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NeuroTech-Neuromuscular Disorders
Novel Muscle System Quantifies Statin-Induced Myopathy Over Weeks

ARTICLE IN BRIEF

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DR. GEORGE TRUSKEY: “Our goal was to create a three-dimensional in vitro model that could reproduce many of the key features of muscle physiology, and on which we could perform measurements over a significant period of time. We wanted to look at functional measures, not just immunohistochemical expression of proteins.”

Researchers developed a novel system to monitor long-term statin-induced myopathy.

A new technique provides a way to monitor contractile forces in human muscle in vitro over several weeks, potentially offering a unique window on the long-term effects of drugs such as statins on muscle physiology. In the system, muscle fibers are linked to stretchable silicone, the same material Hollywood uses to make highly life-like masks. The paper was published in September in the journal Lab on a Chip.

But it may be too soon to know whether the complex system will find wide application in the study of muscle disease, one expert told Neurology Today.

Measurement of muscle force in isolated myotubes has been done in a variety of ways, but each method has had its limitations, said the study's senior author, George Truskey, PhD, professor of biomedical engineering at Duke University in Durham, NC. For instance, myotubes grown in a dish “are somewhat disorganized in structure, and there is a limited window for working with them before they pull themselves off the substrate,” Dr. Truskey said.

“Our goal was to create a three-dimensional in vitro model that could reproduce many of the key features of muscle physiology, and on which we could perform measurements over a significant period of time,” he said. “We wanted to look at functional measures, not just immunohistochemical expression of proteins.”

STUDY DETAILS

To do that, Dr. Truskey and colleagues built a miniature rigid nylon frame, to which they attached one end of a thin sheet of Ecoflex silicone film. This material is widely used for special effects (think Yoda in “Star Wars”), as well as advanced prosthetics, because it stretches easily and then faithfully rebounds to its original length. They grew human myotubes within this frame, allowing the myotubes to attach to the Ecoflex on one end, and to the frame at the other end.

As the myotubes contract, they generate tension, Dr. Truskey explained, and that increases their alignment and furthers their maturation. Contraction also pulls on the Ecoflex, stretching it. The degree of stretch is determined by measuring the displacement of tiny beads embedded in the film, allowing the team to determine the force required for the observed stretch. Unlike most other systems for measurement of myotube contractile force, which bend a cantilever sensor, here the force causes a linear displacement, which is much easier to calibrate to determine the actual force exerted by the cells, Dr. Truskey said.

An additional advantage of the system, he noted, is that it can be used to track the dynamics of either passive or induced force production, and can provide information about parameters such as relaxation time as well.

And because they are long-lived — the myotubes continue to function for up to four weeks — the system can measure the evolution of force over time, and perhaps most importantly, the effects of chronic exposure to drugs.

To explore the utility of the system for detecting myotoxicity, Dr. Truskey exposed the myofibers in their frame to 100 nanomolar cerivastatin, a cholesterol-lowering drug that was withdrawn from the market in 2001 after multiple cases of rhabdomyolysis-induced deaths were reported. The dose was chosen to match the concentration patients were exposed to when they took the drug.

Both treated and control myotubes increased their force output over several days, but the force generated in response to stimulation was less in the treated group, and after four days of treatment, was about half that of the controls. Treatment also led to degradation of sarcomeric alpha-actinin in the myotubes.

Like other statins, cerivastatin blocks cholesterol synthesis by preventing formation of the intermediate mevalonate. Dr. Truskey found that addition of mevalonate to the medium mitigated both the force reduction and the actinin-related effects of cerivastatin on the myotubes, confirming the physiological relevance of the drug-induced force-reduction effect seen in the system.

Had this system been available at the time, might testing cerivastatin's myotoxicity in it have prevented the drug from going to market in the first place? Dr. Truskey is not sure.

“These results would certainly have caused some concern,” he said. “You don't necessarily pick up all the toxic signals in animal studies, and so this might be a way to reveal effects you wouldn't see otherwise.” Lovastatin, he noted, is notably less toxic, and in his prior work, only induced myotoxicity at doses outside its therapeutic window.

The myotube-Ecoflex system can also be used to assess other aspects of muscle physiology, Dr. Truskey said, including calcium transients and oxygen consumption. “We'd also like to be able to replicate the normal glucose uptake you see in skeletal muscle, but that's been more challenging,” in part because it is difficult to mimic the capillary-rich environment of the muscle cell in vivo, “but we are trying to get close.”

Dr. Truskey noted that the major funder of the project, the National Center for Advancing Translational Sciences, also funded a second group of investigators to replicate the system. “There was a learning curve to get to the point that they could set it up and work with it without too much difficulty,” he said, “and then they used it pretty much as we described it.”

EXPERT COMMENTARY

“On first pass, this is a highly attractive system,” commented Steven Baker, MD, associate professor of physical medicine and rehabilitation at McMaster University in Hamilton, Ontario, who researches statin-associated neuromuscular toxicity. “But to apply it more broadly may be challenging, as it is clearly a highly specialized platform that may not be available for many laboratories.”

The demonstration that cerivastatin impairs contractile force, and that mevalonate can mitigate it, was known at least in outline, Dr. Baker pointed out, “but has not been quite this clearly defined before.”

He said he would have preferred to see the system used for a head-to-head comparison of cerivastatin with other clinically available statins, to see how they affect force generation in this model. “Overall I would say this system is not quite ready for application to the mainstream clinical arena, but the field of statin myotoxicity needs to be pushed forward, and I applaud all work in the field. There is certainly more that can be done with this system to explore this important problem.”

LINK UP FOR MORE INFORMATION:

• Zhang X, Hong S, Yen R, et al. A system to monitor statin-induced myopathy in individual engineered skeletal muscle myobundles https://pubs.rsc.org/en/content/articlelanding/2018/lc/c8lc00654g#!divAbstract. Lab Chip 2018;18(18):2787–2796.