Treatment with rGDF11 does not improve the dystrophic muscle pathology of mdx mice
- Fabrizio Rinaldi†1,
- Yu Zhang†1, 2,
- Ricardo Mondragon-Gonzalez†1, 3,
- Jeffrey Harvey4 and
- Rita C. R. Perlingeiro1Email author
© Rinaldi et al. 2016
Received: 24 April 2016
Accepted: 6 May 2016
Published: 14 June 2016
Duchenne muscular dystrophy (DMD) is an inherited lethal muscle wasting disease characterized by cycles of degeneration and regeneration, with no effective therapy. Growth differentiation factor 11 (GDF11), a member of the TGF-β superfamily and myostatin homologous, has been reported to have the capacity to reverse age-related skeletal muscle loss. These initial findings led us to investigate the ability of GDF11 to promote regeneration in the context of muscular dystrophy and determine whether it could be a candidate to slow down or reverse the disease progression in DMD.
Here, we delivered recombinant GDF11 (rGDF11) to dystrophin-deficient mice using the intra-peritoneal route for 30 days and evaluated histology and function in both steady-state and cardiotoxin-injured muscles. Our data confirmed that treatment with rGDF11 resulted in elevated levels of this factor in the circulation. However, this had no effect on muscle contractility nor on muscle histology. Moreover, no difference was found in the number of regenerating myofibers displaying centrally located nuclei. On the other hand, we did observe increased collagen content, which denotes fibrosis, in the muscles of rGDF11-treated dystrophic mice.
Taken together, our findings indicate no beneficial effect of treating dystrophic mice with rGDF11 and raise caution to a potential harmful effect, as shown by the pro-fibrotic outcome.
KeywordsGDF11 Muscle regeneration Duchenne muscular dystrophy Fibrosis
Duchenne muscular dystrophy (DMD), the most common muscular dystrophy in the young, is characterized by progressive muscle wasting, in which skeletal muscle fibers are replaced by nonfunctional fibrotic and connective tissue . DMD is caused by mutations in the X-linked dystrophin gene, which is an essential element for the maintenance of muscle fiber integrity . Dystrophin and its associated proteins play an important role in protecting myofibers from contraction-induced damage. Loss of dystrophin leads to destabilization of the extracellular membrane resulting in rapid and continuous damage of the structural integrity of myofibers . Boys with DMD usually exhibit progressive motor difficulties and muscle wasting, and as a result, patients are wheelchair-bound by their teens, with eventual death due to cardiorespiratory insufficiency . Current therapeutic methods are only palliative as to date no effective treatment is available for DMD, or any type of muscular dystrophy.
Growth differentiation factor 11 (GDF11), a member of the transforming growth factor β (TGF-β) superfamily, has been recently described to promote muscle regeneration in the context of aging . GDF11 is highly homologous to myostatin, another TGF-β member also known as GDF8, sharing 89 % sequence identity throughout the mature active region. Accordingly, GDF11 and myostatin share the same receptors and similar signaling pathways [6, 7]. Despite this likeness, the aforementioned study suggested opposite effects to these ligands on skeletal muscle regeneration. Whereas myostatin has a well-known inhibitory effect on muscle growth and differentiation [8–11], this study demonstrated that reduced circulating levels of GDF11 are linked to age-associated muscle loss, also known as sarcopenia, and that increasing in its levels in old mice, upon a 30-day treatment with GDF11, reversed age-related dysfunction of the mouse skeletal muscle . A similar anti-aging effect for GDF11 has also been reported for the cardiac muscle  and brain , where this growth factor has been demonstrated to reverse age-related cardiac hypertrophy and neurologic defects, respectively.
We tested here whether GDF11 could ameliorate the progression of the muscle wasting phenotype observed in DMD by promoting muscle regeneration. For this, rGDF11 was administered systemically to dystrophin-deficient mdx mice for 30 days. To further test the regenerative ability of GDF11-treated mice, we damaged muscles from a cohort of treated and control mdx mice with cardiotoxin (CTX). Our findings show that increased levels of GDF11 in dystrophic mice have no effect on skeletal muscle regeneration and function. In fact, we observed evidence of fibrosis in GDF11-treated mice, as shown by the higher levels of collagen deposition in the tibialis anterior muscle, raising caution to the use of GDF11 in DMD.
All animal studies were performed according to protocols approved by the University of Minnesota Institutional Animal Care and Use Committee. Five-week-old male mdx mice (C57BL/10ScSn-Dmdmdx/J; stock number 001801), purchased from Jackson Laboratories (Bar Harbor, ME, http://www.jax.org), were treated with GDF11 or vehicle, as detailed below. In a cohort of these mice, we promoted muscle injury by injecting 15 μl of CTX (10 μM, Sigma) into the tibialis anterior (TA) muscle.
rGDF11 dosing and injections
We used the protocol used in the previous report for sarcopenia . rGDF11 stock solution was prepared by dissolving it in water containing 0.1 % BSA and kept at pH 3.8 with HCL, according to manufacturer’s instructions. Dystrophic mice were given a daily single intraperitoneal (IP) injection of either rGDF11 (PeproTech, Inc.) at 0.1 mg/kg or vehicle (water containing 0.1% BSA pH 3.8; at the same volume as GFD11) for 30 days. After 30 days, most mice were analyzed. At this point, a cohort of treated mice was muscle damaged by receiving 15 μl of CTX (10 μM, Sigma) into the TA. Muscle regeneration was evaluated 10 days later. During the 10-day period, this cohort of mdx mice continued to receive daily IP injections of rGDF11 or vehicle.
For histology, the TA and diaphragm muscles were harvested, embedded in OCT compound (Leica Biosystems), and then frozen in isopentane cooled in liquid nitrogen. Serial 10 μm cryosections were collected. For quantification of myofiber size and numbers, sections were stained for laminin. Muscle sections were fixed in 4 % PFA for 30 min, washed three times with PBS, blocked with 5 % BSA in PBS for 1 h at room temperature (RT), and then incubated with rabbit anti-laminin antibody (1:400; Sigma) overnight at 4 °C overnight. The following day, slides were washed three times in PBS and then stained with goat Alexa-555 anti-rabbit secondary antibody (Invitrogen) for 45 min at RT. To stain nuclei, we used ProLong® Gold Antifade reagent along with DAPI (molecular probes). Hematoxylin and eosin (H&E) staining was used for assessment of centrally located nuclei myofibers. Masson’s trichrome staining was utilized to detect collagen content (Polysciences, Inc.). Fiji ImageJ software was used to quantify myofiber size and numbers, as well as muscle cross-sectional area. To quantify the collagen, the colors from the figure were split and a threshold was used to identify the blue staining. Then, the complete muscle section was selected as a region of interest, and the percentage of area depicted by the threshold (equivalent to the collagen area) was measured also using ImageJ software.
Muscle preparation for mechanical studies
For the measurement of contractile properties, mice were anesthetized with ketamine/xylazine (80 mg/kg) and the intact TA muscles were dissected and placed in an experimental organ bath filled with mammalian Ringer solution containing (mM): NaCl 120.5; NaHCO3 20.4; glucose 10; KCl 4.8; CaCl2 1.6; MgSO4 1.2; NaH2PO4 1.2; pyruvate 1.0, adjusted to pH 7.4. The chamber was perfused continuously with 95 % O2–5 % CO2 and maintained at a temperature of 25 °C. The muscles were stimulated by an electric field generated between two platinum electrodes placed longitudinally on either side of the muscle (square wave pulses 25 V, 0.2 ms in duration, 150 Hz). The muscles were adjusted to the optimum length (Lo) for the development of isometric twitch force, and a 5-min recovery period was allowed between stimulations. Optimal muscle length (Lo) and stimulation voltage (25 V) were determined from micromanipulation of muscle length and a series of twitch contractions that produced maximum isometric twitch force. For measuring fatigue time, muscles were stimulated for 1 min and the time for force to decline to 30 % of Fo was measured. In brief, after determination of Lo and measurement of maximum isometric tetanic force, total muscle cross-sectional area (CSA) was calculated by dividing muscle mass (mg) by the product of muscle length (mm) and 1.06 mg/mm3, the density of mammalian skeletal muscle. Specific force (sFo) was determined by normalizing maximum isometric tetanic force (F0) to CSA. All of the mdx mice used for muscle physiology were also analyzed for histopathology.
Western blot analysis
Plasma samples were collected from rGDF11 and vehicle-treated mdx mice, as previously described , and quantified with the Bradford regent (Sigma). Briefly, samples were diluted in Laemmli buffer and boiled for 10 min at 100 °C. Equal amounts (75 μg) of protein from each single mouse were loaded onto 4–20 % gradient mini-PROTEAN® TGX Stain-Free™ gels (BioRad Labs). Proteins were transferred onto polyvinyl difluoride (PVDF) membranes (Millipore), which were then blocked in BSA 5 % Tris-buffered saline (TBS) with 0.05 % Tween-20 (TBST) for 1 h at RT. Membranes were incubated with rabbit anti-GDF11 antibody (1:1000, Abcam) overnight at 4 °C. The following day, these were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (GE healthcare). Proteins were detected by Pierce enhanced chemiluminescence (ECL) substrate (Thermo Scientific).
One-way or two-way analysis of variance (ANOVA) was used to analyze differences. When significant (p < 0.05), this was followed by the Tukey post hoc analysis.
Results and discussion
rGDF11 treatment has no effect on the muscle regeneration of mdx mice
Increased rGDF11 levels have no effect on muscle contractility
Higher collagen content in rGDF11-treated mice
The role of GDF11 in skeletal muscle regeneration has been controversial [8, 12, 15–18]. While the previous report by Sinha et al. demonstrated that systemic injection of rGDF11 reverses age-related dysfunction in the skeletal muscle , more recent studies, published while our study was in progress, contradict this rejuvenating effect [15, 16]. In fact, the latter agree with previous publications describing GDF11 as a negative regulator of myogenesis [17, 18], similar to its homologue, myostatin. Two recent publications by Egerman et al.  and Rodgers and Eldridge  reported that some of the assays previously used to detect GDF11  were not specific, and that GDF11 has an inhibitory effect on muscle regeneration inhibition and that levels of GDF11 actually increase with age. In response to this, Poggioli et al. have recently reported that the apparent age dependent increase in GDF11 levels, reported by Egerman et al. , is due to cross reactivity of the anti-GDF11 antibody with immunoglobulin, which is known to increase with age .
Despite this controversy in the activity of GDF11 in the context of aging, we addressed here whether GDF11 would have the ability to ameliorate the muscle wasting phenotype in the context of muscular dystrophy, by promoting muscle regeneration. Although daily doses of GDF11 treatment for 1 month resulted in elevated levels of this factor in the plasma, we were not able to detect any beneficial effect on the histology or strength in the muscles of treated dystrophic mice. We did not observe differences in terms of numbers of regenerating centrally nucleated myofibers. In contrast, we did observe an increase of collagen content, which denotes fibrosis, in the TA muscle of rGDF11-treated dystrophic mice compared to vehicle-injected controls.
Our study shows no beneficial effect of treating dystrophic mice with rGDF11 and raises caution to a potential harmful effect, as shown by the pro-fibrotic outcome.
analysis of variance
bovine serum albumin
Duchenne muscular dystrophy
growth differentiation factor 11
growth differentiation factor 8
hematoxylin and eosin
- TGF β:
transforming growth factor β
We thank the JB’s Key’s Fund for their generous support for this project. The project was also supported by NIH grant AR055299 (RCRP), the Muscular Dystrophy Center Core Laboratories P30-AR0507220, as well as funding from the MDA (#238127 to RCRP). Y.Z. and R.M-G. were funded by fellowships from the Oversea Study Program of Guangzhou Elite Project-China and CONACyT-Mexico (#394378), respectively.
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