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The structure of cardiac glycogen in healthy

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Contents lists available at SciVerse ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Short communication

The structure of cardiac glycogen in healthy mice

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4

Q1

Quinn A. Besford a , Mitchell A. Sullivan b , Ling Zheng c , Robert G. Gilbert b ,

David Stapleton d , Angus Gray-Weale a,

School of Chemistry, University of Melbourne, Victoria 3010, Australia

Centre for Nutrition and Food Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia
College of Life Sciences, Wuhan University, Wuhan 430072, China
d
Department of Physiology, University of Melbourne, Victoria 3010, Australia

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a r t i c l e

i n f o

a b s t r a c t

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Article history:
Received 16 February 2012
Received in revised form 6 May 2012
Accepted 26 June 2012
Available online xxx

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Transmission electron micrographs of glycogen extracted from healthy mouse hearts reveal aggregate
structures around 133 nm in diameter. These structures are similar to, but on average somewhat smaller
than, the -particles of glycogen found in mammalian liver. Like the larger liver glycogens, these new
particles in cardiac tissue appear to be aggregates of -particles. Free -particles are also present in liver,
and are the only type of particle seen in skeletal muscle. They have diameters from 20 to 50 nm. We discuss
the number distributions of glycogen particle diameters and the implications for the structurefunction
relationship of glycogens in these tissues. We point out the possible implications for the study of glycogen
storage diseases, and of non-insulin dependent diabetes mellitus.
2012 Elsevier B.V. All rights reserved.

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Keywords:
Glycogen
Transmission electron microscopy
Dynamic light scattering

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1. Introduction

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Glycogen is a highly branched polysaccharide, used by mammals

in their livers to control blood glucose [1], and in their muscles as
a ready, local fuel store [2]. The different purposes of glycogen in
these tissues reect their different structures: liver cells contain particles, aggregates of -particles, on average about 150 nm, and
up to about 300 nm, in diameter [3,4]; other cells, skeletal muscle
for example, are known to contain -particles from 20 to 50 nm
in diameter [2]. Until now, it has seemed that the distribution of
sizes in healthy mammal cells follows this simple pattern. Storing
glucose in polymeric form reduces the osmotic stress on the cell
[5], but glycogen is not merely a passive fuel store. It participates
in the regulation of a cells energy supply. For example, different
sized particles of glycogen are made and broken down at different
rates [6], and so the distribution of particle sizes controls glycogens response time to glucose concentrations, and its ability to
buffer glucose concentrations. Further, high muscle glycogen content is correlated with reduced activation of AMP-activated protein
kinase (AMPK) by the AMP mimic, AICA riboside in perfused rat
muscle [7], and by exercise in human muscle [8]. It has also been
argued that glycogen is an anchor point for other macromolecules
[9], which is consistent with the suggestion that glycogen particles
are localised to different parts of the cell, and provide different

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Corresponding author.
E-mail addresses: dis@unimelb.edu.au (D. Stapleton), angusg@unimelb.edu.au
(A. Gray-Weale).

metabolic pools [2]. With the possible exception of AMPK activation, for all of these roles glycogens particle size is essential to its
function, and this reinforces the suggestion that the different role
of glycogen in liver cells dictates its distinctive aggregate -particle
structure. Measuring the structure of glycogen, and in particular its
distribution of particle sizes, is a route to better understanding of
its various roles. Roach has pointed out that glycogen exists as a
continuous distribution of species of different sizes, with the exact
distribution depending on both the synthetic and degradative arms
of glycogen metabolism [10].
Though the heart obtains 6090% of its energy from fatty acids
[11], some comes from carbohydrate [12], with the heart muscle
cells containing about 2% glycogen by volume [9,13]. Pederson et al.
studied mice with the GYS1 gene that encodes an isoform of glycogen synthase disrupted, and found that most GYS1-null animals
died after birth [13]. This work shows that the ability to synthesise glycogen is critical to heart development. The stress responses
of heart muscle glycogen have particular features not reported
in other tissues. Fasting for 48 h decreases both skeletal muscle
and liver glycogen, but increases heart muscle glycogen levels
[14,15]. An early report classes kidney, heart, and stomach glycogens together as they are not reduced after a 48 h fast [16]. Luptak
et al. showed that a lifelong increase in a mouse hearts glucose
uptake, induced by overexpression of the GLUT1 glucose transporter in a mouse heart, improves resistance to ischemic injury
[17]. Tian and Abel showed that mouse hearts decient in GLUT4,
normally the more abundant glucose transporter, are more susceptible to ischemia, and that glycogen stores in fed mice partly
compensate for this effect [18]. In an earlier review, Taegtmeyer

0141-8130/$ see front matter 2012 Elsevier B.V. All rights reserved.
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described as controversial claims that glycogen improves tolerance

to myocardial ischemia [9], but also described high glycogen concentrations in foetal cardiac muscle that allow the heart to maintain
contractions under severe hypoxia [9,19]. In their review, Roach
et al. discuss the importance of both liver and heart accumulations
of glycogen of the foetus before term, which are important because
gluconeogenesis is not well developed at birth [20]. Also, the release
of epinephrine causes a rapid depletion of cardiac glycogen in the
healthy heart [12,21]. These distinctive features of the function and
response of glycogen in heart muscle suggest it may also have a
distinct structure, but no such structure has been reported, to our
knowledge.
Previous studies using transmission electron microscopy (TEM)
on cardiac tissue have said the observable glycogen consists
entirely of -particles [2224]. The cardiac glycogen was not analysed as isolated glycogen, but rather as a species in the cardiac
tissue as a whole [25]. These experiments used low resolution electron microscopy (EM), with dying and extraction techniques that
could lead to a structure unlike that in vivo. It has been shown
that different isolation and extraction techniques for glycogen can
result in up to 100 times difference in quantity [26], suggesting a distortion of the size distribution. Past EM images show
glycogen as a small black species, too small for an accurate size
estimate, or for the detection of any supra-molecular structure
[2224]. Much of this past work used very alkaline dyes, with
saturation occurring after 30 min, however, glycogen should not
be left for extended periods of time as it can degrade [27,28].
The stability of the interaction (or bond) that holds -particles
together into the -particle may not be strong enough to survive
these procedures. The method we use, combining gentle extraction of glycogen and TEM images that make ne structure and
aggregation of glycogen clear, has been applied to glycogen from
various tissues and various species and shown to reveal clearly
the sizes and nature of -particles [4]. Rybicka noted the need
for a revision of the interpretation of experiments on glycogens
ultra-structure [22]. Previous studies have shown that both and
-particles are distributed throughout the cytoplasm, and amongst
the sarcoplasmic and smooth endoplasmic reticulum [23,29,30].
EM work by Jamieson and Palade [23] on atrial muscle cells
showed the glycogen observed is clustered around the sarcoplasmic core.
TEM of hepatic glycogen rst revealed that the highly branched
polymer assembles into -particles [3,4]. In comparison, TEM studies have shown that skeletal muscle glycogen consists exclusively
of -particles [4]. Calder and Geddes reported indirect evidence
of glycogen larger than -particles [31]. They obtained apparent molecular weights from the Svedberg equation, using rat
skeletal muscle glycogen sedimented through sucrose-density gradients. The largest size category considered by Calder and Geddes,
5 108 g mol1 corresponds to a particle under 100 nm in diameter
[32], or to an -particle with about one eighth as many -particles
as the typical liver -particle. TEM, in contrast to sucrose separation, allows identication of non-glycogen cell fragments and other
impurities. Glycogens glucose monomers are joined into linear
chains by -(1,4) links, and branched at -(1,6) links. One possible effect of aggregation into -particles is reduction of the exposed
surface area (i.e. the number of free glucose chains) for degradation
enzymes to work on, as hepatic glycogen maintains a slow release of
glucose for a basal metabolic rate. Rather skeletal muscle glycogen
likely needs a fast degradation during exercise, with a -particle
providing a higher surface area per glucose monomer. Defects in
glycogen synthesising and metabolising enzymes can lead to severe
consequences which are linked with a variety of glycogen storage
diseases [3336].
We report here the distribution of glycogen particle diameters,
and the extent of aggregation of -particles, in the cardiac tissues

of healthy mice. We discuss the relationship between glycogen

structure and the fuel needs of various cell types.

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2. Methods

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We puried murine cardiac and hepatic glycogen using a

recently described non-denaturing method [4] that ensures the
particles remain intact [37]. This method, combined with TEM, has
been applied to a variety of glycogens from various tissues and
species [4]. It clearly shows the differences in the sizes and numbers
of -particles.

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2.1. Purication of glycogen

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The procedure for cardiac glycogen extraction was as previously

reported [4]. Hearts were taken from healthy twelve-week old
mice from the ABSL-III Laboratory of Wuhan University, China, and
homogenised in glycogen isolation buffer (50 mM HEPES; 150 mM
NaCl; 2 mM EDTA; 50 mM NaF; 5 mM sodium pyrophosphate; mini
complete protease inhibitors, Roche Applied Science, Indianapolis,
IN; pH 8.0), centrifuged at 6000 g for 10 min at 4 C, and the resulting supernatant centrifuged at 50, 000 g for 30 min at 4 C. The
supernatant was discarded and the resulting pellet re-dissolved in
a minimal volume of glycogen isolation buffer, and carefully added
on top of a three-stage sucrose gradient (25% (w/v), 50% (w/v), 75%
(w/v), in glycogen isolation buffer), and centrifuged at 300, 000 g
for 120 min at 4 C. The supernatant was discarded and the resulting pellet rapidly washed with 1 ml of glycogen isolation buffer to
remove free sucrose solution, redissolved in 3 ml glycogen isolation
buffer and freeze dried.

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2.2. Transmission electron microscopy

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Murine cardiac and hepatic glycogen was prepared for TEM

as previously described for rat liver and human skeletal muscle
glycogen [4] and examined using a Tecnai 12 Transmission Electron Microscope (FEI, Eindhoven, The Netherlands) at an operating
voltage of 120 kV. Images were recorded using a Megaview III CCD
camera and AnalySIS camera software (Olympus) at a magnication
between 30,000 and 150,000. Images of human skeletal muscle distributions used to construct the distribution in Fig. 2 were
obtained as described previously [4]. Particle diameters were estimated using ImageJ [38]. Diameters were measured along a xed
direction with respect to the edges of the image, against scale bars
in the images. 56 images were used from different regions of the
sample. The distributions in Fig. 2 are insensitive to the size of the
bins used. TEM images were taken from three mice, and none of the
three individual distributions were signicantly different from the
average. Images of human skeletal muscle glycogen were obtained
as described previously [4].

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2.3. Dynamic light scattering

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Dynamic light scattering (DLS) measurements were done on

a Malvern Zetasizer Nano (ZS) instrument, tted with a 4 mW
HeNe laser (633 nm). A standard operating procedure was used
with automatic measuring position and attenuation, with backscattering detection. Samples were analysed at 298 K with plastic
disposable sizing cuvettes containing 30 L of glycogen (pellet dissolved in minimal volume PBS buffer) suspended in 470 L double
distilled water. Automatic laser attenuation and position were used
with the refractive index and viscosity of water, at 25 C. The DLS
software calculates the cumulant mean, or z-average, from a polynomial t to the rst order autocorrelation function. As a check on
our TEM results we report z-average hydrodynamic diameters for

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Fig. 1. Transmission electron microscopy of puried glycogen from mouse heart (images A and C), and from mouse liver (images B and D). Scales are shown at the bottom
right hand corners of each panel. The magnications are the same in panels A and B, and the same in panels C and D. These images are typical. See Table 1 for particle size
statistics taken from these images, and see Fig. 2 for the number distributions of the diameters.

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two of the three samples on which TEM was performed. The width
of the peak quoted is at half maximum.

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3. Results

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Cardiac glycogen particles analysed by negative-staining TEM

include both -particles, and free -particles (see Fig. 1A and C).
These particles closely resemble the particles obtained from liver
tissues (see Fig. 1B and D). The cardiac -particles have an average diameter of 133 nm (see Table 1), with some particles reaching
diameters greater than 200 nm (Fig. 1A). Inspection of the cardiac TEM images reveals the frequent presence of intermediate
sized -particles containing 420 -particles that contribute to
the signicant -particle diameter variability. The identication
of intermediate-sized glycogen -particles is not thought to be a
purication artefact since the same method was used to purify
hepatic glycogen, which TEM showed to be dominated by large
particles.
The number distributions of glycogen particle diameters
obtained from TEM images for mouse heart, mouse liver, and

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Table 1
Mean glycogen particle diameters from transmission electron microscopy,

human skeletal muscle are shown in Fig. 2. Mouse skeletal muscle

glycogen, like the corresponding polymer in humans, has previously been shown to consist only of particles [39]. We assume
after Takeuchi et al. [40] that the maximum diameter of the cardiac
-particles is 50 nm. This denition is also consisted with the radii
measured by multi-angle laser light scattering (MALLS) of a commercial glycogen consisting mostly of -particles, but with a small
population of -particles [32]. Free particles in cardiac glycogen have an average particle diameter of 29 7 nm, whereas the
-particles (one or more -particles) have a broad range of sizes,
with an average diameter of 133 60 nm. Note that the distributions of -particle sizes for both liver and heart tissues are broad, so
that few particles have diameters close to the averages. The cardiac
-particle glycogen diameter number distribution (Fig. 2) falls in
between that observed for mouse hepatic and human skeletal muscle glycogen particles that have been puried by the same method
[4], with 43% of the observed -particles having a diameter greater
than the average of 133 nm. Note that these particles make up 24%
of the total population of particles from heart tissue, both and ,
that we measured. The corresponding gure for the liver sample

dTEM , of glycogen and particles in various tissues, and the widths of the distributions

of diameters about the mean, given as standard deviations,  TEM . These were calculated from the same diameters as were the distributions of Fig. 2. We include also the
percentages of -particles, i.e. particles with diameter greater than 50 nm, that have also diameters greater than 133 nm or 160 nm. Note that the fractions of -particles
larger than the average size is roughly the same for both heart and liver samples.

Type

Number

Mouse heart

181
202

29
133

7
60

43%

28%

Mouse liver

227
738

16
160

4
41

71%

45%

290

33

Human skeletal muscle

dTEM

Tissue

(nm)

 TEM (nm)

>133 nm

Please cite this article in press as: Q.A. Besford, et al., Int. J. Biol. Macromol. (2012), http://dx.doi.org/10.1016/j.ijbiomac.2012.06.037

>160 nm

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0.10

number distribution

cardiac
0.08

liver
skeletal muscle

0.06

0.04

0.02

100

200

300

dTEM / nm
Fig. 2. Number distributions of particle diameters obtained from TEM. The numbers
of diameters for each type of particle are given in Table 1, along with the averages
and standard deviations.

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is remarkably similar: 45% of the particles identied from liver

Q2 tissue are larger than the average size 160 nm (Table 2).
4. Discussion
Murine cardiac glycogen contains a mixture of and -particles.
The distribution of -particle diameters is wider than in liver,
with some aggregate -particles containing only a few -particles.
Cardiac glycogen was previously thought to consist entirely of particles [2224]. For simplicity, we use the term -particle to
describe any aggregate. It remains to be seen if the aggregate particles of intermediate size in the cardiac tissue differ in their binding,
composition, or reactivity from the liver -particles.
Cardiac glycogen has been shown to supply little of the energy
required for basal heart metabolism like excitationcontraction
coupling, though heart muscle does contain relatively large
amounts of glycogen [9]. Rapid breakdown is stimulated during a sudden increase of heart work, stimulated by epinephrine
for example [11,14], or in response to ischemia [9,14,18,19]. Cardiac glycogen therefore serves as a reservoir to provide for rapid
increase in demand for energy [12]. Heart muscle turns over ATP
rapidly, and this turnover must be maintained in the face of reduced
oxygen supply or other stresses [9]. Glycogen must be able to
break down rapidly in response to ischemia or epinephrine, and
the supply should not run out too quickly if the animal is to survive ischemia in particular [9,19]. The presence of large amounts
of glycogen organised into particles smaller than those in the liver
is consistent with the heart muscles need for a large but quickly
available supply. The glycogen particle diameter distributions we
observe for each tissue in Fig. 2 indicate the variation in particle surface areas, and so in the number of exposed non-reducing
ends available to degradation enzymes. This is a generalisation of
the statement by Shearer et al. in their study of skeletal muscle
glycogen that smaller particles are both synthesised and consumed
more rapidly than the larger [41]. If the heart muscles glycogen
were all in the form of free particles, then osmotic stress would
limit the amount that might be stored, or perhaps the response to
Table 2
 
Mean glycogen particle diameters from dynamic light scattering, dDLS , of glycogen
and particles in various tissues, and the widths of the distributions of diameters
about the mean, given as standard deviations,  DLS .
Tissue
Mouse heart
Mouse liver

dDLS

110
149

(nm)

 DLS (nm)
60
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changing glucose concentration might be too fast (see below discussion of NIDDM). For a given amount of glycogen, storage in
particles as large as the livers would slow the cells response to
ischemia. Larger glycogen particles, the aggregates, allow for the
storage of greater amounts of fuel in each cell, while the presence
of the smaller particles, with a greater surface area per glucose unit,
allows for the observed rapid release.
Another possibility arises from the work of Taegtmeyer, who
describe the continuous turnover of myocardial glycogen [9]. This
continuous turnover serves to control the concentration of glucose
in the cell. The rate of release of glucose from glycogen depends
also on enzyme activities, amongst other factors. The function of
the hearts -particles that we describe here could be the control
of the glucose concentration in the cell, a role analogous to that of
the hepatic -particles that control the concentration of glucose in
the blood. A further possible role for myocardial -particles is as
an anchor for other macromolecules [9], either securing them or
bringing them together.
Previous work by Sullivan et al. [42], found that the structure
of hepatic glycogen in mice with non-insulin-dependent diabetes
mellitus (NIDDM) appears to consist primarily of -particles or
poorly assembled -particles, resulting in a poorer storage of glucose for maintaining basal blood glucose levels (BGLs). If this same
impairment of -particle assembly is present in the hearts of
NIDDM patients, one might speculate that it may lead to heart complications. The quantity of cardiac glycogen in NIDDM models can
be up to 50% more than healthy hearts [43]. Since the purpose of
cardiac glycogen only becomes apparent during a rapid increase
in heart action [14,11], we suggest that a possible change in cardiac glycogen structure in NIDDM patients may be correlated to
the increased risk of cardiac dysfunction during periods of strenuous exercise. NIDDM is associated with a marked increase in the
risk of coronary heart disease [44], potentially indicating that heart
complications may be related to, or stem from, a poor storage of
cardiac glucose.
Pompe disease is characterised by an excess of glycogen, particularly in the heart, which often leads to its subsequent failure
[45]. This example suggests that too much cardiac glycogen may
disturb the physiochemical environment of the cardiac tissue. It is
unknown whether this increased quantity is of the healthy and particle sort, or whether there is a much larger particle, or a lack of
-particles with an excess of -particles. Glycogen storage diseases
(GSD) that are unique to the heart, such as types I, III and IV [33],
result in cardiac glycogen of distinctive clinical and chemical manifestations [46]. These distinct differences may involve an inability
to form the healthy -particles, due to a different structure of the
-particles. Our nding of -particle existence in the cardiac muscle suggests that investigation of the mechanism of aggregation of
-particles into -particles would be relevant to the study of these
diseases.

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5. Conclusions

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Cardiac glycogen from healthy mice has been extracted, puried, and imaged via TEM. The images show the glycogen is
assembled into supramolecular -particles, with free -particles
also present. This structure resembles the known characteristics
of liver glycogen, and differs sharply from the previously accepted
view of cardiac glycogen.
Comparison of the structures observed in liver, heart muscle,
and skeletal muscle suggests a relation between structure, particularly the distribution of diameters, and the function of the glycogen
particles. Larger particles allow for the storage of more glucose
without increasing the osmotic pressure, smaller particles have a
greater surface area per glucose monomer and so allow for the more

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rapid release of glucose. This is consistent with the idea that glycogen is not a passive fuel store, but is active in regulating glucose.
It has been suggested that glycogen concentration may be a more
important factor in the regulation of glucose uptake than is insulin
[2].
The presence of -particles in the heart suggests new lines
of investigation into the mechanisms of human diseases such as
NIDDM and glycogen storage diseases. In the case of NIDDM, it will
be interesting to look for a connection between the disregulation of
glucose, the proper formation of -particles and the observed heart
problems during strenuous exercise. The relationship between the
structure and function of glycogen should be further investigated
for its relevance to various GSD. Now that -particles in the heart
have been identied, the effect of each type of GSD on these particles will provide a new perspective on these disease mechanisms.

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Acknowledgements

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This work was supported by the National Health and Medical

Research Council, and by the Melbourne Materials Institute in the
University of Melbourne. Mrs. Lynne Waddington provided valuable advice on and assistance with TEM.

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Please cite this article in press as: Q.A. Besford, et al., Int. J. Biol. Macromol. (2012), http://dx.doi.org/10.1016/j.ijbiomac.2012.06.037

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