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Losing muscle to fat: misdirected fate of a multipotent stem cell drives LGMD2B

Fibro/adipogenic precursors (FAPs) control the onset and severity of disease in limb-girdle muscular dystrophy type 2 (LGMD2B)

Fibro/adipogenic precursors (FAPs) control the onset and severity of disease in limb-girdle muscular dystrophy type 2 (LGMD2B). a) Healthy and/or pre-symptomatic LGMD2B muscle contains resident FAPs. b) After myofiber injury, inflammatory cells invade and trigger FAP proliferation. c) In symptomatic LGMD2B muscle, there is a gradual accumulation of extracellular AnxA2, which prolongs the pro-inflammatory environment, causing excessive FAP proliferation. d) Blocking aberrant signaling due to AnxA2 buildup blocks FAP accumulation and thus preventing adipogenic loss of dysferlinopathic muscle. Credit: “Fibroadipogenic progenitors are responsible for muscle loss in limb girdle muscular dystrophy 2B.” Published online June 3, 2019, in Nature Communications. Marshall W. Hogarth, Aurelia Defour, Christopher Lazarski, Eduard Gallardo, Jordi Diaz Manera, Terence A. Partridge, Kanneboyina Nagaraju and Jyoti K. Jaiswal. https://rdcu.be/bFu9U.

Research led by faculty at Children’s National published online June 3, 2019, in Nature Communications shows that the sudden appearance of symptoms in limb-girdle muscular dystrophy type 2 (LGMD2B) is a result of impaired communication between different cell types that facilitate repair in healthy muscle. Of particular interest are the fibro/adipogenic precursors (FAPs), cells that typically play a helpful role in regenerating muscle after injury by removing debris and enhancing the fusion of muscle cells into new myofibers.

LGMD2B is caused by mutations in the DYSF gene that impair the function of dysferlin, a protein essential for repairing injured muscle fibers. Symptoms, like difficulty climbing or running, do not appear in patients until young adulthood. This late onset has long puzzled researchers, as the cellular consequences of dysferlin’s absence are present from birth and continue through development, but do not impact patients until later in life.

The study found that in the absence of dysferlin, muscle gradually increases the expression of the protein Annexin A2 which, like dysferlin, facilitates repair of injured muscle fiber. However, increasing Annexin A2 accumulates outside the muscle fiber and drives an increase in FAPs within the muscle as well as encourages these FAPs to differentiate into adipocytes, forming fatty deposits. Shutting down Annexin A2 or blocking the adipocyte fate of FAPs using an off-the-shelf medicine arrests the fatty replacement of dysferlinopathic muscle.

“We propose a feed-forward loop in which repeated myofiber injury triggers chronic inflammation which, over time, creates an environment that promotes FAPs to accumulate and differentiate into fat. This, in turn, contributes to more myofiber damage,” says Jyoti K. Jaiswal, MSc, Ph.D., a principal investigator in the Center for Genetic Medicine Research at Children’s National and the study’s senior author.

“Adipogenic accumulation becomes the nucleating event that results in an abrupt decline in muscle function in patients. This new view of LGMD2B disease opens previously unrealized avenues to intervene,” adds Marshall Hogarth, Ph.D., the study’s lead author.

Joyti Jaiswal

“We propose a feed-forward loop in which repeated myofiber injury triggers chronic inflammation which, over time, creates an environment that promotes FAPs to accumulate and differentiate into fat. This, in turn, contributes to more myofiber damage,” says Jyoti K. Jaiswal, MSc, Ph.D.

A research team led by Jaiswal collaborated with Eduard Gallardo and Jordi Diaz Manera, of Hospital de la Santa Creu in Barcelona, Spain, to examine muscle biopsies from people with LGMD2B who had mild to severe symptoms. They found that adipogenic deposits originate in the extracellular matrix space between muscle fibers, with the degree of accumulation tied to disease severity. They found a similar progressive increase in lipid accumulation between myofibers predicted disease severity in dysferlin-deficient experimental models. What’s more, this process can be accelerated by muscle injury, triggering increased adipogenic replacement in areas that otherwise would be occupied by muscle cells.

“Accumulation and adipogenic differentiation of FAPs is responsible for the decline in function for dysferlinopathic muscle. Reversing this could provide a therapy for LGMD2B, a devastating disease with no effective treatment,” predicts Jaiswal as the team continues research in this field.

Promising off-the-shelf drugs include batimastat, an anti-cancer drug that inhibits the extracellular matrix enzyme matrix metalloproteinase. This drug reduces FAP adipogenesis in vitro and lessens injury-triggered lipid formation in vivo. In experimental models, batimastat also increases muscle function.

In addition to Jaiswal, Hogarth, Gallardo and Diaz Manera, other study co-authors include Aurelia Defour, Christopher Lazarski, Terence A. Partridge and Kanneboyina Nagaraju, all of Children’s National.

Financial support for research described in this post was provided by the Muscular Dystrophy Association under awards MDA477331 and MDA277389, the National Institute of Arthritis and Musculoskeletal and Skin Diseases under award R01AR055686 and the National Institutes of Health under awards K26OD011171, R24HD050846 and P50AR060836.

Sen Chandra Sreetama and Jyoti K Jaiswal

Modified glucocorticoid stabilizes dysferlin-deficient muscle cell membrane in experimental models

Sen Chandra Sreetama and Jyoti K Jaiswal

Limb girdle muscular dystrophy type 2B (LGMD2B) – a disease so rare that researchers aren’t even sure how many people it affects – is characterized by chronic muscle inflammation and progressively weakened muscles in the pelvis and shoulder girdle. It can affect able-bodied people during their childbearing years and makes it difficult to tiptoe, walk, run or rise unaided from a squat. Ultimately, many with the muscle-wasting condition require wheelchair assistance. There is no therapy approved by the Food and Drug Administration for this condition.

In a head-to-head trial between the conventional glucocorticoid, prednisolone, and a modified glucocorticoid, vamorolone, in experimental models of LGMD2B, vamorolone improved dysferlin-deficient muscle cell membrane stability and repair. This correlated with increased muscle strength and decreased muscle degeneration, according to a Children’s-led study published online Aug. 27, 2018, in Molecular Therapy. By contrast, prednisolone worsened muscle weakness, impaired muscle repair and increased myofiber atrophy.

“These two steroids differ by only two chemical groups,” says Jyoti K. Jaiswal, MSC, Ph.D., a principal investigator at Children’s National Health System and senior study author. “One made muscle repair better. The other made muscle repair worse or about the same as untreated experimental models. This matches experience in the clinic as patients with LGMD2B experienced increased muscle weakness after being prescribed conventional glucocorticoids, such as prednisolone.”

Healthy muscle cells rely on the protein dysferlin to properly repair the sarcolemmal membrane, a cell membrane specialized for muscle cells that serves a vital role in ensuring that muscle fibers are strong enough and have the necessary resources to contract. Mutations in the DYSF gene that produces this essential protein causes LGMD2B.

Jaiswal likens the plasma membrane to a balloon that sits atop the myofiber, a long cell that when healthy can flex and contract. If, in the process of myofiber contraction, the plasma membrane experiences anything out of sync or overly stressful, it develops a tear that needs to be quickly sealed. An intact balloon keeps air inside; tear it, and air escapes. When the plasma membrane tears, calcium from the outside leaks in, causing the muscle cell to collapse into a ball and die. The body contends with the dead cell by breaking it up into fragments and sending in inflammatory cells to clear the debris.

Lack of dysferlin is associated with increased lipid mobility in the LGMD2B cell membrane

Lack of dysferlin is associated with increased lipid mobility in the limb girdle muscular dystrophy type 2B (LGMD2B) cell membrane, which is further increased by injury and prednisolone treatment, causing failure of these cells to undergo repair. By contrast, vamorolone treatment stabilizes the LGMD2B muscle cell membrane to near healthy cell level, enabling repair of injured cells.

The study team got the idea for the current research project during a previous study of the experimental treatment vamorolone for a different type of muscular dystrophy. “In Duchenne muscular dystrophy (DMD), treatment with vamorolone not only reduced inflammation, but the membranes of muscle fibers were stabilized. That was the team’s ah-hah moment,” he says.

Three different doses of vamorolone were tested on cells derived from patients with LGMD2B with higher cell membrane repair efficacy seen with rising treatment dose. The dysferlinopathic experimental models were treated for three months with daily doses of cherry syrup laced with either 30 mg/kg of vamorolone or prednisolone or cherry syrup alone as the placebo arm.

“Right now there are zero treatments,” he says. People with LGMD2B turn to rehabilitative therapies and movement aids to cope with loss of mobility. Doctors are cautioned not to prescribe steroids. Jaiswal says many patients with LGMD2B grew up doing strenuous exercise, former athletes whose first indication of a problem was muscle cramping and pain. How this progresses to muscle weakness and loss is an area of active research in Jaiswal’s lab. “While additional research is needed, our findings here suggest that modified steroids such as vamorlone may be an option for some patients,” Jaiswal says.

“There is a nuance here: In addition to genomic effects, steroids also have physical effects on the cell membrane which may make some of the approved steroids ‘good’ steroids for dysferlinopathy that could selectively be used for this disease,” adds Sen Chandra Sreetama, lead study author.  Further research could indicate whether vamorolone, which is in Phase II human clinical trials for DMD, or any off-the-shelf drug could slow decline in muscle function for patients with LGMD2B.

Additional Children’s study authors include Goutam Chandra; Jack H. Van der Meulen; Mohammad Mahad Ahmad; Peter Suzuki; Shivaprasad Bhuvanendran; and Kanneboyina Nagaraju and Eric P. Hoffman, both of ReveraGen BioPharma.

Research reported in this news release was supported by the Clark Charitable Foundation; Muscular Dystrophy Association, under award number MDA277389; National Institute of Arthritis and Musculoskeletal and Skin Diseases, under award number R01AR055686; National Institutes of Health (NIH), under award numbers K26OD011171 and R24HD050846; and the District of Columbia Intellectual and Developmental Disabilities Research Center under NIH award number 1U54HD090257.