On a crisp autumn day just over a year ago, pediatric geneticist Ronni Cohn received an unusual shipment from Wisconsin: several crates containing 20 striped ground squirrels. Once fattened on salt-free nuts from his local Trader Joe’s, Cohn made them comfortable inside a refrigerator in the basement of his Hopkins lab, and the squirrels happily got down to the business of hibernating.
And now, just a year into an offbeat research project that has already yielded data and discoveries that this soft-spoken physician-scientist describes as "fascinating, amazing, and stunning," comes a finding that gives him goose bumps.
Cohn is hurrying off to see his patients—many are children with debilitating muscle tone—when he stops by his lab on the fifth floor of the Broadway Research Building to check in with his postdoc, Eva Andres-Mateos. She shows him the results of a recent "western blot," used to detect specific proteins in samples of the squirrels’ muscle tissue. It is apparent that one particular protein—one never before associated with muscle—is much more active when the squirrels are motionless; intriguingly, more of it is produced when the squirrel is hibernating.
"I think we are on the verge," declares Cohn, assistant professor of pediatrics and neurology in the School of Medicine’s McKusick-Nathans Institute of Genetic Medicine. "I think we may have found a new player important for muscle maintenance."
born and educated in Germany—he earned his medical degree in 1996 at University GHS Essen School of Medicine—Cohn remembers being fascinated as a child about bears and hibernation.
A goose bump-inducing finding: Cohn and postdoc Eva Andres-Mateos recently discovered a protein, never before associated with muscle, that is more active when squirrels are motionless. “I think we may have found a new player important for muscle maintenance,” he says.
"Have you ever thought about what it means to hibernate?" he asks. "These ground squirrels, they don’t eat or drink anything for four to six months; their hearts beat four times a minute; they breathe four times a minute. God created these little animals—and many others—to survive like this, without losing skeletal muscle mass. At the end of the four months, they wake up and run and jump around. You and I, we couldn’t do that. What allows them to? I’m trying to take this resource out of nature and make it into something useful not only for patients of mine, but also for ICU patients and aging patients . . . anyone whose muscles are affected by inactivity or other disorders."
Cohn arrived at Hopkins in 2001 to become the first resident to combine both pediatrics and clinical genetics in the Medical Genetics Residency Training Program, a curriculum he completed in 2006, and which he now directs.
"I never planned to become a geneticist," he muses. "I have an inherent love for pediatrics. Given that and my interest in muscle disorders, I thought I would be a pediatric neurologist. But when I heard about this combined clinical genetics program at Hopkins, I realized I could combine my two loves under a new and different umbrella with a much broader perspective—that being genetics."
Cohn recently launched the Center for Hypotonia, a clinic where he sees mostly young patients with decreased muscle tone, a symptom in more than 500 different genetic disorders. His goal is to tailor therapies for patients’ individual needs by combining clinical experience with basic research—work that doesn’t happen without grant money.
Cohn’s discoveries in the lab all imply one fundamental promise: that is, stronger, healthier muscles for his patients, like Gianni Rosier.
In 2008, Cohn applied for a New Innovator award from the National Institutes of Health. As a young investigator who had published extensively for a decade in journals the likes of Cell, Science, Nature, Nature Medicine, and Pediatrics, Cohn wasn’t far enough along with research in his own lab to submit the kind of data needed to garner a so-called RO1 grant—the bread-and-butter money that sustains more seasoned researchers.
"I wouldn’t even have been considered for funding," Cohn recalls. "But I thought that instead of doing nothing in terms of grant writing, why don’t I give the New Innovator award a shot." (The highly sought after grant is bestowed by the NIH upon "exceptionally creative new investigators who propose highly innovative projects that have the potential for unusually high impact.")
"I let myself have fun writing about something really ‘out there,’’’ says Cohn. "I allowed myself to think about doing the kinds of things that you generally can’t afford to do."
Summoning up his lifelong wonder of hibernating creatures, Cohn proposed to investigate the maintenance of skeletal muscle in ground squirrels, a hibernating species bred in Wisconsin. It worked. The idea earned him an award of $1.5 million, flipped a significant switch in his scientific life, and lay what he believes may be the cornerstone of his research career.
His unusual proposal was based on this question: Do hibernating animals such as squirrels regenerate muscle once it’s damaged, and if so, how? Answering this and related questions could have clear implications for humans afflicted with disorders that lead to muscle disuse, deterioration, and scarring.
One of the first things Cohn and Andres-Mateos did in the winter of 2008 was to test a hypothesis by injuring the muscles of a number of the hibernating squirrels while they slept. After sacrificing them, they looked at whether the muscle subsequently repaired itself.
"We have always looked at fibrosis in muscle as an end stage of the kinds of diseases that afflict my patients. Well, our squirrels may be telling us that scar formation in muscle might be able to be limited. That would be unbelievable."
It didn’t. "There was no repair whatsoever," Cohn sighs. "It was disappointing when I saw that if you damage it, it stays damaged."
He was thinking about giving up studying muscle regeneration in the squirrels. But then he noticed something else that gave him pause. Curiously enough, the damage had not progressed into making fibrotic scar tissue which, in a non-hibernating animal such as a human, it surely would have.
That observation was all the ammunition he needed to undertake a new experiment.
In the springtime, 10 days before Cohn planned to allow the remaining four hibernating animals to arouse as they would in nature, he and Andres-Mateos injected two of the squirrels in their calf muscles with snake venom to cause local damage. Then they let everyone sleep soundly—until a wake-up call at 8 a.m. on April 29, when the Plexiglas cages were taken out of the cold, dark refrigerator and were placed in a relatively warm room bathed in light.
Within a couple of hours, the brown and tan striped creatures emerged cautiously from their nests, freeing themselves from shreds of paper towel. One had bits of wood shavings attached to his whiskers. All were twitching, shaking, shivering, and quaking; warming up brains, hearts, and limbs. They blinked at the bright artificial lights of "spring."
"Hola!" Andres-Mateos greeted them. "Welcome back!"
The animals that were so fat and round in September from their high-calorie diet of nuts now were skinny but otherwise apparently healthy.
"Isn’t this fascinating and breathtaking?" Cohn enthused. "You take them out at 8 a.m., and here they are at 10:30, wobbling around, their muscles no worse for having been immobile throughout the winter."
From just looking at them starting to scurry about, it was impossible to tell which of the four animals had been injected with the toxic venom. Their ability to move evidently had not been compromised. But Cohn knew, based on his earlier work, that the microscope would tell another story: one of "lots of damage."
Fast-forward three weeks, then six. Over intervals of time, the scientists examined under microscopes the calf muscle tissue of the now awakened animals that were moving and eating with the renewed vigor of spring. The scientists’ aim was to survey the progress of the damaged tissue, which they knew from their previous experiment did not repair itself during hibernation.
At three weeks, the scientists looked at one of the injected squirrels, and to their surprise, the tissue showed some attempt of muscle regeneration. By six weeks, when they looked at the other squirrel, the damage to the injected muscle was, to Cohn’s utter amazement, "totally repaired."
The essence of Cohn’s research is taking advantage of what biology has to offer medicine, according to David Valle, Henry J. Knott Professor and Director of the Institute of Genetic Medicine: "There’s all kinds of unusual biology in the world, and if we were more aware of how particular living things have solved problems that we would like to solve in medicine, then we should be able to learn from that."
Says Valle, "I’m optimistic that Ronni will learn something that will be quite relevant to his genetic patients with muscle disorders—and to anyone who spends time immobilized. If he makes just the tiniest amount of progress in that, it would be a huge step forward.
"There’s always the chance he’ll hit a real home run. If anyone could do it, it’s Ronni."
The spring and summer of 2009 were busy months in the lab. As Cohn and Andres-Mateos analyzed and interpreted all of the data their squirrels had given them, it became clear to Cohn that muscle maintenance and muscle regeneration in hibernating animals are as complex as his most difficult-to-diagnose patients, whose symptoms range from the subtlest of facial features to obvious behaviors. It was clear that the squirrels, when they hibernate, do not actively regenerate muscle. But curiously enough, neither do they not regenerate.
Cohn suspected that there must be some aspect of regeneration going on, or otherwise the damaged muscles would default, as they do in non-hibernating animals, into making scar tissue, known as fibrosis. His preliminary evidence suggests that the squirrels’ muscle stem cells, known as satellite cells, increase in response to injury, and these eventually lead to tissue repair once the squirrels wake up from hibernation. Characterizing how squirrels accomplish this is among the top three items on Cohn’s to-do list.
"If you have that amount of damage to muscle in humans or mice, they make huge amounts of scar tissue," he says. "But the squirrels don’t do that. And from our research on them, now we have some idea why."
By the fall of 2009, Cohn’s lab had amassed "beautiful evidence" that squirrels activate a molecular pathway during hibernation that increases the production of those powerhouses in cells known as mitochondria. This is the same "endurance exercise" molecular pathway that is active and increased in long-distance runners. Simply put, the hibernating squirrels sleep and don’t move, but on a molecular level, they are doing the same thing as marathoners.
Among humans, the notion is that when this endurance pathway is active, it inhibits a "resistance pathway" that allows for the production of proteins and muscle growth. That’s why you rarely see a muscle-bound marathoner, or a skinny weight lifter, Cohn says: Either one pathway is active, or the other, but not both.
Except in hibernating squirrels, and by extension, other hibernating animals, that is: They seem able to keep both pathways active at the same time.
"What the squirrels are showing us is that nature can combine it all together," Cohn says. "Nature can and does integrate the endurance and resistance exercise pathways. And that’s never been described before."
This finding, according to Cohn, changes the fundamental idea about how muscle is maintained: "The old paradigm requires a balance of synthesis and breakdown versus regeneration. Now we see that mitochondrial function needs to be added to that."
As intriguing as the basic biology is its possible clinical application, Cohn says: "We have always looked at fibrosis in muscle as an end stage of the kinds of diseases that afflict my patients: muscular dystrophy, for instance. The weaker kids get, the more fibrotic tissue they develop. So even with the prospect of new therapies on the horizon, huge concerns remain for physicians like me, caring for patients who already are sitting in wheelchairs and barely able to move. We know that scar tissue has already developed and that fibrosis is an end stage. So what good can we do them?
"Well, our squirrels may be telling us that scar formation in muscle might be able to be limited. That would be unbelievable."
Cohn’s hoard of discoveries—those relating to muscle regeneration, to the endurance/resistance exercise pathway, and most recently to the new "Protein X" that emerged in the western blot test—all imply one fundamental promise: that is, stronger, healthier muscles for his patients.
A rosy-cheeked little girl in shiny black patent leather boots greets Cohn with an effusive hug and a "Hi, Boy!"
She flits and dances among her audience—mother, dad, baby sister, doctor, genetic counselor, and med student, all squeezed inside an exam room in the pediatrics genetic clinic in the Rubenstein Building. The little girl demonstrates extraordinary spelling skills for a 5-year-old with a rare genetic disorder characterized by problems with development: "E-l-e-p-h-a-n-t, Elephant! Y-a-c-h-t, Yacht!"
As appointments go, this one’s smooth sailing. This thriving youngster has a real diagnosis—Williams syndrome—on which her parents can hang their expectations and their worries. Cohn listens to her heart and then welcomes the onslaught of anxious questions that always come from his patients’ families. No matter that nine kids with yet-to-be-determined genetic disorders are on the schedule today, his manner is to take his time to assure this conscientious and concerned mother that a school-related decision she agonized over was absolutely positively what he would have advised her to do, had she consulted him.
His next patient recently had pneumonia, her mother informs. Before Cohn removes the child’s leg braces to check on the degree of her hip dysplasia, he listens to her lungs. Developmentally delayed in both speech and movement, she coos and moans as she attempts to roll over onto her belly.
Cohn is both firm and soothing when the patient’s mother frets about a family doctor and specialist having attached a "cerebral palsy" label to her daughter: "That’s simply not true," Cohn says quietly. "She does not have cerebral palsy. She does not have it."
He wishes he could be as authoritative about what she does have; about what dozens upon dozens of his "little ones" have when they present with all manner of puzzling combinations of physical and cognitive deficits caused by genetic variants.
As the morning wears on, run-of-the-mill pediatric issues (should she get a flu shot?) are as carefully debated and discussed as the most complex questions posed by parents whose casual banter is peppered with cutting-edge genetic jargon.
When Cohn advises that a patient should have a SNP array, a dad counters with exasperation: "At what point do we stop doing all these tests?" The mother nods, adding: "We keep thinking we have the answer—I was convinced it was this or that variant—and then we’re surprised again."
Unflappable, Cohn counsels the couple with the wisdom of a clinician whose 200-plus pediatric patients serve as a near-constant reminder that "cures," genetically speaking, are even more elusive than diagnoses. Although Cohn believes that every one of his patients deserves at least a baseline genetic workup, he proceeds either conservatively or aggressively based on each individual family’s need to know: Some people just have to know. He gently argues for striking a "reasonable" balance which, in this case, in his opinion, does include SNP testing.
Cohn is quiet while they digest his advice. Despite running an hour behind because an unexpected patient was added today, and despite an eagerness to check for messages from his lab where Andres-Mateos is working on proving that Protein X is a player in muscle maintenance, Cohn leans back into his chair and casually crosses his legs.
His relaxed gesture reveals a fashion statement that someone who’s not a kid-loving pediatrician—and anyone who’s not in the midst of the most magical year of his research career—might keep hidden away: At Cohn’s ankles, flop-eared white rabbits emerge playfully from black top hats.
The socks are a fitting metaphor: Having recently received a new shipment of 40 squirrels and just discovered Protein X, Cohn clearly has more than a few new tricks up his sleeves as he delves ever deeper into the mysteries of the muscle wasting diseases that afflict many of his patients.