenerated fibers more fragile?

To test the possibility that it is the presence of freshly regenerated fibers but
not the dystrophin deficiency per se that causes the susceptibility to lengthening
contraction-induced muscle damage, cardiotoxin (10 ?M, 50 ?l), a myotoxic agent, was
injected into TA muscles from mdx mice as described 42]. We found that the force drop following lengthening contractions was not increased
by cardiotoxin injection in mdx and C57 mice (Fig. 6e) (p??0.05), at a time (21-day postinjection) where the regenerating muscle have recovered
its maximal force production (Fig. 6f). This result indicated that recent muscle degeneration/regeneration was not the
cause of the susceptibility to lengthening contraction-induced injury in the mdx mice.

Does dystrophin restoration by exon skipping improve both force drop and muscle excitability?

In order to answer this question, we analyzed the effect of the restoration of dystrophin
protein expression on force drop and muscle excitability following lengthening contractions.
For this purpose, we used AAV-U7snRNA-mediated exon skipping of the Dmd ex23 previously described in mdx mice 10]. Intramuscular injection of mdx TA muscles with AAV1-U7ex23 resulted, 3 weeks later,
in a partial skipping of exon 23 of Dmd (Fig. 7a, b) (p??0.05), without affecting the total level of dystrophin mRNA (Fig. 7c). A partial restoration of dystrophin protein expression was confirmed by Western
blot (Fig. 7d, e).

Fig. 7. Partial restoration of dystrophin expression in the TA muscle from mdx mice injected
with AAV1-U7ex23. a RT-PCR analysis of Dmd exon 23 skipping in mdx mice injected with AAV1-U7ex23. b Quantification of Dmd exon 23 skipping in mdx mice injected with AAV1-U7ex23, c Dystrophin total mRNA level in mdx mice injected with AAV1-U7ex23. d Western blot analysis of dystrophin protein expression in mdx mice injected with
AAV1-U7ex23, e Quantification of dystrophin protein expression in mdx mice injected with AAV1 U7ex23.
U7-ex23 AAV1-U7ex23, Mdx mdx muscle injected with saline, Mdx?+?U7ex23 mdx muscle injected with AAV1-U7ex23, c significant difference with non-injected muscle (p??0.05). ?=?6–10 per group

We found that 3 weeks after AAV1-U7ex23 injection, the force drop following lengthening
contractions was reduced (Fig. 8a) (p??0.05), and specific maximal force was also increased by 19 % (Fig. 8b) (p??0.05), confirming our previous studies 12], 43]. Interestingly, the decrease in CMAP (RMS) following lengthening contractions was
also reduced by AAV1-U7ex23 (Fig. 8a) (p??0.05). Another novel result from this experiment was that the changes in force
were closely related to those of CMAP during both the lengthening contractions and
recovery in both mdx and mdx mice injected with AAV1-U7ex23 (Fig. 8c), with a good correlation between CMAP and absolute maximal force (r2
?=?0.66) (p??0.0001). Together, these results indicate that the beneficial effect of restoration
of dystrophin expression on the force drop following lengthening contractions in mdx
mice was mediated by the prevention of the CMAP impairment, i.e., reduced muscle excitability.

Fig. 8. Effect of partial dystrophin restoration in the TA muscle from mdx mice. a Force and CMAP (RMS) drops following lengthening contractions in mdx mice injected
with AAV1-U7ex23. b Specific maximal force and muscle weight before lengthening contractions in mdx mice
injected with AAV1-U7ex23. c Force and CMAP relationship during lengthening contractions and 20 min recovery in
mdx and mdx mice injected with AAV1-U7ex23. U7-ex23 AAV1-U7ex23, Mdx mdx muscle injected with saline, Mdx?+?U7ex23 mdx muscle injected with AAV1-U7ex23, a significantly different from before lengthening contractions (p??0.05), c significant difference with non-injected muscle (p??0.05). ?=?8–13 per group