Janelle M. Spinazzola

Automated drug screening with contractile muscle tissue engineered from dystrophic myoblasts

Identification of factors that improve muscle function in boys with Duchenne muscular dystrophy (DMD) could lead to an improved quality of life. To establish a functional in vitro assay for muscle strength, mdx murine myoblasts, the genetic homologue of DMD, were tissue engineered in 96‐microwell plates into 3‐dimensional muscle constructs with parallel arrays of striated muscle fibers. When electrically stimulated, they generated tetanic forces measured with an automated motion tracking system. Thirty‐one compounds of interest as potential treatments for patients with DMD were tested at 3 to 6 concentrations. Eleven of the compounds (insulin‐like growth factor‐1, creat‐ine, β‐hydroxy‐β‐methylbutyrate, trichostatin A, lisino‐pril, and 6 from the glucocorticoid family) significantly increased tetanic force relative to placebo‐treated controls. The glucocorticoids methylprednisolone, deflazacort, and prednisone increased tetanic forces at low doses (EC50 of 6, 19, and 56 nM, respectively), indicating a direct muscle mechanism by which they may be benefitting DMD patients. The tetanic force assay also identified beneficial compound interactions (arginine plus deflazacort and prednisone plus creatine) as well as deleterious interactions (prednisone plus creatine inhibited by pentoxifylline) of combinatorial therapies taken by some DMD patients. Since mdx muscle in vivo and DMD patients respond in a similar manner to many of these compounds, the in vitro assay will be a useful tool for the rapid identification of new potential treatments for muscle weakness in DMD and other muscle disorders.—Vandenburgh, H., Shansky, J., Benesch‐Lee, F., Skelly, K., Spinazzola, J.M., Saponjian, Y., Tseng, B.S. Automated drug screening with contractile muscle tissue engineered from dystrophic myoblasts. FASEB J. 23, 3325–3334 (2009). www.fasebj.org

CD82 Is a Marker for Prospective Isolation of Human Muscle Satellite Cells and Is Linked to Muscular Dystrophies

Cell-surface markers for prospective isolation of stem cells from human skeletal muscle have been difficult to identify. Such markers would be powerful tools for studying satellite cell function during homeostasis and in pathogenesis of diseases such as muscular dystrophies. In this study, we show that the tetraspanin KAI/CD82 is an excellent marker for prospectively isolating stem cells from human fetal and adult skeletal muscle. Human CD82+ muscle cells robustly engraft into a mouse model of muscular dystrophy. shRNA knockdown of CD82 in myogenic cells reduces myoblast proliferation, suggesting it is functionally involved in muscle homeostasis. CD82 physically interacts with alpha7beta1 integrin (α7β1-ITG) and with α-sarcoglycan, a member of the Dystrophin-Associated Glycoprotein Complex (DAPC), both of which have been linked to muscular dystrophies. Consistently, CD82 expression is decreased in Duchenne muscular dystrophy patients. Together, these findings suggest that CD82 function may be important for muscle stem cell function in muscular disorders.

Viral expression of insulin-like growth factor I E-peptides increases skeletal muscle mass but at the expense of strength

Insulin-like growth factor I (IGF-I) is a protein that regulates and promotes growth in skeletal muscle. The IGF-I precursor polypeptide contains a COOH-terminal extension called the E-peptide. Alternative splicing in the rodent produces two isoforms, IA and IB, where the mature IGF-I in both isoforms is identical yet the E-peptides, EA and EB, share less than 50% homology. Recent in vitro studies show that the E-peptides can enhance IGF-I signaling, leading to increased myoblast cell proliferation and migration. To determine the significance of these actions in vivo and to evaluate if they are physiologically beneficial, EA and EB were expressed in murine skeletal muscle via viral vectors. The viral constructs ensured production of E-peptides without the influence of additional IGF-I through an inactivating mutation in mature IGF-I. E-peptide expression altered ERK1/2 and Akt phosphorylation and increased satellite cell proliferation. EB expression resulted in significant muscle hypertrophy that was IGF-I receptor dependent. However, the increased mass was associated with a loss of muscle strength. EA and EB have similar effects in skeletal muscle signaling and on satellite cells, but EB is more potent at increasing muscle mass. Although sustained EB expression may drive hypertrophy, there are significant physiological consequences for muscle.

Gamma-sarcoglycan is required for the response of archvillin to mechanical stimulation in skeletal muscle

Loss of gamma-sarcoglycan (γ-SG) induces muscle degeneration and signaling defects in response to mechanical load, and its absence is common to both Duchenne and limb girdle muscular dystrophies. Growing evidence suggests that aberrant signaling contributes to the disease pathology; however, the mechanisms of γ-SG-mediated mechanical signaling are poorly understood. To uncover γ-SG signaling pathway components, we performed yeast two-hybrid screens and identified the muscle-specific protein archvillin as a γ-SG and dystrophin interacting protein. Archvillin protein and message levels were significantly upregulated at the sarcolemma of murine γ-SG-null (gsg(-/-)) muscle but delocalized in dystrophin-deficient mdx muscle. Similar elevation of archvillin protein was observed in human quadriceps muscle lacking γ-SG. Reintroduction of γ-SG in gsg(-/-) muscle by rAAV injection restored archvillin levels to that of control C57 muscle. In situ eccentric contraction of tibialis anterior (TA) muscles from C57 mice caused ERK1/2 phosphorylation, nuclear activation of P-ERK1/2 and stimulus-dependent archvillin association with P-ERK1/2. In contrast, TA muscles from gsg(-/-) and mdx mice exhibited heightened P-ERK1/2 and increased nuclear P-ERK1/2 localization following eccentric contractions, but the archvillin-P-ERK1/2 association was completely ablated. These results position archvillin as a mechanically sensitive component of the dystrophin complex and demonstrate that signaling defects caused by loss of γ-SG occur both at the sarcolemma and in the nucleus.

Mature IGF-I excels in promoting functional muscle recovery from disuse atrophy compared with pro-IGF-IA.

Prolonged disuse of skeletal muscle results in atrophy, and once physical activity is resumed, there is increased susceptibility to injury. Insulin-like growth factor-I (IGF-I) is considered a potential therapeutic target to attenuate atrophy during unloading and to enhance rehabilitation upon reloading of skeletal muscles, due to its multipronged actions on satellite cell proliferation, differentiation, and survival, as well as its actions on muscle fibers to boost protein synthesis and inhibit protein degradation. However, the form of IGF-I delivered may alter the success of treatment. Using the hindlimb suspension model of disuse atrophy, we compared the efficacy of two IGF-I forms in protection against atrophy and enhancement of recovery: mature IGF-I (IGF-IS) lacking the COOH-terminal extension, called the E-peptide, and IGF-IA, which is the predominant form retaining the E-peptide. Self-complementary adeno-associated virus harboring the murine Igf1 cDNA constructs were delivered to hindlimbs of adult female C57BL6 mice 3 days prior to hindlimb suspension. Hindlimb muscles were unloaded for 7 days and then reloaded for 3, 7, and 14 days. Loss of muscle mass following suspension was not prevented by either IGF-I construct. However, IGF-IS expression maintained soleus muscle force production. Further, IGF-IS treatment caused rapid recovery of muscle fiber morphology during reloading and maintained muscle strength. Analysis of gene expression revealed that IGF-IS expression accelerated the downregulation of atrophy-related genes compared with untreated or IGF-IA-treated samples. We conclude that mature-IGF-I may be a better option than pro-IGF-IA to promote skeletal muscle recovery following disuse atrophy.

Pharmacological therapeutics targeting the secondary defects and downstream pathology of Duchenne muscular dystrophy

ABSTRACT Introduction: Since the identification of the dystrophin gene in 1986, a cure for Duchenne muscular dystrophy (DMD) has yet to be discovered. Presently, there are a number of genetic-based therapies in development aimed at restoration and/or repair of the primary defect. However, growing understanding of the pathophysiological consequences of dystrophin absence has revealed several promising downstream targets for the development of therapeutics. Areas covered: In this review, we discuss various strategies for DMD therapy targeting downstream consequences of dystrophin absence including loss of muscle mass, inflammation, fibrosis, calcium overload, oxidative stress, and ischemia. The rationale of each approach and the efficacy of drugs in preclinical and clinical studies are discussed. Expert opinion: For the last 30 years, effective DMD drug therapy has been limited to corticosteroids, which are associated with a number of negative side effects. Our knowledge of the consequences of dystrophin absence that contribute to DMD pathology has revealed several potential therapeutic targets. Some of these approaches may have potential to improve or slow disease progression independently or in combination with genetic-based approaches. The applicability of these pharmacological therapies to DMD patients irrespective of their genetic mutation, as well as the potential benefits even for advanced stage patients warrants their continued investigation.

Absence of γ-sarcoglycan alters the response of p70S6 kinase to mechanical perturbation in murine skeletal muscle.

BackgroundThe dystrophin glycoprotein complex (DGC) is located at the sarcolemma of muscle fibers, providing structural integrity. Mutations in and loss of DGC proteins cause a spectrum of muscular dystrophies. When only the sarcoglycan subcomplex is absent, muscles display severe myofiber degeneration, but little susceptibility to contractile damage, suggesting that disease occurs not by structural deficits but through aberrant signaling, namely, loss of normal mechanotransduction signaling through the sarcoglycan complex. We extended our previous studies on mechanosensitive, γ-sarcoglycan-dependent ERK1/2 phosphorylation, to determine whether additional pathways are altered with the loss of γ-sarcoglycan.MethodsWe examined mechanotransduction in the presence and absence of γ-sarcoglycan, using C2C12 myotubes, and primary cultures and isolated muscles from C57Bl/6 (C57) and γ-sarcoglycan-null (γ-SG-/-) mice. All were subjected to cyclic passive stretch. Signaling protein phosphorylation was determined by immunoblotting of lysates from stretched and non-stretched samples. Calcium dependence was assessed by maintaining muscles in calcium-free or tetracaine-supplemented Ringer’s solution. Dependence on mTOR was determined by stretching isolated muscles in the presence or absence of rapamycin.ResultsC2C12 myotube stretch caused a robust increase in P-p70S6K, but decreased P-FAK and P-ERK2. Neither Akt nor ERK1 were responsive to passive stretch. Similar but non-significant trends were observed in C57 primary cultures in response to stretch, and γ-SG-/- cultures displayed no p70S6K response. In contrast, in isolated muscles, p70S6K was mechanically responsive. Basal p70S6K activation was elevated in muscles of γ-SG-/- mice, in a calcium-independent manner. p70S6K activation increased with stretch in both C57 and γ-SG-/- isolated muscles, and was sustained in γ-SG-/- muscles, unlike the transient response in C57 muscles. Rapamycin treatment blocked all of p70S6K activation in stretched C57 muscles, and reduced downstream S6RP phosphorylation. However, even though rapamycin treatment decreased p70S6K activation in stretched γ-SG-/- muscles, S6RP phosphorylation remained elevated.Conclusionsp70S6K is an important component of γ-sarcoglycan-dependent mechanotransduction in skeletal muscle. Our results suggest that loss of γ-sarcoglycan uncouples the response of p70S6K to stretch and implies that γ-sarcoglycan is important for inactivation of this pathway. Overall, we assert that altered load-sensing mechanisms exist in muscular dystrophies where the sarcoglycans are absent.

Repression of phosphatidylinositol transfer protein α ameliorates the pathology of Duchenne muscular dystrophy

Significance Duchenne muscular dystrophy (DMD) is a genetic X-linked neuromuscular disease characterized by severe muscle degeneration caused by absence of the protein dystrophin. In the golden retriever muscular dystrophy dog model of DMD, two atypical dogs exhibited significantly milder phenotypes compared with their severely affected littermates despite lacking dystrophin. These two notable dogs were found to have decreased expression of phosphatidylinositol transfer protein-α (PITPNA) compared with severely affected dogs. Decreased expression of PITPNA in dystrophin-deficient zebrafish and in human DMD myogenic cells ameliorated several aspects of the dystrophic phenotype, improving muscle structure, increasing survival, and increasing levels of phosphorylated Akt. Our findings present PITPNA as a genetic modifier of DMD and potential target for future therapies. Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by X-linked inherited mutations in the DYSTROPHIN (DMD) gene. Absence of dystrophin protein from the sarcolemma causes severe muscle degeneration, fibrosis, and inflammation, ultimately leading to cardiorespiratory failure and premature death. Although there are several promising strategies under investigation to restore dystrophin protein expression, there is currently no cure for DMD, and identification of genetic modifiers as potential targets represents an alternative therapeutic strategy. In a Brazilian golden retriever muscular dystrophy (GRMD) dog colony, two related dogs demonstrated strikingly mild dystrophic phenotypes compared with those typically observed in severely affected GRMD dogs despite lacking dystrophin. Microarray analysis of these “escaper” dogs revealed reduced expression of phosphatidylinositol transfer protein-α (PITPNA) in escaper versus severely affected GRMD dogs. Based on these findings, we decided to pursue investigation of modulation of PITPNA expression on dystrophic pathology in GRMD dogs, dystrophin-deficient sapje zebrafish, and human DMD myogenic cells. In GRMD dogs, decreased expression of Pitpna was associated with increased phosphorylated Akt (pAkt) expression and decreased PTEN levels. PITPNA knockdown by injection of morpholino oligonucleotides in sapje zebrafish also increased pAkt, rescued the abnormal muscle phenotype, and improved long-term sapje mutant survival. In DMD myotubes, PITPNA knockdown by lentiviral shRNA increased pAkt and increased myoblast fusion index. Overall, our findings suggest PIPTNA as a disease modifier that accords benefits to the abnormal signaling, morphology, and function of dystrophic skeletal muscle, and may be a target for DMD and related neuromuscular diseases.

Reversal of neurobehavioral social deficits in dystrophic mice using inhibitors of phosphodiesterases PDE5A and PDE9A

Duchenne muscular dystrophy is caused by mutations in the DYSTROPHIN gene. Although primarily associated with muscle wasting, a significant portion of patients (approximately 25%) are also diagnosed with autism spectrum disorder. We describe social behavioral deficits in dystrophin-deficient mice and present evidence of cerebellar deficits in cGMP production. We demonstrate therapeutic potential for selective inhibitors of the cGMP-specific PDE5A and PDE9A enzymes to restore social behaviors in dystrophin-deficient mice.

Masticatory muscles of mouse do not undergo atrophy in space

Muscle loading is important for maintaining muscle mass; when load is removed, atrophy is inevitable. However, in clinical situations such as critical care myopathy, masticatory muscles do not lose mass. Thus, their properties may be harnessed to preserve mass. We compared masticatory and appendicular muscles responses to microgravity, using mice aboard the space shuttle Space Transportation System‐135. Age‐ and sex‐matched controls remained on the ground. After 13 days of space flight, 1 masseter (MA) and tibialis anterior (TA) were frozen rapidly for biochemical and functional measurements, and the contralateral MA was processed for morphologic measurements. Flight TA muscles exhibited 20 ± 3% decreased muscle mass, 2‐fold decreased phosphorylated (P)‐Akt, and 4‐ to 12‐fold increased atrogene expression. In contrast, MAs had no significant change in mass but a 3‐fold increase in P‐focal adhesion kinase, 1.5‐fold increase in P‐Akt, and 50‐90% lower atrogene expression compared with limb muscles, which were unaltered in microgravity. Myofibril force measurements revealed that microgravity caused a 3‐fold decrease in specific force and maximal shortening velocity in TA muscles. It is surprising that myofibril‐specific force from both control and flight MAs were similar to flight TA muscles, yet power was compromised by 40% following flight. Continued loading in microgravity prevents atrophy, but masticatory muscles have a different set point that mimics disuse atrophy in the appendicular muscle.— Philippou, A., Minozzo, F. C., Spinazzola, J. M., Smith, L. R., Lei, H., Rassier, D. E., Barton, E. R. Masticatory muscles of mouse do not undergo atrophy in space. FASEB J. 29, 2769‐2779 (2015). www.fasebj.org