1 The Burnham Institute, La Jolla, CA, USA
2 Department of Surgery, University of Tampere, Finland
3 Department of Neuropathology, University of Turku, Finland
We appreciate the keen interest that Chiquet and Flück have shown in
our article (Järvinen et al.,
2003). It is good to discuss the differences in our views in
public, since then other researchers may become aware of some important facts
and even participate in the discussion. In our case, the differences may
actually arise from the apparent confusion regarding the definition and
detection of skeletal muscle injury, rather than the phenomenon itself. This
view is supported by the recent paper by Flück and Chiquet themselves
(Flück et al., 2003
), to
which they also refer in their letter, a study that is in complete agreement
with our findings [i.e. tenascin-C (TN-C) is induced in the skeletal muscle in
response to injury]. Accordingly, we will not comment on their letter further
regarding this particular issue, as there appears to be a mutual understanding
between us. However, a brief comment is clearly warranted regarding their
view, based on findings using the chicken wing loading model
(Flück et al., 2000
), that
mechanical loading alone can induce de novo expression of TN-C in the skeletal
muscle.
First, Flück and Chiquet have clearly not understood the experimental
design of our study correctly
(Järvinen et al., 2003).
By stating in their letter the following, `Järvinen et al. looked only at
a single time point 8 weeks after reloading of an immobilized (atrophied)
muscle. It is possible that an acute burst of TN-C occurs over 8 weeks after
remobilisation', Chiquet and Flück have not paid attention to the fact
that we trained our animals twice a day during the entire 8-week
remobilisation period. Accordingly, the animals we studied had been subject to
two strenuous training sessions on the treadmill within the last 24 hours
before they were sacrificed. Thus, the `acute burst of TN-C' they speculated
to occur in the skeletal muscle in response to mechanical loading should have
also become evident in our study if mechanical loading was indeed able to
induce the production of TN-C.
Furthermore, inspired by the concerns of Chiquet and Flück, we decided to perform additional corroborative experiments on a different set of specimens. Specifically, we carried out TN-C mRNA in situ hybridization analysis on specimens from another experiment (our unpublished data), in which a standardized severe contusion injury was induced in a rat gastrocnemius muscle by a strike with a spring-loaded hammer; the muscle was subsequently subjected to either non-loading (immobilization in a cast) or increased loading (treadmill training). As can be readily seen from the autoradiograph film of the mRNA in situ hybridization experiment (Fig. 1A), only a very faint expression of TN-C was found in the wound tissue in the immobilized muscle and there is no signal in the healthy parts of the muscle. By contrast, the traumatized muscle subjected to increased loading (treadmill training) showed a clear increase in the expression of TN-C in the wound tissue but, more importantly, no signal could be detected in the healthy parts of the skeletal muscle (Fig. 1B). This additional experiment clearly corroborates the findings of our previous study and confirms that mechanical loading indeed regulates the normal expression of TN-C in specific parts of the musculoskeletal tissues but cannot alone (without accompanying injury that disrupts the integrity of the tissue) induce de novo synthesis of TN-C in the skeletal muscle.
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We now turn to the apparent discrepancy of how muscle injury is actually
defined and detected. The authors state that `...TN-C expression after 4 hours
of loading did not correlate with macrophage invasion, which we observed (by
means of a specific antibody) only after 24 hours', presumably trying to
suggest that this would somehow prove that no injury had occurred in response
to training. However, the absence of macrophages at 4 hours does not provide
evidence that TN-C expression could not be attributable to muscle damage.
Rather, one should recall that in healthy muscle (tissue) there should be no
inflammatory cells (neither monocytes nor macrophages) at all. Accordingly,
the existence of inflammatory cells alone is definite proof that muscle damage
occurred during the loading of the chicken wing muscle. As for the timing of
the signs of inflammation, the authors do not take into consideration that
there is always a delay in the tissue reaction to injury. The inability to
detect macrophages by a specific antibody during the first 24 hours after
loading (trauma) is in perfect agreement with the basic principles of acute
wound healing: local injury initially results in tissue edema and exudation of
leukocytes (first neutrophils, followed by monocytes) induced to emigrate
across the endothelium by chemotactic agents released as a result of tissue
trauma. The monocytes, in turn, differentiate into macrophages within 24 to 48
hours after initial trauma, at which point they can be identified by specific
antibodies, as also shown by Flück et al.
(Flück et al., 2000).
Furthermore, in the classic study on segmental necrosis in skeletal muscle
induced by micropuncture, Carpenter and Karpati did not observe any
macrophages (identified by morphology alone and so included monocytes) until
about 8 hours after the puncture (Carpenter
and Karpati, 1989
).
With regards to the statement `...we did not observe... obvious (macro- and microscopic) signs of tissue injury or haemorrhage after 4 hours of loading', this brings up the major problem of early detection of injury. It would be interesting to stain the chicken muscle samples at 4 hours or even earlier after the loading for either intracellular calcium or a sarcolemmal protein desmin, which might show the early injury in myofibers, or extravasated plasma proteins to show the vascular leakage. Most importantly, the authors did not explicitly provide any plausible explanation for the massive and sudden increase in muscle mass that was reported in their experiment. Such a dramatic 50% increase in muscle mass within 4 hours of loading cannot be attributable to anything but muscle trauma and the resulting inflammatory reaction. In the absence of hemorrhage, it was most likely due to the edema that the authors observed and reported - no other plausible explanation exists.
With regards to their statement `In fact, also reloading of rat soleus muscle caused ectopic tenascin-C expression after one day occurring in a patchy manner before signs of muscle fiber injury (central nuclei) were present', it should be pointed out that central nuclei are actually a late manifestation of muscle injury, not an acute response to muscle trauma. After contusion/laceration injury some nuclei become centralized, but this takes place relatively late in the regeneration process, usually when the regenerating myotubes start to fuse with the surviving parts of myofibers.
Thus, on the basis of the facts presented above, loading of the chicken
wing obviously had caused some damage that was not detected by the methods
used by Flück et al. (Flück et
al., 2000), but became evident later (e.g. by the invasion by
macrophages). We want to re-emphasize that our results showing that TN-C
expression is induced by trauma in muscle tissue are in perfect agreement with
many previous studies, including the recent study by Flück and Chiquet
themselves (Flück et al.,
2003
). Accordingly, we believe that the conclusions in our
criticized study (Järvinen et al.,
2003
), which claims that mechanical loading regulates the normal
expression of TN-C in the musculoskeletal tissues, but disruption of the
mechanical integrity of the tissue is required for induction of de novo
synthesis of this protein, are correct. It would be most interesting to
clarify by which mechanism the injured myofibers induce the early production
of TN-C mRNA in endomysial fibroblasts in the chicken wing loading model.
Solving this problem requires such wide expertise in molecular biology as
Flück and Chiquet undeniably have.
References
Carpenter, S. and Karpati, G. (1989). Segmental necrosis and its demarcation in experimental micropuncture injury of skeletal muscle fibers. J. Neuropathol. Exp. Neurol. 48, 154-170.[Medline]
Flück, M., Chiquet, M., Schmutz, S., Mayet-Sornay, M. H.
and Desplanches, D. (2003). Reloading of atrophied rat soleus
muscle induces tenascin-C expression around damaged muscle fibers.
Am. J. Physiol. Regul. Integr. Comp. Physiol.
284,
R792-R801.
Flück, M., Tunc-Civelek, V. and Chiquet, M.
(2000). Rapid and reciprocal regulation of tenascin-C and
tenascin-Y expression by loading of skeletal muscle. J. Cell
Sci. 113,
3583-3591.
Järvinen, T. A., Józsa, L., Kannus, P.,
Järvinen, T. L., Hurme, T., Kvist, M., Pelto-Huikko, M., Kalimo, H. and
Järvinen, M. (2003). Mechanical loading regulates the
expression of tenascin-C in the myotendinous junction and tendon but does not
induce de novo synthesis in the skeletal muscle. J. Cell
Sci. 116,
857-866.