**3. Results**

In all of the muscle samples, SCs were morphologically recognizable as small cells located between the sarcolemma and the myofiber basal lamina; they showed scarce cytoplasm and an ovoid nucleus with an irregular border and abundant heterochromatin clumps (Figure 1).

**Figure 1.** Transmission electron micrographs of satellite cells (SCs) bordering a myofiber (my) in skeletal muscles from euthermic (**A**) and hibernating (**B**) dormice. In both seasonal phases, SC nuclei contain large amounts of heterochromatin (ch). (**B**) The accumulation of lipid droplets (L) in the myofiber is a typical feature of hibernating edible dormice [28]. Bars: 1 μm.

In the LR White embedded samples, the usual RNP structural constituents involved in pre-mRNA transcription and processing were evident in the nucleoplasm (Figure 2): a few perichromatin fibrils (PFs; representing the in situ form of nascent transcripts, as well as of their splicing and 3 end processing [34,35]) and PGs were mainly distributed at the periphery of the heterochromatin clumps, and small clusters of interchromatin granules (IG; representing the storage, assembly and phosphorylation sites for transcription and splicing factors [34]) occurred in the interchromatin space (not shown).

**Figure 2.** Immunoelectron microscopy. SC nuclei from euthermic (**A**,**C**,**E**,**G**) and hibernating (**B**,**D**,**F**,**H**) dormice; immunolabelling for RNA polymerase II (**A**,**B**; arrows), DNA/RNA hybrid molecules (**C**,**D**; arrows), small nuclear RiboNucleoProtein ((Sm)snRNP) core protein (**E**,**F**; arrows), paired box protein 7 (Pax7) (**G**,**H**; arrows) and the myogenic differentiation transcription factor D (MyoD) (**G**,**H**; arrowheads). All antibodies specifically label perichromatin fibrils (PFs) that mostly occur at the periphery of heterochromatin clumps (ch). Perichromatin granules (PGs) are indicated by open arrows (**A**,**B**,**E**,**F**). Gold particles were digitally enhanced to improve their visibility. Bars: 500 nm.

SC nuclei were structurally similar in euthermic and hibernating dormice, and morphological evidence of apoptosis or necrosis was never found in any of the muscle samples examined. No statistically significant difference in the percentage of heterochromatin was found between hibernating and euthermic dormice (Figure 3A). Conversely, PG density increased in hibernating vs. euthermic dormice (Figure 3B).

**Figure 3.** Quantitative evaluation of the percentage of heterochromatin (**A**) and PG density (**B**) (mean ± standard error of the mean (SEM)) in SC nuclei from skeletal muscles of euthermic (eu) and hibernating (hib) dormice. No significant difference was found between euthermia and hibernation for heterochromatin (*p* = 0.091), whereas PG density was significantly higher in hibernating dormice (*p* = 0.002).

The distribution of the immunolabelling for phosphorylated polymerase II, DNA/RNA hybrid molecules, (Sm)snRNP, PAX7 and MyoD was similar in SC nuclei from hibernating and euthermic dormice, being almost exclusively associated with PFs at the edge of the heterochromatin clumps (Figure 2). Quantitative evaluation of the immunolabelling revealed similar densities of all probes in SC nuclei of hibernating and euthermic dormice (Figure 4). Background values were negligible in all the immunolabelling experiments (not shown).

**Figure 4.** Quantitative immunoelectron microscopy. Labelling density (gold particles/μm2) of RNA processing factors in the interchromatin space (mean ± SE) of SC nuclei from skeletal muscles of euthermic (eu) and hibernating (hib) dormice. No significant difference was found between euthermia and hibernation.

#### **4. Discussion**

The absence of morphologically recognizable apoptotic or necrotic nuclei in SCs suggests that hibernation does not negatively affect the viability of the SC pool; this is consistent with findings in hindlimb skeletal muscles of late torpid thirteen-lined ground squirrels showing that the number of SCs does not decrease during deep hibernation in comparison with euthermia [16].

The SCs bordering the myofibers of hibernating dormice were morphologically similar to those found in euthermic animals; in particular, the structural features of their nuclei were typical of quiescent cells with low nuclear activity, i.e., showing abundant clumps of heterochromatin and a few PFs [34,35]. Qualitative analysis was confirmed by quantitative evaluation of heterochromatin, which was comparable in euthermic and hibernating dormice (Figure 3), consistent with previous observations in myonuclei of the same hibernating species [28].

Similarly, the in situ analysis of pre-mRNA transcription and processing factors did not reveal differences in both their intranuclear distribution and amount between euthermic and hibernating dormice.

Activated RNA polymerase II and DNA/RNA hybrid molecules as well as snRNPs were specifically located in PFs. Similar results were obtained in myonuclei of the same edible dormice [28] as well as in the liver and brown adipocytes of hazel dormice [20]. This finding suggests that in the SCs of hibernating dormice, the organization of the mRNA transcription and processing machinery is maintained.

Pax7 (a transcription factor marker of both quiescent and active SC) as well as MyoD (a transcription factor increasing in activated SCs [32,33]), were specifically associated with PFs, i.e., sites of transcription. Such an association was previously observed in murine myoblasts in vitro [36], and now, for the first time, in the SCs of the intact muscle.

The presence of similar amounts of immunolabelling for both Pax7 and MyoD in SC nuclei of euthermic and hibernating edible dormice indicates the absence of changes in their activation state along the hibernation cycle, in accordance with findings by Brooks et al. [16] in ground squirrels. Taken together, these results suggest that SC nuclei do not undergo modification in transcription and early splicing during hibernation. However, accumulation of PGs was found during hibernation in these nuclei. Since PGs are involved in storage/transport of spliced pre-mRNA [34], this finding indicates hibernation-associated changes in pre-mRNA processing and/or a slowdown of intranuclear or nucleus-to-cytoplasm transport of mRNAs [37]. Typically, PG accumulation due to the impairment of pre-mRNA processing is accompanied by PF clustering. For example, PF clustering has been found during ageing in different cell types, inclusive of SCs [30,38]. However, in SC nuclei of hibernating edible dormice, no PF clustering was observed. Indeed, an accumulation of PGs unaccompanied by PF clustering was observed in brown adipocytes of hibernating hazel dormice, and was interpreted as a consequence of continuing transcription and splicing activity paralleled by a reduced export of mature mRNA to the cytoplasm, likely to be promptly used upon arousal [39]. Accumulation of PGs during hibernation also suggests that, in spite of the maintenance of transcription and splicing rate, SC activity undergoes some decrease in hibernating edible dormice. This suggestion is supported by findings in hibernating ground squirrels, where an inhibition of both SC activation and myoblast differentiation were shown [17].

Interestingly, no nuclear bodies were observed during hibernation inside the SC nuclei of edible dormice, whereas myonuclei of the same animals showed some amorphous bodies [28]. Different types of nuclear bodies involved in the storage/assembly of RNA processing factors have been shown to form in various tissues during hibernation [23] and rapidly disappear upon arousal when massive nuclear reactivation takes place [21]. Their absence in SCs during hibernation could be related to the naturally quiescent state of these cells, which present a low metabolic activity even in euthermia.

In conclusion, the SC nuclei of hibernating edible dormice maintain similar transcription and splicing activity as in euthermia, although the nucleus-to-cytoplasm transport undergoes a slowing down. Therefore, the SC activation state is unmodified during hibernation, supporting the idea that skeletal muscle preservation during this seasonal phase, characterized by prolonged inactivity and starvation, is not due to SCs, but rather to the maintenance of some functional activity in myofibers that is able to counteract muscle wasting [17,28].

**Author Contributions:** Conceptualization, writing—review and editing, M.M. and C.Z.; investigation, M.C. and B.C.; writing—original draft preparation, M.M. and B.C.; supervision, C.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
