I The fiber types of severely atrophic muscle fibers with nuclear clumps of human 5-year LMN denervated muscle

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Fiber types of human atrophic myofibers withnuclear clumps

Basic Applied Myology 19 (5&6): 225-228, 2009

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The fiber types of severely atrophic muscle fibers with nuclear clumps of human 5-year LMN denervated muscle

Nicoletta Adami (1), Donatella Biral (2), Silvia Corbianco (1), Sandra Zampieri (1,3), Ugo Carraro (1,2), Helmut Kern (4)

(1) Laboratory of Translational Myology of the Interdepartmental Research Center of Myology University of Padova; (2) C.N.R. Institute of Neuroscience c/o Department of Biomedical Science, Padova, Italy; (3) Division of Rheumatology, Department of Clinical and Experimental Medicine, University of Padova, Italy; (4) L. Boltzmann Institute of Electrical Stimulation and Physical Rehabilitation, and Department of Physical Medicine and Rehabilitation, Wilhelminenspital Vienna, Austria

Abstract

During long-standing lower motor neuron (LMN) denervation of human leg muscles muscle fibers are known to disappear, substituted by adipose and fibrous connective tissue. Here we show that in the loose connective tissue that seems to fill the human denervated muscle after five years of lower motor neuron denervation there are many severely atrophic myofibers, which had lost their myofibrillar apparatus and coil distribution of myonuclei that are relocated in clumps (myonuclear clumps). Immunohistochemistry with anti MHCslow and MHCemb shows that some of these severely atrophic myofibers are of slow type, thus demonstrating that they are surviving adults fibers. The contribution of regenerative myogenesis demonstrated in long-lasting LMN denervation may contribute to extend the presence of muscle fibers and of the severely atrophic muscle fibers with nuclear clumps. These results demonstrate that the human muscle fibers survive permanent lower motor neuron denervation much longer than generally accepted and provide the rationale to plan research aimed to recover long-lasting denervated muscle by combining molecular and cellular approaches with functional electrical stimulation.

Key Words: human muscle; long-standing lower motor neuron denervation; severe atrophy;

myonuclear clumps; spinal cord injury, SCI

Basic Applied Myology 19 (5&6): 225-228, 2009.

I

n rodents between three and seven months after denervation, muscle fibers maintain a stable atrophy that results in a consistent reduction of the muscle weight. The denervated muscle appears to contain numerous severely atrophic muscle fibers, some of which are characterized by the distribution of muscle nuclei in clumps. [1, 7, 8].

In muscle biopsies harvested from human subjects who experienced complete lower motor neuron lesion we observed similar events, but in a more extended period of time (in years not months) [14-16]. Mild atrophy persists up to the second year of denervation, while severe atrophy appears after two-three years

These behaviors are not unique to humans, but seem to be a common feature in mammals larger than rodents [4, 20]. Muscle tissue is then progressively enriched of severely atrophic myofibers, which are depleted of the myofibrillar apparatus. In longitudinal section they

present groups of tens of nuclei (myonuclear clumping) separated by stretches of empty myoplasm.

In transverse sections, these severely atrophic muscle fibers present three or more central nuclei, and typically no contractile structures around. In the present study, our aim is to establish the fiber type of the severely atrophic myofibers present in muscle biopsies harvested from complete LMN denervated quadriceps muscle four years after lumbar-ischiatic spinal cord injury.

Materials and Methods

Patients. We collected muscle biopsies from quadriceps muscles of twenty-two patients who had experienced since one to ten years, traumatic spinal cord injury that caused complete lesion of the lumbar- ischiatic lower motor neurons (complete Conus Cauda Syndrome). Demographic and clinical data are

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Fig. 1 Four years LMN denervated human muscle.

H&E. Some of the severely atrophic myofibers have red remnants of the contractile machinery, while others are empty of any sarcomeric structures.

described in [14, 15]. All subjects enrolled in the study were volunteers who received and signed a detailed informed consent. Clinical and functional assessments, as well as follow-up and muscle biopsies, were performed at the Wilhelminenspital, Vienna (Austria).

Complete denervation of quadriceps muscle was assessed by electrophysiological testing, i.e. by test electrical stimulation, needle electromyography, brain motor control assessment, transcranial and lumbosacral magnetic stimulation [14, 15]. All applicable rules concerning the ethical use of human volunteers were followed during this study (Approval of Ethical Committee, Vienna, Austria: EK-02-068-0702).

Hystological analyses. Light microscopy and morphometry of muscle biopsies were performed at the Interdepartmental Research Center of Myology, University of Padova (Italy), as described in [22]. In brief, muscle biopsies were quenched and in stored in liquid nitrogen. Using conventional techniques, cryosections (10 µm thick) were collected on glass slides and stained with hematoxylin-eosin (H&E). All images were collected using a Zeiss microscope connected to a Leica DC 300F camera under the same conditions that were used to acquire a reference ruler.

Severely atrophic muscle fibers with nuclear clumps were counted in transverse/oblique cryosections [16].

Immunohistochemical analyses. Unfixed muscle sections were labeled for 1 hour at room temperature using mouse monoclonal antibody directed against slow-type myosin heavy chain (MHCs) (Novocastra, Newcastle-upon-Tyne, U.K.) or against embryonic- type myosin heavy chain (MHC-emb) (from Novocastra, NCL-MHCd), 1:20 diluted in Tris- buffered saline (TBS). Sections were rinsed 3x5 min in TBS, and then incubated for 1 hour at room temperature with FITC labeled conjugates directed against mouse IgG (Sigma-Aldrich, St. Louis, USA) 1:200 diluted in 10 % goat serum/TBS. Negative controls were performed by omitting the primary antibodies on samples. After washes, nuclei were counterstained for 5 min at room temperature with Hoechst 33258 (Sigma-Aldrich, St. Louis, USA), sections were coverslipped using mounting medium (Dako, Glostrup, Denmark) and observed under a Zeiss microscope [22].

Results

Figure 1 shows at high magnification H&E stained sections of skeletal muscle biopsies harvested from a denervated subject 4 years after LMN denervation:

many severely atrophic myofibers with nuclear clumps are present. Some have red remnants of the contractile machinery, while others are faint-pink being empty of sarcomeric structures. A few larger, patchy muscle fibers are also present.

Figure 2 shows that many of the severely atrophic myofibers of the 4-year LMN denervated human biopsy are stained by the anti-MHCslow antibody. On

the other hands, by anti-MHCemb immunostaining only a few muscle fibers are positive (Fig. 3).

Discussion

In biopsies harvested from patients affected with complete Conus Cauda syndrome, the muscle tissue, before undergoing adipose and fibrous tissue substitution, progressively shows a severe atrophy of muscle fibers that loses their myofibrillar apparatus and relocates in clumps their nuclei [14, 16]. In long- term paraplegics with complete upper motor neuron lesion from three to twenty years of thoracic level SCI, nuclear clumps were never observed [17].

The severe atrophy characterized by myonuclear clumping appears during the second year from complete and permanent LMN denervation [16]. The early stage of muscle fiber regeneration contains chains of single central nuclei all along the new muscle fiber, which results from the fusion of hundreds/thousands of myoblasts [9, 11, 12, 13, 21, 22]. The myonuclear clumping distinguish, thus, surviving severely atrophic myofibers from the myotubes.

The observations that: 1. after the third year of denervation, 30% of atrophic myofibers show nuclear clumps and 2. that the clumps of nuclei are separated by twice or longer stretches of amyofibrillar cytoplasm (Fig. 1) suggest that almost all the severely atrophic myofibers seen in muscle biopsies from three to five years after permanent LMN injury, present relocation of their nuclei from the coil pattern of normal muscle fibers to nuclear clumps. Embryonic myosin is expressed in myotubes and young muscle fibers and is a sound molecular marker of early myogenesis in both

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- 227 - Fig 2 Immunohistochemistry of 4 years LMN

denervated human muscle. Almost half of the severely atrophic myofibers are stained in green by the anti-MHCslow antibody.

Fig 3 Immunohistochemistry of 4 years LMN denervated human muscle. A few muscle fibers are stained in green by anti-MHCemb. Nuclei are stained by Hoechst 33258.

developing and adult regenerating muscles [10, 13, 24]. When re-innervation of regenerated myofibers occurs, either slow or fast types of adult isomyosins substitute the embryonic myosin 5-7 days post- damage. If not re-innervated, the regenerated myofibers spontaneously express fast type isomyosins [9, 10, 24]. The severely atrophic myofibers positive after anti-MHCslow staining are thus, long-lasting surviving muscle fibers (Fig. 4, A).

Fast-type myofibers are mainly adult surviving myofibers, but the alternative option that they are also regenerated myofibers could not be excluded. Whether their long-lasting presence is the compound result of sparsely repeated muscle fiber regenerations is still an open issue. In support of the last hypothesis, Carraro et al. [10] showed that denervated hemidiaphragm presents ultra structural and molecular characteristics of muscle fiber regeneration after long-term phrenicotomy and also that satellite cell proliferation and differentiation to adult muscle fibers could be repeatedly induced in permanently denervated leg muscles [12, 21]. These pioneering results were confirmed by others [2, 3, 7, 23] and by ourselves in rat [12, 19] and human [11, 14, 22] long-term LMN denervated muscles.

Anyhow, these results demonstrate that human muscle fibers survive LMN denervation much longer than generally accepted and that nuclear clumps are morphologic markers of the long-lasting ability of human muscle fibers to survive in the absence of the nerve.

These results provide the rationale to plan research aimed to recover these severely atrophic myofibers, by combining molecular and cellular approaches with functional electrical stimulation that is able to restore muscle ultra structure and mass in human long-lasting denervated muscle [6, 11, 14-18].

Acknowledgements

Research was supported by funds of the EU Commission Shared Cost Project RISE (Contract n.

QLG5-CT-2001-02191), of the Ludwig Boltzmann Institute of Electrostimulation and Physical Rehabilitation and of The Austrian Ministry of Science; from institutional funds of Italian C.N.R Institute of Neuroscience. The expert assistance of V.

Gobbo is grateful acknowledged.

Corresponding Author

Sandra Zampieri, Interdepartmental Research Center of Myology, Viale G. Colombo 3, I-35121 Padova, Italy Phone: + 39 049 8276359; FAX: +39 049 8276040 E-mails: sanzamp@unipd.it

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