Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) are inherited muscle diseases due to mutations in the genes encoding the extracellular matrix protein collagen (Col) VI. Opening of the cyclosporin A-sensitive mitochondrial permeability transition pore (PTP) is a causative event in disease pathogenesis, and a potential target for therapy. Here, we have tested the effect of N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive cyclophilin inhibitor, in a zebrafish model of ColVI myopathy obtained by deletion of the N-terminal region of the ColVI α1 triple helical domain, a common mutation of UCMD. Treatment with antisense morpholino sequences targeting col6a1 exon 9 at the 1–4 cell stage (within 1 h post fertilization, hpf) caused severe ultrastructural and motor abnormalities as assessed by electron and fluorescence microscopy, birefringence, spontaneous coiling events and touch-evoked responses measured at 24–48 hpf. Structural and functional abnormalities were largely prevented when NIM811—which proved significantly more effective than cyclosporin A—was administered at 21 hpf, while FK506 was ineffective. Beneficial effects of NIM811 were also detected (i) in primary muscle-derived cell cultures from UCMD and BM patients, where the typical mitochondrial alterations and depolarizing response to rotenone and oligomycin were significantly reduced; and (ii) in the Col6a1−/− myopathic mouse model, where apoptosis was prevented and muscle strength was increased. Since the PTP of zebrafish shares its key regulatory features with the mammalian pore, our results suggest that early treatment with NIM811 should be tested as a potential therapy for UCMD and BM.

INTRODUCTION

Deficiency of collagen VI (ColVI) due to mutations of COL6 genes gives rise to three main muscle disorders, Bethlem myopathy (BM, MIM #158810), Ullrich congenital muscular dystrophy (UCMD, MIM #254090) (1) and myosclerosis myopathy (MIM #255600) (2). BM is characterized by axial and proximal muscle weakness, and a hallmark of this disease is the presence of contractures of the interphalangeal joints of the last four fingers (3,4). The clinical features are extremely heterogenous and range from mild to severe myopathy with progressive muscular dystrophy (5). UCMD is a severe muscular dystrophy characterized by early onset, rapidly progressive muscle wasting and weakness, proximal joint contractures and distal joint hyperlaxity. Rapid progression usually leads to early death due to respiratory failure (6,7). Myosclerosis myopathy is characterized by progressive joint contractures, scoliosis, mild girdle and proximal limb weakness and moderate distal weakness. Muscles become thin and sclerotic with woody consistency, causing severe motility restriction (2). About 70 different mutations of the COL6 genes have so far been described in ColVI myopathies, and it is becoming apparent that these disorders represent a clinical continuum (1,8).

ColVI myopathies share a common pathogenesis downstream the lack of ColVI, i.e. (i) mitochondrial dysfunction due to deregulation of the permeability transition pore (PTP), an inner membrane high conductance channel that forms from dimers of the mitochondrial F-ATP synthase (9) and is involved in many degenerative diseases (10); and (ii) defective autophagy impairing clearance of dysfunctional mitochondria (1113). The mitochondrial defect was first identified in the Col6a1−/− mouse model (14) and then detected in cultures from UCMD and BM patients (1517). Genetic inactivation of Ppif [the gene encoding for mitochondrial cyclophilin (CyP) D in the mouse] in Col6a1−/− mice (18) or PTP desensitization with cyclosporin A (CsA) cured the mouse model of the myopathy (14) and gave promising results in a pilot trial on five patients (19,20). However, the immunosuppressive effect of CsA remains a major source of concern for the long-term treatment of patients affected by a disease where respiratory insufficiency, which can be precipitated by pulmonary infections, is a prominent cause of death (21).

Myopathic Col6a1−/− mice have also been successfully cured with D-MeAla3-EtVal4-cyclosporin (Debio025), a non-immunosuppressive derivative of CsA that inhibits CyPs but not calcineurin (22). This finding suggests that, in principle, ColVI myopathies could be cured without exposing the patients to the hazard of immunosuppression. However, the Col6a1−/− mouse is affected by a very mild myopathy with virtually no fibrosis in spite of the total lack of ColVI (23). This is at striking variance with UCMD patients, where fibroadipose substitution is prominent and may not be reversible (21). In a recent important development, the severe form of ColVI myopathy has been modeled in zebrafish by injection of morpholino targeting exon 9 of col6a1, which caused an in-frame deletion of the N-terminal portion of the triple helical domain of ColVI α1 (24), a dominant mutation in UCMD (25,26). Treatment resulted in severe myopathy with early-onset motor deficits, severe ultrastructural changes, mitochondrial abnormalities and increased apoptosis that could be improved by CsA (24). Since the features of the mammalian PTP are indistinguishable from those of zebrafish (27), the latter organism provides a unique opportunity to test whether CyP inhibitors devoid of immunosuppressive activity are effective in the therapy of the severe UCMD-like myopathy. In the present study, we have investigated the effects of N-methyl-4-isoleucine-cyclosporin (NIM811), a CyP inhibitor that desensitizes the PTP to Ca2+ (28), in exon 9 morphant zebrafish, in myopathic Col6a1−/− mice and in cell cultures established from muscle biopsies of UCMD and BM patients.

RESULTS

NIM811 rescues structural and functional abnormalities of ColVI exon 9 morphant zebrafish

To obtain a zebrafish model of ColVI-related myopathies, we used a morpholino specifically directed against the col6a1 exon 9 splicing region, which results in an in-frame deletion of the N-terminal region of the ColVI α1 triple helical domain matching the most frequent mutation of UCMD (24). In wild-type zebrafish embryos, ColVI was expressed at the myotendinous junction (MTJ), which can be identified by β-dystroglycan staining (Fig. 1A and A′). In exon 9 morphants, ColVI expression was severely reduced (Fig. 1B), and the MTJ displayed an aberrant profile (Fig. 1B′). Ninety-seven percent of exon 9 morphant embryos (out of 121 injected eggs) showed hypoplastic and bent bodies at both 24 and 48 hpf (examples are provided in Fig. 1, top right panel), and these morphological abnormalities matched structural myofiber alterations. Indeed, in wild type embryos, myofibers were aligned along the axis of contraction and mitochondria were regularly distributed within the fibers (Fig. 1C–E), while after ColVI depletion the myofibrillar material was partially lost, myofibrils in adjacent myocytes were misaligned and there was no obvious pattern that could be related to the axis of contraction (Fig. 1F) with several twisted myofibrils (Fig. 1F, arrowheads). A marked alteration of mitochondrial distribution matched myofibrillar derangements, with clusters of mitochondria corresponding to the areas of myofibrillar disorganization (Fig. 1G and H).

Figure 1.

Effect of an exon 9 morpholino targeting col6a1 mRNA on ColVI expression, morphology and muscle structure of zebrafish embryos. Top left, frozen sections from control (A and A′) or exon 9 morphant zebrafish embryos (B and B′) were immunolabeled with anti-ColVI (A and B) or anti-β-dystroglycan (β-DG) antibodies (A′ and B′). Bar, 5 μm. Top right, microscopic analysis of control (CTL MO) and exon 9 morphant embryos (Ex9 MO) at 24 and 48 hpf. Bottom, confocal analysis of 48 hpf control (CE) and exon 9 morphant embryos (FH) stained with phalloidin (C and F), Tom20 (D and G). (E and H) Merged images of phalloidin and Tom20 staining. Bar, 30 μm.

Figure 1.

Effect of an exon 9 morpholino targeting col6a1 mRNA on ColVI expression, morphology and muscle structure of zebrafish embryos. Top left, frozen sections from control (A and A′) or exon 9 morphant zebrafish embryos (B and B′) were immunolabeled with anti-ColVI (A and B) or anti-β-dystroglycan (β-DG) antibodies (A′ and B′). Bar, 5 μm. Top right, microscopic analysis of control (CTL MO) and exon 9 morphant embryos (Ex9 MO) at 24 and 48 hpf. Bottom, confocal analysis of 48 hpf control (CE) and exon 9 morphant embryos (FH) stained with phalloidin (C and F), Tom20 (D and G). (E and H) Merged images of phalloidin and Tom20 staining. Bar, 30 μm.

Morphant zebrafish had a severe motor impairment, as measured both on spontaneous coiling events (Fig. 2) and touch-evoked responses (Fig. 3). Treatment with NIM811 fully restored spontaneous coiling events at a concentration of 25 μm, whereas CsA gave a very limited rescue only at 50 μm and FK506, a calcineurin inhibitor that acts through a CyP-independent mechanism (29), was totally ineffective (Fig. 2). Of note, as also reported by Telfer et al. (24), suppression of p53 expression by co-injection of an appropriate morpholino did not improve spontaneous coiling (data not shown), suggesting that the phenotypic effect was not due to an off-site p53 knockdown (30). Touch-evoked responses were also ameliorated by NIM811, marginally affected by CsA and unaffected by FK506, while no treatment was able to rescue the small subset of paralyzed embryos (Fig. 3).

Figure 2.

Effect of NIM811, CsA or FK506 on spontaneous coiling events of col6a1 morphant zebrafish. Spontaneous coiling events were recorded in 24 hpf control and exon 9 morphant embryos in the presence of the indicated concentrations of NIM811, CsA or FK506 added at 21 hpf. Data represent the mean of at least three independent experiments ± SEM. ***P < 0.005. Total embryos analyzed were: controls, 160; exon 9 morphants, 177 untreated, 100 for each NIM811 concentration, 50 for each CsA concentration and 20, 18 and 16 for concentrations of FK506 of 10, 25 and 50 μm, respectively. Abbreviations are as in Figure 1.

Figure 2.

Effect of NIM811, CsA or FK506 on spontaneous coiling events of col6a1 morphant zebrafish. Spontaneous coiling events were recorded in 24 hpf control and exon 9 morphant embryos in the presence of the indicated concentrations of NIM811, CsA or FK506 added at 21 hpf. Data represent the mean of at least three independent experiments ± SEM. ***P < 0.005. Total embryos analyzed were: controls, 160; exon 9 morphants, 177 untreated, 100 for each NIM811 concentration, 50 for each CsA concentration and 20, 18 and 16 for concentrations of FK506 of 10, 25 and 50 μm, respectively. Abbreviations are as in Figure 1.

Figure 3.

Effect of NIM811, CsA or FK506 on touch-evoked escape response of col6a1 morphant zebrafish. Responses evoked by touching 48 hpf embryos with a pipette tip were recorded. Embryos were assigned to four groups based on their escape response: open bars, normal response; hatched bars, minor motility defects; grey bars, embryos responding with coiling only; closed bars, no response. Results are reported as percentage of total embryos analyzed. Where indicated, embryos had been treated with the indicated concentrations of NIM811, CsA or FK506 at 21 hpf. Number of experiments, numerosity for each condition and abbreviations are as in Figure 1.

Figure 3.

Effect of NIM811, CsA or FK506 on touch-evoked escape response of col6a1 morphant zebrafish. Responses evoked by touching 48 hpf embryos with a pipette tip were recorded. Embryos were assigned to four groups based on their escape response: open bars, normal response; hatched bars, minor motility defects; grey bars, embryos responding with coiling only; closed bars, no response. Results are reported as percentage of total embryos analyzed. Where indicated, embryos had been treated with the indicated concentrations of NIM811, CsA or FK506 at 21 hpf. Number of experiments, numerosity for each condition and abbreviations are as in Figure 1.

In order to obtain information on structural muscle organization in vivo, we measured birefringence with polarized light microscopy (31), a technique that allows detection of muscle structural defects in several zebrafish models of muscular dystrophy (32). The appearance of a normal zebrafish is illustrated in Figure 4A, which also provides examples of moderate and severe phenotypes of exon 9 morphant individuals. Treatment with NIM811 allowed recovery of a normal birefringence in ∼50% of exon 9 morphants, while treatment with CsA led to a marginal rescue at 25 μm and to worsening of the severe phenotype at 50 μm, no effect being observed with FK506 (Fig. 4A). Unbiased statistical analysis based on the total birefringence score for all conditions indicates that only NIM811 allowed recovery, the effect being seen at concentrations of 10 μm (Fig. 4B).

Figure 4.

Effect of NIM811, CsA or FK506 on birefringence of col6a1 morphant zebrafish. (A) Birefringence of normal and exon 9 morphant embryos with moderate or severe myopathic phenotype. Bar graph, percentage of birefringence classes after treatment with NIM811, CsA or FK506 (open bars, normal birefringence; grey bars, moderate myopathic phenotype; closed bars, severe myopathic phenotype). (B) Statistical analysis of total birefringence. Data represent the mean of at least three independent experiments ± SEM, ***P < 0.005. Total embryos analyzed were: controls, 43; exon 9 morphants, 43 untreated, 43 for each NIM811 concentration, 30 for each CsA concentration and 15 for each FK506 concentration.

Figure 4.

Effect of NIM811, CsA or FK506 on birefringence of col6a1 morphant zebrafish. (A) Birefringence of normal and exon 9 morphant embryos with moderate or severe myopathic phenotype. Bar graph, percentage of birefringence classes after treatment with NIM811, CsA or FK506 (open bars, normal birefringence; grey bars, moderate myopathic phenotype; closed bars, severe myopathic phenotype). (B) Statistical analysis of total birefringence. Data represent the mean of at least three independent experiments ± SEM, ***P < 0.005. Total embryos analyzed were: controls, 43; exon 9 morphants, 43 untreated, 43 for each NIM811 concentration, 30 for each CsA concentration and 15 for each FK506 concentration.

Exon 9 morphant embryos analyzed at 48 hpf displayed ultrastructural abnormalities, which correlated well with the impaired motor function. The organization of muscle fibers showed areas of myofibrillar disorganization and MTJ derangement (Fig. 5, compare panels A and C) with blebs of sarcolemma (Fig. 5, asterisks in panel C, compare with panel A), basal lamina discontinuities (Fig. 5, arrowheads in panel C), and partial loss of myofibril attachment to the MTJ of sarcolemma in the altered areas; in addition, exon 9 morphants displayed diffuse T-tubule dilations (Fig. 5, arrowheads, compare insets in panels C and A), while the sarcoplasmic reticulum appeared normal. Most mitochondria (M, Fig. 5E, D and F, compare with panel B) appeared enlarged and swollen with reduced number of cristae, detachment of the outer membrane (Fig. 5F, arrowheads) and marked dilation of the intermembrane space (Fig. 5F, asterisk). Strikingly, in vivo treatment with 25 μm NIM811 in fish water rescued the overall organization of muscle fibers including the MTJ derangement, T-tubule dilations and overall mitochondrial morphology as indicated by the lack of intermembrane space dilations and swelling (Fig. 5G–J).

Figure 5.

Ultrastructural analysis of 48 hpf zebrafish embryos. Control (A and B) and exon 9 morphant embryos (CJ) were studied. Note the MTJ derangements with blebs at the sarcolemma (asterisks, C), basal lamina discontinuities (arrowheads, C) and T-tubule dilations (arrowhead, compare insets in C and A) in exon 9 morphants, with mitochondrial (M) abnormalities consisting of swelling (E and D), detachment of the outer membrane (arrowheads, F) and dilation of the intermembrane space (asterisks, F). Treatment of exon 9 morphant embryos with 25 μm NIM811 at 21 hpf (GJ) allowed a striking recovery of MTJ (G) and T-tubule morphology (H) as well as a major improvement of mitochondrial appearance (I and J). Bar, 200 nm.

Figure 5.

Ultrastructural analysis of 48 hpf zebrafish embryos. Control (A and B) and exon 9 morphant embryos (CJ) were studied. Note the MTJ derangements with blebs at the sarcolemma (asterisks, C), basal lamina discontinuities (arrowheads, C) and T-tubule dilations (arrowhead, compare insets in C and A) in exon 9 morphants, with mitochondrial (M) abnormalities consisting of swelling (E and D), detachment of the outer membrane (arrowheads, F) and dilation of the intermembrane space (asterisks, F). Treatment of exon 9 morphant embryos with 25 μm NIM811 at 21 hpf (GJ) allowed a striking recovery of MTJ (G) and T-tubule morphology (H) as well as a major improvement of mitochondrial appearance (I and J). Bar, 200 nm.

NIM811 prevents mitochondrial dysfunction and apoptosis, and rescues muscle force in Col6a1−/− mice

In order to assess the efficacy of NIM811 also in the well-characterized Col6a1−/− mouse model (14,23), we studied the mitochondrial transmembrane potential in situ based on the mitochondrial fluorescence of tetramethylrhodamine methyl ester (TMRM), a probe that accumulates in polarized mitochondria and is released when the transmembrane potential decreases (33). Addition of oligomycin (inhibitor of the mitochondrial F-ATP synthase) to primary cultures derived from diaphragm of Col6a1−/− mice caused the expected decrease of TMRM mitochondrial fluorescence (Fig. 6A, left panel), an anomalous depolarizing response due to PTP opening that can be corrected by treatment with CsA and Debio 025 (14,22). Treatment with NIM811 normalized the response to oligomycin (i.e. prevented mitochondrial depolarization) in the vast majority of cells (Fig. 6A, right panel). Importantly, a similar protective effect was recorded after in vivo treatment of Col6a1−/− mice with NIM811 for 5 days (Fig. 6B). Indeed, the depolarizing response to oligomycin was normalized in the vast majority of flexor digitorum brevis (FDB) fibers prepared from treated mice. If a threshold is arbitrarily set at the initial fluorescence value, 46 out of 144 fibers (32%) depolarized in the placebo group while treatment with NIM811 decreased the number of fibers with depolarizing mitochondria to 35 out of 146 (24%) and 10 out of 126 (8%) at doses of 10 and 25 mg/kg/day, respectively (in each groups, results are pooled for fibers from six individuals) (Fig. 6B). Diaphragm fibers from mice treated with NIM811 for 5 days also showed a remarkable decrease of apoptotic nuclei compared with those from the placebo group both at the 10 and 25 mg/kg/day dose (Fig. 6C). Finally, we measured tetanic (maximal) muscle force in the hindlimbs (34). Col6a1−/− mice treated for 10 days with 25 mg/kg/day NIM811 showed a marked recovery of muscle strength, which matched what we previously observed in CsA-treated mice (11) (Fig. 6D).

Figure 6.

Effect of NIM811 on membrane potential, diaphragm apoptosis and tetanic force development in Col6a1−/− mice. (A) Mitochondrial membrane potential was monitored based on TMRM fluorescence changes of primary cultures established from diaphragms of Col6a1−/− mice; where indicated 6 μm oligomycin (O) and 4 μm FCCP (F) were added to cultures treated with vehicle (left panel) or 0.8 μm NIM811 (right panel) for 30 min; each line reports mitochondrial fluorescence of individual cells. (B) Mice were treated with placebo or NIM811 as indicated in two daily doses for 5 days by gavage, and FDB fibers were isolated, allowed to attach to cover slips and loaded with TMRM, followed by the addition of 6 μm oligomycin; values on the ordinate refer to the percentage of fibers depolarizing after the addition of oligomycin (in each group, results are pooled for fibers from six individuals). (C) Col6a1−/− mice were treated with placebo or NIM811 and diaphragm apoptosis evaluated in four animals per group in 70 random fields. Results are the mean ± SEM, **P < 0.005, ***P < 0.001. (D) Tetanic force was measured by stimulating the gastrocnemius muscle through the sciatic nerve. Results are the mean ± SEM, *P < 0.05. The indicated dose of CsA was administered by gavage.

Figure 6.

Effect of NIM811 on membrane potential, diaphragm apoptosis and tetanic force development in Col6a1−/− mice. (A) Mitochondrial membrane potential was monitored based on TMRM fluorescence changes of primary cultures established from diaphragms of Col6a1−/− mice; where indicated 6 μm oligomycin (O) and 4 μm FCCP (F) were added to cultures treated with vehicle (left panel) or 0.8 μm NIM811 (right panel) for 30 min; each line reports mitochondrial fluorescence of individual cells. (B) Mice were treated with placebo or NIM811 as indicated in two daily doses for 5 days by gavage, and FDB fibers were isolated, allowed to attach to cover slips and loaded with TMRM, followed by the addition of 6 μm oligomycin; values on the ordinate refer to the percentage of fibers depolarizing after the addition of oligomycin (in each group, results are pooled for fibers from six individuals). (C) Col6a1−/− mice were treated with placebo or NIM811 and diaphragm apoptosis evaluated in four animals per group in 70 random fields. Results are the mean ± SEM, **P < 0.005, ***P < 0.001. (D) Tetanic force was measured by stimulating the gastrocnemius muscle through the sciatic nerve. Results are the mean ± SEM, *P < 0.05. The indicated dose of CsA was administered by gavage.

NIM811 prevents mitochondrial dysfunction and apoptosis in cells derived from muscle of UCMD and BM patients

A typical feature of muscle-derived cell cultures from patients with ColVI myopathies is that the addition of oligomycin or rotenone is followed by mitochondrial depolarization that depends on PTP opening (1517,19). Treatment with NIM811 prevented depolarization induced by both oligomycin and rotenone in cultures established from patients affected by UCMD (Fig. 7A and B) and BM (Fig. 7C and D); and it normalized the frequency of muscle apoptotic nuclei (Fig. 7E).

Figure 7.

Effect of NIM811 on oligomycin- and rotenone-induced mitochondrial depolarization and apoptosis in cell cultures from UCMD and BM patients. (AD) Mitochondrial membrane potential was monitored based on TMRM fluorescence changes of primary cultures established from muscle biopsies of UCMD (A and B) or BM patients (C and D). Where indicated by arrows, 6 μm oligomycin (O), 2 μm rotenone (R) and 4 μm FCCP (F) were added in the absence (open symbols) or presence (closed symbols) of 0.8 μm NIM811 added 30 min before the beginning of the recordings (time 0). Data represent the mean of at least six independent experiments ± SEM, and the line marked by one asterisk indicates the time points with P < 0.05 for no treatment compared with NIM811. (E) Primary myoblast cultures from one UCMD patient and one BM patient were plated and scored for the presence of apoptotic nuclei by the TUNEL reaction. Where indicated, cells had been incubated with NIM811 for 2 h. Data are mean of three independent experiments ± SEM, *P < 0.05 and **P < 0.01 for NIM811-treated versus non-treated cells.

Figure 7.

Effect of NIM811 on oligomycin- and rotenone-induced mitochondrial depolarization and apoptosis in cell cultures from UCMD and BM patients. (AD) Mitochondrial membrane potential was monitored based on TMRM fluorescence changes of primary cultures established from muscle biopsies of UCMD (A and B) or BM patients (C and D). Where indicated by arrows, 6 μm oligomycin (O), 2 μm rotenone (R) and 4 μm FCCP (F) were added in the absence (open symbols) or presence (closed symbols) of 0.8 μm NIM811 added 30 min before the beginning of the recordings (time 0). Data represent the mean of at least six independent experiments ± SEM, and the line marked by one asterisk indicates the time points with P < 0.05 for no treatment compared with NIM811. (E) Primary myoblast cultures from one UCMD patient and one BM patient were plated and scored for the presence of apoptotic nuclei by the TUNEL reaction. Where indicated, cells had been incubated with NIM811 for 2 h. Data are mean of three independent experiments ± SEM, *P < 0.05 and **P < 0.01 for NIM811-treated versus non-treated cells.

DISCUSSION

The role of PTP opening in the pathogenesis of ColVI muscular dystrophies (1518) and the therapeutic potential of CsA through inhibition of matrix CyPD are well established: CsA has been successfully used in animal models (14,24) as well as in cells from patients affected by ColVI myopathies (1517,1921). The pharmacology and toxicology of CsA is complex, however, because of the multiple pathways affected by this drug.

The primary intracellular targets of CsA are CyPs, a family of proteins with peptidyl prolyl cis–trans isomerase activity (35), which in humans includes 16 proteins (36). The most abundant is the cytosolic isoform, CyPA, which is responsible for the immunosuppressive effects of CsA. After binding CsA (which leads to inhibition of CyPA), the CsA–CyPA complex targets—and inhibits—the cytosolic phosphatase calcineurin (29), which in turn prevents dephosphorylation—hence nuclear translocation—of NFAT resulting in immunosuppression (37,38). Calcineurin is also inhibited by the complex of FK506 with the FK506-binding proteins, a separate class of intracellular peptidyl prolyl cis–trans isomerases (29).

Calcineurin inhibition prevents mitochondrial translocation of the pro-fission protein Drp1 and mitochondrial fragmentation, resulting in inhibition of autophagy (39). This event is cytoprotective if cells are healthy (40,41), while it is detrimental in ColVI myopathies, where defective removal of dysfunctional mitochondria contributes to disease pathogenesis (1113). It should be stressed that autophagy induction by CsA in the Col6a1−/− mouse—which is beneficial—is not likely to be caused by inhibition of calcineurin because (i) FK506 worsened the incidence of muscle apoptosis in the Col6a1−/− myopathic mouse (14); and (ii) the non-immunosuppressive CsA derivative Debio 025 (Alisporivir) displayed a remarkable therapeutic efficacy (14). Furthermore, while CsA decreased contractile performance of human and rabbit heart muscle preparations (42), Debio 025 was cardioprotective in a mouse model of myocardial infarction (43), suggesting that the toxic effects of CsA are due to inhibition of calcineurin rather than to inhibition of CyPs. While the above findings suggest that patients with ColVI myopathies could be treated with CyP inhibitors devoid of effects on calcineurin, these drugs have only been tested in the adult Col6a1−/− mouse model, which is affected by a very mild myopathy with virtually no fibrosis in spite of the total lack of ColVI (23).

ColVI myopathy has recently been modeled in zebrafish by in-frame deletion of the N-terminal portion of the triple helical domain of ColVI α1 (24), a dominant mutation in UCMD (25,26). This caused severe myopathy with early-onset motor deficits, major ultrastructural changes, mitochondrial abnormalities and increased apoptosis that could be improved by CsA (24). Since the key features of the zebrafish PTP are indistinguishable from those of mammals (27), the fish model provides a unique opportunity to test whether (i) CyP inhibition with NIM811 alone could cure the severe form of the disease; and if so, (ii) whether inhibition of both CyP and calcineurin with CsA would be equally effective, more effective or detrimental. The latter point is of specific importance to the potential treatment of the human disease in pediatric age before onset of fibroadipose substitution, because drugs can be administered during zebrafish development, a stage at which the effects of inhibition of calcineurin and CyPs have not been assessed.

Our results demonstrate that NIM811 is way more effective than CsA in zebrafish lacking ColVI, with recovery from the motor deficit and normalization of muscle structure, which was not observed for CsA (24). This difference between NIM811 and CsA can be traced to the importance of calcineurin in skeletal muscle differentiation, regeneration and fiber type specification, all functions that are crucial to muscle development, metabolism and functional adaptation (44). Since disease progression also depends on the balance between cell death and regeneration, it appears that calcineurin inhibition may have detrimental effects that are not seen after inhibition of CyPs alone. This conclusion is supported by the total lack of protective effects of FK506 both in the Col6a1−/− mouse (14) and in the zebrafish exon 9 morphants (present article).

NIM811 also normalized mitochondrial function in muscle cell cultures from patients affected by BM and UCMD, as well as in cells from the Col6a1−/− mouse, where apoptotic rates were normalized. Of note, recovery of muscle strength in vivo matched what was reported for CsA (11). These data confirm that mitochondrial CyPD is an excellent therapeutic target in ColVI muscular dystrophies. If it is permissible to extrapolate the results obtained in zebrafish to patients, it appears that treatment should be started early, and that lack of calcineurin inhibition is a key advantage of NIM811. Indeed, lack of immunosuppression does not expose patients to increased risk of infections allowing long-term treatment.

NIM811 inhibits all CyP isoforms, and at present it is impossible to tell whether selective inhibition of CyPD could further improve the chances of therapeutic success. The demonstration that isoform-specific inhibitors of CyPs can be synthesized (45) opens new perspectives to the development of CyPD-selective inhibitors, and holds further promise for treatment of diseases where the PTP is involved.

MATERIALS AND METHODS

Zebrafish and embryo maintenance

Adult zebrafish were maintained in the facility of University of Padova containing aerated, 28.5°C-conditioned saline water according to standard protocols. Fish were kept under a 14 h light–10 h dark cycle. For mating, males and females were separated in the late afternoon and the next morning were freed to start courtship, which ended with eggs deposition and fecundation. Eggs were collected, washed with fish water (0.5 mm NaH2PO4, 0.5 mm NaHPO4, 0.2 mg/l methylene blue, 3 mg/l instant ocean) and maintained at 28.5°C in fish water supplemented with an antibiotic-antimycotic cocktail (50 μg/ml ampicillin, 100 units/ml penicillin and 0.1 mg/ml streptomycin, Biochrom, 3.3 μg/ml amphotericin B, Bristol-Myers-Squibb).

Treatment with antisense morpholino

In order to affect ColVI synthesis and secretion, we used a published exon 9 morpholino (24), which targets zebrafish col6a1 mRNA leading to translation of a truncated, dominant negative, version of the ColVI α1 chain. Embryos from Zebrafish wild type incrosses were injected with morpholino at 1–4 cell stage using a WPI pneumatic PicoPump PV820 injector. Morpholino was injected at a concentration of 0.1 mm, corresponding to about 4 ng per embryo.

Ultrastructural analysis

Wild type and exon 9 morphant zebrafish embryos (48 hpf) were fixed with Karnovsky fixative (2.5% glutaraldehyde and 2% paraformaldheyde in 0.1 m cacodylate buffer) for 3 h at 4°C, washed with 0.1 m cacodilate buffer, post-fixed with osmium tetroxide for 2 h and embedded in EPON 812 as previously described (24). Ultrathin sections were stained with uranyl acetate and lead citrate and observed at Philips EM400 operating at 100 kV.

NIM811, CsA and FK506 treatment

Morphant embryos were dechorionated at 20 hpf and then incubated with drugs from 21 hpf until the time of observation (24 or 48 hpf as specified). NIM811 and CsA (Novartis Pharma AG) or FK506 (Sigma, St Louis, MO, USA) were used at 10, 25 and 50 µm and dissolved in 0.01, 0.03 and 0.06% DMSO in fish water. Vehicle control treatment consisted of 0.06% DMSO in fish water.

Immunofluorescence analysis of zebrafish cryosections

Zebrafish embryos (48 hpf) were frozen in isopentane pre-chilled in liquid nitrogen and stored at −80°C. Seven-millimeter-thick frozen sections were immunolabeled with anti-ColVI (Abcam) and anti-β-dystroglycan antibodies (Novocastra) and revealed with TRITC- and FITC-conjugated secondary antibodies, respectively (Dako). Samples were mounted with anti-fading (Molecular Probes) and observed with an epifluorescence Nikon E600 microscope. For confocal analysis (Fig. 1C–H), embryos were fixed with 4% paraformaldehyde in phosphate buffer saline (PBS), washed with PBS and permeabilized with 0.15% Triton X-100. Immunolabeling was performed with a polyclonal anti-Tom20 antibody (Santa Cruz) revealed with a FITC-conjugated anti-rabbit secondary antibody (Dako), and actin was stained with rhodamine-conjugated phalloidin (Sigma). Imaging was performed with an A1-R confocal laser scanning microscope (Nikon, Melville, NY, USA) equipped with 488 and 561 nm laser lines to elicit FITC and TRITC fluorescence, respectively. Each confocal image was obtained by maximum intensity projection of 10 optical sections scanned in the central region of the sample (recorded at z-step size of 300 nm).

Motor activity

Spontaneous coiling rate was recorded as the number of events observed in 15 s for individual embryos at 24 hpf using light microscopy. For the touch escape response assay, we observed the ability to escape after touching embryos with a small tip at 48 hpf. We subdivided embryos into four groups according to their ability to escape: ‘normal’ means embryos with normal motility to swim; ‘motor impairments’ means embryos with minor motility disruptions; ‘only coiling events’ means embryos circling without the ability to escape; ‘paralyzed’ means embryos with no motility.

Birefringence assay

Muscle birefringence was analyzed by taking advantage of muscle fibers anisotropy. Briefly, we placed anesthetized embryos on a glass and analyzed muscle light refraction by using two polarizing filters. The first filter produces the polarized light to illuminate the sample and the second polarizing filter, called the analyzer, restricts detected light to refracted light coming from muscle fibers. In particular, the top polarizing filter was twisted of a 90° angle until the light refracting through the muscle was visible. To perform these experiments, we used a Leica M165FC stereomicroscope. We calculated integrated area of birefringence by using ImageJ software, as described (46). Birefringence values ≥3 × 106 (typical of wild type individuals) were rated as normal, values between 2.9 and 1 × 106 were considered as an indication of mild disease and values ≤1 × 106 were rated as an indication of severe myopathy.

Mice

Col6a1−/− mice (23) had free access to a standard diet and were kept under controlled conditions of temperature and humidity on a 12 h light/12 h dark cycle. Mice were treated with placebo, 10 mg or 25 mg NIM811/kg/day by gavage in two separate doses for 5 and 10 days. Mice were observed every day until the end of the treatment period in order to detect mortality, morbidity or any abnormal clinical event. No abnormal behavioral or digestive signs were found during treatment, nor other signs of poor clinical condition. Body weight measurements at days 1, 3 and 5 displayed small variations that were not significantly different for the various treatment groups. All in vivo experiments were approved by the competent Authority of the University of Padova and authorized by the Italian Ministry of Health.

Muscle isolation

Fibers from FDB muscles were isolated exactly as described previously (33). Fibers were then plated onto glass cover slips coated with laminin (3 µg/cm2) and cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum. During the experiment, FDB fibers were placed in 1 ml Tyrode's buffer and loaded with 20 nm of TMRM as described previously (14).

In vivo muscle mechanics

In vivo determination of force and contraction kinetics of gastrocnemius muscle were carried out as previously described (47).

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay

Seven-micrometer-thick sections of muscle diaphragm were prepared after 4% paraformaldehyde fixation and paraffin embedding. TUNEL was done using the ApopTag in situ apoptosis detection kit. Samples were stained with peroxidase-diaminobenzidine to detect TUNEL-positive nuclei and counterstained with Hoechst 33258 to identify all nuclei, as described previously (14). Total and TUNEL-positive nuclei were determined in randomly selected fibers using a Zeiss Axioplan microscope equipped with a Leica DC 500 camera. In the muscle cultures, TUNEL assays were performed with the Dead End Fluorometric in situ apoptosis detection system (Promega Italia, Milan, Italy). Muscle cultures were permeabilized in methanol–acetone 50:50 at −20°C for 10 min. After drying, slides were washed in PBS, incubated with equilibration buffer for 10 min and treated with buffer containing fluorescent nucleotides, rTdT enzyme and Hoechst 33258 (Sigma) for 1 h at 37°C. SSC solution was used to block the activity of rTdT enzyme, before washing and preparing slides for microscopy analysis.

Patient and mouse muscle-derived cell cultures

BM and UCMD were diagnosed according to the criteria of the European Neuromuscular Center (8). All patients were examined and all underwent a muscle biopsy. All participants provided written informed consent, and approval was obtained from the Ethics Committee of the Rizzoli Orthopedic Institute (Bologna, Italy). Cell cultures were established using enzymatic and mechanical treatment and plating in DMEM plus 20% fetal calf serum (FCS) and antibiotics (penicillin, streptomycin and amphotericin B; Sigma) and then stored in liquid nitrogen. Mouse cell cultures were obtained from diaphragms as previously described (18).

Measurements of mitochondrial membrane potential

Mitochondrial membrane potential was measured by epifluorescence microscopy based on the accumulation of TMRM as described (15). Muscle-derived cell cultures (30 000–35 000 cells) were seeded onto 24 mm diameter round glass cover slips, grown for 2 days in DMEM supplemented with 20% FCS and studied as described after loading with 10 nm TMRM in 1 ml of serum-free DMEM.

Statistical analysis

Data were analyzed using the unpaired Student's t-test except for Figures 2 and 4B, where ANOVA test was used. Values with P < 0.05 were considered significant and data are reported as mean ± SEM.

FUNDING

The study was supported in part by Research Grants from Novartis Pharma AG (Basel) and Telethon, Italy (GGP11082).

ACKNOWLEDGMENTS

NIM811and CsA were supplied by Novartis Pharma AG (Basel, Switzerland).

Conflict of Interest statement. None declared.

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Author notes

These Authors contributed equally to this work.