Much progress but mysteries remain
Back in 1956, investigators at the National Institutes of Health described five members of a three-generation family, all of whom had experienced delayed motor development, with walking not achieved until age 4 or 5, and difficulty climbing stairs, running and changing from a back-lying to a sitting position.
Top photo: the cores in CCD-affected fibers run through the fiber, similar to the way lead runs through a pencil.
Middle and bottom photos: CCD-affected muscle fibers in cross-section at lower and higher microscopic magnification.
Muscle fiber photos courtesy of the Washington University Neuromuscular Disease Center (http://neuromuscular.wustl.edu)
Each of them underwent a biopsy of a thigh muscle and, in every case, the samples showed a previously unknown type of abnormality. If the muscle fibers were viewed in cross-section, areas that appeared to be almost amorphous (formless) could be seen in the central part of the fibers — a few muscle fibers in one case and almost every fiber in the other four cases. Viewed from the side, the amorphous areas ran almost the entire length of the fiber.
By the 1960s, the muscle disorder had received a name based on this phenomenon: central core disease. The cores were found to be devoid of metabolic activity and to lack crucial energy-producing structures called mitochondria.
It makes sense that metabolically inactive areas of muscle fibers would lead to problems with muscle function but, mysteriously, the number of cores isn’t well correlated with disease severity. Some people who have cores in almost all their fibers are minimally affected, while others, with few cores, are very weak.
The picture is further complicated by the fact that most people only undergo one or two muscle biopsies, so any change in the number or nature of the cores over time can’t be observed. In addition, any particular biopsy sample taken at a given time might not be representative of all the muscles.
A complicated dance
Although they disagree on the details, most experts believe cores reflect poor regulation of calcium inside muscle fibers, a conclusion that results from decades of study of muscle physiology, particularly the process called “excitation-contraction coupling” that underlies muscle movement and relaxation.
This process is a complicated dance involving the ebb and flow of many charged particles (ions) across the membrane that surrounds muscle fibers and, in the case of calcium ions, into and out of internal storage areas deep inside the fibers. (See “How Ion Channels Regulate Muscle Contraction,” below.)
“Excitation” refers to the process by which muscle fibers are stimulated by nerve fibers, and “contraction” refers to the process by which microscopic filaments inside each muscle fiber slide over each other, causing muscles to shorten, or contract.
It’s been known since the 1990s that malfunctions of the internal calcium release channel are the basic cause of most cases of CCD. But even now, the precise interactions among calcium release, calcium levels, core formation and muscle malfunction are not completely understood.
Identifying the calcium release channel
In the early 1980s, says muscle biologist Sidney Fleischer, “Researchers in the field were interested in diseases of muscle, but we didn’t know much about the molecular machinery.”
Researchers knew that a rise of calcium concentration in the main compartment of the muscle fiber is the messenger that triggers muscle to contract, and that uptake of calcium from the main part of the fiber back into the storage areas enables muscle to relax, says Fleischer, who received MDA funding to study muscle membranes back then, and who has since retired from his position as a professor in the department of molecular biology at Vanderbilt University in Nashville, Tenn.
Researchers also knew that when muscles relaxed, a molecular pump moved calcium ions into the internal storage compartment, and when muscles contracted, somehow calcium was released from this storage depot into the main compartment of muscle fibers.
However, researchers did not know what mechanism governed this release of calcium during contraction, says Fleischer.
Fleischer and many other scientists — particularly David MacLennan at the University of Toronto in Canada and Kevin Campbell at the University of Iowa (both of whom have received MDA support) — ultimately were able to decipher the calcium release mechanism.
It’s now understood that the internal calcium release channels in a muscle fiber — the “ryanodine receptors” — open in response to signals from different calcium channels, located on special indentations of the muscle fiber’s surface. These surface channels act as sensors of voltage changes.
The internal release of calcium through the ryanodine receptor is the next-to-last step before muscle contraction can occur. It allows calcium ions to surge out from the storage depots and combine with filaments in the fiber that slide over each other, causing muscle contraction.
If the channel doesn’t close back up again almost immediately, calcium will continue to leak out, leading to a prolonged muscle contraction; and the internal calcium stores will become depleted, leaving insufficient calcium for the next contraction.
Enter the geneticists
By the early 1990s, scientists had begun speculating that disorders of calcium release in general, and of the ryanodine receptor in particular, might underlie both central core disease and a dangerous reaction to inhaled anesthesia and certain muscle relaxants known as “malignant hyperthermia susceptibility,” or MHS.
Malignant hyperthermia — which affects many people who have CCD as well as many who don’t — is a phenomenon of prolonged and extreme muscle contraction and very high temperature elevation (hyperthermia) in response to halothane-type inhaled anesthetics and so-called depolarizing muscle relaxants, such as succinylcholine, often given during surgery. (See “Malignant Hyperthermia.")
In 1992, researchers had linked at least some cases of malignant hyperthermia susceptibility to mutations in the RYR1 gene, which carries instructions for the ryanodine receptors in skeletal muscles.
Then, in 1993, MacLennan’s group in Toronto and a separate group of European investigators linked mutations in RYR1 to CCD. Some of the RYR1 mutations cause both disorders, and some appear to cause only one or the other. However, it’s not possible to tell for certain whether or not someone is susceptible to MH if an anesthesia-related reaction hasn’t been experienced. (Susan Iannaccone advises anyone with an RYR1 mutation or a mutation in another gene, known as SEPN1, to assume he or she is at risk for MH.)
|How Ion Channels Regulate Muscle Contraction|
|Acetylcholine leaves the nerve fiber and docks on receptors in the muscle fiber membrane, causing parts of the fiber to become slightly more positively charged.|
|Sodium channels open in response to this small change, permitting a huge flow of positively charged sodium ions to enter the fiber and change the voltage.|
|The voltage change in the fiber is sensed by calcium channels located on indentations of the membrane. They then signal the calcium release channels (ryanodine receptors), which allow calcium to flow out from internal storage areas. The released internal calcium causes the filaments of the muscle fiber to slide over each other (contract).|
|To relax, all the above processes have to reset, with the internal calcium re-entering the storage areas and the release channels closing.|
Today, some 50 mutations in RYR1 have been found to cause CCD. Most are inherited in a dominant fashion, meaning a child needs to inherit the flawed gene from only one parent to show the disease. Occasionally, CCD appears to be inherited in a recessive pattern, meaning a gene mutation from each parent must be inherited before the disease manifests itself in a child.
Mutations in RYR1 that cause CCD can result in either leaky calcium channels, which lead to depletion of calcium from the internal stores and excess calcium outside them; or an inability of the calcium channel to open in response to voltage changes, known as “excitation-contraction uncoupling.”
Cores still a mystery
“The first RYR1 mutations discovered caused calcium leaks,” says muscle biologist Susan Hamilton, a longtime MDA research grantee who specializes in the ryanodine receptor and calcium release.
Scientists, looking for an explanation of the formless “core” that characterizes the disease, hypothesized that a calcium leak might cause a calcium overload, destroying the mitochondria in the center of the muscle fiber but not the mitochondria on the periphery of the fiber, which has systems to deal with calcium overload, explains Hamilton, a professor in the department of molecular physiology and biophysics at Baylor College of Medicine in Houston.
“But lo and behold,” says Hamilton, “other mutations in RYR1 create a block in the channel instead of a leak, so there’s less calcium leaking out, not more.”
As a result, she says, researchers have had to go back and review what they know about core formation. “We don’t know why cores form, and that’s the emphasis of a lot of research.”
In 2009, Hamilton was part of a study in which investigators inserted an RYR1 gene mutation into mice and observed the formation of cores in their muscle fibers over time.
They say they think the mice will provide a powerful new tool for investigations of muscle diseases like CCD.