Myoblots: dystrophin quantification by in-cell western assay for a streamlined development of Duchenne muscular dystrophy (DMD) treatments.

AIMS: New therapies for neuromuscular disorders are often mutation specific and require to be studied in patient's cell cultures. In Duchenne muscular dystrophy (DMD) dystrophin restoration drugs are being developed but as muscle cell cultures from DMD patients are scarce and do not grow or differentiate well, only a limited number of candidate drugs are tested. Moreover, dystrophin quantification by western blotting requires a large number of cultured cells; so fewer compounds are as thoroughly screened as is desirable. We aimed to develop a quantitative assessment tool using fewer cells to contribute in the study of dystrophin and to identify better drug candidates. METHODS: An 'in-cell western' assay is a quantitative immunofluorescence assay performed in cell culture microplates that allows protein quantification directly in culture, allowing a higher number of experimental repeats and throughput. We have optimized the assay ('myoblot') to be applied to the study of differentiated myoblast cultures. RESULTS: After an exhaustive optimization of the technique to adapt it to the growth and differentiation rates of our cultures and the low intrinsic expression of our proteins of interests, our myoblot protocol allows the quantification of dystrophin and other muscle-associated proteins in muscle cell cultures. We are able to distinguish accurately between the different sets of patients based on their dystrophin expression and detect dystrophin restoration after treatment. CONCLUSIONS: We expect that this new tool to quantify muscle proteins in DMD and other muscle disorders will aid in their diagnosis and in the development of new therapies.

PABPN1 gene therapy for oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant, late-onset muscle disorder characterized by ptosis, swallowing difficulties, proximal limb weakness and nuclear aggregates in skeletal muscles. OPMD is caused by a trinucleotide repeat expansion in the PABPN1 gene that results in an N-terminal expanded polyalanine tract in polyA-binding protein nuclear 1 (PABPN1). Here we show that the treatment of a mouse model of OPMD with an adeno-associated virus-based gene therapy combining complete knockdown of endogenous PABPN1 and its replacement by a wild-type PABPN1 substantially reduces the amount of insoluble aggregates, decreases muscle fibrosis, reverts muscle strength to the level of healthy muscles and normalizes the muscle transcriptome. The efficacy of the combined treatment is further confirmed in cells derived from OPMD patients. These results pave the way towards a gene replacement approach for OPMD treatment.

Impact on oxidative phosphorylation of immortalization with the telomerase gene.

Skin fibroblasts are essential tools for biochemical, genetic and physiopathological investigations of mitochondrial diseases. Their immortalization has been previously performed to overcome the limited number of divisions of these primary cells but it has never been systematically evaluated with respect to efficacy and impact on the oxidative phosphorylation (OXPHOS) characteristics of the cells. We successfully immortalized with the human telomerase gene 15 human fibroblasts populations, 4 derived from controls and 11 from patients with diverse respiratory chain defects. Immortalization induced significant but mild modification of the OXPHOS characteristics of the cells with lower rates of oxygen consumption and ATP synthesis associated with their loose coupling. However, it never significantly altered the type and severity of any genetic OXPHOS defect present prior to immortalization. Furthermore, it did not significantly modify the cells' dependence on glucose and sensitivity to galactose thus showing that immortalized cells could be screened by their nutritional requirement. Immortalized skin fibroblasts with significant OXPHOS defect provide reliable tools for the diagnosis and research of the genetic cause of mitochondrial defects. They also represent precious material to investigate the cellular responses to these defects, even though these should afterwards be verified in unmodified primary cells.

The mitotic clock in skeletal muscle regeneration, disease and cell mediated gene therapy.

The regenerative capacity of skeletal muscle will depend on the number of available satellite cells and their proliferative capacity. We have measured both parameters in ageing, and have shown that although the proliferative capacity of satellite cells is decreasing during muscle growth, it then stabilizes in the adult, whereas the number of satellite cells decreases during ageing. We have also developed a model to evaluate the regenerative capacity of human satellite cells by implantation into regenerating muscles of immunodeficient mice. Using telomere measurements, we have shown that the proliferative capacity of satellite cells is dramatically decreased in muscle dystrophies, thus hampering the possibilities of autologous cell therapy. Immortalization by telomerase was unsuccessful, and we currently investigate the factors involved in cell cycle exits in human myoblasts. We have also observed that insulin-like growth factor-1 (IGF-1), a factor known to provoke hypertrophy, does not increase the proliferative potential of satellite cells, which suggests that hypertrophy is provoked by increasing the number of satellite cells engaged in differentiation, thus possibly decreasing the compartment of reserve cells. We conclude that autologous cell therapy can be applied to specific targets when there is a source of satellite cells which is not yet exhausted. This is the case of Oculo-Pharyngeal Muscular Dystrophy (OPMD), a late onset muscular dystrophy, and we participate to a clinical trial using autologous satellite cells isolated from muscles spared by the disease.

Myoblast transfer therapy: is there any light at the end of the tunnel?

Myoblast transfer therapy (MTT) was proposed in the 70's as a potential treatment for muscular dystrophies, based upon the early results obtained in mdx mice: dystrophin expression was restored in this model by intramuscular injections of normal myoblasts. These results were quickly followed by clinical trials for patients suffering from Duchenne Muscular Dystrophy (DMD) in the early 90's, based mainly upon intramuscular injections of allogenic myoblasts. The clinical benefits obtained from these trials were minimal, if any, and research programs concentrated then on the various pitfalls that hampered these clinical trials, leading to numerous failures. Several causes for these failures were identified in mouse models, including a massive cell death of myoblasts following their injection, adverse events involving the immune system and requiring immunosuppression and the adverse events linked to it, as well as a poor dispersion of the injected cells following their injection. It should be noted that these studies were conducted in mouse models, not taking into account the fundamental differences between mice and men. One of these differences concerns the regulation of proliferation, which is strictly limited by proliferative senescence in humans. Although this list is certainly not exhaustive, new therapeutic venues were then explored, such as the use of stem cells with myogenic potential, which have been described in various populations, including bone marrow, circulating blood or muscle itself. These stem cells presented the main advantage to be available and not exhausted by the numerous cycles of degeneration/regeneration which characterize muscle dystrophies. However, the different stem candidates have shown their limits in terms of efficiency to participate to the regeneration of the host. Another issue was raised by clinical trials involving the injection of autologous myoblasts in infacted hearts, which showed that limited targets could be aimed with autologous myoblasts, as long as enough spared muscle was available. This resulted in a clinical trial for the pharyngeal muscles of patients suffering from Oculo-Pharyngeal Muscular Dystrophy (OPMD). The results of this trial will not be available before 2 years, and a similar procedure is being studied for Fascio-Scapulo-Humeral muscular Dystrophy (FSHD). Concerning muscular dystrophies which leave very few muscles spared, such as DMD, other solutions must be found, which could include exon-skipping for the eligible patients, or even cell therapy using stem cells if some cell candidates with enough efficiency can be found. Recent results concerning mesoangioblasts or circulating AC133+ cells raise some reasonable hope, but still need further confirmations, since we have learned from the past to be cautious concerning a transfer of results from mice to humans.

Glucose transporter gene expression in freshly isolated and cultured rat pneumocytes.

Alveolar epithelium in situ takes up luminal glucose by cotransport with sodium. Cultured alveolar type II pneumocytes have only sodium-independent glucose uptake. It is unclear which isoforms are responsible for glucose transport in these cells and why sodium-glucose cotransport activity disappears during culture. GLUT1, GLUT4, GLUT5 and SGLT1 mRNA were detected in freshly isolated rat alveolar type II cells by reverse transcriptase-polymerase chain reaction. We show that SGLT1 mRNA was 90% lower in cells cultured in plastic wells for 2 or 4 days than in freshly isolated cells. mRNAs coding for the facilitated transporters were reduced from 40% (GLUT1) and 75% (GLUT4 and GLUT5) in cultured cells. Cells cultured at the air-liquid interface better preserved their phenotype as attested by significantly higher surfactant-associated protein mRNA levels. However, these cells had no higher GLUT1 and SGLT1 gene expression. Thus, alveolar type II cells lose sodium-glucose cotransport activity in part because of a decrease in mRNA levels. These changes in gene expression and/or mRNA stability may be an additional consequence of the shift towards the type I cell phenotype observed in cultured type II pneumocytes.

A method for measuring the oxygen consumption of intact cell monolayers.

This report describes an open-air method for measuring the O(2) consumption (QO(2)) of intact monolayers of cultured cells. This method is based on Fick's second law of diffusion. It requires only a micromanipulator and a miniature O(2) electrode to measure the PO(2) gradient in the culture medium in the well. It was compared with the conventional oxygraph chamber method. Both methods gave the same value for QO(2) in freshly isolated rat type II cells: 166 +/- 15.3 nmol. h(-1). 10(6) cells(-1) for the open-air method and 151 +/- 11.6 nmol. h(-1). 10(6) cells(-1) for the oxygraph chamber method (n = 11 experiments). But the open-air method gave significantly larger values for QO(2) in cells cultured for 2 days (236 +/- 8.8 nmol. h(-1). 10(6) cells(-1)) than the oxygraph method (71 +/- 15.2 nmol. h(-1). 10(6) cells(-1); P < 0.001; n = 12 experiments). This suggests that the way cells are detached from their substratum to be placed in the oxygraph chamber affects their QO(2). The open-air method may be useful for studies on the metabolic properties of monolayers because the cells do not risk being damaged.