Of Mice and Men...and Muscle (part 3)

Read my previous posts on myostatin here and here.

Meet the whippets (click for larger version):

Whippets are a breed of dog developed specifically for racing. They were officially recognized as a breed in the late 1800s and can reportedly run up to 35 mph.

Take another look at the photos of whippets above. See anything interesting? If you look close, you might notice that the dogs get bigger from left to right. The heavily muscled dogs in the right column are referred to as ‘bully’ whippets by breeders. Apparently, whippet breeders brought this trait to the attention of the National Human Genome Research Institute at NIH. Knowing about the role of myostatin mutations in producing other examples of double muscling, the researchers went looking for mutations in the myostatin gene of whippets.

The results are pretty nicely summed up by the pictures. Wild type whippets (lacking any mutation in the myostatin gene) are on the far left, heterozygotes (dogs that possess one copy of a normal myostatin gene and one copy with a mutation that produces a malfunctioning protein) in the middle, and individuals (bullies) with two copies of the mutation on the right.

If that was the end of the story, it would be interesting to scientists (and useful to dog breeders), but not much else. What makes this more newsworthy is that the researchers were able to connect the genetics with athletic performance.

I don’t know anything about dog racing, but racing whippets are apparently divided into four classes: A, B, C, D. Think of it like the system used in professional baseball in the US (or like soccer leagues in the rest of the world). The fastest dogs are A, the slowest in D. It turns out that dogs with the mutation make up a disproportionate number of dogs in the faster classes. We now have a quantitative connection between genes and performance. (I should point out that the faster dogs almost always possess only one copy of the mutation, not two. I don’t think dogs with two copies of the mutation generally are raced. Muscle bound perhaps?)

This brings me back to the boy in Germany who also has two mutated copies of the myostatin gene. His mother, who was a professional athlete and comes from a family with several members noted for their strength, has one copy of the mutation.

Given that high level athletes have shown themselves willing to do lots of things to gain an advantage over the competition, and knowing that drugs are in the pipeline that would inhibit myostatin, how long will it be before athletes are trying to build muscle by blocking their myostatin? Or how long before the national Olympic training programs or college scouts start screening for these mutations to guarantee that funds for training are spent on athletes with the best genes?

As the authors of the whippet research note:

“Our findings have implications for competitive and professional sports. Here, we show that a disruption in the function of the MSTN [myostatin] gene increases an individual’s overall athletic performance in a robust and measurable way. …

The potential to increase an athlete’s performance by disrupting MSTN either by natural or perhaps artificial means could change the face of competitive human and canine athletics. Given the poorly understood consequences for overall health and well-being, caution should be exercised when acting upon these results.”

References:

1. Mosher, D.S. et al. (2007). "A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs." PLoS Genetics 3(5): e79 (doi:10.1371/journal.pgen.0030079)

Of Mice and Men...and Muscle (part 2)

For part 1 of my post on myostatin, go here.

From the beginning, it was apparent to researchers that myostatin might provide treatment options for musculodegenerative diseases like muscular dystrophy. In 2002, researchers showed that by blocking activity of the protein produced by the myostatin gene with an antibody, mice from a strain used as a mouse model of Duchenne muscular dystrophy (known as mdx mice) had greater body weight, muscle mass, and muscle strength than mdx mice that did not receive the antibody. The same research group repeated the research in a study published in 2005 using a different method to block myostatin and got similar results.

[Note on figure: EDL = extensor digitorum longus. In humans, this muscle is located in your shin and is one of several muscles that acts in dorsiflexion of foot. (To do this, stand with your feet flat on ground and raise the ball of your foot off the floor.)]

Unfortunately, research on other strains of mice wasn’t so positive. Using a strain of mice (referred to as dy^W/dy^W) that acts as a model of a different kind of muscular dystrophy, researchers in southern California found that, although the mice without myostatin had more muscle, they suffered from the same clinical problems that the control mice did. Even worse, it appeared that a decrease in fat due to the lack of myostatin (lack of myostatin makes muscles bigger with less fat) caused increased mortality in the mice. So, not only did blocking myostatin not help, it actually made things worse.

A year later, another research group (this time from Ohio and Chicago) examined the effects of blocking myostatin in a third strain of mice. This strain, known as scgd ^-/^-, lacks a molecule called δ-sarcoglycan, a protein that sticks out from the cell membrane and forms part of a complex of molecules that helps stabilize the cell membrane of muscle cells. In humans, a similar defect produces limb-girdle muscular dystrophy, a very rare type of MD.

The outcome of this research emphasized the variability in blocking myostatin. Using mice that had received an antibody blocking myostatin beginning at 4 weeks old and other mice that began receiving the antibody at 20 weeks old, the researchers found that the younger mice received greater benefits than did the older mice. Also, reaction to the treatment varied depending on the particular muscle group under consideration. For example, the quadriceps (thigh muscle) was larger in the 20 week old mice that received the antibody blocking myostatin, but the gastrocnemius (calf muscle) was smaller than untreated mice.

Finally, to cloud the water even more, a recent study published in the Proceedings of the National Academy of Sciences indicated that bigger muscles may not mean stronger muscles. This study simply blocked the myostatin gene in otherwise normal mice, then tested the force of contraction of the muscles. It turns out that the force of contraction was not significantly different between mice without myostatin and control mice. And, since the muscles of the mice lacking myostatin were bigger, the force per unit mass of muscle was actually less than the force of contraction in control mice.

This research throws a cloud over the research described above that found mice with blocked myostatin had muscles that contracted with greater force than mice without blocked myostatin. The main difference between the two studies is the mice: normal mice without myostatin in the PNAS study, and mdx mice without myostatin in the other.

The upshot of all of this is that the effect of blocking myostatin seems to depend on age of the intervention, the strain of mice receiving the intervention, and the particular muscle under consideration.

Despite all this, pharmaceutical companies have completed clinical trials in humans (phase I/II). According to clinicaltrials.gov, Wyeth has completed phase I and II on stamulumab (MYO-029). The MDA website notes that the data are currently being analyzed.

We’ll have to wait and see if inhibiting myostatin turns out to be a workable treatment for any types of MD. But there’s one other aspect of myostatin’s usefulness that I haven’t really touched on. It will bring us back to the baby with mutations in his genes for myostatin. Stay tuned, sports fans…

References:

1. Bogdanovich, S. et al. (2002). "Functional improvement of dystrophic muscle by myostatin blockade." Nature 420: 418-421. (Nov. 28)
2. Bogdanovich, S. et al. (2005). "Myostatin propeptide-mediated amelioration of dystrophic pathophysiology." FASEB J 19: 543-549.
3. Li, Z. et al. (2005). "Elimination of myostatin does not combat muscular dystrophy in dy mice but increases postnatal lethality." American Journal of Pathology 166(2): 491-497.
4. Parsons, S.A. et al. (2006). "Age-dependent effect of myostatin blockade on disease severity in a murine model of limb-girdlemuscular dystrophy."American Journal of Pathology 168(6): 1975-1985.
5. Amthor, H. et al. (2007). "Lack of myostatin results in excessive muscle growth but impaired force generation." PNAS 104(6): 1835-1840 (Feb. 6)

See links to abstracts of papers in text of post.

Of Mice and Men...and Muscle (part 1)

In 2004, a group of German researchers reported on a child that they had been following for over four years. The baby came to the attention of the researchers because several hours after his birth he developed stimulus-induced myoclonus (muscle twitching or jerking; an example is a hiccup).

The child was also abnormally muscular. His quadriceps had twice the cross-sectional area as that of children of a comparable age and sex. His subcutaneous fat was half as thick.

In 1997, only three years before the child’s birth, researchers at the Johns Hopkins University School of Medicine had discovered a gene that, when expression of the gene was blocked, caused abnormally large muscle growth in mice. Mice lacking a functioning copy of the gene had muscles 2-3 times the mass of mice that did have a functioning copy. The gene, initially called GDF-8, belongs to the TGF-β gene family. (GDF stands for growth and differentiation factor; TGF is short for transforming growth factor.) The members of this family of genes regulate growth and differentiation. Because the normal function of GDF-8 was to limit muscle growth, the researchers named it myostatin (myo for muscle, statin because it seemed to inhibit muscle growth).

In their own words:

“GDF-8 null animals are significantly larger than wild-type animals and show a large and widespread increase in skeletal muscle mass. Individual muscles of mutant animals weigh 2-3 times more than those of wild-type animals, and the increase in mass appears to result from a combination of muscle cell hyperplasia and hypertrophy. These results suggest that GDF-8 functions specifically as a negative regulator of skeletal muscle growth.”

[In the photo, taken from McPherron et al (1997), normal (wild-type) mouse on top, mouse without myostatin on bottom.]

This discovery was perhaps a bit more than just another brick in the wall of molecular biology and gene function. As the scientists noted, manipulation of either the gene, its expression, or its signaling pathway in cells had potential implications for treatment of diseases like muscular dystrophy or the muscle wasting that occurs in patients immobilized for long periods or those suffering from cancer (cancer cachexia). However, they also commented that it might have agricultural implications (think chicken breast and beef cattle).

In fact, within months, of the discovery of myostatin, several research groups had sequenced the gene in cattle and identified the mutations that produced two breeds noted for their extraordinary muscle: Belgian blues and Piedmontese. In these cattle, the phenomenon was known as “double muscling.” In both breeds, mutations in the myostatin gene prevented the animals from produce enough (or any) functioning protein from the myostatin gene. As a result, the negative regulation of muscle growth was lacking and muscle was more massive. The meat of these breeds is reported to be more tender than other meat because it apparently has less connective tissue. On the downside, calves generally have to be delivered by C-section because they are so large at birth.

[A Belgian Blue, from McPherron and Lee (1997).]

But the research on myostatin hasn't been all about agriculture. It also has been an active subject of research related to Duchenne muscular dystrophy. I’ll discuss some of that research soon.

References:

1. Schuelke, M. et al. (2004). "Myostatin mutation associated with gross muscle hypertrophy in a child." New England Journal of Medicine (June 24) 350: 2682-8. (I think it's available free with registration?)
2. McPherron, A.C. et al. (1997). "Regulation of skeletal muscle mass in mice by a new TGF-β superfamily member." Nature (May 1) 387: 83-90. (subcription required)
   
3. McPherron, A.C. and SJ Lee (1997). "Double muscling in cattle due to mutations in the myostatin gene." Proc. Nat'l Acad. Sci. (USA) 94: 12457-61.
   

Hormone replacement therapy and coronary heart disease

As I’ve written in a previous post, the Women’s Health Initiative (WHI) examined the risks of heart disease, breast cancer, and osteoporosis in post-menopausal women. One aspect of the WHI was randomized trials investigated the effects of hormone therapy (either estrogen alone or estrogen plus progestin) on the health of post-menopausal women. The study was shut down early due to a high number of adverse effects. From the data that was collected while the study was ongoing came the somewhat surprising result that women receiving the hormone therapy were not at any reduced risk for non-fatal myocardial infarction (heart attacks) or coronary artery disease. This was surprising because it had long been thought that estrogen supplied some sort of cardioprotective function (this was the explanation for why pre-menopausal women have lower risk of heart attacks than men of similar age). Follow-up analyses after the WHI trial ended showed that the effect of estrogen on the heart seemed to be time dependent – the earlier the estrogen was administered, the more cardioprotective the result. In women aged 50-59, there was a fairly notable beneficial effect of hormone therapy. In older women, there was either no benefit, or, in the 70-79 cohort, perhaps some detriment to the hormone therapy.

A recent study in the New England Journal of Medicine looked at atherosclerotic calcification of women in the 50-59 year-old cohort using computed tomography, comparing women who had received hormones and those that hadn’t. The women studied had been part of the initial WHI study (estrogen only trial - so all the women lacked a uterus). Technicians at a central lab scored all the scans for calcification. In the end, women that had received estrogen during the WHI trial had less calcification than women that received a placebo.

The authors conclude:

The new findings from WHI-CACS [CACS = Coronary Artery Calcium Study; the name of the follow-up analysis] indicate that estrogen therapy initiated in women at 50 to 59 years of age is related to a reduced plaque burden in the coronary arteries and a reduced prevalence of subclinical coronary artery disease, providing support for the hypothesis that estrogen therapy may have cardioprotective effects in younger women.

The authors also provide a potential explanation for why estrogen might be cardioprotective in recently post-menopausal women (50-59 years old), but have negative effects on older women (70-79):

It is possible that estrogen could reduce coronary-artery calcium scores but still increase the risk of clinical CHD [coronary heart disease] events, owing to adverse effects on thrombosis and plaque rupture, which are more likely in older women with advanced stages of atherosclerosis. Such a duality of effects would not necessarily apply to younger women with lower burdens of atherosclerosis.

But nobody should get carried away with hormone replacement therapy; it still has significant risks. Any decision to initiate hormone therapy is still a balanace between those risks and the benefits:

In the meantime, hormone therapy should not be initiated (or continued) for the express purpose of preventing cardiovascular disease in either younger or older postmenopausal women. The current recommendations from many organizations that hormone therapy be limited to the treatment of moderate-to-severe menopausal symptoms, with the lowest effective dose used for the shortest duration necessary, remain appropriate.

References:

1. Manson, J.E. et al. (2007). "Estrogen Therapy and Coronary Artery Calcification." New England Journal of Medicine 365: 2591-25602. (Available for free after 6 months)

2. WHI steering committee (2004). "Effects of Conjugated Equine Estrogen in Post-menopausal Women with Hysterectomy." Journal of the American Medical Association 291(14): 1701-1712. (Free with registration)

Red Desert

If you look at a map of Wyoming, you’ll notice a big empty area in the southern part of the state between Rawlins and Rock Springs – few paved roads, fewer towns, no national parks, no national forests. From Highway 28 and 287 on the north to the Colorado border on the south, there’s little for a map maker to do that isn’t connected to I-80 in that part of Wyoming. But get away from the interstate, and you’ll see a little known jewel known as the Red Desert.

Some of the most spectacular areas include Adobe Town, Kilpecker Dunes, the Pinnacles, Oregon Buttes, and Honeycomb Buttes. But the most impressive thing about the Red Desert is its expansiveness. Don’t confuse expansiveness with emptiness, however. This area is home to wild horses, elk, and pronghorn aplenty. Unfortunately, it’s also home to some big league oil, gas, and coal reserves.

Much of the Red Desert is managed by the Bureau of Land Management (BLM), and in these days of high fossil fuel prices and devil-may-care development policies, the BLM isn’t doing much to stand in the way of the drilling rigs.

Within the last month, the BLM approved the drilling of 2000 wells along the Atlantic Rim (located along the southeastern edge of the Red Desert). The majority of these wells are for natural gas (specifically, coal-bed methane). Coal-bed methane is natural gas that occupies the interstices of a coal seam. The methane is held in place by water pressure. To get the methane out, gas companies drill down and pump off some of the water. The reduction in water pressure allows the methane to escape and be captured by the wells. Normally, the water is just released into surface waters (which can often cause problems due to saline water), but conservationists were able to get the BLM to require reinjection of the water on the Atlantic Rim.

One potential problem on the Atlantic Rim is methane seeps. These can occur naturally, but there is some evidence that pumping by test wells in the area has led to the formation of new seeps. These are dangerous because the methane is flammable but odorless and colorless. Be careful where you light your camp stove.

Larger concerns relate to wildlife in the area. Some of the areas of highest use by mule deer, elk, pronghorn, and sage grouse are frequently the same areas targeted for drilling. And, although the oil and gas industry claims that the disturbance to wildlife will cease once the wells quit producing, the scars of development tend to be long lasting in an area that gets less than 10” of precipitation a year. Wagon tracks from stagecoach lines used 150 years ago are still visible in places.

For more information on the Red Desert (and pictures that are better than mine) try these sites:

Friends of the Red Desert
Biodiversity Conservation Alliance
National Wildlife Federation
Wyoming Outdoor Council
An article about coal-bed methane in Orion
An article about the Red Desert at NWF

Maybe not the best photographs of all time, but here are some that I took from a recent trip to the Red Desert:

Adobe Town:

And another Adobe Town (from the North Rim of Adobe Town):

The White Mountain Petroglyphs:

And Honeycomb Buttes:

Photos from Arapaho NWR

As promised (about a month ago), here are a few photos from the restoration work at Arapaho National wildlife Refuge.

This is one of the exclosures. One mature willow was already present; the newly planted willows are just thin vertical lines.


Here's a view of the northern part of the refuge (looking west). The meandering stream is the Illinois River.

Glucagon regulation, part 2

Getting back to glucagon regulation. The paper I mentioned at the end of my last post on this topic was written by MacDonald and colleagues, and published online in PLoS Biology: “A K_ATP Channel-Dependent Pathway within α Cells Regulates Glucagon Release from Both Rodent and Human Islets of Langerhans”.

The title seems to imply that the article is just about what ion channel is involved in the regulation of glucagon release. It goes beyond this however, suggesting that blood glucose alone is enough to regulate glucagon (as the thing that influences the ion channel’s activity).

“We have now compared insulin and glucagon release and α- and β-cell Ca²⁺ responses in intact mouse, rat, and human pancreatic islets. We show that glucose retained the ability to suppress glucagon release from isolated islets during blockade of the Zn²⁺ and GABA paracrine pathways, and in the absence of stimulated insulin secretion or β-cell Ca²⁺ responses. Thus we now provide evidence in both rodent and human islets supporting the direct (intrinsic) glucose regulation of glucagon release from pancreatic α-cells.”

Recall that the review I discussed previously favored the ‘paracrine/endocrine’ hypothesis – specifically emphasizing the possible role of insulin (the β-cell ‘switch-off’ hypothesis). In fact, that review repeatedly downplayed any direct role for glucose in the regulation of glucagon:

“A direct inhibitory action of glucose on α-cell secretion seems to be of little physiological significance…”

In brief, MacDonald and colleagues systematically set out to (1) test whether glucose levels were sufficient to regulate glucagon secretion and (2) examine the electrophysiology of a-cells under different experimental conditions.

To demonstrate that glucose can directly regulate glucagon (without paracrine intermediaries), MacDonald measured glucagon secretion by mouse and rat islets at two different concentrations of glucose. In addition, they selectively blocked two known paracrines: GABA and Zn²⁺ ions. (Both of these chemicals inhibit glucagon release.) The results of these experiments are shown in the figure below.

To understand this figure, you need to know that Ca²⁺-EDTA removes Zn²⁺ from solution. Actually, the Zn²⁺ is still there, but it’s bound to the EDTA, so we’d say it is not biologically available. Similarly, SR-95531 blocks the activity of GABA. The pluses and minuses below the x-axis of each graph indicate the presence (+) or absence (-) of the EDTA and the SR-95531.

We can work through the graph in the following way (taking only the mouse data for simplicity). The first two bars basically show data from islets at low (left bar) and high (right bar) glucose. Based on normal physiology, we’d expect glucagon to be high when glucose is low and vice versa. Since neither EDTA nor SR-95531 are present, the reduction in glucagon secretion could be due to either glucose, GABA, or Zn²⁺. The next pair of bars again shows the response at two different concentrations of glucose, but now we’ve prevented Zn²⁺ from acting by adding EDTA to the mix. Even without Zn²⁺, however, we still see a reduction in glucagon production at high glucose concentrations. So, Zn²⁺, isn’t what’s causing the reduction. In the third pair of bars, the only difference is a change in glucose concentration – both Zn²⁺ and GABA have been blocked. What can we conclude? Glucose alone is sufficient to reduce glucagon secretion. Notice that the drop in glucagon secretion isn’t as great this time. This might signal that the presence of GABA magnifies the effect of glucose on glucagon secretion.

These data contradict the conclusions of the review paper, but they don’t deal with the β-cell ‘switch-off’ hypothesis. However, a subsequent experiment (panel A and B of Figure 2) shows that glucagon secretion (filled circles) increases well before insulin drops (open circles) to any significant degree (this is particularly apparent in the mouse data). Conclusion: it isn’t a drop in insulin that switches on the secretion of glucagon.

A similar effect can be seen in human islets (presented in Figure 5B in a slightly different format).

The evidence for the central role of K_ATP channels can be seen in Figure 2 above. Diazoxide is a chemical that opens the channels, and tolbutamide blocks them. Panels 2A and 2B show that glucagon secretion rises as the amount of diazoxide rises (and as more K_ATP channels open). Beyond a certain point, however, increased activity of the K_ATP channels actually leads to a reduction in glucagon secretion. (I’ll try to explain the authors’ interpretation of this in a second.)

For brevity and simplicity, I’ll skip the experiments that investigated ion channels for Na⁺ and Ca²⁺, and just say that the authors conclude that both channels involved are active at intermediate membrane potentials. As the membrane becomes either hyperpolarized (more negative than usual) or too depolarized (more positive than usual) these channels become inactive and close). The effect of this is inhibition of glucagon secretion.

Having said that, I’ll jump to the conceptual model that MacDonald and crew devise to explain what they think is going on. (As an aside, this is one of the things I really liked about this paper. Although the authors might be wrong in their interpretation of what the results of these experiments mean, the model will provide other researchers with some specific things to test.) The model is summarized graphically in Figure 10.

Panel A basically depicts what they think is going on in normal α-cells: at low glucose, the K_ATP channels are mostly open (as are the Na⁺ and Ca²⁺ channels - shown in the red and blue lines) and glucagon secretion is high (solid line at bottom of panel). As glucose levels rise, the K_ATP channels begin to close (due to the increase of ATP in the cell from glucose metabolism), and the cell membrane begins to depolarize (i.e., becomes less negative because fewer K⁺ ions are leaving the cell – keeping more positive charges in the cell makes the inside less negative). At some point the membrane is sufficiently depolarized that the Na⁺ and Ca²⁺ channels close, inhibiting glucagon release.

The other three panels basically explain what they think is happening to produce some of the results they saw in their other experiments. For example, if diazoxide opens more K_ATP channels, then why does glucagon secretion decline at high levels of diazoxide? The idea is that as more and more K_ATP channels open, the cell membrane becomes hyperpolarized (more negative because more K⁺ ions are leaving the cell). This hyperpolarized state causes the Na⁺ and Ca²⁺ channels to close, reducing glucagon secretion.

I have to say that the one thing that I don’t really understand about all this is the connection between the Na⁺ and Ca²⁺ channels and action potentials of the α-cell membrane that ultimately trigger the release of glucagon (by exocytosis). I would assume that other channels that are either not voltage sensitive or sensitive to different voltages (different from the particular Na⁺ and Ca²⁺ studied in these experiments) are also active. But experience suggests that I’m probably wrong.

References:

1. 1. MacDonald, P.E., et al. (2007). "A K_ATP Channel-Dependent Pathway within α Cells Regulates Glucagon Release from Both Rodent and Human Islets of Langerhans” PLoS Biology. Published May 15, 2007.
2. Gromada, J., Franklin, I., and Wollheim, C.B. (2007) “α-Cells of the Endocrine Pancreas: 35 Years of Research but the Enigma Remains.” Endocrine Reviews 28(1):84-116. (Author manuscript pdf free here.)