Endoplasmic reticulum stress does not mediate palmitate-induced insulin resistance in mouse and human muscle cells.

AIMS/HYPOTHESIS: Recent experiments in liver and adipocyte cell lines indicate that palmitate can induce endoplasmic reticulum (ER) stress. Since it has been shown that ER stress can interfere with insulin signalling, our hypothesis was that the deleterious action of palmitate on the insulin signalling pathway in muscle cells could also involve ER stress. METHODS: We used C2C12 and human myotubes that were treated either with palmitate or tunicamycin. Total lysates and RNA were prepared for western blotting or quantitative RT-PCR respectively. Glycogen synthesis was assessed by [¹4C]glucose incorporation. RESULTS: Incubation of myotubes with palmitate or tunicamycin inhibited insulin-stimulated protein kinase B (PKB)/ v-akt murine thymoma viral oncogene homologue 1 (Akt). In parallel, an increase in ER stress markers was observed. Pre-incubation with chemical chaperones that reduce ER stress only prevented tunicamycin but not palmitate-induced insulin resistance. We hypothesised that ER stress activation levels induced by palmitate may not be high enough to induce insulin resistance, in contrast with tunicamycin-induced ER stress. Indeed, tunicamycin induced a robust activation of the inositol-requiring enzyme 1 (IRE-1)/c-JUN NH2-terminal kinase (JNK) pathway, leading to serine phosphorylation of insulin receptor substrate 1 (IRS-1) and a decrease in IRS-1 tyrosine phosphorylation. In contrast, palmitate only induced a very weak activation of the IRE1/JNK pathway, with no IRS1 serine phosphorylation. CONCLUSIONS/INTERPRETATION: These data show that insulin resistance induced by palmitate is not related to ER stress in muscle cells.

Discrepancies between the fate of myoblast xenograft in mouse leg muscle and NMR label persistency after loading with Gd-DTPA or SPIOs.

1H-NMR (nuclear magnetic resonance) imaging is regularly proposed to non-invasively monitor cell therapy protocols. Prior to transplantation, cells must be loaded with an NMR contrast agent (CA). Most studies performed so far make use of superparamagnetic iron oxide particles (SPIOs), mainly for favorable detection sensitivity. However, in the case of labeled cell death, SPIO recapture by inflammatory cells might introduce severe bias. We investigated whether NMR signal changes induced by preloading with SPIOs or the low molecular weight gadolinium (Gd)-DTPA accurately monitored the outcome of transplanted cells in a murine model of acute immunologic rejection. CA-loaded human myoblasts were grafted in the tibialis anterior of C57BL/6 mice. NMR imaging was repeated regularly until 3 months post-transplantation. Label outcome was evaluated by the size of the labeled area and its relative contrast to surrounding tissue. In parallel, immunohistochemistry assessed the presence of human cells. Data analysis revealed that CA-induced signal changes did not strictly reflect the graft status. Gd-DTPA label disappeared rapidly yet with a 2-week delay compared with immunohistochemical evaluation. More problematically, SPIO label was still visible after 3 months, grossly overestimating cell survival (<1 week). SPIOs should be used with extreme caution to evaluate the presence of grafted cells in vivo and could hardly be recommended for the long-term monitoring of cell transplantation protocols.

Differentiation potential of human muscle-derived cells towards chondrogenic phenotype in alginate beads culture.

OBJECTIVE: The aim of this study was to evaluate the differentiation potential of two populations of muscle-derived cells (CD56- and CD56+) towards chondrogenic phenotype in alginate beads culture and to compare the effect of transforming growth factor beta 1 (TGFbeta1) on the differentiation process in these populations. METHODS: Muscle CD56- and CD56+ cells were cultured in alginate beads, in a chondrogenic medium, containing or not TGFbeta1 (10 ng/ml). Cultures were maintained for 3, 7, 14 or 21 days in a humidified culture incubator. At harvest, one culture of each set was fixed for alcian blue staining and aggrecan detection. The steady-state level of matrix macromolecules mRNA was assessed by real-time polymerase chain reaction (PCR). Protein detection was performed by western-blot analysis. The binding activity of nuclear extracts to Cbfa1 DNA sequence was also evaluated by electrophoretic mobility shift assays (EMSA). RESULTS: Chondrogenic differentiation of both CD56+ and CD56- muscle-derived cells was improved in alginate scaffold, even without growth factor, as suggested by increased chondrogenesis markers expression during the culture. Furthermore, TGFbeta1 enhanced the differentiation process and allowed to maintain a high expression of markers of mature chondrocytes. Of importance, the combination of alginate and TGFbeta1 treatment resulted in a further down-regulation of collagen type I and type X, as well as Cbfa1 both expression and binding activity. CONCLUSIONS: Thus, alginate scaffold and chondrogenic medium are sufficient to lead both populations CD56+ and CD56- towards chondrogenic differentiation. Moreover, TGFbeta1 enhances this process and allows to maintain the chondrogenic phenotype by inhibiting terminal differentiation, particularly for CD56- cells.

Myoblast xenotransplantation as a tool to evaluate the appropriateness of nanoparticular versus cellular trackers.

Myoblast transplantation is being considered as a potential strategy to improve muscle function in myopathies; hence, it is important to identify the transplanted cells and to have available efficient reagents to track these cells. We first validated a human to mouse xenotransplantation model warranting the complete and rapid rejection of the cells. We then used this model to assess the appropriateness of a nanoparticle reagent to track the transplanted cells. Human myoblasts were loaded with ferrite nanoparticles and injected into the tibialis muscle of immunocompetent mice. Upon collection and histological analysis of muscle sections at different time points, we observed the total disappearance of the human cells within 6 days while ferrite particles remained detectable and colocalized with mouse infiltrating and neighboring cells at the injection site. These results suggest that the use of exogenous markers such as ferrite nanoparticles may lead to false-positive results and misinterpretation of cell fate.

Normal growth and regenerating ability of myoblasts from unaffected muscles of facioscapulohumeral muscular dystrophy patients.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disease characterized by a typical regional distribution, featuring composed patterns of clinically affected and unaffected muscles. No treatment is available for this condition, in which the pathophysiological mechanism is still unknown. Autologous transfer of myoblasts from unaffected to affected territories could be considered as a potential strategy to delay or stop muscle degeneration. To evaluate the feasibility of this concept, we explored and compared the growth and differentiation characteristics of myoblasts prepared from phenotypically unaffected muscles of five FSHD patients and 10 control donors. According to a clinically approved procedure, 10(9) cells of a high degree of purity were obtained within 16-23 days. More than 80% of these cells were myoblasts, as demonstrated by labeling of the muscle markers CD56 and desmin. FSHD myoblasts presented a doubling time equivalent to that of control cells; they kept high proliferation ability and did not show early telomere shortening. In vitro, these cells were able to differentiate and to express muscle-specific antigens. In vivo, they participated to muscle structures when injected into immunodeficient mice. These data suggest that myoblasts expanded from unaffected FSHD muscles may be suitable tools in view of autologous cell transplantation clinical trials.

Myoblast transplantation: clinical trials and perspectives. Mini-review.

Myoblast transplantation (MT) is proposed to challenge the repair of striated muscles in various pathological conditions. Indeed, myoblasts are of easy procurement and expansion from small muscle biopsies. They are naturally able to participate to skeletal muscle regeneration through cell-cell fusion, leading to the formation of new or hybrid muscle fibers. In an autologous context, MT relies on the localized increased muscle generative capacity brought by large number of myoblasts : cells may be transferred from a clinically unaffected area to the disabled territory. In an heterologous context, MT further allows to transfer a non-pathologic, donor genome into the disabled recipient muscle fibers. Autologous approaches are then developed as localized repairs are mandated, while heterologous approaches are used in the context of generalized, advanced degenerative diseases. Whatever the immunological situation, however, MT is hampered by several issues, mainly the poor survival and the limited migratory ability of the myoblasts. Some solutions are emerging, which recently allowed the set-up of new clinical trials in dedicated indications.

Laminin alpha2 deficiency and muscular dystrophy; genotype-phenotype correlation in mutant mice.

Deficiency of laminin alpha2 is the cause of one of the most severe muscular dystrophies in humans and other species. It is not yet clear how particular mutations in the laminin alpha2 chain gene affect protein expression, and how abnormal levels or structure of the protein affect disease. Animal models may be valuable for such genotype-phenotype analysis and for determining mechanism of disease as well as function of laminin. Here, we have analyzed protein expression in three lines of mice with mutations in the laminin alpha2 chain gene and in two lines of transgenic mice overexpressing the human laminin alpha2 chain gene in skeletal muscle. The dy(3K)/dy(3K) experimental mutant mice are completely deficient in laminin alpha2; the dy/dy spontaneous mutant mice have small amounts of apparently normal laminin; and the dy(W)/dy(W) mice express even smaller amounts of a truncated laminin alpha2, lacking domain VI. Interestingly, all mutants lack laminin alpha2 in peripheral nerve. We have demonstrated previously, that overexpression of the human laminin alpha2 in skeletal muscle in dy(2J)/dy(2J) and dy(W)/dy(W) mice under the control of a striated muscle-specific creatine kinase promoter substantially prevented the muscular dystrophy in these mice. However, dy(W)/dy(W) mice, expressing the human laminin alpha2 under the control of the striated muscle-specific portion of the desmin promoter, still developed muscular dystrophy. This failure to rescue is apparently because of insufficient production of laminin alpha2. This study provides additional evidence that the amount of laminin alpha2 is most critical for the prevention of muscular dystrophy. These data may thus be of significance for attempts to treat congenital muscular dystrophy in human patients.

[Myocardial implantation of muscle cells]. / Implantation myocardique de cellules musculaires.

THE DEVELOPMENT OF CELL THERAPY: The stakes in the management of heart failure have become such that new therapeutic strategies have to be developed. Among the cell, molecular and genetic approaches aimed at reinforcing the deficient heart muscle by restoring its functional potential, cell therapy is the favored option in clinical application perspectives. IN THE FIELD OF ISCHEMIC HEART FAILURE: All the experimental data have shown that implantation of contractile cells in the post-infarction areas led to improved cardiac function. INTERESTING PRELIMINARY RESULTS: For ethical and immuno-biological reasons, the successful transplantation of autologous skeletal myoblasts has led our team to conduct a phase I clinical trial. Although the results of this study are preliminary with regard to cardiac function, they suggest the validity of the cell transplantation concept and allow one to hope that this new treatment method will have its place among the therapeutic arms of heart failure.

Cell transplantation for post-ischemic heart failure.

Post-ischemic heart failure is becoming a major issue for public health in occidental countries and therapeutical options are limited. Therefore cell transplantation was developed as an alternative strategy to improve cardiac structure and function. This review describes the multiple cell types and clinical trials considered for use in this indication. The transplantation of fetal or neonatal cardiomyocytes has proven to be functionally successful, but ethical as well as technical reasons make their clinical use limited. Recent reports, however, suggested that adult autologous cardiomyocytes could be prepared from stem cells present in various mesenchymal tissues. Alternatively, endothelial progenitors originating from bone marrow or peripheral blood could promote the neoangiogenesis within the scar tissue. Finally, the transplantation of skeletal muscle cells (SMC) in the infarcted area improved myocardial function, in correlation with the development of skeletal muscle tissue in various animal models. The latter results paved the way for the development of a first phase I clinical trial of SMC transplantation in patients with severe ischemic heart failure. It required the scale-up of human cell production according to Good Manufacturing Procedures, it started in June 2000 in Paris and was terminated in November 2001, and it was followed by several others. The results were encouraging and prompted the onset of a blinded, multicentric phase II clinical trial for SMC transplantation. Meanwhile, clinical trials also evaluate the safety and efficacy of various cells types originating from the bone marrow.

Regeneration of the myocardium: a new role in the treatment of ischemic heart disease?

Intramyocardial cell grafting aims to limit the consequences of the loss of contractile function of a damaged left ventricle. Its functional efficacy is suggested by a wealth of experimental data using multiple evaluation techniques in different animal species. Intramyocardial injections of cultured fetal cardiomyocytes after infarction increase the ejection fraction. Cultured autologous skeletal myoblasts, which do not raise immunologic, ethical, tumorigenesis, or donor availability problems, improve ventricular function to a similar extent. The presence of connexin-43 is demonstrated between fetal (but not myoblast) grafted cells and host myocytes. Thus, the mechanisms of this beneficial effect (direct systolic effect, paracrine factors, passive girdling effect, and decrease in wall stress) remain controversial. These encouraging results have opened the way to the first clinical trial in patients with low ejection fractions, akinetic and nonviable postinfarction scars, and indications for coronary artery bypasses in remote, viable, and ischemic areas. Large-scale cell expansion allows a yield of >10(9) myoblasts from a single human muscular biopsy. Cultured autologous myoblasts are directly administered by multiple injections within and around the infarcted area during open-chest surgery. Preliminary postoperative observations show an improvement in ejection fraction, reappearance of a systolic thickening of the grafted scars, and a new-onset metabolic viability within this area. Thus, this new procedure might become a useful adjunct to current treatments of severe ischemic heart failure.

Is skeletal myoblast transplantation clinically relevant in the era of angiotensin-converting enzyme inhibitors?

BACKGROUND: There is compelling experimental evidence that autologous skeletal muscle (SM) cell transplantation improves postinfarction cardiac function. This study assessed whether this benefit is still manifested in the clinically relevant setting of a treatment by ACE inhibitors. METHODS AND RESULTS: A myocardial infarction was created in 99 rats by coronary artery ligation. They were divided into 4 groups. Two groups did not receive any drug and were intramyocardially injected 7 days after the infarct with either culture medium alone (control rats, n=16) or autologous SM cells (2.3x10(6) myoblasts) previously expanded ex vivo for 7 days (myoblasts, n=24). Two other groups received the ACE inhibitor perindoprilat (1 mg. kg(-1). d(-1)), started the day of the infarct and continued uninterruptedly thereafter, and underwent time-matched procedures, that is, they were intramyocardially injected at 7 days after infarction with either culture medium alone (ACE inhibitors, n=22) or autologous SM cells (2.5x10(6) myoblasts) previously expanded ex vivo for 7 days (ACE inhibitors+myoblasts, n=37). Left ventricular function was assessed by 2D echocardiography. At the end of the 2-month study, left ventricular ejection fraction (%, mean+/-SEM) was increased in all groups (myoblasts, 37.4+/-1.2; ACE inhibitors, 31.6+/-1.7; ACE inhibitors+myoblasts, 43.9+/-1.4) compared with that in control rats (19.8+/-0.7) (P<0.0001). The improvement in ejection fraction was similar in the ACE inhibitor and the myoblast groups (31.6+/-1.7 versus 37.4+/-1.2, P=0.0636). However, in the ACE inhibitor+myoblast group, this improvement was greater than that seen in hearts receiving either treatment alone (43.9+/-1.4 versus 31.6+/-1.7 in the ACE inhibitor group and 43.9+/-1.4. versus 37.4+/-1.2 in the myoblast group, P<0.0001 and P=0.0084, respectively). CONCLUSIONS: These data provide further support for the clinical relevance of autologous SM cell transplantation in that its cardioprotective effects are additive to those observed with ACE inhibitors.

[Transplantation of normal or genetically modified myoblasts for the treatment of hereditary or acquired diseases]. / La transplantation de myoblastes normaux ou génétiquement modifiés pour le traitement de maladies héréditaires ou acquises.

The clinical trials of myoblast transplantation in Duchenne Muscular Dystrophy (DMD) patients produced disappointing results. The main problems responsible for these poor results have since then been identified and partially resolved. One of them was related to the use of an inadequate immunosuppression and, since then, immunosuppression with FK506 has permitted successful myoblast transplantation not only in mice but also in monkeys. The requirement for a sustained immunosuppression may be eventually avoided by developing a state of tolerance to the allogeneic cells or by autologous transplantation of genetically corrected myoblasts or stem cells. The rapid death of 75-80% of the injected myoblasts during the first five days has also contributed to the limited success of the early trials. This death was due to an inflammatory reaction and has been compensated in animal experiments by the injection of a larger number of cells (30 millions per cc). Finally, the myoblasts migrated only 0.5 mm away from their site of injection. This problem is currently compensated in animal experiments by injecting the myoblasts at every mm. The number of injections required may eventually be reduced by transfecting myoblasts with one or several metalloproteinase genes. The very good results obtained during the last two years in primates permit us to undertake a new phase I clinical trial to verify that myoblast transplantation can lead to the formation of muscle fibers expressing normal dystrophin in muscles of DMD patients.

[Transplantation of autologous skeletal myoblasts in ischemic cardiac insufficiency]. / Transplantation de myoblastes squelettiques autologues dans l'insuffisance cardiaque ischémique.

Despite medical therapeutic advances, congestive heart failure (CHF), which is the common ultimate consequence of many primary cardiovascular diseases, remains a major and growing public health problem. Although orthotopic heart transplantation is the gold standard, there is now growing evidence that one therapeutic option could be cellular cardiomyoplasty. Autologous adult skeletal myoblast transplantation seems to be the most clinically relevant, compared with other cell types, in that it avoids immunosuppression therapy, availability and ethical issues. Previous experimental studies have documented the efficacy of myoblast transplantation in improving function of infarcted myocardium. Although the mechanisms involved in this improvement are not elucidated, it has been demonstrated convincingly enough to consider ripping to clinical trials.

Electrotransfer of naked DNA in the skeletal muscles of animal models of muscular dystrophies.

The electrotransfer of naked DNA has recently been adapted to the transduction of skeletal muscle fibers. We investigated the short- and long-term efficacy of this methodology in wild-type animals and in mouse models of congenital muscular dystrophy (dy/dy, dy(2J)/dy(2J)), or Duchenne muscular dystrophy (mdx/mdx). Using a reporter construct, the short-term efficacy of fiber transduction reached 40% and was similar in wild-type, dy/dy and dy(2J)/dy(2J) animals, indicating that ongoing muscle fibrosis was not a major obstacle to the electrotransfer-mediated gene transfer. Although the complete rejection of transduced fibers was observed within 3 weeks in the absence of immunosuppression, the persistency was prolonged over 10 weeks when transient or continuous immunosuppressive regimens were used. Using therapeutic plasmids, we demonstrated that electrotransfer also allowed the transduction of large constructs encoding the laminin alpha2 chain in dy/dy mouse, or a chimeric dystrophin-EGFP protein in mdx/mdx mouse. The correct sarcolemmal localization of these structural proteins demonstrated the functional relevance of their expression in vivo, with a diffusion domain estimated to be 300 to 500 microm. However, degeneration-regeneration events hampered the long-term stability of transduced fibers. Given its efficacy for naked DNA transfer in these models of muscular dystrophies, and despite some limitations, gene electrotransfer methodology should be further explored as a potential avenue for treatment of muscular dystrophies.

[Autologous skeletal myoblast transplantation for cardiac insufficiency. First clinical case]. / Greffe de myoblastes squelettiques pour insuffisance cardiaque. Premier cas chez l'homme.

The authors report the first intramyocardial transplantation of autologous skeletal myoblasts in a patient with severe ischaemic cardiac failure. The encouraging result after eight months' follow-up underlines the potential of this new approach.

Factors affecting functional outcome after autologous skeletal myoblast transplantation.

BACKGROUND: This study assessed the extent to which the initial degree of functional impairment and the number of injected cells may influence the functional improvement provided by autologous skeletal myoblast transplantation into infarcted myocardium. METHODS: One week after left coronary artery ligation, 44 rats received into the infarcted scar, autologous skeletal myoblasts expanded in vitro for 7 days (mean, 3.5 x 10(6), n = 21), or culture medium alone (controls, n = 23). Left ventricular function was assessed by two-dimensional echocardiography. RESULTS: When transplanted hearts were stratified according to their baseline ejection fraction, a significant improvement occurred at 2 months in the less than 25% (from 21.4% to 37%), 25% to 35% (from 29% to 43.8%), and in the 35% to 40% (from 37.2% to 41.7%) groups, compared to controls (p = 0.048, 0.0057, and 0.034, respectively), but not in the more than 40% stratum. A significant linear relationship was found between the improvement in ejection fraction and the number of injected myoblasts, both at 1 and 2 months after transplantation (p < 0.0001). CONCLUSIONS: Autologous myoblast transplantation is functionally effective over a wide range of postinfarct ejection fractions, including in the sickest hearts provided that they are injected with a sufficiently high number of cells.

Myoblast transplantation for heart failure.

Intramyocardial skeletal muscle transplantation has been shown experimentally to improve heart function after infarction. We report success with this procedure in a patient with severe ischaemic heart failure. We implanted autologous skeletal myoblasts into the postinfarction scar during coronary artery bypass grafting of remote myocardial areas. 5 months later, there was evidence of contraction and viability in the grafted scar on echocardiography and positron emission tomography. Although this result is encouraging, it requires validation by additional studies.

Intramyocardial transplantation of autologous myoblasts: can tissue processing be optimized?

BACKGROUND: Autologous skeletal myoblast (SM) transplantation improves function of infarcted myocardium, but pretransplantation cultures remain a complex process. This study assessed whether it could be optimized by muscle preconditioning with the local anesthetic bupivacaine or even bypassed with the use of the so-called mince technique. METHODS AND RESULTS: Muscle preconditioning consisted of intramuscular injections of the tibialis anterior of rats, 2 days before harvest. After 7 days of culture, the number of available myoblasts was significantly increased compared with nonconditioned controls (1 683 147 versus 85 300, P:=0.0013). The mince technique was then assessed. A myocardial infarction was created in 66 rats by coronary artery ligation. One week later, rats were reoperated on and intramyocardially injected with culture medium alone (controls, n=23), autologous cultured SM (3.5 x 10(6), n=21), or autologous muscle minced into a fine slurry, which was immediately transplanted (n=22). All muscles had been preconditioned. Left ventricular function was assessed by 2D echocardiography. Whereas end-diastolic volumes expanded over time in all groups, left ventricular ejection fraction (%, mean+/-SEM) was increased only in the cultured SM-transplanted group at 1 (P:=0. 0006) and 2 months (P:=0.0008) versus baseline (37.52+/-1.92 and 40. 92+/-2.17 versus 30.34+/-1.74), with a significant additional benefit between 1 and 2 months (P:=0.0069). CONCLUSIONS: Cell culture remains mandatory for SM transplantation to be successful but, in a clinical perspective, this process can be made more expeditious by preharvest muscle conditioning with bupivacaine, which greatly enhances the baseline cell yield.

Functional EGFP-dystrophin fusion proteins for gene therapy vector development.

Transfection and transduction studies involving the use of the full-length dystrophin (11 kb) or the truncated mini-gene (6 kb) cDNAs are hampered by the large size of the resulting viral or non-viral expression vectors. This usually results in very low yields of transgene-expressing cells. Moreover, the detection of the few transgene-expressing cells is often tedious and costly. For these reasons, expression vectors containing the enhanced green fluorescent protein (EGFP) fused with the N-termini of mini- and full-length human dystrophin were constructed. These constructs were tested by transfection of Phoenix cells with Effectene, resulting after 48 h in a green fluorescent signal in 20% of cells. Analysis of the cell extracts by immunoblotting with the use of a monoclonal antibody specific to the dystrophin C-terminus confirmed the expression of EGFP-mini- (240 kDa) and EGFP-full-length human dystrophin (450 kDa) fusion proteins. Moreover, following the in vivo electroporation of the plasmids containing the EGFP-mini- and full-length dystrophin in mouse muscles, both fluorescent proteins were observed in cryostat sections in their normal location under the plasma membrane. This indicates that the fusion of EGFP to dystrophin or mini-dystrophin did not interfere with the normal localization of the protein. In conclusion, the fusion of EGFP provides a good tool for the search of the best methods to introduce mini- or full-length dystrophin cDNA in the cells (in vitro) or muscle fibers (in vivo) for the establishment of a treatment by gene therapy of Duchenne muscular dystrophy patients.

Muscular transverse relaxation time measurement by magnetic resonance imaging at 4 Tesla in normal and dystrophic dy/dy and dy(2j)/dy(2j) mice.

Muscular transverse relaxation times values were measured in vivo in normal mice (strain C57BL6/J, n=14) and in murine models of human congenital muscular dystrophy (dy/dy, n=9; dy(2j)/dy(2j), n=8). A single-slice multi-echo sequence was used. Gastrocnemius/soleus complex, thigh and buttock muscles were studied. Muscular transverse relaxation times values were compared between different muscle groups in each type of animal and between animal groups. Differences were observed between normal and dy(2j)/dy(2j) mice from 3 to 12 weeks of age, and between normal and dy/dy mice at 6 weeks. In specific age ranges, the values of muscular transverse relaxation times in two dystrophic models are different from those in normal mice, and could thus be used as an index of modifications in dystrophic muscle to evaluate therapies.