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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.

Jyoti Jaiswal and Adam Horn

Antioxidants could thwart muscle repair

Science Signaling cover image 05Sept17

Science Signaling features a Research Article that describes the pathway by which mitochondria transduce the increase in cytosolic Ca2+ caused by plasma membrane injury into a ROS-dependent repair response. The image shows ROS production and actin polymerization as detected by fluorescent reporters near a plasma membrane injury site in a skeletal myofiber in an intact bicep of an experimental model. Credit: Adam Horn and Jyoti Jaiswal, M.S.C., Ph.D. Children’s National Health System and The George Washington University School of Medicine and Health Sciences

Reactive oxygen species (ROS) are a biological double-edged sword. These atoms, molecules or molecular fragments containing oxygen that is poised for chemical reactions, are a key part of the immune response, used by immune cells to kill potentially dangerous invaders such as bacteria. However, too much ROS – which also are produced as a normal part of cellular metabolism – can cause extreme damage to normal, healthy cells.

Because oxidative damage has been linked with cancer, many people make a concerted effort to consume antioxidants in food and as concentrated supplements. These compounds can neutralize ROS, stemming cellular damage. Taking antioxidants also has been thought to stem the muscle soreness from exercise since ROS are produced in excess during hard physical activity.

However, a new study led by researchers from Children’s National Health System finds that taking antioxidants could thwart the processes that repair muscle fibers. According to the study published Sept. 5, 2017 in Science Signaling and featured on the journal’s cover, oxidative species are crucial signals that start the process of repairing muscle fibers.

Cellular powerhouses known as mitochondria help injured muscle cells (myofibers) repair by soaking up calcium that enters from the site of injury and using it to trigger increased production of reactive oxygen species. Loading up mitochondria with excess antioxidants inhibits this signaling process, blocking muscle repair, exacerbating myofiber damage and diminishing muscle strength.

“Our results suggest a physiological role for mitochondria in plasma membrane repair in injured muscle cells, a role that highlights a beneficial effect of reactive oxygen species,” says Jyoti K. Jaiswal, M.S.C., Ph.D., principal investigator in the Center for Genetic Medicine Research at Children’s National Health System, associate professor of genomics and precision medicine at The George Washington University School of Medicine and Health Sciences and senior study author. “Our work highlights the need to take a nuanced view of the role of reactive oxygen species, as they are necessary when they are present at the right place and right time. Indiscriminate use of antioxidants actually could harm an adult with healthy muscles as well as a child with diseased muscle.”

Antioxidants are widely used by Baby Boomers with muscles that ache from a grueling workout or newborns diagnosed with muscular dystrophy. Jaiswal and Children’s National colleagues understand that their results buck conventional wisdom that antioxidants generally benefit muscle recovery.

“It is still a common belief within the fitness community that taking antioxidant supplements after a workout will help your muscles recover better. That’s what people think; that’s what I thought,” says Adam Horn, lead study author, a graduate student at The George Washington University who works with Jaiswal at Children’s National. “What we’ve done is figure out that mitochondria need to produce a very specific oxidative signal in response to muscle damage in order to help injured muscles repair.”Jyoti Jaiswal and Adam Horn

The oxidative signals produced by mitochondria are delicately balanced by the antioxidant defenses in healthy cells. This balance can be disrupted in diseases such as Duchenne muscular dystrophy, which is caused by the lack of a muscle-specific protein, dystrophin. Lack of dystrophin makes the muscle cell plasma membrane more vulnerable to injury. In an experimental model of Duchenne muscular dystrophy, the muscles at birth are seemingly normal but, within weeks, show obvious muscle damage and progressive weakness.

“What changes? One of the things that changes in the third and fourth week of life of this experimental model is mitochondrial functionality,” Jaiswal adds. “They end up with many dysfunctional mitochondria, which compromise repair of injured myofibers. This permits chronic and excessive oxidation of the myofibers and disruption of the proper oxidant-antioxidant balance.”

In this case, a dose of antioxidants may restore that proper balance and help to reverse muscle damage and progressive weakness.

As a next step, the research team is examining oxidation in healthy and diseased muscle to understand how the oxidant-antioxidant balance is disrupted and how it could be restored efficiently by using existing supplements. In one such study funded by the National Institutes of Health, the team is looking at the potential benefit of vitamin E supplements for patients with muscular dystrophy.

“Antioxidant supplements are made from extracts of bark, sap, chocolate and other compounds so they’re all different,” Jaiswal says. “Knowing which ones can restore balance under a specific circumstance has the potential to help the body maintain proper cellular signaling ability, which will keep muscles healthy and working properly.”


The response of actin protein following injury to a pair of muscle fibers in an intact biceps muscle.

mitochondria

Mitochondria key for repairing cell damage in DMD

mitochondria

A research team led by Jyoti K. Jaiswal, M.S.C., Ph.D., found that dysfunctional mitochondria prevent repair of muscle cells in Duchenne muscular dystrophy.

What’s known

Duchenne muscular dystrophy (DMD), one of the most severe forms of muscular dystrophy, is caused by a defect in the dystrophin gene. The protein that this gene encodes is responsible for anchoring muscle cells’ inner frameworks, or cytoskeletons, to proteins and other molecules outside these cells, the extracellular matrix. Without functional dystrophin protein, the cell membranes of muscle cells become damaged, and the cells eventually die. This cell death leads to the progressive muscle loss that characterizes this disease. Why these cells are unable to repair this progressive damage has been unknown.

What’s new

A research team led by Jyoti K. Jaiswal, M.S.C., Ph.D., a principal investigator in the Center for Genetic Medicine Research at Children’s National Health System, investigated this question in two experimental models of DMD that carry different mutations of the dystrophin gene. The researchers monitored the effects of the lack of functional dystrophin protein in these preclinical models on the level and function of muscle cell. They found that mitochondria – organelles that act as powerhouses to supply the chemical energy to drive cellular activities – are among the first to be affected. They found that the decline in mitochondrial level and activity over time in these experimental models preceded the onset of symptoms. The research team also looked at the ability of the experimental models’ muscle cells to repair damage. As the muscle cell mitochondria lost function, the cells’ ability to repair damage also declined. Efforts to increase mitochondrial activity after these organelles became dysfunctional did not improve muscle repair. This suggests that poor muscle repair may not be caused by a deficit in energy production by mitochondria.

Questions for future research

Q: Does similar mitochondrial dysfunction occur in human patients with DMD?
Q: How can the mitochondrial dysfunction be prevented?
Q: Is there a way to reverse mitochondrial dysfunction to better preserve the ability of muscle cells to repair from DMD-related damage?

Source: “Mitochondria mediate cell membrane repair and contribute to Duchenne muscular dystrophy.” Vila, M.C., S. Rayavarapu, M.W. Hogarth, J.H. Van der Meulen, A. Horn, A. Defour, S. Takeda, K.J. Brown, Y. Hathout, K. Nagaraju and J.K. Jaiswal. Published by Cell Death and Differentiation February 2017.