Muscle Wasting in Muscular Dystrophy Due to Defective Muscle Stem Cells, But Can Be Treated with Blood Pressure Drug


By utilizing a mouse model of Duchenne muscular dystrophy (DMD), researchers at Stanford University School of Medicine have compared gene expression differences between muscle stem cells from DMD mice and muscle stem cells from non-DMD mice. Muscle stem cells from DMD mice express connective-tissue genes associated with fibrosis and muscle weakness as opposed to those from non-DMD mice.

DMD mice, just like their human counterparts, experience progressive muscle degeneration and accumulate connective tissue within the muscle as they age. This new study strongly suggests that the stem cells that surround the muscle fibers might be responsible for this defect. During the course of the disease, muscle stem cells in DMD mice become less able to make new muscle and instead begin to express genes involved in the formation of connective tissue. Excess connective tissue causes scarring (a condition called fibrosis), and these excess scars can accumulate in other organs besides muscle, including the lungs, liver and heart. In the skeletal muscles of people with muscular dystrophy, scarring impairs muscle function and leads to increasing weakness and stiffness, which are hallmarks of the disease.

In addition to this discovery, Thomas Rando, professor of neurology at Stanford University Medical School, and his colleagues showed that these abnormal changes in muscle stem cells could be prevented in laboratory mice by giving the animals a drug that is already approved for use in humans. This drug blocks a signaling pathway involved in the development of fibrosis. Of course more work is required, but scientists are hopeful that a similar approach may one day help treat children with muscular dystrophy.

“These cells are losing their ability to produce muscle, and are beginning to look more like fibroblasts, which secrete connective tissue,” said Dr. Rando. “It’s possible that if we could prevent this transition in the muscle stem cells, we could slow or ameliorate the fibrosis seen in muscular dystrophy in humans.”

Rando and his coworkers published their findings in Science Translational Medicine. Rando, who is the senior author of this paper, is also the director of the Glenn Laboratories for the Biology of Aging and is also the founding director of the Muscular Dystrophy Association Clinic at Stanford. Rando’s former postdoctoral scholar Stefano Biressi, who is presently at the Centre for Integrative Biology at the University of Trento in Italy, is the lead author of this paper.

DMD is a truly devastating disease that affects about 1 in every 3,600 boys born in the United States. The hallmark of this disease is the severe, progressive muscle weakness that confines patients to a wheelchair by early adolescence and eventually leads to paralysis. Mutations in the dystrophin gene cause DMD. The dystrophin gene encodes the Dystrophin protein, which connects muscle fibers to the surrounding external matrix, which stabilizes the fibers, enhances their strength and prevents their injury. Mutations in the dystrophin gene cause production of defective copies of the dystrophin protein. Without functional copies of Dystrophin, the unanchored muscle is unstable, weak, and subject to constant injury. DMD patients are almost always boys because the dystrophin gene is located on the X chromosome. Girls must inherit two faulty copies of the dystrophin gene to contract DMD, which is unlikely because male carriers often die in early adulthood.

By decelerating the fibrotic activity of muscle stem cells in DMD patients, it is possible to delay or even fix the scarring observed in human DMD patients. Normally, muscle stem cells are stimulated when muscles are damaged, and they divide into new cells, some of which form new muscle. In DMD mice, however, muscle stem cells the lack a functional copy of the dystrophin gene slowly begin to resemble fibroblasts instead of muscle-making stem cells.

In this study, Biressi and Rando used a strain of laboratory mice in which the muscle stem cells express a glowing protein when they are treated with a drug called tamoxifen. These glowing mice were then mated with another mouse strain that had a defective copy of the dystrophin gene. These DMD mice now had muscle stem cells that glowed when treated with tamoxifen, which allowed Biressi, Rando and others to trace the movements and activities of muscle stem cells. They discovered that the expression of myogenic genes associated with the regeneration of muscle in response to injury was nearly completely lacking in many of the muscle stem cells in the mice after just 11 months. However, the expression of fibrotic genes had increased compared with that of control animals. The muscle stem cells from the DMD animals were also oddly located, since instead of being nestled next to the muscle fibers where they normally are found, they had begun to move away into the spaces between tissues.

Such increased fibrosis is also observed during normal aging and this process is governed by signaling proteins, which include the Wnt and TGF-beta protein families. Wnt plays a critical role in embryonic development and cancer; TGF-beta controls cell division and specialization. Rando and Biressi hypothesized that inhibiting the Wnt/TGF-beta pathway in DMD would inhibit fibrosis in the animals’ muscles.

To do this, they turned to a blood pressure medicine called losartan. Losartan inhibits the expression of the genes for TGF-beta types 1 and 2, and therefore, might interrupt the signaling pathway that leads the muscle stem cells astray. When DMD mice were treated with losartan, the drug prevented the muscle stem cells from expressing fibrosis-associated genes and partially maintained their ability to form new muscle.

“This scar tissue, or fibrosis, leaves the muscle less elastic and impairs muscle function,” Rando said. “So we’d like to understand why it happens, and how to prevent it. It’s also important to limit fibrosis to increase the likelihood of success with other possible therapies, such as cell therapy or gene therapy.”

TGF-beta-1 is an important signaling molecule throughout the body. Therefore, researchers are now working to find ways to specifically inhibit TGF-beta-2, which is involved in the transition of the muscle stem cells from muscle makers to scar producers. They’re also interested in learning how to translate the research to other diseases.

“Fibrosis seems to occur in a vicious cycle,” Rando said. “As the muscle stem cells become less able to regenerate new muscle, the tissue is less able to repair itself after damage. This leads to fibrosis, which then further impairs muscle formation. Understanding the biological basis of fibrosis could have a profound effect on many other diseases.”

Reducing the Heart Scar After a Heart Attack


After a heart attack, inflammation in the heart kills off heart muscle cells and fibroblasts in the heart make a protein called collagen, which forms a heart scar. The heart scar does not contract and does not conduct electrochemical signals. The scar will contract over time, but its presence can lead to abnormal heart rhythms, also known as arrhythmias. Arrythmias can be fatal, since they can cause a heart attack. To prevent a heart attack, physicians will treat heart attack patients with a group of drugs called beta-blockers that slow down the heart rate and protect the heart from the deleterious effects of norepinephrine (secreted by the sympathetic nerve inputs to the heart). An alternative treatment is digoxin or digitalis, which is a chemical found in foxglove. Digitalis inhibits ion pumps in heart muscle cells and slows the heart and the force of its contractions. Digitalis, however, interacts with a whole shoe box fill of drugs, has a very long half-life, and is hard to dose. Therefore it is not the first choice.

Given all this, helping the heart to make a smaller heart scar is a better strategy for treating a heart after a heart attack. To accomplish this, you need to inhibit the heart fibroblasts that make the heart scar in the first place. Secondly, you must move something into the place of the dead cells. Otherwise, the heart could burst or scar tissue will move into the area anyway.

To that end, Yigang Wang and his colleagues at the University of Cincinnati Medical Center in Ohio have published an ingenious paper in which they tried two different strategies to reduce the size of the heart scar, which concomitantly increased the colonization of the heart by induced pluripotent stem cells engineered to express a sodium-calcium exchange pump.

Previously, Wang and his colleagues used a patch to heal the heart after a heart attack. The patch consisted of endothelial cells, which make blood vessels, induced pluripotent stem cells engineered to make a sodium-calcium exchange pump called NCX1, and embryonic fibroblasts. This so-called tri-cell patch makes new blood vessels, establishes new heart muscle, and the foundational matrix molecules to form a platform for beating heart muscle.

In order to get these cells to spread throughout the injured heart, Wang and others used a reagent that specifically inhibits heart fibroblasts. They used a small non-coding RNA molecule. A group of microRNAs called miR-29 family are downregulated after a heart attack. As it turns out, these microRNAs inhibit a group of genes that involved in collagen deposition. Therefore, by overexpressing miR-29 microRNAs, they could prevent collagen deposition and reduce scar formation.

The experimental design in this paper is rather complex. Therefore, I will go through it slowly. First, they tried to overexpress miR-29 microRNAs in cultured heart fibroblasts and sure enough, they inhibited collagen synthesis. Cells overexpressing miR-29 made less than a third of the collagen of their normal counterparts. When they placed these fibroblasts into the heart and induced heart attacks, again, they made significantly less collagen when they were expressing miR-29.

Then they used their miR-29 RNAs by injecting them directly into the heart before inducing a heart attack, and then after the heart attack, they applied the tri-patch. Their results were significant. The scar size was smaller (almost one-third the size of the controls), and the density of blood vessels was much higher in the tri-patched hearts treated with miR-29. The induced pluripotent stem cells differentiated into heart muscle cells and spread throughout the heart. Heart function measures also consistently went up too.  The echiocardiograph before more normal, the ejection fraction went up, the % shortening of the heart muscle fibers was increased, and the relaxation phase of the heart (diastole) also was not so puffy (see graphs and figures below).

(A): M-mode echocardiograph data in three groups. (B): Quantification analysis for heart function. Quantitative data for LVDd (B-1), LVDs (B-2), EF (B-3), and FS (B-4) 4 weeks after Tri-P implantation. *p,0.05 vs. Ctrl+MI+Tri-P group; {p,0.05 vs. Anti-29b+MI+Tri-P group. LVDd, left ventricular enddiastolic diameters; LVDs, left ventricular end-systolic diameters; EF, ejection fraction index; FS, fractional shortening. All values expressed as mean 6 SEM. n = 6 for each group. (C): Two-D mode echocardiograph data in three groups, analyzed by long-axis and short-axis views. *p,0.05 vs. Ctrl+MI+Tri-P group; {p,0.05 vs. miR-29b+MI+Tri-P group. Ctrl, control mimic pretreatd rat with Tri-cell patch graft; miR-29b, miR- 29b mimic pretreated rat with Tri-cell patch graft; Anti-29b, miR-29b inhibitor pretreated rat with Tri-cell patch graft. White dotted lines indicate endocardium and epicardium.
(A): M-mode echocardiograph data in three groups. (B): Quantification analysis for heart function. Quantitative data for LVDd (B-1), LVDs (B-2), EF (B-3), and FS (B-4) 4 weeks after Tri-P implantation. *p,0.05 vs. Ctrl+MI+Tri-P group; {p,0.05 vs. Anti-29b+MI+Tri-P group. LVDd, left ventricular enddiastolic diameters; LVDs, left ventricular end-systolic diameters; EF, ejection fraction index; FS, fractional shortening. All values expressed as mean 6 SEM. n = 6 for each group. (C): Two-D mode echocardiograph data in three groups, analyzed by long-axis and short-axis views. *p,0.05 vs. Ctrl+MI+Tri-P group; {p,0.05 vs. miR-29b+MI+Tri-P group. Ctrl, control mimic pretreatd rat with Tri-cell patch graft; miR-29b, miR-29b mimic pretreated rat with Tri-cell patch graft; Anti-29b, miR-29b inhibitor pretreated rat with Tri-cell patch graft. White dotted lines indicate endocardium and epicardium.

There is a cautionary note to this study. Inhibiting collagen formation after a heart attack could create soft fragile regions of the heart that are subject to rupture should the vascular systolic pressure increase. While that threat was not observed in this study, human hearts, which are much larger, would be much more susceptible to such a mishap. Therefore, while this study is interesting and suggest a strategy in humans, it requires more testing and refinement before anyone can even think about applying it to humans.