Costameric proteins in human skeletal muscle during muscular inactivity

Abstract

Costameres are regions that are associated with the sarcolemma of skeletal muscle fibres and comprise proteins of the dystrophin-glycoprotein complex and vinculin-talin-integrin system. Costameres play both a mechanical and a signalling role, transmitting force from the contractile apparatus to the extracellular matrix in order to stabilize skeletal muscle fibres during contraction and relaxation. Recently, it was shown that bidirectional signalling occurs between sarcoglycans and integrins, with muscle agrin potentially interacting with both types of protein to enable signal transmission. Although numerous studies have been carried out on skeletal muscle diseases, such as Duchenne muscular dystrophy, recessive autosomal muscular dystrophies and other skeletal myopathies, insufficient data exist on the relationship between costameres and the pathology of the second motor nerve and between costameric proteins and muscle agrin in other conditions in which skeletal muscle atrophy occurs. Previously, we carried out a preliminary study on skeletal muscle from patients with sensitive-motor polyneuropathy, in which we analysed the distribution of sarcoglycans, integrins and agrin by immunostaining only. In the present study, we have examined the skeletal muscle fibres of ten patients with sensitive-motor polyneuropathy. We used immunofluorescence and reverse transcriptase PCR to examine the distribution of vinculin, talin and dystrophin, in addition to that of those proteins previously studied. Our aim was to characterize in greater detail the distribution and expression of costameric proteins and muscle agrin during this disease. In addition, we used transmission electron microscopy to evaluate the structural damage of the muscle fibres. The results showed that immunostaining of alpha 7B-integrin, beta 1D-integrin and muscle agrin appeared to be severely reduced, or almost absent, in the muscle fibres of the diseased patients, whereas staining of alpha 7A-integrin appeared normal, or slightly increased, compared with that in normal skeletal muscle fibres. We also observed a lower level of alpha 7B- and beta 1D-integrin mRNA and a normal, or slightly higher than normal, level of alpha 7A-integrin mRNA in the skeletal muscle fibres of the patients with sensitive-motor polyneuropathy, compared with those in the skeletal muscle of normal patients. Additionally, transmission electron microscopy of transverse sections of skeletal muscle fibres indicated that the normal muscle fibre architecture was disrupted, with no myosin present inside the actin hexagons. Based on our results, we hypothesize that skeletal muscle inactivity, such as that found after denervation, could result in a reorganization of the costameres, with alpha 7B-integrin being replaced by alpha 7A-integrin. In this way, the viability of the skeletal muscle fibre is maintained. It will be interesting to clarify, by future experimentation, the mechanisms that lead to the down-regulation of integrins and agrin in muscular dystrophies.

Fig. 2

Compound panel showing immunohistochemical findings in normal human skeletal muscle. Skeletal muscle fibres were immunolabelled with antibodies against vinculin (A), talin (B), α7A-integrin (C), α7B-integrin (D), β1D-integrin (E) and agrin (F). All tested proteins showed a costameric distribution and a normal immunostaining pattern.

Fig. 1

Compound panel showing immunohistochemical findings in normal human skeletal muscle. Skeletal muscle fibres were immunolabelled with antibodies against α-sarcoglycan (A), β-sarcoglycan (B), γ-sarcoglycan (C), δ-sarcoglycan (D) and dystrophin (E). All tested proteins showed a costameric distribution and a normal immunostaining pattern.

Fig. 3

Compound panel showing immunostaining of longitudinal sections of skeletal muscle fibres of patients with sensitive-motor polyneuropathy. The sections were immunolabelled with antibodies against α-sarcoglycan (A), β-sarcoglycan (B), γ-sarcoglycan (C), δ-sarcoglycan (D) and dystrophin (E). Sarcoglycans were detectable along the sarcolemma; dystrophin showed a normal immunostaining pattern.

Fig. 4

Compound panel showing immunostaining of longitudinal sections of skeletal muscle fibres of patients with sensitive-motor polyneuropathy. The sections were immunolabeled with antibodies against proteins of the vinculin–talin–integrin system and muscle agrin: (A) vinculin; (B) talin; (C) α7A-integrin; (D) α7B-integrin; (E) β1D-integrin; (F) muscle agrin. α7A immunostaining appeared to be normal, or slightly increased; immunofluorescence for α7B-integrin, β1D-integrin and muscle agrin was severely reduced and nearly absent.

Fig. 6

Display profiles of longitudinal sections of human skeletal muscle fibres of patients with sensitive-motor polyneuropathy. (A) α7A-integrin; (B) α7B-integrin; (C) β1D-integrin; (D) muscle agrin. The α7A-integrin fluorescence peaks were of normal magnitude, while α7B-integrin, β1D-integrin and muscle agrin fluorescence intensities were absent.

Fig. 5

Display profiles of longitudinal sections of normal human skeletal muscle fibres: (A) α7A-integrin; (B) α7B-integrin; (C) β1D-integrin; (D) muscle agrin. All analysed samples showed fluorescence peaks with normal values.

Fig. 7

Transverse sections of normal human skeletal muscle (A) and skeletal muscle from patients with sensitive-motor polyneuropathy (B) observed by transmission electron microscopy. In B, the normal geometry of the thick and thin filaments is absent and the normal muscular architecture is disordered.

Fig. 8

RT-PCR. (A) Control sample. Representative 1.5% agarose gel electrophoresis showing RT-PCR products amplified from one of the unaffected control individuals. In each lane an aliquot of the corresponding β-actin amplification reaction was included as a control to ensure equal quantities of input RNA. Lane 1: α7A-integrin and β-actin; lane 2: α7B-integrin and β-actin; lane 3: β1D-integrin and β-actin; lane 4: 100-bp ladder. (B) Representative 1.5% agarose gel electrophoresis showing RT-PCR products amplified from a sample from one of the affected patients. Lane 1: α7A-integrin and β-actin; lane 2: α7B-integrin and β-actin; lane 3: β1D-integrin and β-actin; lane 4: 100-bp ladder.