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T.P.39 Building muscle with myostatin inhibitors
864
Abstracts / Neuromuscular Disorders 22 (2012) 804–908
T.P.37 Changes in muscle expression of PGC-1a and related signalling molecules in the mdx mouse: Possible impact on adaptive fiber phenotype transition and implication for drug studies G.M. Camerino, M. Cannone, R.F. Capogrosso, A. De Luca University of Bari, Dept. Bioscience, Biotechnology and Pharmacological Sciences, Bari, Italy In Duchenne muscular dystrophy (DMD), the absence of dystrophin causes a complex pathogenetic cascade also involving sarcolemmal fragility and metabolic distress. Increasing evidences in mdx mouse model outline the potential interest of therapeutic strategies to reinforce pathways leading to activation of peroxisome proliferator-activated receptor (PPAR) c coactivator 1a (PGC-1a), a key modulator of oxidative metabolism and mitochondrial biogenesis. Accordingly, we recently found beneficial effects of resveratrol, a PGC-1a activator via sirtuin 1 (Sirt1), in exercised mdx mice. To better understand the basal regulation of this mechano-sensitive metabolic pathway in dystrophic skeletal muscle we performed real-time PCR experiments in gastrocnemius (GC) muscles of mdx mice at different ages. We found slightly higher levels of PGC-1a mRNA in mdx mice of 8 weeks of age vs. age-matched C57BL/10 (wt) mice. The increase in PGC-1a became highly significant at 16 weeks of age and was paralleled by a 40% rise in Sirt1 mRNA. Focusing on 16 weeks of age, we then assessed the gene expression of PPARs since PGC-1a cooperates with members of this receptor family to regulate expression of mitochondrial and b-oxidation enzymes. Interestingly, the expression of PPAR-d was markedly higher in mdx GC muscle vs. wt one, while PPAR-c had a comparable expression in the two genotypes. Afterwards the impact of the above changes on phenotype and glycolytic/oxidative metabolism of dystrophic muscle has been evaluated on genes expression of myosin heavy chain (MHC) isoforms. A significant increase of slow-oxidative type I MHC and a decrease of fast-glycolytic IIb MHC were found in mdx GC muscle vs. wt ones. Then a modification of PGC-1a pathway, which likely triggers a protective slow-oxidative myofiber program, naturally occurs in the mdx mouse muscle and may modulate the outcome of physiological stimuli and of pharmacological strategies (Supported by Duchenne Parent Project NL). http://dx.doi:10.1016/j.nmd.2012.06.202
T.P.38 Effects of steroid hormones on myostatin expression and on genes of muscle regeneration pathway A.L.F. Santos, P.C. Calyjur, D. Zilberztajn, P.C.G. Onofre-Oliveira, P.C.M. Martins, L.U. Yamamoto, M. Vainzof Universidade de Sa˜o Paulo, Centro de Estudos do Genoma Humano, Sa˜o Paulo, Brazil Myostatin is an important negative regulator of skeletal muscle growth, while decanoato de nadrolone, an anabolic steroid, is a strong positive effector. Inhibition of myostatin has been tested as an approach for treatment neuromuscular diseases. In order to investigate the possible interaction between myostatin and anabolic steroids, as a therapeutic strategy, we studied myostatin expression in the quadriceps femoris of normal mice treated with DecadurabolinÒ (D), flutamide (F), an antagonist of the androgen receptor, and Decadurabolin administration, post flutamide treatment (FD), as compared to controls, treated with saline (S). We also studied the relative expression of the genes, myogenin, MyoD and Myf5, involved in the pathway of muscle regeneration. We observed significant increase in the body mass in the (D) and (FD) groups, and a decrease in the group (F), when compared to groups (S). Real-time PCR quantitative analysis for myostatin expression
showed no statistically significant differences between the studied groups. On the other hand, the groups (D) and (FD) showed a significant decrease in the expression of myogenin, MyoD and Myf5, while animals of the group (F) showed a significant increase in the expression of these genes. We conclude that administration of anabolic steroid, or its inhibition did not alter the expression of the myostatin gene, despite the increase or decrease in the body mass observed in group (D), (FD) and (F). However, the blockade of androgen receptor by flutamide, clearly stimulate the regeneration cascade, by increasing the expression of genes related to proliferation (MyoD and Myf5) and cell differentiation (myogenin). Additional studies will elucidate the possible role of other pathways in this stimulus for regeneration. Financial support: FAPESP-CEPID, CNPq-INCT, FINEP, ABDIM. http://dx.doi:10.1016/j.nmd.2012.06.203
T.P.39 Building muscle with myostatin inhibitors P. Bialek 1, J. Owens 1, M. St. Andre 1, D. Marsden 1, M. Denz 2, P. Yaworsky 3, C. Morris 1 1 Pfizer Research, Orphan & Genetic Diseases, Cambridge, United States; 2 Pfizer Research, Global BioTherapeutics, Cambridge, United States; 3 Pfizer Research, Inflammation & Remodelling, Cambridge, United States Therapeutic inhibition of myostatin (GDF8) has been considered one obvious approach that could provide significant benefit in indications such as sarcopenia, cachexia and the muscular dystrophies. Several approaches have been taken to develop muscle building therapies by neutralizing GDF8 activity. A highly efficacious, but non-selective ActRIIb receptor fusion (ACE-031) showed promising results, however the program stalled due to safety liabilities. In contrast, the selective antibody MYO-029 failed to show improvements on efficacy endpoints. In light of these data, it has been important to gain confidence in this mechanism by learning from the past and by continued preclinical testing of GDF8 inhibitor effects. We have been able to show that ActRIIbFc was able to attenuate muscle atrophy associated with glucocorticoids, cancer cachexia, immobilization, and tendon transection in preclinical studies. The role of satellite cells versus protein synthesis are still not resolved with respect to muscle mass increases observed after treatment with inhibitors. To examine one aspect, the mTOR inhibitor, rapamycin, was used to inhibit muscle growth during ActRIIbFc treatment. The results showed the anabolic response was maintained in the presence of rapamycin, suggesting other pathways may be involved in the muscle growth observed. Further, gene expression studies looked for other candidate pathways and found only a small number of highly regulated genes. ActRIIbFc was shown to attenuate Murf1/MafBx expression, suggesting a link to that pathway. Concerns around the role of myostatin on specific force and tendon stiffness led us to examine GDF8 / mice and mice treated with inhibitors. Only the GDF8 / showed reduced specific force while tendon function was unchanged in all animals. Continued study of the GDF8 pathway has provided further support that inhibitors have significant potential to improve muscle function in a number of indications associated with muscle frailty. http://dx.doi:10.1016/j.nmd.2012.06.204
T.P.40 Implication of SIRT1 and its downstream pathways in dystrophic process S. Messina, G.L. Vita, M.G. De Pasquale, A. Cama, A. Licata, S. Romeo, A. Ciranni, C. Lo Giudice, A. Toscano, M. Aguennouz, G. Vita
Abstracts / Neuromuscular Disorders 22 (2012) 804–908
T.P.37 Changes in muscle expression of PGC-1a and related signalling molecules in the mdx mouse: Possible impact on adaptive fiber phenotype transition and implication for drug studies G.M. Camerino, M. Cannone, R.F. Capogrosso, A. De Luca University of Bari, Dept. Bioscience, Biotechnology and Pharmacological Sciences, Bari, Italy In Duchenne muscular dystrophy (DMD), the absence of dystrophin causes a complex pathogenetic cascade also involving sarcolemmal fragility and metabolic distress. Increasing evidences in mdx mouse model outline the potential interest of therapeutic strategies to reinforce pathways leading to activation of peroxisome proliferator-activated receptor (PPAR) c coactivator 1a (PGC-1a), a key modulator of oxidative metabolism and mitochondrial biogenesis. Accordingly, we recently found beneficial effects of resveratrol, a PGC-1a activator via sirtuin 1 (Sirt1), in exercised mdx mice. To better understand the basal regulation of this mechano-sensitive metabolic pathway in dystrophic skeletal muscle we performed real-time PCR experiments in gastrocnemius (GC) muscles of mdx mice at different ages. We found slightly higher levels of PGC-1a mRNA in mdx mice of 8 weeks of age vs. age-matched C57BL/10 (wt) mice. The increase in PGC-1a became highly significant at 16 weeks of age and was paralleled by a 40% rise in Sirt1 mRNA. Focusing on 16 weeks of age, we then assessed the gene expression of PPARs since PGC-1a cooperates with members of this receptor family to regulate expression of mitochondrial and b-oxidation enzymes. Interestingly, the expression of PPAR-d was markedly higher in mdx GC muscle vs. wt one, while PPAR-c had a comparable expression in the two genotypes. Afterwards the impact of the above changes on phenotype and glycolytic/oxidative metabolism of dystrophic muscle has been evaluated on genes expression of myosin heavy chain (MHC) isoforms. A significant increase of slow-oxidative type I MHC and a decrease of fast-glycolytic IIb MHC were found in mdx GC muscle vs. wt ones. Then a modification of PGC-1a pathway, which likely triggers a protective slow-oxidative myofiber program, naturally occurs in the mdx mouse muscle and may modulate the outcome of physiological stimuli and of pharmacological strategies (Supported by Duchenne Parent Project NL). http://dx.doi:10.1016/j.nmd.2012.06.202
T.P.38 Effects of steroid hormones on myostatin expression and on genes of muscle regeneration pathway A.L.F. Santos, P.C. Calyjur, D. Zilberztajn, P.C.G. Onofre-Oliveira, P.C.M. Martins, L.U. Yamamoto, M. Vainzof Universidade de Sa˜o Paulo, Centro de Estudos do Genoma Humano, Sa˜o Paulo, Brazil Myostatin is an important negative regulator of skeletal muscle growth, while decanoato de nadrolone, an anabolic steroid, is a strong positive effector. Inhibition of myostatin has been tested as an approach for treatment neuromuscular diseases. In order to investigate the possible interaction between myostatin and anabolic steroids, as a therapeutic strategy, we studied myostatin expression in the quadriceps femoris of normal mice treated with DecadurabolinÒ (D), flutamide (F), an antagonist of the androgen receptor, and Decadurabolin administration, post flutamide treatment (FD), as compared to controls, treated with saline (S). We also studied the relative expression of the genes, myogenin, MyoD and Myf5, involved in the pathway of muscle regeneration. We observed significant increase in the body mass in the (D) and (FD) groups, and a decrease in the group (F), when compared to groups (S). Real-time PCR quantitative analysis for myostatin expression
showed no statistically significant differences between the studied groups. On the other hand, the groups (D) and (FD) showed a significant decrease in the expression of myogenin, MyoD and Myf5, while animals of the group (F) showed a significant increase in the expression of these genes. We conclude that administration of anabolic steroid, or its inhibition did not alter the expression of the myostatin gene, despite the increase or decrease in the body mass observed in group (D), (FD) and (F). However, the blockade of androgen receptor by flutamide, clearly stimulate the regeneration cascade, by increasing the expression of genes related to proliferation (MyoD and Myf5) and cell differentiation (myogenin). Additional studies will elucidate the possible role of other pathways in this stimulus for regeneration. Financial support: FAPESP-CEPID, CNPq-INCT, FINEP, ABDIM. http://dx.doi:10.1016/j.nmd.2012.06.203
T.P.39 Building muscle with myostatin inhibitors P. Bialek 1, J. Owens 1, M. St. Andre 1, D. Marsden 1, M. Denz 2, P. Yaworsky 3, C. Morris 1 1 Pfizer Research, Orphan & Genetic Diseases, Cambridge, United States; 2 Pfizer Research, Global BioTherapeutics, Cambridge, United States; 3 Pfizer Research, Inflammation & Remodelling, Cambridge, United States Therapeutic inhibition of myostatin (GDF8) has been considered one obvious approach that could provide significant benefit in indications such as sarcopenia, cachexia and the muscular dystrophies. Several approaches have been taken to develop muscle building therapies by neutralizing GDF8 activity. A highly efficacious, but non-selective ActRIIb receptor fusion (ACE-031) showed promising results, however the program stalled due to safety liabilities. In contrast, the selective antibody MYO-029 failed to show improvements on efficacy endpoints. In light of these data, it has been important to gain confidence in this mechanism by learning from the past and by continued preclinical testing of GDF8 inhibitor effects. We have been able to show that ActRIIbFc was able to attenuate muscle atrophy associated with glucocorticoids, cancer cachexia, immobilization, and tendon transection in preclinical studies. The role of satellite cells versus protein synthesis are still not resolved with respect to muscle mass increases observed after treatment with inhibitors. To examine one aspect, the mTOR inhibitor, rapamycin, was used to inhibit muscle growth during ActRIIbFc treatment. The results showed the anabolic response was maintained in the presence of rapamycin, suggesting other pathways may be involved in the muscle growth observed. Further, gene expression studies looked for other candidate pathways and found only a small number of highly regulated genes. ActRIIbFc was shown to attenuate Murf1/MafBx expression, suggesting a link to that pathway. Concerns around the role of myostatin on specific force and tendon stiffness led us to examine GDF8 / mice and mice treated with inhibitors. Only the GDF8 / showed reduced specific force while tendon function was unchanged in all animals. Continued study of the GDF8 pathway has provided further support that inhibitors have significant potential to improve muscle function in a number of indications associated with muscle frailty. http://dx.doi:10.1016/j.nmd.2012.06.204
T.P.40 Implication of SIRT1 and its downstream pathways in dystrophic process S. Messina, G.L. Vita, M.G. De Pasquale, A. Cama, A. Licata, S. Romeo, A. Ciranni, C. Lo Giudice, A. Toscano, M. Aguennouz, G. Vita