Myostatin

[Michael Scally MD]

Does Myostatin Induce Insulin Resistance?

Rodgers BD. Does Myostatin Induce Insulin Resistance? J Biol Chem. 2014;289(30):21203. http://www.jbc.org/content/289/30/21203.long

Bonala et al. (1) report that high glucose or fat increases myostatin protein expression in muscle and surprisingly liver (cells and tissue) and that myostatin induces insulin resistance.

Their myostatin antiserum used for Western blotting and possibly ELISAs (not discussed) recognizes a reported 26-kDa protein under reducing conditions despite a predicted and empirically determined 12.5-kDa mass (2, 3).

This group previously reported that the antiserum recognizes the same 26-kDa protein in mstn null muscle extracts (4).

Zimmers et al. (3) also identified this protein in wild-type and mstn null mice, although their antiserum detected only a 12.5-kDa band in wild-type mice.

Moreover, several studies have demonstrated the lack of myostatin mRNA in liver.

These findings together suggest that Bonala et al. (1) did not quantify myostatin as their antiserum appears to cross-react with an unknown protein.

There is also concern with their use of recombinant myostatin made in bacteria, which cannot form the critical disulfide necessary for myostatin maturation. These recombinants may function as dominant negatives and were reported to inhibit C2C12 proliferation whereas different sources of recombinant myostatin generated in eukaryotes stimulate it (5).

In fact, concentrations used by Bonala et al. (1) were supraphysiological, 100-fold higher than the functional concentrations of recombinant myostatin made in eukaryotes, which greatly exceeds the nanomolar affinity range of ActRIIb.

This use of highly questionable reagents in turn questions many of the authors’ conclusions. It also adds confusion to the field, and unless the reagents can be finally validated, their use should be discontinued.

 

[pumpingiron22]

Wednesday, January 8, 2014

New Approach to Myostatin-Related Muscle Growth





Muscle wastage is a core killer of the elderly and this is not observed in any other species who typically sustain muscle integrity to almost the end. Thus an outright remedy is very desirous and will lead to substantially more people surviving to the century mark.


The good news is that we may have a simple and safe fix for this where the body gets to do what it was supposed to do. This continues to be independent of actual life extension research as that is a different problem. This is the problem we should not have anyway.


This also eliminates natural bone deterioration as well. That has always been about healthy muscles stimulating bone growth and renewal.


A New Approach to Myostatin-Related Muscle Growth


https://www.fightaging.org/archives/2013/12/a-new-approach-to-myostatin-related-muscle-growth.php


Blocking myostatin has been shown to boost muscle growth and regeneration in various species. There are even a few natural human myostatin mutants, but that is a rare genetic occurrence. For some years now researchers have been investigating means to manipulate myostatin levels and related signaling as a therapy for age-related loss of muscle mass and strength, as well as for various other medical conditions in which wasting of muscles plays a role. This, like many forms of modern medical research, aims to produce a form of compensatory change, potentially beneficial but in no way addressing root causes to prevent progression of the underlying condition.


Researchers here have moved on past myostatin and further down the chain of signals and molecular mechanisms to find a novel place to intervene in order to boost muscle growth in mice and humans. So far results are promising: if this treatment turns out to produce few to no side-effects, it is the sort of thing that everyone could benefit from. That said, again, it doesn't address root causes of degenerative muscle loss with aging - something that needs to be accomplished in order to reliably and most effectively extend healthy life.


New Compound Could Reverse Loss of Muscle Mass in Cancer and Other Diseases

Quote:

The new compound (BYM338) acts to prevent muscle wasting by blocking a receptor that engages a cellular signaling system that exists to put the brakes on muscle development when appropriate. But sometimes those brakes are activated inappropriately, or are stuck on.


A variety of signals can activate the receptor. Prior to development of BYM338, compounds developed to block these molecules were blunt instruments, either trapping all incoming signals (which stimulated muscle growth but also caused harmful side effects) or blocking just a single receptor activator (providing only tepid growth stimulation.) BYM338 was designed to be in the Goldilocks zone (just right.)


In the study the compound boosted muscle mass 25 to 50 percent and increased strength in animal models. Those gains were significantly superior to those of compounds that blocked a single receptor activator. Clinical trials are currently underway. Preliminary data on the antibody was promising enough to have it designated a breakthrough therapy by the US Food and Drug Administration for sporadic inclusion body myositis, a rare muscle wasting disease with no approved therapies.


Here is a link to the paper - the PDF format full version is also presently available if you'd like to wade in to the details.


An Antibody Blocking Activin type II Receptors Induces Strong Skeletal Muscle Hypertrophy and Protects from Atrophy

Quote:

The myostatin/Activin type II receptor (ActRII) pathway has been identified as critical in regulating skeletal muscle size. Several other ligands, including GDF11 and the Activins, signal through this pathway, suggesting that the ActRII receptors are major regulatory nodes in the regulation of muscle mass.


We have developed a novel, human anti-ActRII antibody ("Bimagrumab", aka BYM338) to prevent binding of ligands to the receptors, and thus inhibit downstream signaling. BYM338 enhances differentiation of primary human skeletal myoblasts, and counteracts the inhibition of differentiationinduced by myostatin or Activin A.


BYM338 dramatically increases skeletal muscle mass in mice, beyond sole inhibition of myostatin as detected by comparing the antibody with a myostatin inhibitor. A mouse version of the antibody induces enhanced muscle hypertrophy in myostatin-mutant mice, further confirming a beneficial effect on muscle growth through blockade of ActRII ligands beyond myostatin inhibition alone.

 

[Michael Scally MD]

[Mice] Combined Strategies for Maintaining Skeletal Muscle Mass and Function in Aging: Myostatin Inactivation and AICAR-Associated Oxidative Metabolism Induction
http://biomedgerontology.oxfordjournals.org/content/early/2014/09/16/gerona.glu147.abstract

Myostatin (mstn) blockade, resulting in muscle hypertrophy, is a promising therapy to counteract age-related muscle loss. However, oxidative and mitochondrial deficit observed in young mice with myostatin inhibition could be detrimental with aging.

The aim of this study was
(a) to bring original data on metabolic and mitochondrial consequences of mstn inhibition in old mice, and
(b) to examine whether 4-weeks of AICAR treatment, a pharmacological compound known to upregulate oxidative metabolism, may be useful to improve exercise capacity and mitochondrial deficit of 20-months mstn KO versus wild-type (WT) mice.

Our results show that despite the enlarged muscle mass, the oxidative and mitochondrial deficit associated with reduced endurance running capacity is maintained in old mstn KO mice but not worsened by aging.

Importantly, AICAR treatment induced a significant beneficial effect on running limit time only in old mstn KO mice, with a marked increase in PGC-1α expression and slight beneficial effects on mitochondrial function.

We showed that AICAR effects were autophagy-independent.

This study underlines the relevance of aged muscle remodelling by complementary approaches that impact both muscle mass and function, and suggest that mstn inhibition and aerobic metabolism activators should be co-developed for delaying age-related deficits in skeletal muscle.

 

[Michael Scally MD]

Dubois V, Laurent MlR, Sinnesael M, et al. A satellite cell-specific knockout of the androgen receptor reveals myostatin as a direct androgen target in skeletal muscle. The FASEB Journal 2014;28(7):2979-94. http://www.fasebj.org/content/28/7/2979.abstract

Androgens have well-established anabolic actions on skeletal muscle, although the direct effects of the androgen receptor (AR) in muscle remain unclear.

We generated satellite cell-specific AR-knockout (satARKO) mice in which the AR is selectively ablated in satellite cells, the muscle precursor cells.

Total-limb maximal grip strength is decreased by 7% in satARKO mice, with soleus muscles containing ∼10% more type I fibers and 10% less type IIa fibers than the corresponding control littermates. The weight of the perineal levator ani muscle is markedly reduced (−52%).

Thus, muscle AR is involved in fiber-type distribution and force production of the limb muscles, while it is a major determinant of the perineal muscle mass.

Surprisingly, myostatin (Mstn), a strong inhibitor of skeletal muscle growth, is one of the most androgen-responsive genes (6-fold reduction in satARKO) through direct transcription activation by the AR. Consequently, muscle hypertrophy in response to androgens is augmented in Mstn-knockout mice.

Our finding that androgens induce Mstn signaling to restrain their own anabolic actions has implications for the treatment of muscle wasting disorders.

 

[Michael Scally MD]

Padhi D, Higano CS, Shore ND, Sieber P, Rasmussen E, Smith MR. Pharmacological Inhibition of Myostatin and Changes in Lean Body Mass and Lower Extremity Muscle Size in Patients Receiving Androgen Deprivation Therapy for Prostate Cancer. J Clin Endocrinol Metab 2014;99(10):E1967-E75. http://press.endocrine.org/doi/abs/10.1210/jc.2014-1271

Context: Myostatin is a negative regulator of muscle growth. Androgen deprivation (ADT) is associated with muscle loss and increased body fat, and currently available therapies have limited efficacy to treat this complication. The antimyostatin peptibody (AMG 745/Mu-S) markedly attenuated muscle loss and decreased fat accumulation in orchiectomized mice.

Objective: The objective of the study was to evaluate the safety, pharmacokinetics, and muscle efficacy of AMG 745 in men undergoing ADT for nonmetastatic prostate cancer.

Methods: This was a randomized, blinded, placebo-controlled, multiple-dose, phase 1 study of AMG 745 given for 28 days. The end point of percentage change from baseline in lean body mass (LBM) as assessed by dual x-ray absorptiometry was prespecified.

Results: Rates of adverse events (AMG 745 vs placebo) were the following: diarrhea (13% vs 9%), fatigue (13% vs 4%), contusion (10% vs 0%), and injection site bruising (6% vs 4%). Exposure increased linearly from 0.3 mg/kg to 3 mg/kg. AMG 745 significantly increased LBM in the 3 mg/kg vs the placebo groups on day 29 by 2.2% (+/-0.8% SE, P = 0.008); in exploratory fat mass analysis, a decrease of -2.5% (+/-1.0% SE, P = 0.021) was observed. Pharmacodynamic changes in muscle and fat were maintained at follow-up, 1 month after day 29.

Conclusion: Four weekly sc doses of AMG 745 were well tolerated and were associated with increased LBM and decreased fat in the men receiving ADT for nonmetastatic prostate cancer. Results support further investigation of AMG 745 in clinical settings with muscle loss and atrophy.

 

[Michael Scally MD]

O'Connell KE, Guo W, Serra C, et al. The effects of an ActRIIb receptor Fc fusion protein ligand trap in juvenile simian immunodeficiency virus-infected rhesus macaques. Faseb J. http://www.fasebj.org/content/early/2014/11/25/fj.14-257543.abstract

There are no approved therapies for muscle wasting in children infected with human immunodeficiency virus (HIV), which portends poor disease outcomes.

To determine whether a soluble ActRIIb receptor Fc fusion protein (ActRIIB.Fc), a ligand trap for TGF-beta/activin family members including myostatin, can prevent or restore loss of lean body mass and body weight in simian immunodeficiency virus (SIV)-infected juvenile rhesus macaques (Macaca mulatta).

Fourteen pair-housed, juvenile male rhesus macaques were inoculated with SIVmac239 and, 4 wk postinoculation (WPI) treated with intramuscular injections of 10 mg kg-1 wk-1 ActRIIB.Fc or saline placebo. Body weight, lean body mass, SIV titers, and somatometric measurements were assessed monthly for 16 wk. Age-matched SIV-infected rhesus macaques were injected with saline.

Intervention groups did not differ at baseline. Gains in lean mass were significantly greater in the ActRIIB.Fc group than in the placebo group (P < 0.001). Administration of ActRIIB.Fc was associated with greater gains in body weight (P = 0.01) and upper arm circumference than placebo. Serum CD4+ T-lymphocyte counts and SIV copy numbers did not differ between groups. Administration of ActRIIB.Fc was associated with higher muscle expression of myostatin than placebo.

ActRIIB.Fc effectively blocked and reversed loss of body weight, lean mass, and fat mass in juvenile SIV-infected rhesus macaques.

 

[Michael Scally MD]

Pfizer Initiates Phase 2 Study of PF-06252616 in Duchenne Muscular Dystrophy
http://www.pfizer.com/news/press-re...of_pf_06252616_in_duchenne_muscular_dystrophy

Pfizer Inc. (NYSEFE) announced today enrollment of the first patient in a multicenter Phase II clinical trial of the investigational compound PF-06252616 in boys with Duchenne muscular dystrophy (DMD), a genetic disorder characterized by progressive muscle degeneration and weakness.

PF-06252616 is an experimental, infused, anti-myostatin monoclonal antibody. Myostatin is a naturally occurring protein in muscles that helps control muscle growth; it is believed that blocking the activity of myostatin may have potential therapeutic application in treating muscle wasting diseases such as DMD.

The phase 2 clinical trial will evaluate the safety, tolerability and efficacy of PF-06252616 in boys aged 6 to <10 years old diagnosed with DMD regardless of genotype. Based on the proposed mechanism of action of PF-06252616, Pfizer is exploring whether there is the potential to increase muscle mass and function in boys with DMD who are weak and have lost muscle.

PF-06252616 was granted Orphan Drug designation in July 2012 and Fast Track Designation in November 2012 by the U.S. Food and Drug Administration (FDA). The FDA’s Fast Track Designation is a process designed to facilitate the development and expedite the review of new drugs and biologics intended to treat or prevent serious conditions and that address an unmet medical need.ii The European Medicines Agency (EMA) granted the investigational candidate Orphan Medical Product designation in February 2013.

 

[Michael Scally MD]

Highlights
· Cardiac MSTN activation occurs rapidly after cardiac ischemia.
· Skeletal muscle MSTN remains unchanged after cardiac ischemia.
· Skeletal muscle SMAD2,3, atrogin-1 and MuRF1 are increased after cardiac ischemia.
· Circulating MSTN is consistently increased after cardiac ischemia.
· Increased serum MSTN after ischemia can activate atrophy in the skeletal muscle.

Castillero E, Akashi H, Najjar M, et al. Cardiac Myostatin Upregulation Occurs Immediately After Myocardial Ischemia and is Involved in Skeletal Muscle Activation of Atrophy. Biochem Biophys Res Commun. https://www.sciencedirect.com/science/article/pii/S0006291X14022232

Myostatin (MSTN), a negative regulator of muscle growth and size, is increased after acute myocardial infarction(AMI) but timing of upregulation after injury is not known. In this study, we investigated the timing of the MSTN/AKT/p38 pathway activation in heart and skeletal muscle after AMI, as well as the potential effect of cardiac injury-related MSTN endocrine signaling on skeletal muscle and other circulating growth factors.

METHODS: Coronary artery ligation was performed in C57BL/6 mice at age 8 weeks to induce AMI. Mice were sacrificed at different time points (10m, 1h, 2h, 6h, 12h, 24h, 1 week, 2 weeks, 1 months and 2 months) after surgery (n=3 per time point, n=18 total).

RESULTS: Cardiac and circulating MSTN upregulation occurred as early as 10 minutes after AMI. Two months after AMI, increased cardiac MSTN/SMAD2,3 and p38 together with decreased IGF-1/AKT signaling suggest an anti-hypertrophic profile. In skeletal muscle, an absence of local MSTN increase was accompanied by increased MSTN-dependent SMAD2,3 signaling, suggestive of paracrine effects due to cardiac-derived MSTN. Protein degradation by the ubiquitin-proteasome system in the skeletal muscle was also evident. Serum from 24h post-MI mice effectively induced a MSTN-dependent increase in atrogin1 and MuRF1.

CONCLUSION: Our study shows that cardiac MTSN activation occurs rapidly after cardiac ischemia and may be involved in peripheral protein degradation in the skeletal muscle by activating atrogin1 and MuRF1.

 

[Michael Scally MD]

Mendias CL, Bakhurin KI, Gumucio JP, Shallal-Ayzin MV, Davis CS, et al. Haploinsufficiency of myostatin protects against aging-related declines in muscle function and enhances the longevity of mice. Aging Cell. http://onlinelibrary.wiley.com/doi/10.1111/acel.12339/full

The molecular mechanisms behind aging-related declines in muscle function are not well understood, but the growth factor myostatin (MSTN) appears to play an important role in this process.

Additionally, epidemiological studies have identified a positive correlation between skeletal muscle mass and longevity.

Given the role of myostatin in regulating muscle size, and the correlation between muscle mass and longevity, we tested the hypotheses that the deficiency of myostatin would protect oldest-old mice (28-30 months old) from an aging-related loss in muscle size and contractility, and would extend the maximum lifespan of mice.

We found that MSTN+/- and MSTN-/- mice were protected from aging-related declines in muscle mass and contractility. While no differences were detected between MSTN+/+ and MSTN-/- mice, MSTN+/- mice had an approximately 15% increase in maximal lifespan.

These results suggest that targeting myostatin may protect against aging-related changes in skeletal muscle and contribute to enhanced longevity.

 

[Michael Scally MD]

Biesemann N, Mendler L, Wietelmann A, et al. Myostatin Regulates Energy Homeostasis in the Heart and Prevents Heart Failure. Circ Res. http://circres.ahajournals.org/content/115/2/296.full

RATIONALE: Myostatin is a major negative regulator of skeletal muscle mass and initiates multiple metabolic changes including enhanced insulin sensitivity. However, the function of Myostatin in the heart is barely understood although it is upregulated in the myocardium in several pathological conditions.

OBJECTIVE: Here, we aimed to decipher the role of Myostatin and Myostatin-dependent signaling pathways for cardiac function and cardiac metabolism in adult mice. To avoid potential counter-regulatory mechanisms occurring in constitutive and germ-line based Myostatin mutants we generated a mouse model that allows Myostatin inactivation in adult cardiomyocytes.

METHODS AND RESULTS: Cardiac MRI revealed that genetic inactivation of Myostatin signaling in the adult murine heart caused cardiac hypertrophy and heart failure partially recapitulating effects of the age-dependent decline of the Myostatin paralogue GDF11.

We found that Myostatin represses AMPK activation in the heart via TAK1 thereby preventing a metabolic switch towards glycolysis and glycogen accumulation. Furthermore, Myostatin stimulated expression of Rgs2, a GTPase activating protein that restricts Gaq and Gas signaling and thereby protects against cardiac failure. Inhibition of AMPK in vivo rescued cardiac hypertrophy and prevented enhanced glycolytic flow as well as glycogen accumulation after inactivation of Myostatin in cardiomyocytes.

CONCLUSIONS: Our results uncover an important role of Myostatin in the heart for maintaining cardiac energy homeostasis and preventing cardiac hypertrophy.

 

[Michael Scally MD]

Cohen TV, Kollias HD, Liu N, Ward CW, Wagner KR. Genetic disruption of Smad7 impairs skeletal muscle growth and regeneration. J Physiol. http://onlinelibrary.wiley.com/doi/10.1113/JP270201/abstract

The TGF-beta family of growth factors plays essential roles in mediating cellular growth and differentiation.

Myostatin is a muscle-specific member of the TGF-beta superfamily and a negative regulator of muscle growth. Myostatin inhibitors are currently being pursued as therapeutic options for muscle disorders.

Smad7 inhibits intracellular myostatin signalling via Smads2/3, and thus presents a means of regulating myostatin and potentiating muscle growth.

We investigated the functional loss of Smad7 on muscle in vivo by examining muscle growth and differentiation in mice deficient in Smad7 (Smad7-/- ). Smad7-/- mice showed reduced muscle mass, hypotrophy, and hypoplasia of muscle fibres, and increased oxidative fibre types.

Examination of muscle strength showed reduced force generation in vivo and ex vivo, compared to wild-type controls. Analysis of muscle regeneration showed a delay in recovery, likely due to decreased activation, proliferation, and differentiation of satellite cells, confirmed in vitro. Additionally, myostatin expression was upregulated in Smad7-/- muscle.

Our findings suggest that increased Smad2/3 signalling in the absence of Smad7 inhibition impedes muscle growth and regeneration. Taken together, our experiments demonstrate that Smad7 is an important mediator of muscle growth in vivo.


Our studies enhance the understanding of in vivo TGF-beta pathway modulation and suggest that Smad7 may be an important therapeutic target for muscle disorders.

 

[Michael Scally MD]

[Note: Lilly Research Laboratories, Eli Lilly and Company]

Smith RC, Cramer MS, Mitchell PJ, Capen A, Huber L, et al. Myostatin Neutralization Results in Preservation of Muscle Mass and Strength in Preclinical Models of Tumor Induced Muscle Wasting. Mol Cancer Ther. http://mct.aacrjournals.org/content/early/2015/04/22/1535-7163.MCT-14-0681.abstract

Skeletal muscle wasting occurs in a great majority of cancer patients with advanced disease and is associated with a poor prognosis and decreased survival.

Myostatin functions as a negative regulator of skeletal muscle mass and has recently become a therapeutic target for reducing the loss of skeletal muscle and strength associated with clinical myopathies.

We generated neutralizing antibodies to myostatin in order to test their potential use as therapeutic agents to attenuate the skeletal muscle wasting due to cancer. We show that our neutralizing anti-myostatin antibodies significantly increase body weight, skeletal muscle mass and strength in non-tumor bearing mice with a concomitant increase in mean myofiber area.

The administration of these neutralizing antibodies in two preclinical models of cancer induced muscle wasting (C26 colon adenocarcinoma and PC3 prostate carcinoma) resulted in a significant attenuation of the loss of muscle mass and strength with no effect on tumor growth.

We also show that the skeletal muscle mass and strength preserving effect of the antibodies is not affected by the co-administration of gemcitabine, a common chemotherapeutic agent, in both non-tumor bearing mice and mice bearing C26 tumors.

Additionally, we show that myostatin neutralization with these antibodies results in the preservation of skeletal muscle mass following reduced caloric intake, a common comorbidity associated with advanced cancer.

Our findings support the use of neutralizing anti-myostatin antibodies as potential therapeutics for cancer induced muscle wasting.

 

[Michael Scally MD]

Babcock LW, Knoblauch M, Clarke MS. The Role of Myostatin and Activin Receptor IIB in the Regulation of Unloading Induced Myofiber Type-Specific Skeletal Muscle Atrophy. J Appl Physiol (1985). http://jap.physiology.org/content/early/2015/07/20/japplphysiol.00762.2014

Chronic unloading induces decrements in muscle size and strength. This adaptation is governed by a number of molecular factors including myostatin, a potent negative regulator of muscle mass. Myostatin must first be secreted into the circulation and then bind to the membrane-bound activin receptor IIB (actRIIB) to exert its atrophic action. Therefore, we hypothesized that myofiber type-specific atrophy observed after hindlimb suspension (HLS) would be related to myofiber type-specific expression of myostatin and/or actRIIB.

Wistar rats underwent HLS for 10 days, after which the tibialis anterior (TA) was harvested for frozen cross sectioning. Simultaneous multi-channel immunofluorescent (IF) staining combined with differential interference contrast (DIC) imaging was employed to analyze myofiber type-specific expression of myostatin, actRIIB and myofiber type cross sectional area (CSA) across fiber types, myonuclei and satellite cells.

HLS induced significant myofiber type-specific atrophy in MHC-IIx (p < 0.05) and MHC-IIb myofibers (p < .05). Myostatin staining associated with myonuclei was less in HLS rats compared to controls, while satellite cell staining for myostatin remained unchanged.

In contrast, the total number myonuclei and satellite cells per myofiber was reduced in HLS compared to AMBU rats (p < .01). Sarcoplasmic actRIIB staining differed between myofiber types (I < IIa < IIx < IIb) independent of loading conditions. Myofiber types exhibiting the greatest cytoplasmic staining of actRIIB corresponded to those exhibiting the greatest degree of atrophy following HLS.

Our data suggest that differential expression of actRIIB may be responsible for myostatin induced myofiber type selective atrophy observed during chronic unloading.

 

[Michael Scally MD]

Lee YS, Lehar A, Sebald S, Liu M, Swaggart KA, et al. Muscle hypertrophy induced by myostatin inhibition accelerates degeneration in dysferlinopathy. Hum Mol Genet. http://hmg.oxfordjournals.org/content/early/2015/07/22/hmg.ddv288.abstract

Myostatin is a secreted signaling molecule that normally acts to limit muscle growth. As a result, there is extensive effort directed at developing drugs capable of targeting myostatin to treat patients with muscle loss.

One potential concern with this therapeutic approach in patients with muscle degenerative diseases like muscular dystrophy is that inducing hypertrophy may increase stress on dystrophic fibers, thereby accelerating disease progression.

To investigate this possibility, we examined the effect of blocking the myostatin pathway in dysferlin-deficient (Dysf-/-) mice, in which membrane repair is compromised, either by transgenic expression of follistatin in skeletal muscle or by systemic administration of the soluble form of the activin type IIB receptor (ACVR2B/Fc).

Here, we show that myostatin inhibition by follistatin transgene expression in Dysf-/- mice results in early improvement in histopathology but ultimately exacerbates muscle degeneration; this effect was not observed in dystrophin-deficient (mdx) mice, suggesting that accelerated degeneration induced by follistatin transgene expression is specific to mice lacking dysferlin. Dysf-/- mice injected with ACVR2B/Fc showed significant increases in muscle mass and amelioration of fibrotic changes normally seen in 8 month old Dysf-/- mice.

Despite these potentially beneficial effects, ACVR2B/Fc treatment caused increases in serum CK levels in some Dysf-/- mice, indicating possible muscle damage induced by hypertrophy.

These findings suggest that depending on the disease context, inducing muscle hypertrophy by myostatin blockade may have detrimental effects, which need to be weighed against the potential gains in muscle growth and decreased fibrosis.

 

[Michael Scally MD]

Ohsawa Y, Takayama K, Nishimatsu SI, Okada T, Fujino M, et al. The Inhibitory Core of the Myostatin Prodomain: Its Interaction with Both Type I and II Membrane Receptors, and Potential to Treat Muscle Atrophy. PLoS One. 2015;10(7):e0133713. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133713

Myostatin, a muscle-specific transforming growth factor-beta (TGF-beta), negatively regulates skeletal muscle mass. The N-terminal prodomain of myostatin noncovalently binds to and suppresses the C-terminal mature domain (ligand) as an inactive circulating complex.

However, which region of the myostatin prodomain is required to inhibit the biological activity of myostatin has remained unknown. We identified a 29-amino acid region that inhibited myostatin-induced transcriptional activity by 79% compared with the full-length prodomain.

This inhibitory core resides near the N-terminus of the prodomain and includes an alpha-helix that is evolutionarily conserved among other TGF-beta family members, but suppresses activation of myostatin and growth and differentiation factor 11 (GDF11) that share identical membrane receptors.

Interestingly, the inhibitory core co-localized and co-immunoprecipitated with not only the ligand, but also its type I and type II membrane receptors. Deletion of the inhibitory core in the full-length prodomain removed all capacity for suppression of myostatin.

A synthetic peptide corresponding to the inhibitory core (p29) ameliorates impaired myoblast differentiation induced by myostatin and GDF11, but not activin or TGF-beta1. Moreover, intramuscular injection of p29 alleviated muscle atrophy and decreased the absolute force in caveolin 3-deficient limb-girdle muscular dystrophy 1C model mice. The injection suppressed activation of myostatin signaling and restored the decreased numbers of muscle precursor cells caused by caveolin 3 deficiency.

Our findings indicate a novel concept for this newly identified inhibitory core of the prodomain of myostatin: that it not only suppresses the ligand, but also prevents two distinct membrane receptors from binding to the ligand.


This study provides a strong rationale for the use of p29 in the amelioration of skeletal muscle atrophy in various clinical settings.

 

[Michael Scally MD]

Latres E, Pangilinan J, Miloscio L, et al. Myostatin blockade with a fully human monoclonal antibody induces muscle hypertrophy and reverses muscle atrophy in young and aged mice. Skelet Muscle 2015;5:34. http://www.skeletalmusclejournal.com/content/5/1/34

BACKGROUND: Loss of skeletal muscle mass and function in humans is associated with significant morbidity and mortality. The role of myostatin as a key negative regulator of skeletal muscle mass and function has supported the concept that inactivation of myostatin could be a useful approach for treating muscle wasting diseases.

METHODS: We generated a myostatin monoclonal blocking antibody (REGN1033) and characterized its effects in vitro using surface plasmon resonance biacore and cell-based Smad2/3 signaling assays.

REGN1033 was tested in mice for the ability to induce skeletal muscle hypertrophy and prevent atrophy induced by immobilization, hindlimb suspension, or dexamethasone. The effect of REGN1033 on exercise training was tested in aged mice.

Messenger RNA sequencing, immunohistochemistry, and ex vivo force measurements were performed on skeletal muscle samples from REGN1033-treated mice.

RESULTS: The human monoclonal antibody REGN1033 is a specific and potent myostatin antagonist. Chronic treatment of mice with REGN1033 increased muscle fiber size, muscle mass, and force production.

REGN1033 prevented the loss of muscle mass induced by immobilization, glucocorticoid treatment, or hindlimb unweighting and increased the gain of muscle mass during recovery from pre-existing atrophy. In aged mice, REGN1033 increased muscle mass and strength and improved physical performance during treadmill exercise.

CONCLUSIONS: We show that specific myostatin antagonism with the human antibody REGN1033 enhanced muscle mass and function in young and aged mice and had beneficial effects in models of skeletal muscle atrophy.

 

[Michael Scally MD]

Generation of Gene-Modified Goats Targeting MSTN and FGF5 via Zygote Injection of CRISPR/Cas9 System

Genome-editing technologies rely on the use of engineered nucleases to induce cellular DNA repair mechanisms and introduce programmable, site-specific genetic modifications in diverse systems.

These programmable endonucleases include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and, most recently developed, the clustered regularly interspaced short palindromic repeats CRISPR-associated 9 (Cas9) system.

The CRISPR/Cas9 system uses short, single-guide RNAs (sgRNA) that recognize the target DNA, then programmed the Cas9 towards targets that are complementary to the first 20 nucleotides of the sgRNA2.

Compared with ZFNs and TALENs, the RNA-guided CRISPR/Cas9 system demonstrates its precious, versatile and robust merits for targeted genome editing in a variety of species, including model organisms, as well as crops and animals that are crucial to agriculture.

Wang X, Yu H, Lei A, et al. Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system. Scientific Reports 2015;5:13878. http://www.nature.com/articles/srep13878

Recent advances in the study of the CRISPR/Cas9 system have provided a precise and versatile approach for genome editing in various species.

However, the applicability and efficiency of this method in large animal models, such as the goat, have not been extensively studied.

Here, by co-injection of one-cell stage embryos with Cas9 mRNA and sgRNAs targeting two functional genes (MSTN and FGF5), we successfully produced gene-modified goats with either one or both genes disrupted.

The targeting efficiency of MSTN and FGF5 in cultured primary fibroblasts was as high as 60%, while the efficiency of disrupting MSTN and FGF5 in 98 tested animals was 15% and 21% respectively, and 10% for double gene modifications.

The on- and off-target mutations of the target genes in fibroblasts, as well as in somatic tissues and testis of founder and dead animals, were carefully analyzed.

The results showed that simultaneous editing of several sites was achieved in large animals, demonstrating that the CRISPR/Cas9 system has the potential to become a robust and efficient gene engineering tool in farm animals, and therefore will be critically important and applicable for breeding.

 

[Michael Scally MD]

First Gene-Edited [Myostatin] Dogs Reported in China
http://www.technologyreview.com/news/542616/first-gene-edited-dogs-reported-in-china/

[Zou Q, Wang X, Liu Y, et al. Generation of gene-target dogs using CRISPR/Cas9 system. J Mol Cell Biol. http://jmcb.oxfordjournals.org/content/early/2015/10/12/jmcb.mjv061.long]

Scientists in China say they are the first to use gene editing to produce customized dogs. They created a beagle with double the amount of muscle mass by deleting a gene called myostatin.

The dogs have “more muscles and are expected to have stronger running ability, which is good for hunting, police (military) applications,” Liangxue Lai, a researcher with the Key Laboratory of Regenerative Biology at the Guangzhou Institutes of Biomedicine and Health, said in an e-mail.
...

The dog researchers took much the same approach, directly introducing the gene-editing chemicals—a DNA snipping enzyme, Cas9, and a guide molecule that zeroes in to a particular stretch of DNA—into more than 60 dog embryos.

Their objective was to damage, or knock out, both copies of the myostatin gene so that the beagles’ bodies would not produce any of the muscle-inhibiting protein that the gene manufactures.

In the end, of 65 embryos they edited, 27 puppies were born, but only two, a female and a male, had disruptions in both copies of the myostatin gene. They named the female Tiangou, after the “heaven dog” in Chinese myth. They named the male Hercules.

Lai and his colleagues reported that in Hercules, the gene editing was incomplete, and that a percentage of the dog’s muscle cells were still producing myostatin.

But in Tiangou, the disruption of myostatin was complete and the beagle “displayed obvious muscular phenotype” characteristics—for example, her thigh muscles were large compared to those of her litter mates.

 

[Michael Scally MD]

Yano S, Nagai A, Isomura M, et al. Relationship between Blood Myostatin Levels and Kidney Function: Shimane CoHRE Study. PLoS One 2015;10(10):e0141035. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0141035

OBJECTIVES: Myostatin (MSTN), a member of TGF-beta superfamily, is produced in the skeletal muscle to inhibit myocyte differentiation. MSTN expression is increased in the skeletal muscle in patients with chronic kidney disease (CKD), which may play a role in the pathogenesis of sarcopenia or in the protein energy wasting (PEW).

This observation implies that the plasma MSTN level may be correlated with kidney function. Thus, we conducted a cross-sectional study to evaluate the association between the plasma MSTN concentration and the estimated glomerular filtration rate (eGFR).

SUBJECTS AND METHODS: Subjects were 781 participants of a health examination performed in a rural area in Japan. Among them, 124 subjects were selected by stratified random sampling according to eGFR. Creatinine clearance (ClCr) by the Cockcroft-Gault equation was used as a measure of kidney function. Plasma concentration of MSTN was determined by ELISA.

RESULTS: The plasma MSTN level was not different between men (3.42+/-1.61 ng/mL) and women (3.27+/-1.43 ng/mL). In a simple regression analysis, the MSTN level was significantly correlated with eGFR (r = -0.25, p<0.01) and ClCr (r = -0.20, p<0.05) but not with age and BMI. In a multiple linear regression analysis, the MSTN level showed a negative correlation with eGFR (standardized beta = -0.31, p<0.01) and ClCr (standardized beta = -0.35, p<0.01) under the adjustment with age, sex, BMI and LDL-C. Weak correlation was observed between the MSTN level and BMI / the serum LDL-C level.

When the subjects were stratified into 4 groups according to eGFR, MSTN was significantly greater in the groups with the lowest and the 2nd lowest eGFR (3.55+/-1.79 and 3.76+/-1.75 ng/mL, respectively) than the level in the group with the highest eGFR (2.77+/-0.85 ng/mL).

CONCLUSION: Plasma MSTN level was elevated in an early stage of CKD, which could be involved in the progression of sarcopenia.

 

[HuckingFuge]

I wish they would get on with the research, instead of playing around.