In the human, true myocutaneous junctions of skeletal muscle fibers are limited to the face

Abstract Within the panniculus carnosus‐associated skeletal muscles in the human, the palmaris brevis and the platysma showed myotendinous/myofascial junctions with clear distance to the corium and the specific connection collagen type XXII. The orbicularis oris muscle, in contrast, contained bundles of striated muscle fibers reaching the corium at two distinct levels: the predominant inner ending was connected to the elastic network of the inner corium and the outer ending was within the more superficial collagen network. At both locations, the striated muscle fibers showed brush‐like cytoplasmic protrusions connecting a network which was not oriented toward the muscle fibers. Collagen type XXII was not present.


| INTRODUC TI ON
The majority of skeletal muscle fibers are covered by a muscle fascia that separates individual or groups of muscles from the skin. The individual fiber terminations contact either the inner aspect of this fascia (myofascial junctions) or form myotendinous junctions at the tendons as intermediates to the final osseous insertions.
A small number of skeletal muscles, however, are named to insert directly in the dermal or subcutaneous layer of the skin. They are summarized as panniculus carnosus muscle and include various muscles of the face and trunk (reviewed by Naldaiz-Gastesi et al., 2018). While myotendinous junctions have been studied in some detail, almost no information exists about the morphology of myocutaneous junctions. To clarify this specific connection we studied the orbicularis oris, palmaris brevis, and platysma muscle in the human.

| Tissue preparation
Muscle specimens of the orbicularis oris, palmaris brevis, and platysma muscle were collected from 11 human cadavers. They were part of the donor program of the Department of Anatomy in Dresden (Germany) and had given in their lifetime a written consent that their body might be used in purpose of science and education after death. There were 6 male and 5 female cadavers, age range was between 70 and 96 years, lacking neuroor myopathies in their medical history as far as documented. All cadavers were fixed 2-4 days post mortem with a mixture of formalin and alcohol and then processed for embedding in paraffin wax.

| Histology and immunohistochemistry
Serial sections (5 μm thick) of each specimen were performed in different planes and selected sections stained with hematoxylin and eosin (H&E), Goldner trichrome, or Sirius red solution.
For immunohistochemistry, consecutive sections of H&E stained sections containing myocutaneous junctions were dewaxed, rehydrated, and irradiated with microwaves in 0.01 M sodium citrate buffer (pH 6.0) for 2 × 5 min at 800 W to unmask the antigens. After washing in PBS, the sections were treated with 0.3% hydrogen peroxide for 10 min and blocked in normal mouse serum for 15 min at 37°C followed by washing in PBS. The primary polyclonal rabbit antibody against Collagen XXIIA1 (aa 181-273; Creative Diagnostics; dilution 1:100) was incubated over night at 4°C. After washing in PBS, an appropriate biotinylated secondary antibody was added and incubated for 15 min at 37°C, followed by washing and incubation with a VECTASTAIN ® Elite ABC mouse kit (PK 6101, PK 6102 Vector Laboratories Inc.). Visualization of peroxidase activity was realized by adding 3,3-diaminobenzidine for 8 min.
The sections were examined on Zeiss Jenamed2 microscope (Carl Zeiss AG) and images were recorded by using a Digital Sight DS-Fi1 camera (Nikon AG).

| Semi-thin sections
From selected regions of the orbicularis oris muscle small specimens (3 × 3 × 3 mm) were post-fixed in glutaraldehyde and embedded in Epon. Serial semi-thin sections were performed and stained with F I G U R E 1 (a,b) HE staining of the platysma (PL) and its connection to the skin. A. Note the location of the platysma in the hypodermis (HD) with notable distance to the reticular dermis (RD). Myotendinous junctions (shown in b; the muscle fiber marked by an asterisks) are marked with arrowheads. Note the subcutaneous retinacula (collagen fiber strands) connecting the muscle with the reticular dermis. (c,d) Sirius red staining of the myotendinous junction of the palmaris brevis muscle. (c) Note the clear endings of the individual muscle fibers (green) at collagen fiber bundles (red), marked by arrowheads. (b) Higher magnification shows the parallel finger-like protrusions of the muscle fibers (asterisks) toward the tendon-like dense collagen fibers toluidine blue to reconstruct the myocutaneous junction in more detail.

| RE SULTS
Serial sections through the palmaris brevis and platysma muscle revealed that both muscles do generally not form true myocutaneous junctions. Instead, they insert into connective tissue fasciae of either the hypothenar muscles (in case of the palmaris brevis muscle) or the superficial body fascia (in case of the platysma; Figure 1a). These insertions show myotendinous junctions and formation of multiple small tendons connecting the individual muscle fibers and the corresponding fascia (Figure 1b-d). The muscle fibers always ended in a clear distance to the dermis. The fascia of the muscles connected to the dermis via subcutaneous retinacula (Figure 1a). Myocutaneous junctions were therefore limited in this study to the orbicularis oris muscle.

| Myocutaneous junctions of the orbicularis oris muscle
At certain places throughout the circumference of the orbicularis oris muscle, small groups of 5-15 striated muscle fibers branched off from the main muscle body, ran through the connective tissue strains of the subcutaneous layer (hypodermis), and reached the reticular dermis of the adjacent skin (Figure 2a). The skin architecture showed some specifics: the epidermal layer (mean thickness 41 ± 4 µm) was connected to the papillary dermis (mean thickness 31 ± 2 µm) which showed a flat appearance and followed the hair follicles toward the hypodermis. The reticular dermis (mean thickness 670 ± 85 µm) showed a resistance for staining resulting in a unique paleness (Figure 2a). In the inner third of the reticular dermis, the muscle fibers formed finger-like protrusions which were brush-like connected to the surrounding collagen tissue  (Figure 3b,c). Mainly, the bundles of collagen and the elastic fibers showed no specific arrangement toward the muscle fibers (Figure 3b). At places, however, a dense elastic network was seen around single muscle fibers just prior to their brush-like ending ( Figure 3c).

F I G U R E 3
Toluidine blue-stained semi-thin sections of striated muscle fibers (asterisks) in the dermis (orbicularis oris muscle).

| Collagen XXII
At the myotendinous junction of the palmaris brevis muscle, intense staining of collagen XXII was present (Figure 4a). In contrast, the myocutaneous junctions of the orbicularis oris muscle showed no presence of collagen XXII at their interface ( Figure 4b).

| DISCUSS ION
Summarizing our findings, true myocutaneous junctions exist in the human but are limited to the mimic muscles. Here, muscle fibers reach the dermis and insert in the fibro-elastic network without locally modifying its three-dimensional structure. This connection is different from myotendinous junctions and lacks collagen type XXII, a specific myotendinous junction protein.
Although it is a long known fact that the mimic muscles insert into the skin, not much attention was paid to a close morphology of this region. The term 'myocutaneous junction' in analogy to 'myotendinous junction' has not yet been established, although it seems appropriate since there are morphological differences between both junctions, for the first time described here. The limitation to facial mimic muscles, excluding the platysma, might underline the exclusiveness of this group of skeletal muscles including their lack of muscle-specific sensory organs which are present in the platysma (May et al., 2018).
It is tempting to speculate that the changes in muscle length usually sensed by muscle spindles in skeletal muscles can specifically be transduced by changes in skin tension to the facial mimic muscles via the myocutaneous interface superseding muscle spindles in this unique situation.
Surprisingly, the connective tissue of the dermis was not substantially modified by the insertion of the muscle fibers. More specific, the collagen and elastic components did not orient themselves toward the finger-like protrusions of the muscle fibers which entered the network perpendicularly. The single muscle fibers seem not to create enough mechanical force to influence their surroundings (compare Huijing, 1999). This is in contrast to the suggested summation of several parallel muscle fibers at myotendinous or even myo-myonal junctions (Zenker et al., 1990). This mechanical difference might also explain the lack of collagen type XXII at the myocutaneous junctions.
An interesting observation noted only in the skin containing myocutaneous junctions was the virtually unstained reticular dermis. This observation is hitherto unmentioned in the literature and awaits further investigation.
Interestingly, not much attention was paid to the insertion of the panniculus carnosus/cutaneus trunci muscle in animals and its associated muscles in human. Here, we could show that muscles associated with the panniculus carnosus in man are more connected to the body fascia than to the dermis. This seems also true for the rat (Theriault & Diamond, 1988), although not morphologically demonstrated. Therefore, these muscles differ from mimic muscles showing at least in part true interaction with the skin dermal layers.

ACK N OWLED G EM ENTS
The late donors are especially recognized for their individual support of science. The authors thank Doreen Streichert for technical help with the semi-thin sections. No author has any conflicts of interest.

AUTH O R CO NTR I B UTI O N S
CAM took tissue samples, performed the evaluation, and prepared the manuscript. SB performed the staining and reviewed the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All slides are available to view. Otherwise there is no statistic data block.