Adv Physiol Educ 43: 476–485, 2019;
doi:10.1152/advan.00094.2019.
HISTORICAL PERSPECTIVES
Calcium and muscle contraction: the triumph and tragedy of Lewis Victor
Heilbrunn
Jack A. Rall
Department of Physiology and Cell Biology, College of Medicine, Ohio State University, Columbus, Ohio
Submitted 17 July 2019; accepted in final form 10 September 2019
Rall JA. Calcium and muscle contraction: the triumph and tragedy
of Lewis Victor Heilbrunn. Adv Physiol Educ 43: 476 – 485, 2019;
doi:10.1152/advan.00094.2019.—Lewis Victor Heilbrunn has been
called the pioneer of Ca2⫹ as an intracellular regulator (Campbell AK.
Cell Calcium 7: 287–296, 1986; Campbell AK. Intracellular Calcium,
2015). In 1947, he was the first to provide convincing evidence that
Ca2⫹ triggered muscle contraction (Heilbrunn LV, Wiercinski FJ. J
Cell Comp Physiol 29: 15–32, 1947). Yet his work was met mostly
with silence and neglect. One wonders why. Heilbrunn was a general
physiologist who believed in the uniformity of nature with regard to
movement. He believed that “. . . the theory of what makes cells
divide should not be very different from the theory of what makes
muscle contract . . .” (Heilbrunn LV. The Dynamics of Living Proto-
plasm, 1956). He did not believe that one could understand how the
living machine worked by investigating its parts. He believed that, to
understand life, one must study the dynamics of living protoplasm.
The origin and evolution of Heilbrunn’s thought process regarding the
role of Ca2⫹ as a physiological activator will be traced back to the
1920s. The ways in which he tested the Ca2⫹ hypothesis in sea urchin
eggs in the 1920s and 1930s will be explored. This work shaped
Heilbrunn’s thinking about the role of Ca2⫹ in muscle contraction.
Importantly, why he and his results were ignored for years will be
examined. It turned out that being right was not enough. Bad luck and
a stubborn belief in an outmoded scientific philosophy contributed to
the neglect.
marine biological laboratory; muscle contraction; sea urchin egg
division
INTRODUCTION
Lewis Victor Heilbrunn (1892–1959) (Fig. 1) received a
PhD under the guidance of the pioneering embryologist Frank
R. Lillie (1870 –1947) at the University of Chicago in 1914 at
the age of 22 yr (60). During 1917 and 1918, he was in the Air
Service of the United States Army and part of the American
Expeditionary Forces that fought on the Western Front during
World War I. From February of 1918 until Armistice Day on
November 11, 1918, he flew reconnaissance missions with the
88th aero squadron1 and was awarded a silver citation with two
Address for reprint requests and other correspondence: J. A. Rall, Dept. of
Physiology and Cell Biology, College of Medicine, Ohio State University,
1645 Neil Ave, 302 Hamilton Hall, Columbus, OH 43210 (e-mail: Rall.1
@osu.edu).
1
The 88th Aero Squadron was a Corps Observation Squadron that con-
ducted short-range visual reconnaissance and short-range photographic mis-
sions. These missions required the pilot to fly between altitudes of 50 m to
5,000 m under all sorts of weather conditions. It has been said that the pilot
“. . . had to keep to a carefully prescribed straight and level course, a process
476 1043-4046/19 Copyright © 2019 The American Physiological Society
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oak leaves for heroism in the face of the enemy (Fig. 2). After
demobilization, he taught for 8 yr at the University of Michi-
gan and then spent a year in Europe as a Guggenheim fellow.
In 1929, he was appointed to the staff of the Department of
Zoology at the University of Pennsylvania, where he remained
throughout his career. Beginning with his graduate student
days, Heilbrunn spent his summers doing research and teach-
ing at the Marine Biological Laboratory (MBL) at Wood Hole,
Massachusetts. Like Lillie, he was dedicated to the mission of
the MBL and was elected a member of the corporation in 1915,
a trustee in 1931, and to the executive committee in 1934. Well
over 50 scientists took their PhD degrees under Heilbrunn’s
direction. He produced numerous peer-reviewed manuscripts
and generated two monographs and a textbook of general
physiology that went through three editions. By all accounts,
he was a prolific and impeccable scholar. He could be generous
and thoughtful but also critical and stubborn. Steinbach (60) in
an affectionate obituary stated:
Heilbrunn was a man of strong opinions and had an ability
to state them forcefully and clearly. His was a tough mind with
little inclination to compromise. His criticisms were stated as
clearly as were his praises, and his critics were frequently as
vocal as his supporters.
Heilbrunn was guided throughout his career by a scientific
philosophy that dictated his experimental approach and opin-
ions. He believed that, to understand life, one must study the
dynamics of living protoplasm. The origin of the word proto-
plasm dates to the middle of the 19th century (26). The word
protoplasm comes from the Greek, protos, first, and plasma, a
thing formed, and means literally “the first creation.” In 1868,
Thomas Henry Huxley (1825–1895) called it the “physical
basis of life.” The idea that protoplasm was a single entity, “the
living substance,” persisted into the early 20th century. The
word protoplasm was in general use before the techniques of
electron microscopy and differential centrifugation helped to
elucidate the detailed structures and specific function of dis-
crete intracellular organelles. It is a word rarely used today.
Heilbrunn (36) believed that it was imperative to study
protoplasm when it was alive and not after it was dead. Thus to
Heilbrunn it seemed “rather obvious” that, to understand pro-
toplasm as a living machine, one must study the machine itself
and not depend on speculation as to what the machine might be
that required tremendous discipline, precision, and steady nerves. To achieve
mission success required the pilot be a top performer, possessing great skill and
a sense of orientation” (17). These attributes also describe well Heilbrunn’s
personality and approach to science.
 PIONEER OF CALCIUM AND MUSCLE CONTRACTION
Fig. 1. Portrait of Lewis Victor Heilbrunn (1892–1959) in 1928 at the Marine
Biological Laboratory. [From History of the Marine Biological Laboratory,
with permission. https://hpsrepository.asu.edu/handle/10776/3105, 2007-01-28.]
like based on its chemical nature. He did not believe that
purified substances isolated from living cells could possibly
duplicate the behavior of living protoplasm. To understand the
physical properties of living protoplasm, Heilbrunn investi-
gated the viscosity of protoplasm and how it changed with cell
activation or inhibition. This so-called colloidal chemical ap-
proach to the study of protoplasm became, in time, strongly
criticized with the ascension of biochemistry. Particularly crit-
ical was Florkin (18) when he stated that this approach gener-
ated “an enormous amount of useless literature.” Nonethe-
less, Heilbrunn never wavered from the study of the phys-
ical machine via measurement of viscosity changes of
protoplasm (37).
Heilbrunn was strong willed and unbending in his criticism
of the reductionist approach to science. He was a general
physiologist who believed in the uniformity of nature with
regard to movement and liked to say that “. . . the theory of
what makes cells divide should not be very different from the
theory of what makes muscle contract . . .” (36). He investi-
gated the physical rather than chemical nature of protoplasm.
How this stubborn adherence to an eventually outmoded ex-
perimental approach led Heilbrunn to correctly propose Ca2⫹
as the physiological activator of cell division and muscle
contraction will be explored.
Calcium and Cell Division
In 1915, Heilbrunn studied the initiation of cell division in
the eggs of the sea urchin arbacia in experiments predomi-
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477
nantly done at the MBL. The sea urchin is an exceptional
experimental model organism for the study of developmental
biology. Sea urchin eggs are transparent, uniform in diameter
(~70 ␮m), and can be manipulated easily in the research
laboratory. These eggs can be fertilized easily, and then they
develop rapidly and synchronously. Heilbrunn (27) showed
that agents that incited the egg to divide caused an increase in
the viscosity of the cell protoplasm. He measured the qualita-
tive change in viscosity of the protoplasm by observing the
movement of granules through the cell caused by an increase in
centrifugal force generated by a hand centrifuge. The force
exerted to push the granules through the cell was taken as a
measure of the viscosity of the protoplasm. Eggs that had been
treated in various ways were placed in one tube of the centri-
fuge, and untreated eggs were placed into the other tube. After
centrifugation, a microscopic examination revealed any differ-
ences that may have existed between normal and treated eggs.
When normal unfertilized eggs were centrifuged, the proto-
plasmic materials rapidly became separated into layers or
zones. As the viscosity of the protoplasm increased, stratifica-
tion became more difficult, and, in a thoroughly coagulated
egg, no stratification was observed. The cells were not dam-
aged by the centrifugation. Heilbrunn (27) concluded that:
. . . the only physico-chemical effect which all parthenoge-
netic agents possess in common is the production of a gelati-
nization or coagulation within the egg. Hence I regard this
gelatinization (or coagulation) as the direct cause of the initi-
ation of development.
He thus provided the first experimental evidence that cell
division involved a gelation or increase in protoplasmic vis-
cosity.
Because of the importance of measuring protoplasmic vis-
cosity, Heilbrunn wanted to determine the absolute viscosity of
the protoplasm. This measurement was much more difficult
than measuring changes in viscosity. It required some assump-
tions and corrections. Most important was Heilbrunn’s tacit
assumption that the protoplasm of the sea urchin egg was a
structureless Newtonian fluid.2 To determine absolute viscos-
ity, the speed of movement of granules through the egg must be
measured with a given centrifugal force, the radius and specific
gravity of the granules and specific gravity of the medium must
be estimated, and then the viscosity of the medium could be
calculated (28). The approximate value for the viscosity of the
granule-free protoplasm of the arbacia egg was ~2 cP (29). He
concluded that the intergranular material in the arbacia egg had
a viscosity approximately twice that of water (1 cP). This result
countered some earlier reports that the protoplasm was a gel.
Clearly it was a fluid, a sol. Heilbrunn (29) stated: “We must
now admit that protoplasm, in some instances at least, is in no
way the highly viscous, slimy material that the morphologists
would have us believe.” Heilbrunn (37) always believed that
the measurement of protoplasmic viscosity was the most im-
2
Later Robert D. Allen (1), studying amoeba cytoplasm, concluded that it
was not possible to provide a single absolute value of protoplasmic viscosity
because the cell interior was not a structureless fluid and it exhibited non-
Newtonian behavior. The same argument would hold for the sea urchin egg
and the muscle fiber. Quantitative viscometry appeared to be ruled out by the
obvious signs of non-Newtonian behavior. Qualitative estimates of changes in
cell viscosity could still be useful.
478 PIONEER OF CALCIUM AND MUSCLE CONTRACTION

Fig. 2. Photograph of the pilots of the 88th

Aero Squadron taken in November of 1918
(22). Heilbrunn is third from the right stand-
ing behind the Salmson 2A2 biplane. Note
the Bucking Broncho insignia of the 88th
Aero Squadron on the plane. [From the San
Diego Air and Space Museum Archive,
Philip Babcock Collection no. 000051; pub-
lic domain.]

portant way to study the physical properties of living proto- released interacted with the cell protoplasm and caused a
plasm. clotting reaction there. This hypothesis was clearly put forward
The question then became: What caused the increased pro- in his 1928 monograph (32).
toplasmic viscosity observed when the sea urchin egg was Heilbrunn believed throughout his research career that the
stimulated to divide? In a paper read at a symposium of the surface precipitation reaction was “in large measure responsi-
American Society of Naturalists in 1925, Heilbrunn hinted at ble for the action of the living machine” (36) and “is a
an experiment that would shape his thinking on this question characteristic of living substance generally” (34). He thought
throughout the rest of his career (30). When a sea urchin egg that the surface precipitation reaction was “a form of clotting”
was broken by applied pressure, the protoplasm flowed out. A
precipitation process occurred involving the formation of a film
at the surface of the naked protoplasm as it interacted with the
medium. The precipitation process could be studied under
various chemical and physical conditions. With regard to this
process, Heilbrunn (30) noted:
During the past summer a few crude preliminary experi-
ments were made in the case of the sea-urchin egg. As far as
they have gone they indicate that the formation of a surface
membrane in this cell is favored by the presence of calcium
salts and hindered or prevented in their absence.
He viewed this process as a sort of coagulation process that
must occur at the boundary of the protoplasm to prevent its
diffusion throughout the surrounding medium. He followed up
these preliminary experiments in 1927 with a thorough study of
what he called the “surface precipitation reaction” (Fig. 3) and
its relation to Ca2⫹ (31). The reaction did not occur if the cell
was broken in a NaCl or MgCl2 solution or in Ca2⫹-free sea
water, where Ca2⫹ was bound by an oxalate solution. Appar-
ently a certain amount of Ca2⫹ was necessary for the reaction
to proceed. Heilbrunn believed that washing for a minute in
isotonic NaCl solution or treatment for a minute with oxalated
sea water could not remove the Ca2⫹ from the egg interior.
Therefore, the Ca2⫹ within the egg must not be in an available Fig. 3. The surface precipitation reaction in the egg of arbacia. As a result of
form. With regard to the marine eggs, these eggs can be a break or a tear of a sea urchin egg of arbacia, the protoplasm emerges from
induced to divide by various artificial means, such as strong the cell and forms a discrete droplet containing many vacuoles, which does not
diffuse through a Ca2⫹-containing medium. Heilbrunn called this reaction of
salt solutions, heat, ultraviolet light radiation, or acids. Heilb- naked protoplasm with Ca2⫹ “the surface precipitation reaction.” He specu-
runn hypothesized that all of these agents caused a release of lated that the large vacuoles formed within the exuded protoplasm also were
Ca2⫹ from a bound form within the cell. The Ca2⫹ that was formed by the same surface precipitation reaction. [From Heilbrunn (31)].

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 PIONEER OF CALCIUM AND MUSCLE CONTRACTION
induced by Ca2⫹ (34). In this latter respect, Heilbrunn believed
that the clotting of protoplasm was analogous to the clotting of
blood. It had been known since the late 19th century that blood
clotting required Ca2⫹ (2, 21). Thus Heilbrunn linked together
the role of Ca2⫹ in the well-known phenomenon of blood
clotting with the viscosity changes in protoplasm.
What evidence did Heilbrunn have that the intracellular free
Ca2⫹ increased when the sea urchin egg was activated to
divide? In 1937, Daniel Mazia3, a graduate student in the
Heilbrunn laboratory, measured ultrafilterable (free) and non-
ultrafilterable Ca2⫹ (bound) in suspensions of broken fertilized
and unfertilized sea urchin eggs (45). He found that the total
Ca2⫹ content of the egg suspensions was unchanged with
fertilization, but that the free Ca2⫹ increased with fertilization.
Thus Mazia concluded: “It can then be postulated that on
fertilization, the free Ca concentration within the cell goes
above the threshold for the reaction, and the viscosity increase
takes place.” But he also added: “Concerning the actual mech-
anism by which activation causes Ca release, little can be said
at this time.” Mazia acknowledged Heilbrunn’s guidance and
attributed the main ideas of the work to him. This was the first
study to provide quantitative evidence to support the hypoth-
esis that fertilization in sea urchin eggs resulted from an
increase in intracellular Ca2⫹. This pioneering study has been
cited over 200 times.
Where was the Ca2⫹ stored within the sea urchin egg? Based
on earlier work in his laboratory on amoeba locomotion,
Heilbrunn proposed that the Ca2⫹ was stored in the cortex just
under the cell membrane of the sea urchin egg. In the sea
urchin egg, the cortical layer or cortex was demonstrated by
Moser (46) in the Heilbrunn laboratory. The granules in the
cortex were not readily displaced by centrifugal force, and for
this reason the cortex of these cells was considered to be a gel.
But, Heilbrunn admitted (36), there was no direct evidence that
the Ca2⫹ actually was stored in the cortex of the sea urchin egg
and released on egg fertilization.
To summarize Heilbrunn’s thinking, the bulk of the proto-
plasm of the sea urchin egg is a fluid (sol), not a gel. The outer
part of the protoplasm consists of a rigid gellike cortex. Ca2⫹
is stored in the cortex in a bound form. Chemical and physical
agents can free the Ca2⫹ in the cortex from its bound form. The
surface precipitation reaction is the basic reaction of proto-
plasm to Ca2⫹. As the Ca2⫹ enters the cell interior, it causes
the protoplasm to undergo a clotting reaction, which increases
protoplasmic viscosity. In a marine egg, the clotting reaction
results in cell division. Since he did not have the direct proof
that he needed, he continued to search for this proof but, in the
process, never wavered from the belief that he was correct.
Like the reconnaissance pilot that he was in WWI, Heilbrunn
3
Daniel Mazia (1912–1996) was one of Heilbrunn’s most distinguished
former students (15). He, along with Katsuma Dan, isolated the mitotic
apparatus or mitotic spindle in 1952. Mazia was a member of both the National
Academy of Sciences and the American Academy of Arts and Sciences. He
served as President of both the American Society for Cell Biology (in 1976)
and the International Federation for Cell Biology (from 1976 to 1982).
Heilbrunn trained other leaders in cell biology, including H. Burr Steinbach
(1905–1981) and Paul R Gross, who became directors of the MBL; Robert Day
Allen (1927–1986), developer of video-enhanced contrast microscopy in cell
and molecular biology; and Katsuma Dan (1904 –1996) and his wife Jean
Clark Dan (1910 –1978). Usually Heilbrunn did not put his name on the
primary publications of his students.
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479
kept “to a carefully prescribed straight and level course”.1
Finally, he stated that, “The main conclusion from our work is
that calcium is not only important for the rigidity of animals
and cells, it is the prime instigator of vital activity” (34).
It was not until after Heilbrunn’s death in 1959 (see below)
that the advent of Ca2⫹ ionophores (61), Ca2⫹ buffers (24, 66),
and intracellular Ca2⫹ indicators (50, 55) proved conclusively
that increases in intracellular Ca2⫹ concentration triggered egg
cell activation. Whereas many of these studies were first
carried out on sea urchin eggs, it is now established that Ca2⫹
acts as the trigger for egg activation in practically all species.
For a recent review of the activation of egg cell at fertilization,
see Parrington et al. (49).
Calcium and Muscle Contraction
Sydney Ringer (1836 –1910) in London was the first person
to systematically investigate the effect of removal of particular
ions from the solution bathing muscle tissues on structure and
function. His experiments showed that the isolated frog heart
would stop beating if extracellular Ca2⫹ was removed from the
medium (57).
In the late 1930s, Heilbrunn turned his attention to investi-
gating the hypothesis that intracellular, not extracellular, Ca2⫹
was the physiological regulator of skeletal muscle contraction.
Heilbrunn was a general physiologist. In 1946, he was one of
the founding members and the first president of the Society of
General Physiologists (52).4 He authored a book entitled: “An
Outline of General Physiology” that went through three edi-
tions (35).5 He believed that “. . . the theory of what makes
cells divide should not be very different from the theory of
what makes muscle contract. . .” (36). According to his previ-
ous views, cells typically consist of a cortex that is a gel and a,
more or less, fluid interior. Among its other constituents, the
cortex contains Ca2⫹ in a bound form. On stimulation, Ca2⫹ is
released from the cortex and enters the cell interior. There it
produces gelation or clotting. This clotting reaction is essen-
tially the same type of reaction that occurs when naked proto-
plasm is exposed to Ca2⫹, i.e., a surface precipitation reaction.
In the case of muscle, Heilbrunn proposed that this clotting
reaction led to muscle contraction.
His first substantive paper to test the role of Ca2⫹ in muscle
contraction appeared in 1940 (33). Single muscle fibers with
cut ends were isolated from frog skeletal muscle and placed in
a Ringer solution with a 1.4 mM CaCl2 concentration. A
“plug” appeared at the cut surface. Heilbrunn believed that this
“plug” was analogous to the surface precipitation reaction seen
in other cells when Ca2⫹ interacted with the exposed proto-
plasm.
When the isolated muscle fiber was placed in an isosmotic
solution of CaCl2 (90 mM), a sudden change occurred. The
fiber shortened rapidly until in a minute or two it was only
4
The Journal of General Physiology, established in 1918, has had a long
association with the MBL. Since the Society of General Physiologists was
founded in 1946, the Society and Journal of General Physiology have main-
tained an informal association with each other and with the MBL.
5
In an effort typical of Heilbrunn’s character and drive, in the first edition
of his general physiology textbook, he states that he was unhappy with other
texts on general physiology, so he wrote his own. The third edition of the book
ran over 800 pages and, by Heilbrunn’s own count, contained ~4,300 refer-
ences (35).
480 PIONEER OF CALCIUM AND MUSCLE CONTRACTION
~30% of its original length. Figure 4 displays a series of
photographs of an isolated fiber segment in an isosmotic CaCl2
solution. The individual photographs were taken at 10-s inter-
vals. If instead an isolated fiber was suspended over two small
glass rods, then it was possible to apply a CaCl2 solution either
to the cut ends or to the uninjured surface along the length of
the fiber. If the fiber ends were exposed to the isosmotic CaCl2
solution, shortening occurred at about the same rate as it did
when the entire fiber was immersed. On the other hand, if the
CaCl2 solution was applied along the sides of the fiber, with the
ends in Ringer solution, no rapid shortening occurred. This
effect of Ca2⫹ also was observed in muscle fibers from snakes,
turtles, mammals, and in invertebrate muscle, including crabs
and insects (35). Thus the conclusions were that an increase of
Ca2⫹ concentration within the muscle fiber caused contraction,
and the Ca2⫹ was likely released from an intracellular store. In
analogy with the conclusions with the sea urchin egg, the
intracellular store was proposed to be the cortex of the muscle
fiber. The results seemed obvious, and the conclusions justi-
fied, but the experiments were criticized because the CaCl2
concentration in the solution utilized to generate muscle short-
ening was far beyond physiological limits (16).6
To further test the hypothesis that Ca2⫹ was the physiolog-
ical initiator of muscle contraction, Heilbrunn and Floyd J.
Wiercinski (1912–1996), a former graduate student in the
Heilbrunn laboratory and now a research collaborator, con-
ducted a series of experiments in 1947 that were both thorough
and elegant for the time (38). At the outset they stated their
point of view clearly:
Many types of stimulation cause a muscle fiber to contract.
We believe that essentially all these diverse types of stimula-
tion cause a release of calcium ion from the surface or outer
6
Total muscle cell calcium content is in the low millimolar range (25).
Fig. 4. Shortening of an isolated muscle fiber in an
isosmotic CaCl2 solution (90 mM). The succession of
stages is from left to right. The first photograph was
taken in Ringer solution; the second, 10 s after the
addition of an isosmotic CaCl2 solution. Subsequent
pictures were taken at 10-s intervals. [From Heilbrunn
(33), with permission from The University of Chicago
Press Journals.]
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region of the cell and that this calcium then enters the cell and
produces the response.
To establish direct contact between a given solution and the
protoplasm, they injected various cation solutions into the
interior of muscle fibers. Frog muscle fibers with cut ends (~1
mm long) were placed on a glass coverslip, excess fluid was
removed, and the coverslip was inverted and placed over a
moist chamber. This “hanging drop” arrangement allowed a
micropipette with a tip diameter of ~2 ␮m to be inserted
through the open side of the chamber and the solution to be
tested to be pressure injected into the fiber.7
They injected isotonic solutions of varying ionic composi-
tions into the frog fiber segments and monitored the resulting
extent of segment shortening in at least 10 fibers for each
perturbation. A Ringer solution containing Na⫹, K⫹, and 1.3
mM Ca2⫹ caused in every fiber a large (average 44%) and
rapid (complete in ~6 s) fiber shortening (see Fig. 5). In
contrast, when a Ringer solution where Mg2⫹ was substituted
for Ca2⫹ was injected into the fibers, essentially no length
change was observed. Nor did a solution of MgCl2 alone
initiate any contraction. A KCl solution (126 mM) caused a
small variable amount of shortening (average ⬍10%) that took
minutes to be complete. The majority of the fibers injected with
KCl exhibited no shortening at all. A NaCl solution (123 mM)
resulted in only 4% shortening in 1–2 min, but, once again,
most fibers showed no shortening at all immediately following
injection.
Because of the previous criticism by Fenn (16) of the
nonphysiological level of Ca2⫹ (90 mM) exposed to the pro-
toplasm, they attempted to determine the minimum injected
Ca2⫹ concentration that would cause a large and rapid muscle
7
This technique and many other micromanipulation techniques were devel-
oped by Robert Chambers during the early 20th century to study living cells.
See Chambers and Chambers (8) for a description of results and techniques.
 PIONEER OF CALCIUM AND MUSCLE CONTRACTION 481

Rieser concluded that this was probably due to damage on

injection of the oil drop. In the 10 experiments reported where
the oil drop did move, 3 produced high values for protoplasmic
viscosity that Rieser reasoned were likely due to fiber injury.
For the other 7 cases where the ~17-␮m-diameter oil drop
moved more rapidly, the resulting estimate of absolute fiber
viscosity was ~29 cP. This value of absolute viscosity was
higher than that observed in the sea urchin egg (2 cP) or water
(1 cP), but was consistent with the conclusion that the skeletal
muscle protoplasm was a sol and not a gel. The plan was to
employ the oil drop technique to assess changes in protoplas-
mic viscosity on muscle activation, but no results were pub-
lished in this regard.
Rieser (56) performed another type of experiment, where he
Fig. 5. A table of results from microinjection of an isotonic Ringer solution attempted to fill the total cross section of the muscle fiber with
into cut fiber segments isolated from the adductor magnus muscle of Rana oil. The injected oil never occupied the entire width of the
pipiens. Note the rapid and extensive muscle fiber shortening after the
injection. In contrast, when a Ringer solution where Mg2⫹ was substituted for fiber. There was always a region directly beneath the sarco-
Ca2⫹ was injected into the fibers, no length change was observed (not shown). lemma that the oil could never displace. This outer region had
To determine fiber segment length in micrometers, multiply by 11.5. [From a thickness of ~10 ␮m. Rieser concluded that this outer region
Heilbrunn and Wiercinski (38),with permission from John Wiley and Sons of the muscle fiber was similar to the gel-like cortex of other
Inc.]
types of cells.
These results reinforced Heilbrunn’s hypothesis for the
shortening. That minimum Ca2⫹ concentration was ~0.2 mM physiological role of Ca2⫹ as the mediator of muscle contrac-
CaCl2. Since the dilute CaCl2 solution was strongly hypotonic, tion. The supposition was that Ca2⫹ was stored at rest in the gel
they did a pure distilled water control that, in most instances, cortex of the muscle fiber and, on stimulation, was released
caused no shortening of the fibers. Since the CaCl2 solution into the fluid core of the fiber to cause a viscosity change,
injected into the fiber was diluted by the protoplasm of the
muscle, one could assume that the effective CaCl2 concentra-
tion was even ⬍0.2 mM.
Of the cations, Ca2⫹, Mg2⫹, K⫹, and Na⫹, present in any
quantity in muscle, only Ca2⫹ injection caused a rapid and
extensive contraction. This was a massive study with hundreds
of muscle fiber segments injected with clear and persuasive
results. Many years after this landmark study, Wiercinski
recalled, “It was fantastic to watch. We injected the calcium,
and the fibre instantly pulled into a mass” (7). The negative
results with K⫹ (compared with Ca2⫹) were especially impor-
tant since Albert Szent-Gyorgyi (62) had proposed a model of
muscle contraction based on biochemical studies of isolated
proteins, wherein contraction was induced by small changes in
K⫹ concentration. This model could not be confirmed in living
fibers and thus was clearly wrong. In all, these experiments
provided strong support for what was called the Ca2⫹ release
theory of muscle contraction.
Because of the importance of changes in protoplasmic vis-
cosity as an indicator of the action of Ca2⫹ as an activator in
sea urchin eggs, Heilbrunn wanted to have a measure of
protoplasmic viscosity in skeletal muscle fibers. After many
procedures failed, graduate student Peter Rieser in the Heilb-
runn laboratory adapted the micromanipulative technique to
the rising sphere method of viscometry for measurement of
muscle fiber viscosity (56). A single small oil drop was
injected into the protoplasm of a frog muscle fiber in a
Ca2⫹-free Ringer solution and observed with the microscope
tilted so that the stage was vertical (Fig. 6). Because the oil
drop had a lower specific gravity than the fiber protoplasm, it
rose slowly upward through the fiber protoplasm. The velocity
of rise of the oil drop was determined by noting the distance Fig. 6. An oil drop injected into the interior of a frog muscle fiber was utilized
to estimate skeletal muscle viscosity (56). The speed at which the oil drop
traveled in a given time. Absolute viscosity was calculated moved through the protoplasm was taken, with assumptions, as a measure of
from Stokes law with various assumptions.2 The experiments skeletal muscle viscosity, which averaged 29 cP. [From Heilbrunn (36), with
were tedious because, in most cases, the oil drop did not move. permission from Elsevier Books.]

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482 PIONEER OF CALCIUM AND MUSCLE CONTRACTION
leading to muscle contraction. Rieser’s results also fit with
Heilbrunn’s belief that muscle fiber protoplasm had the same
essential physical structure as in the cell of a sea urchin egg,
i.e., it was composed of fluid protoplasm surrounded by a stiff
cortex. Nonetheless Heilbrunn (36) had to admit that:
We do not know that this calcium comes from the cortex.
Also there is no great amount of evidence to show that the free
calcium, which we assume to have come all or in large part
from the cortex, finds its way into the cell interior.
Later in a brief report in 1955, Niedergerke (48) also found
that microinjection of Ca2⫹ into a frog muscle fiber resulted in
local contraction and partial relaxation. But rather than invok-
ing a clotting of proteins, Niedergerke suggested that the Ca2⫹
activated a link of the contractile cycle. He did not follow up
on this brief report.
Scientific Reception of the Calcium Hypothesis in Muscle
Even though the Heilbrunn and Wiercinski (38) study in
1947 is now considered to be a landmark in the history of the
investigation of the role of Ca2⫹ in muscle contraction,
strangely the results did not influence the course of the history
of investigation into the role of Ca2⫹ in muscle contraction.
The role of Ca2⫹ in muscle contraction essentially was redis-
covered years later in the 1960s (see below). One wonders why
this landmark study was ignored. According to Google
Scholar, the paper was cited only ~16 times between 1947 and
Heilbrunn’s death in 1959 (see below). Heilbrunn (36) la-
mented that “the theory suffers more from being disregarded
than from an actual attacks on it.” Wiercinski (65) many years
later called the disregard the “hush of neglect.” To be sure, the
study was ahead of its time in the clarity of thought about the
role of Ca2⫹ as the physiological activator of muscle contrac-
tion, but there were other reasons to explain its lack of
influence. The reasons are of two general types: one was
scientific and the other related to Heilbrunn’s scientific philos-
ophy and personality.
The scientific problem with Heilbrunn’s Ca2⫹ activator
hypothesis in muscle related to the simple question: Could
Ca2⫹ released on stimulation from a storage site in the cortex
beneath the sarcolemma of a muscle fiber diffuse rapidly
enough throughout the fiber to explain the time course of
muscle contraction? This question was addressed by the fa-
mous muscle physiologist A. V. Hill (1886 –1977) in a theo-
retical paper (39) published a year after the Heilbrunn and
Wiercinski (38) paper. Hill was a Nobel Laureate who was
trained as a mathematician [see Rall (53) for details]. He was
one of the first muscle biophysicists. As a Nobel Laureate, he
had great stature in the scientific community. In fact, it was
said that Hill’s word was “instant law” (59). Hill’s conclusion
was equivocal. He (39) stated:
If the twitch of a muscle fibre is assumed to involve the
contraction of the whole of its contents, diffusion from the
outer surface could not be fast enough to account for observed
speeds of contraction.. . . If, however, in a twitch the outer
region only, say one-half of the fibre, were involved, which
agrees with the known fact that the ratio of twitch tension to
tetanus tension is usually considerably less than unity, then
diffusion over the shorter distances involved would be amply
quick enough.
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So it seemed possible that diffusion of Ca2⫹ from the muscle
cortex could explain the speed of muscle contraction in a
twitch.
Hill followed this paper with another paper in 1949 (40) that
was very important in the history of muscle research and
devastating for Heilbrunn’s Ca2⫹ hypothesis. According to the
prevailing view of muscle mechanics, the isometric twitch
(response to a single stimulus) was less in magnitude than an
isometric tetanus (response to repeated stimulation), not be-
cause of partial activation, but rather because of the need to
stretch the passive elastic component of the muscle that is in
series with the contractile component, e.g., muscle tendons. As
the muscle responds to a single stimulus, the contractile com-
ponent begins to stretch the elastic component, but, before
maximum force can be reached, the muscle is turned off, and
thus a twitch is of lesser amplitude than a tetanus. Hill circum-
vented this issue by rapidly stretching a frog skeletal muscle
after a single stimulus but just before force began to rise. The
muscle resisted the stretch soon after the stimulation, with a
force equal to the maximum force in a tetanus but at a rate 10
times faster than the rate of force development in the normally
activated twitch. Thus the muscle was rapidly and maximally
activated throughout its whole cross section on stimulation.
Now Hill (40) stated that the “present results allow a perfectly
clear decision to be reached.” That decision was: “It is quite
impossible, therefore, to explain the rapid development of full
activity in a twitch by assuming that it is set up by the arrival
at any point of some substance diffusing from the surface:
diffusion is far too slow.” To be clear Hill never claimed that
Ca2⫹ could not be the activator of muscle contraction. Rather,
he simply showed that the activator could not be a substance
that diffused from the surface of a muscle fiber. Nonetheless, in
the minds of many scientists, this study was the “death knell”
of Heilbrunn’s Ca2⫹ hypothesis. It was now clear, Heilbrunn
must be wrong.
Heilbrunn (36) attempted to counter Hill’s conclusion. He
first emphasized that Hill’s argument applied only to stretched
muscles and not to muscles contracting normally. He went on
to further state that the agitation of the protoplasm due to
stretching of the muscle might accelerate diffusion. In 1952,
Alexander Sandow (58) wrote an influential review, wherein
he defined the term “excitation-contraction coupling” for the
first time. He was persuaded by the results of Heilbrunn and
Wiercinski (38) but was also aware of the Hill (40) criticism.
As a solution to the dilemma, he proposed a form of “exchange
diffusion” to accelerate the transport of Ca2⫹ into the center of
a muscle fiber. These were weak arguments that never gained
much traction with critics.
Other Factors Contributing to Neglect of Heilbrunn’s
Calcium Hypothesis
There were other factors that contributed to Heilbrunn’s
Ca2⫹ hypothesis being set aside. One factor was Heilbrunn’s
outmoded scientific philosophy. Heilbrunn’s experiments on
Ca2⫹ and muscle contraction came at a time when there was
natural tension between traditional muscle physiologists and
emerging muscle biochemists. With the exciting advances in
biochemistry in the 1940s and in electron microscopy in the
1950s, emphasis started to shift away from the physiological
studies. Nonetheless, throughout his career, Heilbrunn was
 PIONEER OF CALCIUM AND MUSCLE CONTRACTION
committed to the idea that changes in the state of living
protoplasm held the key to cellular events, such as movement
and cell division. In 1956, he generated a monograph entitled
The Dynamics of Living Protoplasm (36). But to many scien-
tists, arguments about protoplasm sounded like a return to the
mysterious vital force that had impeded scientific investigation
of the chemistry of life in the 19th century. One of the founders
of biochemistry, Frederick Gowland Hopkins (1861–1947),
rejected the vague concept of “protoplasm.” Hopkins in 1933
(19) described the bias against the biochemist when he stated,
“the old taunt that when the chemist touches living matter it
immediately becomes dead matter.” Heilbrunn as late as 1958
(37) in his last major publication still seemed to adhere to the
“old taunt” when he stated,
And the student of protoplasmic viscosity and of the proto-
plasmic colloid is never much interested in the behavior of
what may once have been a living cell.. . . To understand the
living colloid we must make studies of it while it is alive and
intact, and not a jumble of unassembled and damaged parts.
He lamented the fact that cytology became largely a study of
dead material, and that electron microscopy, by its very nature,
was not able to provide information on protoplasm in a living
condition (36).
Another factor contributing to the neglect of Heilbrunn’s
ideas was his sometimes sarcastic personality. He was not a
muscle physiologist and did not attend the usual meetings
frequented by muscle physiologists. He was not a member of
that scientific “club.” Heilbrunn’s most sarcastic and unfortu-
nate remarks were reserved for the muscle biochemists and
their belief in ATP and at the time its confusing proposed dual
roles in contraction and relaxation. Heilbrunn (36) stated:
One thing is certain, adenosine triphosphate, or ATP, is a
powerful word in all our modern thinking about mus-
cle. . .words seem to have taken the place of the incantations
used by magicians in the days before modern science. Whereas
the old magicians were content with their abracadabra, we now
have much more impressive words like adenosine triphosphate.
Adenosine triphosphate—and the muscle contracts; adenosine
triphosphate and it relaxes again.
He also directed his attack at Albert Szent-Gyorgyi (1893–
1986), a Nobel Laureate and Director of the Institute of Muscle
Research and fellow investigator at the MBL. With regard to
the glycerol extracted model of muscle contraction that Szent-
Gyorgyi developed (63), Heilbrunn (36) stated:
But let us return to the model experiments. All of them
involve a fundamental error. For in every single model that has
been chosen . . . the inanimate material believed to simulate the
living muscle is a gel. But . . . the interior protoplasm of muscle
fibers is a fluid sol.. . . To take a dried gel, to find that some
substances cause it to shorten– others to lengthen–all gives no
real information concerning the protoplasmic colloids of the
muscle fiber.. . . Obviously the living muscle does not behave
like glycerinated fibers do, and it is the living muscle rather
than the dead muscle that we are primarily concerned with.
Despite the Heilbrunn criticism, Szent-Gyorgyi’s glycerol
extracted model of muscle contraction, or a variation thereof,
has been utilized in thousands of experiments on muscle. It
thus was an understatement when Setsuro Ebashi (10) stated
that “The fact that Heilbrunn (1952) did not seem to appreciate
the role of ATP may have discredited the concept that Ca itself
is important at the molecular level.”
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483
Furthermore, despite evidence for the existence of muscle
myofibrils in fixed preparations in the middle of the 19th
century (5), Heilbrunn never seemed to believe that the myo-
fibrils existed in living muscle. In as late as 1958 (37), he asked
the rhetorical question: “Could it perhaps be possible that it is
only after treatment with the coagulative agents used in the
fixation that precedes sectioning that both fibrils and striations
appear as artefacts through the fiber?” He reasoned that it
would be difficult to understand how a droplet of oil could pass
up through the center of the muscle fiber without disturbing the
striations (see Rieser above). Also, he often noted (e.g., Refs.
34, 36, 37) that, over 100 yr ago, the famous German physi-
ologist Wilhelm F. Kuhne (44) saw a small nematode worm
swimming in a frog muscle fiber and suggested that the
protoplasm was fluid. Of course Heilbrunn’s views were com-
pletely contrary to the striations observed in living muscle
fibers by Andrew F. Huxley and Niedergerke (41) and the
striations observed in isolated myofibrils by Hugh E. Huxley
and Hanson (43), which formed the basis for the sliding
filament model of muscle contraction in 1954. That Heilbrunn
seemed to ignore these results was alarming and likely con-
tributed to the ignoring of his ideas about Ca2⫹ by others.
It was such views coupled with Hill’s criticism that hindered
the general acceptance of Heilbrunn’s Ca2⫹ hypothesis based
on the effects of Ca2⫹ on muscle protoplasm. Heilbrunn and
his views became increasingly isolated as science progressed in
the 1950s. Paul R. Gross (23), a former Heilbrunn student,
described Heilbrunn as a lonely man of contrasts. It turned out
that being right about the essential role of calcium as the
activator of muscle contraction was not enough. Bad luck and
a stubborn belief in an outmoded scientific philosophy contrib-
uted to the neglect of Heilbrunn’s discoveries. Clearly he often
was his own worst enemy.
Setsuro Ebashi and the Calcium Hypothesis in Muscle
Later, the role of Ca2⫹ in muscle contraction was rediscov-
ered primarily through the experiments of Annemarie Weber
(1923–2012) and Setsuro Ebashi (1922–2006) and his col-
leagues in Japan. In 1959, Weber (64) showed that Ca2⫹ in the
micromolar range regulated the ATPase activity of isolated
myofibrils. In 1961, Ebashi (9) showed that a minute amount of
Ca2⫹ was essential for actomyosin superprecipitation and the
associated acceleration of ATP splitting. Actomyosin super-
precipitation was considered to be a biochemical model of
muscle contraction. In the same paper, he described the ATP-
dependent Ca2⫹ binding of a vesicular relaxing factor isolated
from muscle that became known as the sarcoplasmic reticulum.
This latter work subsequently was published in full in 1962
(14). These experiments helped clarify the dual role of ATP in
muscle contraction and relaxation. In the discussion of the
paper, Ebashi combined the physiological evidence of Heilb-
runn and Wiercinski (38), the structural evidence of the sarco-
plasmic reticulum (4, 51), and the inward spread of activation
(42) with his biochemical evidence into a coherent hypothesis
for the role of Ca2⫹ in muscle contraction and relaxation.
Ebashi (9) proposed:
In resting muscle calcium is highly concentrated in the
endoplasmic reticulum; the concentration of calcium inside the
myofibril is too low to cause the shrinking of contractile
protein. When the muscle is excited, the concentrated calcium
484 PIONEER OF CALCIUM AND MUSCLE CONTRACTION
in some portion of the endoplasmic reticulum is released by the
electrical influence due to the depolarization of the muscle
membrane, and the calcium thus released causes in turn the
shrinking of the actomyosin system. When the excitation is
over, calcium release ceases and the liberated calcium is recap-
tured by the endoplasmic reticulum (the relaxing factor); con-
sequently, the shrunken actomyosin-system is restored to its
relaxed state.
This brilliant hypothesis eliminated the criticism of A. V.
Hill (40) regarding Ca2⫹ diffusion since it was proposed that
the Ca2⫹ was stored and released throughout the muscle fiber
and not released from a cortex near the muscle fiber surface.
The Ebashi hypothesis has guided research in excitation-con-
traction coupling for decades. The work of Heilbrunn and
Wiercinski (38) was crucial in Ebashi’s thinking, because they
demonstrated a physiological role for Ca2⫹ in contraction of
living muscle.
Attention quickly shifted to understanding the mecha-
nism of Ca2⫹ action, and from 1963 to 1968 Ebashi and his
colleagues discovered, characterized, and named the first
intracellular Ca2⫹ binding protein, troponin, and determined
that it was located on the thin actin-containing filaments of
muscle [see Rall (53) for a survey of these experiments and
the classic review by Ebashi and Endo (13)]. Their results
provided a plausible mechanism for the role of Ca2⫹ as a
muscle activator. Of course it is a major irony that Heilb-
runn’s Ca2⫹ hypothesis was confirmed and extended to the
molecular level by biochemists working with “dead” proto-
plasmic components.
Somehow, with the evolving new and exciting mechanistic
discoveries of the role of Ca2⫹ in muscle contraction and
relaxation, Heilbrunn’s work was forgotten by many people.
Looking back over his own contribution to the Ca2⫹ hypoth-
esis, Ebashi (12) has stated:
Although experiments similar to the those done by Heilb-
runn . . . were carried out by several researchers about that
time, he was the only one to affirm that Ca2⫹ was the physi-
ologic inducer of contraction.. . . Hill’s comment in 1949 . . .
accepted widely as a denial of the positive role of Ca2⫹ in
excitation-contraction coupling, exerted strong influence on
researchers in the 1950’s.
Of course the diffusion problem was eliminated with the
discovery of the sarcoplasmic reticulum that stored Ca2⫹
throughout the muscle fiber and the discovery of the transverse
tubular system that conducted the electrical stimulation from
the surface into the fiber to cause the release of Ca2⫹ within a
few micrometers of where it would act. Eventually intracellular
Ca2⫹ indicators proved directly that the free Ca2⫹ concentra-
tion increased with stimulation, leading to force development
in intact muscle fibers (3, 54). For a recent historical overview
of the structure and function of excitation-contraction cou-
pling, see Franzini-Armstrong (20). None of this information
was known at the time of the Heilbrunn and Wiercinski (38)
landmark experiments, which showed that, of all the ions tested
by injection into muscle fibers, only Ca2⫹ had a strong and
repeatable effect on contraction. With the rediscovery of the
role of Ca2⫹ in muscle contraction came the rediscovery of the
Heilbrunn and Wiercinski publication (38) that now has been
cited nearly 400 times.
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In the End
Recently, I asked a muscle physiologist for whom I have a
deep respect: “Who was the first person to discover the role of
Ca2⫹ in muscle contraction?” The person said without hesita-
tion: “Setsuro Ebashi.” In contrast, in 1988, Ebashi (11) stated:
“It is well known that the first person who clearly declared the
essential role of Ca2⫹ in contractility was Heilbrunn. . .” Hei-
lbrunn never lived to receive the credit that he deserved as he
died at 67 yr of age in an automobile accident on October 24,
1959. He was on his way back to Philadelphia from a series of
lectures given at southern colleges and universities when the
car he was driving collided with a truck in Virginia (47).
Despite Ebashi’s comment, Heilbrunn’s role in the history of
the discovery of the role of Ca2⫹ in muscle contraction appar-
ently is not as well known as it should be. This paper was
written in part to help correct that deficiency.
ACKNOWLEDGMENTS
The author thanks the Department of Physiology and Cell Biology at Ohio
State University for continued support.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author.
AUTHOR CONTRIBUTIONS
J.A.R. conceived and designed the research; analyzed data; interpreted
results of research; prepared figures; drafted the manuscript; edited and revised
the manuscript; and approved final version of the manuscript.
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