Referee 3's Review (Bruno Allard):

This paper extends a precedent study, carried out by the same authors, which suggested a possible functional link between dystrophin and the dihydropyridine receptor (DHPR) operating as L-type Ca2+ channel in skeletal muscle. Confocal immuno-fluorescence images obtained using an elegant technique on non-fixed muscle fibres present here convincing evidence for co-localization of dystrophin and DHPR. Co-localization was significantly reduced in dystrophin-deficient fibres from young animals while in a sub-population of revertant fibres from older animals, displaying a wild-type-like dystrophin staining, co-localization was partially restored. Reduced co-localization was well correlated with reduced L-type Ca2+ current amplitude investigated in parallel with two-microelectrode voltage clamp technique. This study is of undeniable interest for researchers involved in studies related to the understanding of the pathophysiology of Duchenne dystrophy. It would be useful to look in the future for the existence of a molecular interaction between DHPR and the dystrophin complex.

Several points must be nevertheless clarified by the authors before acceptation:

- Peak Ca2+ currents amplitudes are expressed in nA and the authors claim that, because capacitances were shown to be similar in the 3 strains in their preceding study (ref. 25), there is no need to normalize the current to the capacitance. However, the authors showed in their previous study (ref. 25) that the ratio between capacity and fibre surface area was indeed similar, but that the size of the fibres was smaller in mdx (see methods of ref. 25). Hence, to not normalize currents to the size or capacity of the fibres should under-estimate the current amplitude in mdx muscle fibres. The maximal Ca2+ current amplitude is dependent on the voltage-dependence of activation. A possible shift of the activation curve of the current in mdx muscle might also somehow distort the data.

- Authors aimed to measure the percentage of dystrophin co-localized with respect to DHPR. However they mention in the methods section p11 that the count of co-localized pixels was divided by the pixel count of DHPR as reference. Under these conditions, they measure the percentage of DHPR co-localized with dystrophin and not the percentage of dystrophin co-localized with DHPR. In dystrophin-deficient fibres, one should expect a drastic reduction of co-localization simply due to the absence of dystrophin.

- Assuming that about one third of dystrophin is co-localized with DHPR in wild-type muscle as indicated by the authors, one can expect milder effects on Ca2+ currents in dystrophin-deficient fibres from young animals than the about two third reduction described here and in the previous study (ref. 25). Moreover, given that dystrophin is poorly expressed in t-tubules as compared to DHPR, a large fraction of DHPR should not be affected by the absence of dystrophin.

- Because the authors developed a new immuno-fluorescence technique, it is important that they compare qualitatively the staining they obtained for dystrophin and DHPR with the one described by others on fixed tissues.

- It would be useful that the authors further comment on the consequences of their results for the understanding of the pathophysiology of Duchenne dystrophy.
Minor points:

- In Fig. 3, it seems that DHPR is less expressed in the surface membrane of MinD fibres as compared to wild-type and mdx fibres.

- Could the authors mention how they cope with the successive washings of the fibres for immuno-staining which may be expected to lead to the loss of the fibres.

- Please mention to what correspond the dotted lines in Fig. 3B.

- It seems more appropriate to show the working model of DHPR / dystrophin interaction at the end of the paper.

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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.