Glycogenosome accumulation in the arrector pili muscle in Pompe disease

We examined 6 adult patients (2 males, 4 females; mean age 48,5 years) with biochemically and genetically confirmed Pompe disease. At the time point of biopsy 2 of the patients had been receiving ERT and 4 had not yet started ERT. In 2 of the latter patients, a second biopsy was performed after commencing ERT. All patients gave their informed consent. The study was approved by the local ethics committee of Martin-Luther-University Halle (Saale). Skin biopsies from 6 healthy individuals obtained from the diagnostic skin biopsy archive of the Institute of Neuropathology, RWTH Aachen University, were used as controls.

A 3 mm skin punch biopsy was taken from the standard location of 10 cm proximally of the lateral malleus [14] to screen for glycogen deposits and vacuoles in smooth muscle cells of the arrector pili muscles by light and electron microscopy. Archived diagnostic skeletal muscle biopsies obtained from six adult patients (2 male; 4 female; mean age 43.7 years) with genetically confirmed, untreated Pompe disease were also analysed by light and electron microscopy. These were used to compare the ultrastructural morphological changes in skeletal and arrector pili muscle with special focus on glycogen accumulation and vacuolisation within skeletal muscle fibers, endothelial cells and smooth muscle cells of the small intramuscular blood vessels. Skeletal muscle biopsies prior to ERT were not available for any of the patients undergoing skin punch biopsy.

Cryostat sections of formalin fixed, Tissue-Tek O.C.T. compound (Sakura Finetek Europe, Zoeter Woude, The Netherlands) embedded tissues were stained with hematoxylin and eosin (H&E), periodic acid Schiff stain (PAS) and with anti-p62 (Abcam, mouse monoclonal antibody, visualized with HRP conjugated secondary anti-mouse antibody, Cell Signalling Technology). For electron microscopy, glutaraldehyde-fixed tissue specimens were post-fixed with 1% OsO₄ in 0.1 M cacodylate buffer containing 50 mM K₃Fe (CN)₆ and embedded in epoxy resin. Ultramicrotome cut semithin sections from the resin blocks were stained with toluidine blue and paraphenylene diamine. Ultrathin sections were contrast enhanced with uranyl acetate and lead citrate and examined with a Philips EM 400 T electron microscope and images were captured using a Morada digital camera as previously described [15].

The glycogen content of arrector pili smooth muscle cells was assessed semiquantitatively using electron micrographs of randomly selected fields on the cross sections of the muscles. The individual smooth muscle cells were then manually examined, counted and assigned to five categories according to their glycogen content (see legend of Figure 1). We counted 849 cells from controls and 1511 cells from Pompe disease patients. The chi-squared test was used to compare the patient and control group regarding the normal and abnormal glycogen accumulations.

Clinical characteristics of the adult Pompe patients are given in Table 1. The mean age at examination was 48.5 years. All patients had proximal paresis of arms and legs. No patient was wheelchair-bound or depended on nocturnal non-invasive ventilation. For the two patients (P4 and P5) who had received ERT after the first skin biopsy, the second skin biopsies were obtained 19 and 20 months after ERT, respectively.

In PAS and H&E stained paraffin and cryostat sections, in semithin sections stained with toluidine blue and by electron microscopy we observed vast intermyofibrillar and subsarcolemmal glycogen accumulations (Figure 2A) associated with autophagic vacuoles in all Pompe patient-derived muscle biopsies (Figure 2B). Increased and abnormal glycogen accumulations were also found in fibroblasts in the vicinity of endomysial capillaries, in smooth muscle cells of intramuscular arterioles and in small arteries (Figure 2C).

Light microscopy of PAS stains did not reveal any differences in arrector pili smooth muscles from controls and Pompe patients (Figure 3A-B), which could result from the inadequacy of our tissue preparation for the proper visualization of glycogen accumulation. The toluidine blue staining of the semithin sections showed the membrane bound nature of the glycogen deposits and accumulations of autophagic vacuoles in Pompe disease cases (Figure 3C-D). The autophagic origin of these vacuoles was confirmed by p62 staining (Figure 3E-F). The role of p62 is to link the ubiquitinated cargos to the autophagy machinery for autophagic degradation; p62 is widely used as a marker for accumulation of autophagic vacuoles [16]. However, unequivocal differences could only be demonstrated by electron microscopic examination. Arrector pili smooth muscle cells from Pompe disease patients showed vastly increased glycogen storage in glycogenosomes associated with prominent, pleomorphic, mostly granular accumulations in autophagic vacuoles (Figure 2F-I). In contrast, control smooth muscle cells also showed glycogen accumulations but these were less pronounced, not as densely packed and not membrane-bound. Such granular glycogen deposits were in many cases loosely associated with sub-cellular structures, such as mitochondria or plasma membrane and endoplasmic reticulum, but were not associated with prominent autophagic vacuoles (Figure 2D-E).

Quantitative analysis confirmed that although many smooth muscle cells (78%) in controls contained glycogen, the typical form of glycogen was as small granular deposits. Only 7% of cells showed an elevated number of deposits and less than 1% contained membrane-bound glycogen. No glycogen deposits could be found in 22% of control cells. In contrast, 37% of Pompe disease patient-derived arrector pili smooth muscle cells contained abnormal forms of glycogen; among these 11% showed accumulation of autophagic vacuoles filling a significant part of the cytoplasm. The distribution of these different forms of glycogen deposits was significantly different between patients and controls (p > 0,0001) (Figure 1A).

We also compared the pre- and post-ERT biopsies in two cases. In the pooled quantification data of these two cases we saw a decrease in the number of cells containing glycogenosomes (33.1% vs. 29.7%) and also in those severely affected by autophagy (from 9.0% to 5.7%). The distribution for the two treated patient can be seen in Table 2. Due to the small number of subjects (n = 2) and the high individual variation in patients, a comprehensive statistical analysis of the patterns of intracellular glycogen deposition before and after ERT was not possible, even though the decrease in cells affected by autophagy compared to all other cells was significant (p = 0.003) (Figure 1B).