HUMAN MUSCLE DOES REGENERATE!

--An Asa Zook Dialog

Asa Zook is a fictional philosopher of science who occasionally serves as a resource person on factual science for an imaginary popular science writer. Here, the writer engages Zook in discussion about the regeneration of muscle.

ZOOK: Please!

WRITER: Sorry Doc. I know you detest prattle. But I just can't break this parler petite habit, heh! heh! -- if you'll forgive my C-minus French. Anyway, I came by to see you about muscle regeneration -- regeneration in mammalian muscle. Your name popped up in my preliminary literature search. I was surprised but delighted to find, after a little digging in some ancient archives, that you were once active in that line of research. That's you, isn't it?

ZOOK: Most likely.

WRITER: First off, let me make sure I've got this right. This stuff on mice and rats. That applies to people, too, doesn't it.

ZOOK: Would it make a difference?

WRITER: There's a lot of ignorance about animal research -- especially among editors, some of whom don't seem to believe in Evolution. But it'll be easier to slip in some hard science if there's a medical angle. Ergo, let me ask again, do the findings on other mammals apply directly to human muscle?

ZOOK: Very much so. In fact, the microscopic signs of regeneration in mammalian skeletal muscle -- myotubes as the new muscle fibers are called -- were first observed in degenerative human muscle tissue.

WRITER: When was that, Doc?

ZOOK: In the Berlin typhoid epidemic of 1864. Typhoid can cause a particular degeneration -- Zenker's waxy degeneration, it is called -- and is especially evident at autopsy, immeditely upon laying back the abdominal skin: in the rectus abdominis muscles, which appear wax-like . A pathologist and anatomist, Heinrich von Waldeyer found myobutes in microscopic sections of muscle that exhibited waxy degeneration.

WRITER: How in the world did he know it was regeneration? I mean, if nobody had seen it before....

ZOOK: Regenerative myobutes closely resemble those of newly differentiating embryonic muscle. One also sees myotubes in muscular dystrophy, incidentally. They are very easy to spot in a field of adult muscle fibers-- especially in a cross section -- because they are of reduced diameter and tend to be basophilic.*

WRITER: Baso what?

ZOOK: Basophilic. An indication that they are still making new proteins. They're usually darker than adult fibers.

WRITER Ah-so-ca! Anyhow, I was nosing through my paper's morgue a few days ago and ran across a freelanced article that had bought but never published. Here, let me read from the manuscript:

"If New Orleans is the soul center of music Americana, certainly Al Hurt has breathed his spirit into the roil of that delta city. But a sad world now waits, wondering if live sound shall every again pour from Al's great and plentiful horn...

WRITER: The editor's notes say, "Too sophisticated!"

ZOOK: For whom?

WRITER: The reader. It's always the reader who's too damn dumb for science. But it's really the excuse rather than the real reason, especially in this case. Let me continue some with the piece:

"On Sunday, February 8, 1970, as he rode in the Mardi Gras Parade, lending his magic to the swag cadence of New Orleans jazz, a flying piece of hate in the physical form of a hurtling brick smashed Al's lower lip and ripped through the very muscle that translated Al's music his mind to our world life.

"Is there even a chance the damaged lip muscle can repair itself? Or, as far as science knows, is the great show over forever?"

"I'd use my words to pray for luck, except for widespread misinformation on the subject of muscle regeneration. Famous, best-selling textbooks flat-footedly declare that muscle doesn't regenerate. Many doctors read those words in medical school and blend the misguided belief into how they practice medicine.

"But the truth is this. Human muscle does regenerate! The conventional experts are just plain wrong....

WRITER: I think the piece would be outright rejected even today. -- And for the same fundamental reasons.

ZOOK: Protecting the reader?

WRITER: Nooo, Doc. That's the ass cover, not the antecedent. It really has to do with how science filters into the mainstream of culture. Here, look at what Isaac Asimov wrote in a Sunday supplement back around the time of the Hirt news peg:

"Other cells...as in nerve and muscle, do not multiply at all in the adult, under any circumstances."[2]

ZOOK: I have known pathologists who held the same misconception, as I might myself, except for my own research in the area.

WRITER: A big gun writer lifts his information directly from a textbook and passes it along like an inscription on the Holy Grail. Time and Newsweek science editors and TV news doctors -- and Ann Landers -- pick it up and pass it on to the rest of us slobs.

And the textbooks! The textbook writer probably heard some blowhard at a scientific meeting expound a B. S. theory of cancer premised on the non-regeneration of muscle....

Like....Look, Doc. I mean, millions read Asimov -- or the London Times -- including science writers. Scheist has a way of looping back and picking up umph through a second, third, fourth cycle....and so on, until the whole world takes it as the gospel of the gods instead of the crap of the caribou, which it really is. But once a prejudice gets stuck in the collective belief, it's like a starb jammed in the cerebrum: pull it out and all the brain's copntents come with out it.

ZOOK: I believe the misconceptions about muscle regeneration are both understandable and correctable. In time, truth supplants misguided popular notions. But your verbal assault....

WRITER: Doc, I wanted you to know "Where I'm Calling From," to borrow a thought from a great writer.[3]

ZOOK: Raymond Carver?

WRITER: Hey Doc! I didn't know you messed with fiction. Anyway, I wanted to explain to you what's in the back drop - what's making me tick on this assignment.

You see, Doc I believe deep in my ear ossicles that society needs science -- maybe more than at any time in history. I think C. P. Snow's cultural gap has become a Grand Canyon of ignorance threatening to swallow us all up. In the abstract, though, the underlying reasons are just too much for my pea-sized intellect. But maybe I can make a tiny contribution by focusing on the small. On a particular.

ZOOK: Such as mammalian muscle regeneration?

WRITER: Right on, Doc! First off, though, can you help me out on how muscle regeneration could possibly escape the text writers?

ZOOK: Muscle regeneration is easily missed, microscopically. Following injury -- in contrast to degenerative conditions -- the process is rhythmic, quick and vulnerable to competing events, such as scar formation: And it is usually confined to a small region.

WRITER: You're saying it's either over before one finds it or that scarring beats it to the punch?

ZOOK: One other important reason why it is often missed, even today: in the early phases, one must know when --within a few hours -- and where -- within a few millimeters -- to look in order to find it -- histologically as well as biochemically.

WRITER: Speaking of when and where: when and how did you get interested in it?

ZOOK: In the 1950's.

WRITER: Forever-and-a half-ago, to somebody my age. ZOOK: But some of the research of the era is quite valid and useful even today. WRITER: What was the big philosophical deal? I mean, there's always some theoretical implication in what you tackle, right?

ZOOK: In general, phase information in biological systems. But specifically, in the case of muscle regeneration, the pivotal issue, for me, was amitosis, the alleged multiplication of myotube nuclei by budding.

WRITER: Like random soap bubbles?

ZOOK: Division without a mitotic apparatus -- without the mechanism for the orderly replication and segregation of chromosomes. Skeletal muscle fibers are multi-nucleated cells joined end to end, each fiber containing scores of linearly elongated nuclei. In spite of the vast numbers of nuclei in each fiber, one never sees mitosis among muscle fiber nuclei; nor in myotubes of regeneration -- or of embryonic development for that matter. How did the thousands of new muscle nuclei come to be? That was, perhaps, the key question about muscle differentiation in the Forties and Fifties. To some investigators the answer was by amitosis.[4]

WRITER: That sounds absolutely inconceivable! I mean you'd need an orderly duplication and sorting of chromosomes -- the carriers of the genes -- to make new muscle molecules, n'est pas?

ZOOK: You say that because you are young, because you grew up with the double helix in your elementary school books and you know as well as you know the geography of North America that the genes orchestrate differentiation, operating through transcription and translation. But the age of molecular and cell biology was just being born, then. It was a time when biologists spoke of chemical events of the cell as existing apart from cell structure. Although I was personally skeptical even at the time, the then extant possibility of amitosis was what attracted me to muscle regeneration. I saw it as an issue I would have to resolve for myself.

Divided opinion had existed in the immediately preceding generation about the importance of the nucleus to heredity, let alone cell differentiation. One school of thought, dominated by embryologists, believed that the cytoplasm carries the heredity wherewithal for the differentiation of the major attributes of protoplasm. The other, led especially by geneticists, believed that the chromosomes --the nuclei -- carry the plans for differentiation. To those inclined towards the cytoplasm amitosis was perfectly logical, especially if you could find no evidence of mitosis.

WRITER: Still sounds wacko to me!

ZOOK: Perhaps. But remembers, cells can live without a nucleus--

WRITER: Example?

ZOOK: The mammalian red blood cell.

WRITER: Ugh! I never thought of that!

ZOOK: There are others. In nuclear transplantation, for example, the first step, typically, is to remove the host nucleus. The host cell obviously does not die while its awaing the donor nucleus. Too, it is the cytoplasm where most cell differentiation goes on. And to some students of development a few generations ago, the genes were merely carriers of particular -- and relatively trivial -- traits. Decorations! Cosmetics, instead of vital attributes.

Today, we know that we need new classes of proteins for a cell to acquire specialized attributes, that specific genes -- DNA sequences -- must be activated, to serve as guide for the manufacture of specific RNA -- transcription; and that RNA will guide proteins construction -- transcription. Still many cytoplasmic factors come into play. RNA can act as a gene carrier. Mitochondria possess DNA. And I could go through a long list of specifics about cytoplasmic entities in inheritance that complicate the picture even today, let alone fifty or sixty years ago.

At any rate, if we do not need a nucleus to make new muscle -- as some believed -- then the numbers of chromosomes in a cells would be irrelevant, and amitosis would fit the paradigm rather well. Several distinguished biologists of the day believed that only amitosis could account for the many new muscle nuclei in the embryo as well as in the regenerate[5].

Three then young investigators -- Lash, Holtzer and Swift[6] pursued the issue through laboratory experiments, instead of as a polemical treatment of descriptive data. Recall that in the Cell Cycle[7] mitosis follows DNA synthesis. It was upon DNA replication in cells of wounded muscle that Lash et al focused their attention.

WRITER: Autoradiography?

ZOOK: No. Intracellular radio isotope tracing methods -- tritium-labeled thymidine -- were a few years away, too. Hewson Swift, who was even then a pioneer of cell biology had perfected a cytospectrophotometer...

WRITER: A what?

ZOOK: A light meter fitted to a microscope.

WRITER: To aim at cells?

ZOOK: Are you familiar with the Feulgen stain?

WRITER: You mean where you can get DNA to turn that dye -- what is it, fuchsin -- from clear to magenta? I remember that from a history of science course I took. Kind of old fashioned, eh?

ZOOK: The amount of dye taken up by a nucleus -- a detectable with a microphotometer -- will depend on the amount of DNA in that nucleus, obviously. Among cells normally undergoing division, the DNA per nucleus increases up to double the resting level -- ideally to tetraploidy -- and then, with mitosis, drops back to diploidy.

Lash and his colleagues conducted such analyses in injured mouse muscle. They found no evidence of DNA replication going in the multi-nucleated myotubes nor in injured, mature muscle fibers. They did find it among free, mono-nucleated cells of the wound coagulum.

WRITER: Mono-nucleated?

ZOOK: Singly nucleated, in contradistinction to the multi-nucleated myotubes. But, of critical significance, Lash et al observed that the mono-nucleated cells replicate in waves. And the waves of replication preceded dramatic increases in both the number of myotubes and the numbers of nuclei per myotube. The myoblasts -- the cells that would actually become the myotubes -- Lash et al proposed were among the proliferative mono-nucleated cells of the wound coagulum; the latter, cells would have completed division prior to joining end to end to produce the mult-nucleated myotubes.

WRITER: Guilt by association, eh.

ZOOK: John Stuart Mill's concomitant variation, one of the primary sources of empirical scientific knowledge.

WRITER: And so that was that, eh?

ZOOK: I personally believed that Lash et al made a strong case for mitosis among wound coagulum mono-nucleated cells and, by implication, severely weakened the amitosis hypothesis.

Yet uncertainties existed. Some histology textbooks of the era continued to present amitosis, as did some investigators of muscle regeneration. The hypothesis of Lash et al -- that the myoblasts derived from the pool of dividing cells of the wound coagulum -- required additional testing, which soon came along.

WRITER: How?

ZOOK: Are you familiar with the alkaloid, colchicine?

WRITER: The old gout remedy that blocks mitosis?

ZOOK: Colchicine inhibits the formation of the mitotic spindle. Now one famous anatomist of the day had reported that colchicine had not prevented regeneration, as you would predict if Lash et al were right[5]. But he had applied colchicine at 4 days. Another investigator believed the treatment missed the period Lash et al considered crucial. When he timed the colchicine administration deliberately to miss the mitoses Lash et al had observed, myotubes arrived on schedule. But when colchicine struck the mitotic peaks in question, myotube formationalmost totally failed.[8]

WRITER: Colchicine knocked out regeneration?

ZOOK: Only when injected so as to arrest the waves of replication Lash et al had observed.

WRITER: Could you call the injections that missed the waves the controls?

ZOOK: You could -- some of the controls. The investigator also injected the vehicle...

WRITER: The what?

ZOOK: A jargon for the solvent -- the solvent in which the colchicine was dissolved -- saline or distilled water, neither of which inhibited regeneration.

WRITER: Where did he make the injections?

ZOOK: When injected into the peritoneal cavity or into the veins, only small amounts of colchicine reached the muscles. From the literature, and from preliminary experiments, the investigator estimated that well above the lethal doses of systemically applied colchicine would be required to affect mitosis in the wound coagulum. Now the wound was in the belly of a muscle of a leg -- the anterior tibial muscle, to be precise -- next to the shin. He inserted the needle down near the ankle, alongside the tendon, slipped up behind the muscle belly and slowly infused the fluid from behind. WRITER: How did he know the stuff was getting into the wound? ZOOK: For visual confirmation that the fluid had reached the entire depths of the wound, he let some ooze onto the surface through the wound in the skin, which had been pressed shut but not sutured. He was trying to lavage the wound coagulum with the test fluid. WRITER: La... what was that again? ZOOK: Lavage. He was trying to wash the wound area with colchicine; his thinking was that if colchicine has a difficult time getting from the blood stream into muscle, it ought to have difficulty going from muscle to blood stream. Thus a relatively small amount, injected locally, would saturate the wound for about than a day, he estimated.

WRITER: How did he really know when to zap the cells?

ZOOK: He did not know, not at first. Whether a single dose of colchicine would hit or miss the target was highly problematic. He decided to use two injections, spaced 24 hours apart.

WRITER: Hit 'em again, hit 'em again, harder, harder!

ZOOK: Working with two knowns -- instead of unknowns -- vastly simplifies the algebra. I will be glad to demonstrate the theory....

WRITER: Spare me theory, Doc! I don't need my nap this early in the day! But I can see the point in casting two nets.

ZOOK: The investigation also brought out the highly proliferative nature of the wound coagulum, which some investigators actually disputed at the time. By the way, I believe there is an important lesson here for anyone who is trying to interdict proliferation, experimental or clinical.

Mitosis, per se, is fairly brief -- 1 to 2 hours -- and random examination of a fixed slide, even a rapidly growing tumor, seldom presents a true picture of the extent of cell replication going on in a tissue. Colchicine poisons the mitotic spindle and arrests mitosis at metaphase. Thus instead of vanishing after 1 or 2 hours, the mitotic figures pile up at the scene.

Colchicine -- and the more efficient demecolcine, [16]today -- has been employed to arrest and thus amplify mitosis. In the studies I'm referring to, myotube-arresting doses of colchicine, when appropriately timed, rendered the wound coagulum utterly packed with arrested mitotic figures -- perhaps five to six orders of magnitude greater than anyone had previously suspected.

WRITER: So did that do it?

ZOOK: The colchicine experiments produced the results predicted by the Lash et al hypothesis.

WRITER: The hypothesis passed the pragmatic test of truth!

ZOOK: I liked the design of the experiments. But uncertainties existed: the specific action of colchicine had not yet been worked out. Perhaps it poisoned some other aspect of cell growth or prevented differentiation. Ambiguities notwithstanding, I tentatively accepted the hypothesis that the myoblasts derived from the highly proliferative mon-nucleated cells of the wound coagulum. But I readily conceded the need for better evidence.

WRITER: What did clinched it for you?

ZOOK: Some investigators discounted the importance of replication to myogenesis, asserting that myotubes were impervious to X-irradiation, which has a severely negative effect on DNA replication. But, when timed to hit the cells Lash et al had observed, X-rays are at least as devastating as colchicine in preventing the scheduled arrival of myotubes.[9]

But what convinced me was a biochemical study in the journal Nature.[10] The experiments involved an antibiotic, phleomycin, that can selectively arrest DNA synthesis. Instead of looking at myotubes, microscopically -- subjectively -- the workers analytically investigated the synthesis of actomyosin, the main contractile protein complex of the muscle cell.

WRITER: How?

ZOOK: By the incorporation of the amino acid lysine labeled with carbon-14. They chemically extracted actomyosin and used a scintillation counter to determine the uptake of the tagged amino acid.

WRITER: Say, that would prove muscle regeneration depends on opening up the genes and making brand new proteins, no? ZOOK: Prior to the analysis I am referring to, one could have arrived at that inference only indirectly, and then after a few assumptions. But the phleomycin study parenthetically established as fact that new actomyosin synthesis occurs in the regeneration of mammalian muscle. It is not an issue today, of course, but it was a potential point of contention, until 1965. WRITER: By the way, what about evidence of transcription -- of messenger RNA synthesis in muscle regeneration? You'd have to have that too, right?

ZOOK: There is a great deal today, of course. But even by the 1960's it was known that the antibiotic actinomycin D, which blocks messenger RNA synthesis, severely inhibits regeneration in mouse muscle -- if its administration is appropriately timed.[11]

WRITER: So this was proof that the genes are directly involved in muscle regeneration?

ZOOK: Another study in the same period also showed that the number of myotubes varies with pedigree.[12]

WRITER: I got you off the track, I'm afraid. You were talking about blocking DNA. How did the workers do that?

ZOOK: By the incorporation of tritiated thymidine into DNA of the wound. They extracted the DNA and quantitatively measured its radioactivity. WRITER: Wait a minute, Doc. What's all this quantitative stuff? ZOOK: It is a matter of reliability, as well as credibility. Concering the latter, one can train technicians to carry out the extractions and operate the analytical machines, and then statistically evaluate how consistently they perform, one run to the next -- even with no idea whatsoever about the implications in the data. Experimenter bias is a major problem in scientific research. Quantitative analyses permit controls that hedge against that.

But before the investigators did anything with phleomycin, they produced a statistically valid quantitative profile of DNA synthesis during the period from before wounding until the first appearance of myotubes. With side experiments, they demonstrated the need to control for difference in the time of day of treatments and of pulse labeling with isotopes; they also produced analytical correction factors for the data. The ancillary data let them accurately design the phleomycin challenges.

WRITER: How so?

ZOOK: First, they found no signs of actomyosin synthesis in uninjured muscle. Thus the wound itself, whose timing they could accurately plot, would set the chain of regenerative events into motion.

They knew the starting point -- the moment they inflicted the wound. They discovered three waves of DNA synthesis between injury and the interval prior to the messenger RNA synthesis for the contractile protein complex.

WRITER: Can you give me some times here, Doc?

ZOOK: The three waves of DNA replication are crowded into interval between 38 and 68 hours after injury; the period of sensitivity to actinomycin D is just after that, at about 72 hours. Myotubes become abundant at day 4 and contractile protein synthesis is intense at day five.

Concerning DNA synthesis, the investigators found that waves I and III could be accurately reproduced in quantity and on several occasions. The middle wave, which might have been two waves jammed together, they decided to ignore while fixing their attention on waves I and III, which they had precisely plotted.

WRITER: And phleomycin's effects?

ZOOK: When applied before wounding, it had no effect on either DNA or actomyosin synthesis. But it severely inhibited DNA synthesis during waves I and III.

WRITER: Actomyosin? Say you're grinning like a well fed fox, Doc.

ZOOK: During wave III, it had modest effects -- as though a certain percentage of myogenic cells had been culled from the population. But when applied during wave I, phleomycin virtually eliminated actomyosin synthesis.

WRITER: In other words, phleomycin pole-axed muscle regeneration if it flattened wave I?

ZOOK: If by 'pole-axed' you mean 'completely inhibited the molecular signs of muscle differentiation,' then yes! Poleaxed! An excellent word in this context.

WRITERS: So that proves the Lash et al hypothesis?

ZOOK: The phleomycin studies demonstrated a strong correlation between wave I and the first wave of myotubes. But the investigators were very conservative. Something essential to myogenesis happens during wave I, they observed. The myogeneic genes are known today.[13] It is quite possible that myogenesis or its activating factors are set into motion as a concomitant of wave I in muscle regeneration. It may be that phleomycin totally destroyed the cell pool for the initial round of myotube formation. It is also possible that the replicating cells released myogenic inductive agents or cofactors in wave I. But they would go no further than to say that replication is a concomitant variation of the activition of myogenesis -- in regenerating mammalian skeletal muscle.

WRITER: Damn, that frustrates me. Why can't you people ever come to a final conclusion on a subject.

ZOOK: The value of a good scientific hypothesis is in its predictiveness -- in its capacity to foretell of the future, what experience cannot tell us in the present. The phleomycin experiments led to the hypothesis that replication is an important covariate of the activation of muscle regeneration.

WRITER: Further predictions? Not proof?

ZOOK: Not proof. Predictions!

WRITER: What other predictions?

ZOOK: The hypothesis successfully predicted that elevating replication would enhance regeneration in dystrophic as well as injured muscle.[14] WRITER: No sh....kidding?

ZOOK: Again, timing the dosage was critical.

WRITER: Boy, that timing business seems to come up, again and again.

ZOOK: To me it is the most important aspect of everything I've said today. But that is another story.

WRITER: Is that an invitation to come again.

ZOOK: Before you go, can you tell me what happened to the musician whose lip was injured?

WRITER: He still blows a horn, which means that his soul as well as his lip regenerated. When he's in New Orleans you can catch him at Patout's Restaurant on Bourbon Street -- upstairs in Jelly Roll's room .[17] Also, he was on a giant float during the half-time ceremonies at Superbowl XXX.

ZOOK: At the what?

WRITER: A championship football game!

ZOOK: I'm glad he recovered.

For another Zook dialog, go here

go to the ShuffleBrain main menu