Level of muscle regeneration in limb-girdle muscular dystrophy type 2I relates to genotype and clinical severity

<p>Abstract</p> <p>Background</p> <p>The balance between muscle regeneration and ongoing degeneration is a relationship that greatly influences the progression of muscular dystrophy. Numerous factors may influence the muscle regeneration, but more information about the relationship between genotype, clinical severity and the ability to regenerate is needed.</p> <p>Methods</p> <p>Muscle biopsies were obtained from the tibialis anterior muscle, and frozen sections were stained for general histopathological and immunohistological evaluation. Differences between groups were considered statistical significant at <it>P </it>< 0.05 using Student's unpaired <it>t</it>-test.</p> <p>Results</p> <p>We found that all patients with limb-girdle muscular dystrophy type 2I (LGMD2I) had a large number of internally nucleated fibers, a sign of previous regeneration. The level of expression of muscle-specific developmental proteins, such as neonatal myosin heavy chain (nMHC) and myogenin, was related to the clinical severity. Additionally, we found that the majority of nMHC-positive fibers did not stain positively for utrophin in patients who were compound heterozygous for the L276I mutation, suggesting that the predominant form of regeneration in these patients is fiber repair rather than formation of new fibers. Double staining showed that many smaller nMHC-positive fibers were positive for antibodies against the glycosylation on α-dystroglycan, suggesting that such glycosylation may be a result of muscle regeneration.</p> <p>Conclusion</p> <p>Severely affected patients with LGMD2I have a high level of muscle degeneration, which leads to a high rate of regeneration, but this is insufficient to change the imbalance between degeneration and regeneration, ultimately leading to progressive muscle wasting. Detailed information regarding the level and rate of muscle regeneration and potential obstructions of the regenerative pathway should be of use for future therapies involving satellite-cell activation.</p

Navigating the 'paradox of openness' in energy and transport innovation: insights from eight corporate clean technology research and development case studies

Using an inductive case study approach drawn from original interview data, this article investigates the innovation approaches among a sample of international energy companies, or corporate firms. It first presents a conceptual framework synthesized from the business studies, entrepreneurship, evolutionary economics, innovation studies, management science, organization studies, political science, and sociology literature. This framework suggests that corporate approaches to clean technology innovation will cut across the four dimensions of organizational multiplicity and stakeholder involvement, information sharing, coordination and control, and market orientation. It then explores how eight firms—the Algal Carbon Conversion Flagship and Aurora Algae (biofuel), DONG and Statoil (carbon capture and storage), Tesla and Volkswagen (electric vehicles), and Siemens and Vestas (offshore wind turbines)—approach clean technology development with “open innovation” attributes mixed with “closed” attributes. Although the study finds striking similarities among the particular approaches embraced by each corporate actor, it also notes that approaches are technology and firm specific, and the potential for different permutations leads to an almost endless number of possible stylistic combinations. The innovation profiles depicted also reveal conflict and competition among various stakeholders, the implication being that corporate innovation in the energy sector remains a conflicted, disjointed, and messy process

Calpain 3 is important for muscle regeneration: Evidence from patients with limb girdle muscular dystrophies

<p>Abstract</p> <p>Background</p> <p>Limb girdle muscular dystrophy (LGMD) type 2A is caused by mutations in the CAPN3 gene and complete lack of functional calpain 3 leads to the most severe muscle wasting. Calpain 3 is suggested to be involved in maturation of contractile elements after muscle degeneration. The aim of this study was to investigate how mutations in the four functional domains of calpain 3 affect muscle regeneration.</p> <p>Methods</p> <p>We studied muscle regeneration in 22 patients with LGMD2A with calpain 3 deficiency, in five patients with LGMD2I, with a secondary reduction in calpain 3, and in five patients with Becker muscular dystrophy (BMD) with normal calpain 3 levels. Regeneration was assessed by using the developmental markers neonatal myosin heavy chain (nMHC), vimentin, MyoD and myogenin and counting internally nucleated fibers.</p> <p>Results</p> <p>We found that the recent regeneration as determined by the number of nMHC/vimentin-positive fibers was greatly diminished in severely affected LGMD2A patients compared to similarly affected patients with LGMD2I and BMD. Whorled fibers, a sign of aberrant regeneration, was highly elevated in patients with a complete lack of calpain 3 compared to patients with residual calpain 3. Regeneration is not affected by location of the mutation in the <it>CAPN3 </it>gene.</p> <p>Conclusions</p> <p>Our findings suggest that calpain 3 is needed for the regenerative process probably during sarcomere remodeling as the complete lack of functional calpain 3 leads to the most severe phenotypes.</p

Muscle Atrophy Reversed by Growth Factor Activation of Satellite Cells in a Mouse Muscle Atrophy Model

<div><p>Muscular dystrophies comprise a large group of inherited disorders that lead to progressive muscle wasting. We wanted to investigate if targeting satellite cells can enhance muscle regeneration and thus increase muscle mass. We treated mice with hepatocyte growth factor and leukemia inhibitory factor under three conditions: normoxia, hypoxia and during myostatin deficiency. We found that hepatocyte growth factor treatment led to activation of the Akt/mTOR/p70S6K protein synthesis pathway, up-regulation of the myognic transcription factors MyoD and myogenin, and subsequently the negative growth control factor, myostatin and atrophy markers MAFbx and MuRF1. Hypoxia-induced atrophy was partially restored by hepatocyte growth factor combined with leukemia inhibitory factor treatment. Dividing satellite cells were three-fold increased in the treatment group compared to control. Finally, we demonstrated that myostatin regulates satellite cell activation and myogenesis <i>in vivo</i> following treatment, consistent with previous findings <i>in vitro</i>. Our results suggest, not only a novel <i>in vivo</i> pharmacological treatment directed specifically at activating the satellite cells, but also a myostatin dependent mechanism that may contribute to the progressive muscle wasting seen in severely affected patients with muscular dystrophy and significant on-going regeneration. This treatment could potentially be applied to many conditions that feature muscle wasting to increase muscle bulk and strength.</p></div

Protein Turnover and Cellular Stress in Mildly and Severely Affected Muscles from Patients with Limb Girdle Muscular Dystrophy Type 2I

Patients with Limb girdle muscular dystrophy type 2I (LGMD2I) are characterized by progressive muscle weakness and wasting primarily in the proximal muscles, while distal muscles often are spared. Our aim was to investigate if wasting could be caused by impaired regeneration in the proximal compared to distal muscles. Biopsies were simultaneously obtained from proximal and distal muscles of the same patients with LGMD2I (n = 4) and healthy subjects (n = 4). The level of past muscle regeneration was evaluated by counting internally nucleated fibers and determining actively regenerating fibers by using the developmental markers embryonic myosin heavy chain (eMHC) and neural cell adhesion molecule (NCAM) and also assessing satellite cell activation status by myogenin positivity. Severe muscle histopathology was occasionally observed in the proximal muscles of patients with LGMD2I whereas distal muscles were always relatively spared. No difference was found in the regeneration markers internally nucleated fibers, actively regenerating fibers or activation status of satellite cells between proximal and distal muscles. Protein turnover, both synthesis and breakdown, as well as cellular stress were highly increased in severely affected muscles compared to mildly affected muscles. Our results indicate that alterations in the protein turnover and myostatin levels could progressively impair the muscle mass maintenance and/or regeneration resulting in gradual muscular atrophy

Effect of alternating hepatocyte growth factor and leukemia inhibitory factor treatment on protein synthesis pathway during hypoxia.

<p>Western blots of tibialis anterior homogenates from hypoxia induced atrophic mice shows activation of protein synthesis pathway after repeated alternating injections with hepatocyte growth factor and leukemia inhibitory factor (N = 13, red) compared to PBS (N = 14, dark blue). This is based on evaluation of the phosphorylation of components from the protein synthesis pathway; pPI3K [p55α] (A), pPDK1 (B), pmTOR (C), pp70S6K (D), p4E-BP1 (E) and peIF4E (F). RU = Relative Units. Error bars are SD; Statistical significance was determined by a two-tailed Student's t–test. *denotes significant (<i>P</i><0.05). Data are representative of one experiment.</p

Hepatocyte growth factor activates protein synthesis pathway and increases protein expression of myogenic factors.

<p>In order to establish if HGF initiated the protein synthesis and myogenesis pathways and the timeframe in vivo, we injected a total of 21 normoxic mice (N = 3 for each time point) with hepatocyte growth factor (20 ng HGF/g body weight) and sacrificed them at time points between 0 and 48 h. A western blot of components of the protein synthesis and myogenesis pathways demonstrates how the signal activates the different proteins with time and eventually leads to an increased expression of myogenic factors (A). Two mice were given a 10 times higher dose of hepatocyte growth factor (200 ng HGF/g body weight) and sacrificed after 60 min ( = 60 minH). Western blots of tibialis anterior homogenates (N = 3 for each time point) shows activation of protein synthesis pathway through phosphorylation of PDK1 (B)/mTOR (C)/Akt (D)/p70S6K (E), and increased protein expression of myogenic factors MyoD (F) and myogenin (G). A 200 ng HGF/g body weight dose of hepatocyte growth factor yields a diminished response compared to a 20 ng HGF/g body weight dose RU = Relative Units. Error bars are SD. Thin dividing lines on the blot are for clarification only whereas the bold red line symbolizes that samples were run on two separate gels. One-way ANOVA with Bonferroni <i>post hoc</i> correction for multiple comparisons was used to assess differences among groups for each dependent variable. *denotes significant (<i>P</i><0.05). Data are representative of one independent experiment.</p

The hypoxic protocol and verification of atrophy model.

<p>In order to create a muscle atrophy model, we let the mice stay in a chamber with a gradually more hypoxic atmosphere until it reaches approx. 7.5%. Oxygen content in the hypoxic chamber was monitored during the hypoxic protocol (•) and time points for alternating intraperitoneal injections (▴) with hepatocyte growth factor = H and leukemia inhibitory factor = L (A). Hypoxia did not change body composition (Lean/fat) measured by quantitative MRI of PBS-injected normoxic mice (N = 8, light blue), PBS-injected hypoxic mice (N = 8, dark blue) (B). Hypoxia did not change muscle water content based on dry weight: wet weight ratio in tibialis anterior (TA) of PBS injected normoxic mice (N = 8, light blue), PBS injected hypoxic mice (N = 8, dark blue) (C). Hypoxia induced loss of muscle protein measured as total soluble protein in muscle homogenates per wet weight of PBS-injected normoxic mice N = 8 (light blue) and PBS injected hypoxic mice (N = 8, dark blue) (D). Error bars are SD; Statistical significance was determined by a two-tailed Student's t-test. *denotes significant (<i>P</i><0.05). Data are representative of one independent experiment.</p