Francis Bacou

Transplantation of adipose tissue-derived stromal cells increases mass and functional capacity of damaged skeletal muscle.

The regenerating skeletal muscle environment is capable of inducing uncommitted progenitors to terminally differentiate. The aim of this work was to determine whether adipose tissue-derived stromal cells were able to participate in muscle regeneration and to characterize the effect on muscle mass and functional capacities after transplantation of these cells. Adipose tissue stromal cells labeled with Adv cyto LacZ from 3-day-old primary cultures (SVF1) were autotransplanted into damaged tibialis anterior muscles. Fifteen days later, β-galactosidase staining of regenerated fibers was detected, showing participation of these cells in muscle regeneration. Two months after SVF1 cell transfer, muscles were heavier, showed a significantly larger fiber section area, and developed a significantly higher maximal force compared with damaged control muscles. These results are similar to those previously obtained after satellite cell transplantation. However, SVF1 transfer also generated a small amount of adipose tissue localized along the needle course. To minimize these adipose contaminants, we transferred cells from 7-day-old secondary cultures of the SVF1, containing only a small proportion of already engaged preadipocytes (SVF2). Under these conditions, no adipose tissue was observed in regenerated muscle but there was also no effect on muscle performances compared with damaged control muscles. This result provides further evidence for the existence of progenitor cells in the stromal fraction of freshly isolated adipose tissue cells, which, under our conditions, keep some of their pluripotent properties in primary cultures.

EXPRESSION OF SPECIFIC WHITE ADIPOSE TISSUE GENES IN DENERVATION-INDUCED SKELETAL MUSCLE FATTY DEGENERATION

Denervation of skeletal muscle results in rapid atrophy with loss of contractile mass and/or progressive degeneration of muscle fibers which are replaced to a greater or lesser degree by connective and fatty tissues. In this study, we show that denervated rabbit muscles are transformed into a white adipose tissue, depending on their fiber types. This tissue does express LPL, G3PDH and particularly the ob gene, a white adipose tissue‐specific marker, and does not express the brown adipose tissue molecular marker UCP1 mRNA.

Comparative evolution of muscular dystrophy in diaphragm, gastrocnemius and masseter muscles from old male mdx mice

X chromosome-linked muscular dystrophic mdx mouse lacks the sarcolemmal protein dystrophin and represents a genetic homologue of human Duchenne muscular dystrophy (DMD). The present study analysed some aspects of pathological processes such as fibrosis, frequency of centralized nuclei, presence of degenerative or regenerative fibres, expression of utrophin and associated protein complexes, and myosin heavy chain isoforms in three muscles [diaphragm (DIA), gastrocnemius (GTC) and masseter (MAS)] from old male mdx mice. All parameters investigated comparatively in these pathological muscles provided evidence that the MAS mdx muscle presents a slight deterioration pattern in comparison to that of DIA and GTC muscles. Utrophin and associated proteins are present in many cell clusters with continuous membrane labelling in MAS muscle. Respective proportions of myosin heavy chain isoforms, measured by electrophoresis/densitometry, showed only slight change in GTC muscle, significant evolution in DIA muscle but drastic isoform conversions in MAS muscle. These results highlighted the difference in deterioration susceptibility of various muscles to muscular dystrophy. The reason why this occurs in MAS muscles is still obscure and discussed in terms of the comparative developmental origins of these muscles.

Differential expression of myosin heavy chain mRNA and protein isoforms in four functionally diverse rabbit skeletal muscles during pre- and postnatal development

Myosin heavy chains (hcs) are the major determinant in the speed of contraction of skeletal muscle, and various isoforms are differentially expressed depending on the functional activity of the muscle. Using the rapid amplification of cDNA ends (3′ RACE) method, we have characterised the 3′ end of the embryonic, perinatal, type 1, 2a, 2x, and 2b myosin hc genes in rabbit skeletal muscle and used them as probes in RNase protection assays to quantitatively monitor their expression in different type of skeletal muscles just before and after birth. SDS PAGE was used to study the changes in the expression level of their respective protein and to determine the relative abundance of each myosin hc isoform in the muscles studied. The results show that for each anatomical muscle, the developmental changes in myosin hc gene expression at the mRNA level correlate strongly to those observed at the protein level. By studying their developmental expression in four functionally diverse skeletal muscles (semimembranosus proprius, diaphragm, tibialis anterior, and semimembranosus accessorius), it was shown that all muscles express the embryonic, perinatal, and type 1 isoform during prenatal development up to the E27 stage. In the diaphragm, low levels of the type 2a and 2x transcripts, which are adult fast isoforms, were also detected at the E27 stage. During the first week of postnatal growth the myosin hc transition leading to the expression of the adult isoforms is complex, and as many as five different myosin heavy chains are concurrently expressed in some muscles at around birth. As the animal matures, individual muscles become adapted to perform highly specialised functions, and this is reflected in the myosin hc composition within these muscles. Accordingly, the expression of the type 1 isoform, and the sequence of appearance and the expression levels of the type 2 isoforms, were exclusively dependent on the muscle type and largely reflect the functional activity of each muscle during the postnatal growth period. Dev. Dyn. 1998;211:193–203.

EXPRESSION OF MYOSIN HEAVY CHAIN AND OF MYOGENIC REGULATORY FACTOR GENES IN FAST OR SLOW RABBIT MUSCLE SATELLITE CELL CULTURES

SummaryWe investigated the myogenic properties of rabbit fast or slow muscle satellite cells during their differentiation in culture, with a particular attention to the expression of myosin heavy chain and myogenic regulatory factor genes. Satellite cells were isolated from Semimembranosus proprius (slow-twitch muscle; 100% type I fibres) and Semimembranosus accessorius (fast-twitch muscle; almost 100% type II fibres) muscles of 3-month-old rabbits. Satellite cells in culture possess different behaviours according to their origin. Cells isolated from slow muscle proliferate faster, fuse earlier into more numerous myotubes and mature more rapidly into striated contractile fibres than do cells isolated from fast muscle. This pattern of proliferation and differentiation is also seen in the expression of myogenic regulatory factor genes. Myf5 is detected in both fast or slow 6-day-old cell cultures, when satellite cells are in the exponential stage of proliferation. MyoD and myogenin are subsequently detected in slow satellite cell cultures, but their expression in fast cell cultures is delayed by 2 and 4 days respectively. MRF4 is detected in both types of cultures when they contain striated and contractile myofibres. Muscle-specific myosin heavy chains are expressed earlier in slow satellite cell cultures. No adult myosin heavy chain isoforms are detected in fast cell cultures for 13 days, whereas cultures from slow cells express neonatal, adult slow and adult fast myosin heavy chain isoforms at that time. In both fast and slow satellite cell cultures containing striated contractile fibres, neonatal and adult myosin heavy chain isoforms are coexpressed. However, cultures made from satellite cells derived from slow muscles express the slow myosin heavy chain isoform, in addition to the neonatal and the fast isoforms. These results are further supported by the expression of the mRNA encoding the adult myosin heavy chain isoforms. These data provide further evidence for the existence of satellite cell diversity between two rabbit muscles of different fibre-type composition, and also suggest the existence of differently preprogrammed satellite cells.

Anal sphincter reconstruction by dynamic graciloplasty after abdominoperineal resection for cancer

PURPOSE: Chronic low-frequency electrical stimulation can safely transform fatiguing muscle into fatigue-resistant muscle. This fundamental discovery was used to reconstruct the anal sphincter. Dynamic graciloplasty was found to be effective in the treatment of fecal incontinence. Our study was undertaken to investigate the oncologic, functional, and quality of life results of dynamic graciloplasty anal reconstruction after an abdominoperineal resection for carcinoma. METHODS: Between April 1993 and April 1996, nine patients (4 males) with a median age of 51.2 (range, 29–69) years underwent an abdominoperineal resection for carcinoma (4 had a rectal adenocarcinoma and 5 had an epidermoidal anal tumor) and an anal sphincter reconstruction with electrically stimulated graciloplasty. Oncologic and functional results were evaluated after a mean follow-up of 32 (range, 14–59) months. A quality of life questionnaire was filled out by seven patients. RESULTS: Sphincter reconstruction required the same hospitalization period as abdominoperineal resection. Two patients died from evolutive disease. Three patients were operated on twice, one for immediate colonic necrosis, two for colonic perforation after enema. One of them refused the graciloplasty and had an abdominoperineal resection. Six patients were dysfunctioned. The mean resting pressure was 24±10 mmHg, and the mean pressure during stimulation was 95±25 mmHg. Five patients were continent for solids and liquid; four wore less than three pads per day, and one wore more than three. Four patients used enemas twice a week; one patient had spontaneous evacuation. The quality of life questionnaire showed that the mean scores for social interaction, symptoms, and psychological and physical states were 2.1, 2.2, 2.4, and 2.7, respectively. The mean value was 1.5 CONCLUSIONS: Total anorectal reconstruction with dynamic graciloplasty is an oncologically safe procedure. Functional results improve with time, but careful patient selection guarantees a successful functional outcome. Technical progress is necessary to improve the quality of life.

Aldehyde dehydrogenase activity promotes survival of human muscle precursor cells.

Aldehyde dehydrogenases (ALDH) are a family of enzymes that efficiently detoxify aldehydic products generated by reactive oxygen species and might therefore participate in cell survival. Because ALDH activity has been used to identify normal and malignant cells with stem cell properties, we asked whether human myogenic precursor cells (myoblasts) could be identified and isolated based on their levels of ALDH activity. Human muscle explant‐derived cells were incubated with ALDEFLUOR, a fluorescent substrate for ALDH, and we determined by flow cytometry the level of enzyme activity. We found that ALDH activity positively correlated with the myoblast‐CD56+ fraction in those cells, but, we also observed heterogeneity of ALDH activity levels within CD56‐purified myoblasts. Using lentiviral mediated expression of shRNA we demonstrated that ALDH activity was associated with expression of Aldh1a1 protein. Surprisingly, ALDH activity and Aldh1a1 expression levels were very low in mouse, rat, rabbit and non‐human primate myoblasts. Using different approaches, from pharmacological inhibition of ALDH activity by diethylaminobenzaldehyde, an inhibitor of class I ALDH, to cell fractionation by flow cytometry using the ALDEFLUOR assay, we characterized human myoblasts expressing low or high levels of ALDH. We correlated high ALDH activity ex vivo to resistance to hydrogen peroxide (H2O2)‐induced cytotoxic effect and in vivo to improved cell viability when human myoblasts were transplanted into host muscle of immune deficient scid mice. Therefore detection of ALDH activity, as a purification strategy, could allow non‐toxic and efficient isolation of a fraction of human myoblasts resistant to cytotoxic damage.

Inhibition of myoblast differentiation by Sfrp1 and Sfrp2

Secreted Frizzled-related proteins (Sfrps) are extracellular regulators of Wnt signalling and play important roles in developmental and oncogenic processes. They are known to be upregulated in regenerating muscle and in myoblast cultures but their function is unknown. Here, we show that the addition of recombinant Sfrp1 or Sfrp2 to C2C12 cell line cultures or to primary cultures of satellite cells results in the inhibition of myotube formation with no significant effect on the cell cycle or apoptosis. Even though at confluence, treated and untreated cultures are identical in appearance, analyses have shown that, for maximum effect, the cells have to be treated while they are proliferating. Furthermore, removal of Sfrp from the culture medium during differentiation restores normal myotube formation. We conclude that Sfrp1 and Sfrp2 act to prevent myoblasts from entering the terminal differentiation process.

Wnt4 activates the canonical β-catenin pathway and regulates negatively myostatin: functional implication in myogenesis

Expression of Wnt proteins is known to be important for developmental processes such as embryonic pattern formation and determination of cell fate. Previous studies have shown that Wn4 was involved in the myogenic fate of somites, in the myogenic proliferation, and differentiation of skeletal muscle. However, the function of this factor in adult muscle homeostasis remains not well understood. Here, we focus on the roles of Wnt4 during C2C12 myoblasts and satellite cells differentiation. We analyzed its myogenic activity, its mechanism of action, and its interaction with the anti-myogenic factor myostatin during differentiation. Established expression profiles indicate clearly that both types of cells express a few Wnts, and among these, only Wnt4 was not or barely detected during proliferation and was strongly induced during differentiation. As attested by myogenic factors expression pattern analysis and fusion index determination, overexpression of Wnt4 protein caused a strong increase in satellite cells and C2C12 myoblast differentiation leading to hypertrophic myotubes. By contrast, exposure of satellite and C2C12 cells to small interfering RNA against Wnt4 strongly diminished this process, confirming the myogenic activity of Wnt4. Moreover, we reported that Wnt4, which is usually described as a noncanonical Wnt, activates the canonical β-catenin pathway during myogenic differentiation in both cell types and that this factor regulates negatively the expression of myostatin and the regulating pathways associated with myostatin. Interestingly, we found that recombinant myostatin was sufficient to antagonize the differentiation-promoting activities of Wnt4. Reciprocally, we also found that the genetic deletion of myostatin renders the satellite cells refractory to the hypertrophic effect of Wnt4. These results suggest that the Wnt4-induced decrease of myostatin plays a functional role during hypertrophy. We propose that Wnt4 protein may be a key factor that regulates the extent of differentiation in satellite and C2C12 cells.

Changes in mass and performance in rabbit muscles after muscle damage with or without transplantation of primary satellite cells.

Changes in morphology, metabolism, myosin heavy chain gene expression, and functional performances in damaged rabbit muscles with or without transplantation of primary satellite cells were investigated. For this purpose, we damaged bilaterally the fast muscle tibialis anterior (TA) with either 1.5 or 2.6 ml cardiotoxin 10–5 M injections. Primary cultures of satellite cells were autotransplanted unilaterally 5 days after muscle degeneration. Two months postoperation, the masses of damaged TAs, with or without transplantation, were significantly larger than those of the controls. Furthermore, damaged transplanted muscles weighed significantly more than damaged muscles only. The increase in muscle mass was essentially due to increased fiber size. These results were independent of the quantity of cardiotoxin injected into the muscles. Maximal forces were similar in control and 2.6 ml damaged TAs with or without satellite cell transfer. In contrast, 1.5 ml damaged TAs showed a significant decrease in maximal forces that reached the level of controls after transplantation of satellite cells. Fatigue resistance was similar in control and 1.5 ml damaged TAs independently of satellite cell transfer. Fatigue index was significantly higher in 2.6 ml damaged muscles with or without cell transplantation. These changes could be explained in part by muscle metabolism, which shifted towards oxidative activities, and by gene expression of myosin heavy chain isoforms, which presented an increase in type IIa and a decrease in type I and IIb in all damaged muscles with or without cell transfer. Under our experimental conditions, these results show that muscle damage rather than satellite cell transplantation changes muscle metabolism, myosin heavy chain isoform gene expression, and, to a lesser extent, muscle contractile properties. In contrast, muscle weight and fiber size are increased both by muscle damage and by satellite cell transfer.