Hindawi Publishing Corporation
Journal of Biomedicine and Biotechnology
Volume 2011, Article ID 393740, 11 pages
AdherentPrimaryCultures of MouseIntercostal MuscleFibers
Patrick Robison,ErickO.Hern´ andez-Ochoa,and MartinF.Schneider
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene Street,
Baltimore, MD 21201, USA
Correspondence should be addressed to Erick O. Hern´ andez-Ochoa, firstname.lastname@example.org
Received 26 April 2011; Accepted 24 May 2011
Academic Editor: Robert J. Bloch
Copyright © 2011 Patrick Robison et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Primary culture models of single adult skeletal muscle fibers dissociated from locomotor muscles adhered to glass coverslips are
routine and allow monitoring of functional processes in living cultured fibers. To date, such isolated fiber cultures have not been
established for respiratory muscles, despite the fact that dysfunction of core respiratory muscles leading to respiratory arrest is
the most common cause of death in many muscular diseases. Here we present the first description of an adherent culture system
for single adult intercostal muscle fibers from the adult mouse. This system allows for monitoring functional properties of these
living muscle fibers in culture with or without electrical field stimulation to drive muscle fiber contraction at physiological or
pathological respiratory firing patterns. We also provide initial characterization of these fibers, demonstrating several common
techniques in this new model system in the context of the established Flexor Digitorum Brevis muscle primary culture model.
The bodies of vertebrates include hundreds of skeletal mus-
cles, each involved in performing different motor tasks at
specific anatomical locations. This variety in skeletal muscles
and their functions implies genotypic and phenotypic diver-
sity among skeletal muscle fibers. In fact, the preferential
expression of different muscular protein isoforms and there-
fore the existence of distinct muscle fiber phenotypes is one
of the main determinants of the muscle performance [1, 2].
Not only does this diversity manifest in multiple muscle
contraction coupling properties, the sensitivity of the con-
tractile apparatus to Ca2+, mechanical power output, short-
ening velocity, and rate of ATP hydrolysis are also known
to vary greatly from fiber to fiber [1, 4–7]. The intercostal
muscles are an exemplary case of such a heterogeneous mus-
cle population, making them an attractive model for com-
The mechanical functions of the intercostal muscles
during ventilation are highly complex. There are two major
division criteria for intercostal fibers: anatomical and func-
tional. Anatomical divisions are based on location on the
internal or external side of the ribs; functional divisions are
based on participation in expiratory or inspiratory respi-
ration . The external intercostals and the parasternal
intercostals function in inspiratory breathing, whereas the
internal intercostals have an expiratory function .
breathing at rest , the intercostal muscles may contribute
c.a. 40% of the volume shift by movement of the thoracic
wall . During intensified breathing, the contribution
of the intercostals becomes more prominent [12, 13]. The
lateral internal intercostals are also recruited for a variety
of nonventilatory functions including the cough reflex and
speech as well as for postural support .
fibers are slow-type and 40% fast-type fibers [14, 15]. As a
result of this diversity, the same preparation can be used to
address many aspects of muscle fiber-type physiology. Also,
due to their involvement in the mechanics of ventilation
and the fact that respiratory pathology is common in sev-
eral diseases (e.g., muscular dystrophy, chronic obstructive
2Journal of Biomedicine and Biotechnology
pulmonary disease, amyotrophic lateral sclerosis, hereditary
polyneuropathies, autoimmune conditions like myasthenia
a model of high clinical relevance.
Indeed, in cases where diagnosis and treatment for mus-
critical factor in the majority of fatalities arising from mus-
cular dystrophy [16, 17]. While the vast majority of studies
evaluating the function of the respiratory muscles have used
the diaphragm muscle as model of study, information on
the intercostal muscles, in particular at cellular level, is more
These muscles are also of relevance in Chronic Obstruc-
tive Pulmonary Disorder (COPD), given their critical role in
manipulating the configuration of the ribs  and therefore
the overall morphology of the thorax. In addition to the me-
chanical reorganization of the intercostal muscles that results
from the inflated thorax common to COPD patients, clear
changes in the expression of myosin heavy chain isoforms
have been documented .
Here we describe in detail the isolation and preliminary
characterization of isolated adherent intercostals muscle
fibers in the common laboratory mouse based on techniques
and methods previously developed by our laboratory for use
on other muscles [19–25]. The loading of fluorescent Ca2+
indicators combined with electrophysiological approaches,
including electrical field stimulation of the cultured muscle
the study of spatiotemporal aspects of excitation-contraction
coupling and biological processes such as excitation-tran-
scription coupling. Due to the difficulty encountered in
mercial glass-bottom culture dishes, we also include detailed
methods for the construction and preparation of glass-bot-
tom dishes optimized for such muscle fiber studies. These
dishes have improved the reliability and yield of attached
fibers in our preparations several fold. The methods pre-
sented here can be modified to allow culture of intercostal
fibers from animal at various ages or from animal models
with specific genetic background. The protocol described
here is intended more generally to provide a flexible new
primary cell culture of the as yet poorly characterized, but
clinically relevant muscular group, the intercostals.
2.1. Animal Use. All animals were euthanized by CO2expo-
sure followed by cervical dislocation before removal of the
muscles according to protocols approved by the University of
Maryland Institutional Animal Care and Use Committee.
2.2. Dish Construction. Dishes are constructed from plastic
Petri dishes with glass coverslips and sealed using parafilm.
washed with 70% ethanol (Supplemental Figures 1(a)–1(e)
available online at doi:10.1155/2011/393740). Multiple wells
may be cut per dish to increase plating area (Supplemental
Figure 1(f)); drilling larger holes is not recommended as we
slip as a template (SupplementalFigure 2). An unused pencil
eraser is useful to hold the coverslip in place, but should
be covered with a piece of scotch tape to minimize transfer
of oils to the glass. Coverglass is then heated lightly on
a hotplate with the parafilm facing up until the parafilm
over coverslips to seal each well and then parafilm is allowed
to cool (Supplemental Figures 2(e)–2(g)). Parafilm from the
center of the well is cut out with a scalpel and removed. The
seal should be tested with water or media prior to use.
Prior to use, dishes are sterilized by UV irradiation and
spotted with approximately 6µL of cold mouse laminin
(1mg/mL). Laminin is immediately covered with 100µL
MEM and the bottom of each well is lightly scratched
with the pipette tip to ensure full coverage (Supplemental
is removed from the wells and dishes are rinsed twice with
MEM and covered with 1mL MEM plus 100µL MEM per
2.3. Dissection. Following cervical dislocation (see animal
carried out in a sterile laminar flow hood to prevent contam-
rior extension, and forepaws are pinned at maximum lateral
extension with the ventral face of the mouse up (Figure
1(b)). Skin is cut, posterior to anterior, from the lower
abdomen to the throat (Figure 1(c)), and a lateral incision is
the skin (Figure 1(d)). Skin is removed by gently raking the
scalpel against the interface between skin and subcutaneous
tissue (Figures 1(e) and 1(f)) until the vertebral column is
nearly exposed. An incision is made at the anterior end of
the abdominal cavity, exposing the liver (Figure 1(g)). When
the liver is pulled back, the diaphragm is exposed, although
difficult to visualize. The thoracic cavity may be punctured
by following the dorsal face of the sternum with a scalpel,
causing the thoracic cavity to inflate (Figure 1(h)). This
makes the diaphragm easier to see and remove by cutting
along the posterior internal edge of the rib cage (Figure 1(i)).
Once the diaphragm is free, an incision is made along the
ventral face of the sternum and the superficial muscles of the
thorax are removed in a similar fashion to the skin (Figures
1(j)–1(l)). This exposes the intercostal muscles which are
then immediately bathed in MEM to prevent drying. One
half of the rib cage is then excised by using scissors to
cut along the sternum (Figure 1(m)) and thoracic vertebrae
(Figure 1(n)). The half rib cage is then placed in MEM to
rinse away excess debris (Figure 1(o)).
2.4. Digestion and Fiber Isolation. The media used to rinse
the ribs is removed and replaced with MEM containing
2mg/mL collagenase and 5mg/mL dispase which has been
passed through a 0.2µm filter. The ribs are then incubated
at 37◦C and 5% CO2for 3 hours (older mice may require
Journal of Biomedicine and Biotechnology3
Figure 1: Critical Steps in Gross Dissection of Intercostal Muscles. (a) Cervical dislocation following CO2asphyxiation and 70% ethanol
wash. (b) Pinning mouse, ventral face up. (c) First incision from posterior abdomen to throat. (d) Skinning: pinning back initial flap. (e)
Skinning: cutting subcutaneous connective tissue. (f) Skinning: continue removal to vertebral column. (g) Opening incision at anterior end
of abdominal cavity. (h) Exposed posterior face of thoracic cavity (after puncture). (i) Removal of diaphragm. (j) Incision along ventral face
of sternum. (k) Removal of pectoral and other muscle groups. (l) Full exposure of ribs. (m) Severing ribs (ventral face). (n) Severing ribs
(dorsal face). (o) Isolated ribs transferred to HBSS for imaging. Scale is centimeter ruler.
longer incubation). Following digestion, the ribs are trans-
ferred into MEM supplemented with 10% FBS and the pro-
cedure is continued under a dissection microscope. Excess
vasculature and fragments of nonintercostal muscle frag-
ments are removed with forceps. A single band of intercostal
muscle is isolated with its flanking ribs (Figure 2(a), white
arrows) by pulling the flanking ribs away from the next
nearest (Figure 2(a), black arrows).
The isolated rib pair and the intercostal band are trans-
ferred to a clean 60mm dish containing 7mL of MEM sup-
plemented with 10% FBS (Figure 2(b)). Fibers are triturated
using a large bore flame polished pipette (Figure 2(c),
Supplemental Figure 3) until a significant population of
isolated fibers is obtained (Figure 2(d)). If muscle tissue
detaches completely from ribs, a smaller bore pipette may be
used. The dish is gently swirled to aggregate fibers (Figure
2(e)). If large amounts of debris are floating on the surface
of the media, it may be removed. A wide bore micropipette
tip is used to gather 8–12 healthy fibers per well from the
periphery of the fiber cluster. Fibers are plated directly onto
the glass surface in each well of the dishes prepared earlier.
Fibers may be damaged or stick to the interior of the tip.
These are removed and replaced; however, overplating can
cause fibers to mutually interfere with attachment and is to
with forceps if it is possible to do so without disturbing
healthy fibers. After several minutes to allow initial attach-
ment, dishes are transferred to the incubator overnight to
complete attachment. We note that fibers often attach near
the periphery of the dish even when plated initially in the
following dissociation of the fibers mitigates gross morpho-
logical changes, proliferation of secondary cells, and some
changes in calcium signaling [26, 27].
2.5. Digestion Conditions of Nonintercostal Fibers. Dispase is
omitted from the digestion of both soleus and FDB muscles.
FDBs (but not soleus) may be fully digested in as little as 2.5
hours, particularly if the mouse used is young. Apart from
removal of bone (not required for either soleus or FDB),
plating may proceed exactly as with the intercostal fibers,
although a smaller bore pipette may be used for trituration if
4Journal of Biomedicine and Biotechnology
Figure 2: Dissociation. Following digestion, ribs are transferred into MEM containing 10% FBS and gentamycin (a). Rib pair flanking the
desired muscle (white arrows) is isolated (b) and transferred into clean 60mm Falcon Petri dish containing 7mL MEM with 10% FBS and
gentamycin. Fibers are triturated (c) until a significant population of fibers are isolated (d). Fibers may be aggregated in the center of dish
(e) by shaking gently in a circular motion. FinnTip 250 Wide pipette tips are used to plate fibers (f) to prevent damage from fluidic shearing.
Brightfield images of an adhered fiber at low (g) and high (h) magnification. Scale bars are approximately 1mm (d, e) or 50µm (g, h).
2.6. Indo1 Ratiometric Recordings. Cultured FDB or inter-
costal fibers were loaded with Indo-1 AM (Invitrogen, Eu-
gene, OR) at a bath concentration of 2µM for 30min at
22◦C. Then, the fibers were washed thoroughly with L-
15 media to remove residual Indo-1 AM and incubated at
22◦C for another 30min to allow dye conversion. The cul-
ture dish was mounted on an Olympus IX71 inverted
microscope and viewed with an Olympus 60x/1.20NA water
immersion objective. Fibers were illuminated at 360nm, and
the fluorescence emitted at 405 and 485nm was detected
pled at 10Hz using a built-in AD/DA converter of a EPC10
amplifier and the acquisition software Patchmaster (HEKA,
Instruments). Field stimulation (square pulse, 14V × 1ms)
was produced by a custom pulse generator through a pair
of platinum electrodes. The electrodes were closely spaced
(0.5mm) and positioned directly above the center of the
objective lens, to achieve semilocal stimulation. Only fibers
exhibiting reproducible and consistent responses to field
stimulation of alternate polarity were used for the analysis.
2.7. Fluo-4 AM Ca2+Recordings. Fluo-4 AM (Invitrogen,
Eugene, OR) loading, high speed line scan x-t imaging on
a Zeiss LSM 5 Live confocal system, and image analysis
were all performed as previously described . Fibers were
stimulated using parallel platinum field electrodes, with
acquisition of the Fluo4 confocal line scan image synchro-
nized to the field stimulus to generate a temporal profile.
2.8. Exogenous Protein Expression and Imaging. The recom-
binant adenovirus of Glut4-GFP was kindly provided to our
laboratory by Dr. Jeffrey Pessin. The expression plasmid of
NFATc1 cDNA was a gift from Dr. Gerald R. Crabtree. The
construction of recombinant adenoviruses of NFATc1-GFP
as well as the procedures for infection with recombinant
adenoviruses of single muscle fibers has been previously
described . Cultures were maintained in MEM in a 37◦C,
5% CO2incubator until expression levels were appropriate
for imaging, then transferred into Liebovitz L-15 (GIBCO)
for microscope work. Images were taken using an Olympus
IX70 inverted microscope equipped with an Olympus Flu-
oview 500 laser scanning confocal imaging system using an
Olympus 60x/1.2NA water immersion objective. A 488nm
excitation wavelength and 505nm long-pass emission filter
were used to visualize GFP.
2.9. Data Analysis. Line-scan Fluo4 images were processed
using LSM examiner (Carl Zeiss, Jena). GFP and Glut4-
GFP confocal images were processed using ImageJ (NIH,
Bethesda, MD, USA; http://rsb.info.nih.gov/ij/). Indo1 and
fluo4 Ca2+signals and statistical analysis were conducted
using Origin pro 8 (OriginLab Corporation, Northampton,
MA, USA). Summary data were reported as mean ±
SEM. Statistical significance was assessed using parametric
unpaired two sample t-test. Differences were considered
significant when P value < 0.05.
Indo1 data was reported as the ratio of fluorescence
emission at 405nm to emission at 485nm (indo1 ratio)
which correspond to the peak values of the calcium bound
and unbound forms, respectively, or the difference in indo1
ratio between peak and resting values (Δindo1 ratio). Fluo4
data was reported as the difference between fluorescence
intensity and an average value of fluorescence taken when
the fiber was at rest, normalized to the resting value (ΔF/F0).
Journal of Biomedicine and Biotechnology5
Indo1 ratio at rest
Indo1 ratio at rest
Δindo1 ratio at peak
Figure 3: Intercostal fibers exhibit decreased peak amplitude of the Indo-1 Ca2+transient following a single action potential. (a) Average
Indo-1 ratio Ca2+transients from FDB (red trace; n = 9) and Intercostal (ITC; green trace; n = 11) fibers. Isolated fibers were stimulated
with field electrodes at time zero and emission ratio was examined. (b) Bar plot summarizing resting Indo-1 ratio averages of FDB and ITC
fibers. No significant differences in resting indo1 ratio were detected between groups (P = 0.12). (c) Traces from panel (a), expressed as
Δindo-1 ratio (Indo1 ratio—Indo1 ratio at rest). (d) Bar plot representation of peak ΔIndo-1 ratio shown in (c), FDB = 0.61 ± 0.04, n = 9;
ITC = 0.40 ± 0.04, n = 11;∗P = 0.0015. Error bars in (d) are the SE values. ITC fibers displayed significantly reduced Ca2+transients when
compared with FDB fibers.
Data from the NFATc1-GFP fusion protein was reported
as the ratio of fluorescence intensity in the nucleus to the
intensity in the cytoplasm (N/C) in order to correct for
variations in expression level between fibers. Raw data for
analysis by subtracting the intensity measured in a region
outside the muscle fiber.
of two overlapping sheets (inner and outer) of muscle fibers
with roughly perpendicular orientations. Each muscle spans
diagonally between two ribs from the vertebral column to
6 Journal of Biomedicine and Biotechnology
Figure 4: Fluo4 Ca2+transients elicited by field stimulation in intercostal and FDB fibers. Representative line-scan images of Ca2+transient
responses in FDB (a) and intercostal (b) fibers to a 5sec 10Hz train of field stimulation applied at time zero, performed with nonratiometric
images of FDB and intercostals fibers to indicate the location of the scan line, scale bar is 20µm. Note that fluo-4 signal at rest in these fibers
is very low. Traces under the images are the time courses of the fluo4 Ca2+transients. The white lines in the time domain images indicate
the region of interest drawn at the center of the fiber and used to calculate the change in fluorescence in response to field stimulation.
Data presented as ΔF/F0. Fluorescence was detected with a Zeiss LSM 5 Live ultrafast confocal system based on an Axiovert 200M inverted
microscope. Fibers were imaged with a 63x/NA 1.2 water immersion objective lens. Excitation for fluo-4 was provided by the 488nm line of
a 100mW diode laser, and emitted light was collected at >510nm.
muscle and ribs are not readily appreciable at the macro-
scopic scale (Figure 1).
3.2. Isolating Intercostal Fibers. The initial attempt to isolate
and culture intercostal muscle fibers was based on our
existing protocols for isolating and culturing mouse Flexor
Digitorum Brevis (FDB) fibers [21–25], which are main-
an initial development and optimization of dissection and
enzymatic treatment methods, we achieved a high fraction
of viable isolated intercostal fibers. These fibers maintained
clear striations and twitch response for several days, but
did not reliably form adherent cultures, even after extensive
experimentation with a variety of substrates derived from
noted that customized glass-bottomed dishes which we had
designed with smaller diameter wells using a different glass
substrate seemed to facilitate attachment in our FDB model
(seeSupplementalFigures 1and 2).Platingintercostalfibers
in these dishes dramatically improved the attachment rate
and reliability of the culture.
After fiber isolation, our procedure yields a heteroge-
neous mix of internal and external intercostal fibers with
a successful attachment rate around 50%. Isolated intercos-
tal fibers (Figure 2) are similar to FDBs in width, but 4-5
times the length. This additional length makes the fibers
Journal of Biomedicine and Biotechnology7
Figure 5: Comparison of electrically evoked Ca2+transients between intercostal and FDB fibers. Average fluo4 Ca2+transient from FDB
((a), red trace; n = 9) or intercostals fibers ((b), ITC, green trace; n = 9) in response to 5sec 10Hz train field stimulation. Traces in (c)
were normalized to the amplitude of the initial response. At the end of the 5sec train, ITC fibers demonstrated significantly smaller relative
decrease of Ca2+transient amplitude compared to FDB counterparts (P < 0.05). ITC muscle fibers exhibit decreased peak amplitude of the
fluo-4 Ca2+transients in response to a single field stimulus; however, the intercostals are able to maintain consistent levels of calcium during
repetitive stimulation. Fluorescence was detected as in Figure 4.
susceptible to bends when they are fully attached (see Figure
1(g)). Almost all of the whole fibers that attach to the glass
display clear striations (Figure 6) and respond to external
electrical field stimulation administered either as single
pulses or repetitive trains with twitching similar to that of
FDBs (see supplemental videos 1 and 2).
In order to examine the general applicability of these
dishes, we also used them to plate fibers from the slow-type
soleus muscle, which also displays poor adherence charac-
teristics . Although we used a less stringent digestion
procedure, plating was carried out exactly as for intercostals.
The exceptional length of the soleus fibers causes them to
interfere with each other and permits the formation of many
bends and kinks which prevent complete attachment. None-
theless, the fibers were responsive to electrical stimulation
and adhered securely enough to withstand repeated twitch-
ing (see supplemental video 3) more than 24 hours after
plating. Successful attachment rate was variable between the
wells plated, but overall comparable to the intercostals.
3.3. Ca2+Homeostasis and Electrically Evoked Ca2+Transients
in Intercostal Fibers. Primary cultured intercostal fibers
8 Journal of Biomedicine and Biotechnology
(e) (f)(g) (h)
either GFP (a–d) or a Glut4-GFP fusion construct (e–h). Scale bars are 50µm (b, d) or 25 µm (f, h). Images were acquired as in Figure 4
for panels (a–d) or with an Olympus FluoView 500 confocal system, built on an Olympus IX71 inverted microscope for panels (e–h). Fibers
were imaged viewed with a UPlan Apo 60x/NA 1.20 water-immersion objective lens using 488 nm excitation (Multiline Argon laser) for GFP
with emission detected with 505nm long-pass filter. Photobleaching and photodamage were reduced by operating the lasers at the lowest
possible power that still provided images with satisfactory signal to noise ratio. Brightfield images in panels (b, d, f, h) were adjusted for
improved contrast using ImageJ.
retain many properties of native fibers, including contrac-
tions and Ca2+transients in response to electrical field
stimuli (Figures 3, 4, and 5; see Supplemental Video 1). A
brief comparative study between intercostal fibers and the
established FDB isolated fiber model was also conducted
(Figures 3, 4, and 5). First, we sought to evaluate Ca2+
handling properties in single, intact indo1-loaded intercostal
(n = 8) and FDB (n = 16) muscle fibers stimulated by an
action potential (AP) elicited by a 1ms electric field stimulus
not significantly different between intercostal (0.45 ± 0.02;
n = 11) and FDB fibers (0.49 ± 0.01; Figures 3(a) and 3(b);
n = 9, P = 0.12), the peak of the indo1 Ca2+transient
in intercostal fibers than in the FDB fibers (intercostal = 0.40
± 0.04, FDB = 0.61 ± 0.04, P = 0.0015, Figures 3(c) and
3(d)). These results suggest that intercostal fibers exhibit a
decreased AP-evoked Ca2+release.
To allow better temporal resolution of the Ca2+tran-
sients, we next monitored fluo-4 transients during repetitive
speed (100µs/line) confocal microscope in line scan mode.
Figure 4 illustrates representative x-t confocal images and
corresponding fluo4 Ca2+transients, expressed as ΔF/F0,
from an FDB (Figure 4(a)) and an intercostal (Figure 4(b))
fiber during a 5-second 10Hz train. Line-scan fluorescence
images were obtained along a line perpendicular to the
long axis of the fiber (Figures 4(a) and 4(b), top panels).
Successive vertical lines in each image reveal the time course
(left to right; middle panels) at 100µs resolution of the flu-
orescence signal along the scanned line before and during
the repetitive stimulation. Bottom panels in Figures 4(a) and
4(b) show the time course of the fluo4 fluorescence. Figure 5
shows average responses from FDB (red trace; Figure 5(a))
and intercostal (green trace; Figure 5(b)) fibers stimulated
by a 5-second 10Hz train. Consistent with the Indo-1
data (Figure 3), fluo4 Ca2+transients appear suppressed
in intercostal fibers (Figure 5(b)) in comparison to those
observed in FDB fibers (Figure 5(a)).
However, the intercostals are able to maintain a more
consistent level of calcium release during repetitive stimu-
lation (Figure 5(b)). By normalizing the traces elicited by
the 10Hz trains to the amplitude of the first fluo4 transient
in their respective train, the relative differences in Ca2+
transient summation as well as the properties of decaying
phase during the train can be appreciated (Figure 5(c)). Both
intercostal and FDB fibers showed an initial increase in the
peak amplitude during the 10Hz train protocol, exceeding
the peak value of the initial stimulation until 2 and 0.5
seconds, respectively (Figure 5(c)). Additionally, the relative
amplitude of the Ca2+transients during the 10Hz train
declines more slowly in intercostal fibers (Figure 5(c)).
Unexpectedly, subpopulations reflecting an anticipated
difference between fiber types were not observed in the
intercostal fibers. However, we suspect that this is not due to
genuine homogeneity, but rather a limitation of the calcium
ion equilibration kinetics of fluo-4 in distinguishing such
sub-populations, which may be quite similar.
Journal of Biomedicine and Biotechnology9
Figure 7: NFATc1-GFP distribution in intercostal and FDB fibers. FDB (a–c) and intercostal (d–f) fibers expressing NFATc1-GFP imaged
at rest after 3 days in culture. Scale bar, 5µm. Nuclei in intercostal fibers exhibit significantly elevated levels of NFATc1 when compared to
FDBs cultured under the same conditions (g), N/C ratio of NFATc1-GFP in ITC = 1.38 ± 0.05, n = 102 nuclei versus 0.51 ± 0.01, n = 42
nuclei, in FDB fibers, P < 0.05). Fluorescence was detected as in Figure 6.
Ca2+-signaling pathways at the cellular and subcellular levels
by using ultrafast confocal imaging combined with Ca2+-
sensitive fluorescent probes.
3.4. Expression of Exogenous Proteins. In order to assess
the viability of the intercostal cultures for imaging studies
utilizing fluorescent proteins, fibers in culture were exposed
GFP. Exposure to virus was carried out overnight, beginning
immediately after plating. Vector dosage was calibrated by
expression levels in similarly treated FDB fibers. Cultures
were maintained in an incubator to allow expression prior
to imaging. Expression of GFP was robust after 48 hours and
did not appear to damage the fibers, which maintained clear
striations (Figures 6(b) and 6(d)).
We also exposed other cultures to a viral vector express-
ing a Glut4-GFP fusion protein. Expression continued
through 96 hours (Figure 6(g)). Glut4-GFP was expressed in
intercostal fibers in puncta both around the nucleus and in
lines parallel to the axis of the fiber (Figures 6(e) and 6(g)).
This distribution is similar to what we observe in FDB fibers
infected with the same construct, as well as the distribu-
tion of the native transporter characterized by immunoflu
orescence , although we observe weaker striations,
presumably due to lack of exposure to insulin during the cul-
ture period reducing the Glut4 present in the transverse
Finally, we exposed the fibers to a viral construct ex-
pressing a NFATc1-GFP fusion construct. NFATc1 is a
transcription factor canonically regulated by the calcium-
dependent phosphatase calcineurin [29, 30]. Typically
resting FDB fibers exhibit low translocation of NFATc1 into
10Journal of Biomedicine and Biotechnology
GFP at rest (Figures 7(d), 7(e), and 7(f)) when compared
to FDB fibers (Figures 7(a), 7(b), and 7(c)). The resting
nuclear/cytosolic (N/C) ratio of NFATc1 was significantly
elevated in intercostal fibers (Figure 7(g)) compared to FDB
(1.38 ± 0.05, n = 102 nuclei versus 0.51 ± 0.01, n = 42
nuclei, resp.; P < 0.05).
Adult skeletal muscle fiber culture currently focuses on only
a handful of the myriad available muscles in the body, ex-
trapolating from the few established culture systems like the
FDB to the body as a whole. While it is generally true that
various skeletal muscles resemble one another, there is sig-
nificant divergence among them with respect to their physi-
ology and the expression levels and isoforms of many muscle
specific and metabolic proteins [1, 5–7]. This is dramatically
illustrated by the high specificity of pathology in many
genes (e.g., the disproportionate targeting of facial, shoul-
der, and limb muscles in facioscapulohumeral muscular
dystrophy or the extremities in distal muscular dystrophy
The intercostal fiber cultures introduced here themselves
appear to be nearly as versatile as the more established FDB
cultures. While we have not yet used intercostal cultures
in applications requiring large numbers of adherent fibers,
they have proven viable for a wide variety of single fiber
experiments, even after several days in culture. Previous
work related to the physiology of intercostal muscles comes
from studies utilizing organ culture of intact muscle fiber
bundles [31–35]. Whereas intercostal muscle fiber bundles
offer the advantage of preserving nerve-muscle interactions,
as with many organ culture systems it may not be well suited
for single cell electrophysiology and high spatial resolution
imaging. Furthermore, the ability to mimic stimulation
patterns that may be experienced by the muscle during
stressed states in vivo (such as hyperventilation or coughing)
at the single muscle fiber level offers the opportunity to
isolate the response of the muscle from that of the sur-
rounding tissue in a manner not possible with organ culture
methods. A variant of the single intercostal fiber isolation
was described previously and was used to study satellite
cell differentiation cocultured with nonadherent intercostal
fibers derived from goats . While these methods are
valuable, the availability of adherent single fiber models
for imaging and electrophysiology substantially expands our
ability to study these muscle types.
The value of studying each skeletal muscle as a separate
tissue is demonstrated by our unexpected observation of
fibers. It is clear that studies using only a few particular
skeletal muscles risk overlooking interesting phenomena.
Given the complexity of transcriptional regulation, the het-
erogeneous nature of the physiology and molecular biology
of various skeletal muscles and the diverse tissues which
surround them, we suspect that examining a wider variety
of muscles specifically relevant to individual diseases in
controlled in-vitro settings will prove fruitful.
Our culturing of intercostal muscles aims to do this
not only by providing a particular model system specifically
relevant to the pathology of COPD and the respiratory
complications of the muscular dystrophies [16, 17], but also
by improving the general techniques of adherent isolated
muscle fiber culture. If these techniques are optimized for
poorly adhering muscle types, generating cultures from any
muscle in the body may become substantially easier, and
using the most relevant tissue in a given disease model may
This work was supported by NIH-NIAMS Grants R01-
AR055099, R01-AR056477. P. Robison was supported by
the Interdisciplinary Training Program Muscle Biology T32-
 R. Bottinelli and C. Reggiani, “Human skeletal muscle fibres:
molecular and functional diversity,” Progress in Biophysics and
Molecular Biology, vol. 73, no. 2–4, pp. 195–262, 2000.
 R. I. Close, “Dynamic properties of mammalian skeletal
muscles,” Physiological Reviews, vol. 52, no. 1, pp. 129–197,
 J. C. Calder´ on, P. Bola˜ nos, S. H. Torres, G. Rodr´ ıguez-Arroyo,
and C. Caputo, “Different fibre populations distinguished by
their calcium transient characteristics in enzymatically disso-
ciated murine flexor digitorum brevis and soleus muscles,”
Journal of Muscle Research and Cell Motility, vol. 30, no. 3-4,
pp. 125–137, 2009.
 J. C. Calder´ on, P. Bola˜ nos, and C. Caputo, “Myosin heavy
chain isoform composition and Ca2+transients in fibres
from enzymatically dissociated murine soleus and extensor
digitorum longus muscles,” Journal of Physiology, vol. 588, no.
1, pp. 267–279, 2010.
 R. L. Moss, G. M. Diffee, and M. L. Greaser, “Contractile
properties of skeletal muscle fibers in relation to myofibrillar
protein isoforms,” Reviews of Physiology, Biochemistry &
Pharmacology, vol. 126, pp. 1–63, 1995.
 D. Pette and R. S. Staron, “Cellular and molecular diversities
of mammalian skeletal muscle fibers,” Reviews of Physiology
Biochemistry and Pharmacology, vol. 116, pp. 1–76, 1990.
 S. Schiaffino and C. Reggiani, “Molecular diversity of myofib-
rillar proteins: gene regulation and functional significance,”
Physiological Reviews, vol. 76, no. 2, pp. 371–423, 1996.
 B. Polla, G. D’Antona, R. Bottinelli, and C. Reggiani, “Respi-
ratory muscle fibres: specialisation and plasticity,” Thorax, vol.
59, no. 9, pp. 808–817, 2004.
 A. De Troyer, P. A. Kirkwood, and T. A. Wilson, “Respiratory
action of the intercostal muscles,” Physiological Reviews, vol.
85, no. 2, pp. 717–756, 2005.
 A. Taylor, “The contribution of the intercostal muscles to the
effort of respiration in man,” The Journal of Physiology, vol.
151, pp. 390–402, 1960.
 D. Faithfull, J. G. Jones, and C. Jordan, “Measurement of the
relative contributions of rib cage and abdomen/diaphragm to
tidal breathing in man,” British Journal of Anaesthesia, vol. 51,
no. 5, pp. 391–398, 1979.
Journal of Biomedicine and Biotechnology 11 Download full-text
 G. H. Koepke, E. M. Smith, A. J. Murphy, and D. G. Dick-
inson, “Sequence of action of the diaphragm and intercostal
muscles during respiration. I. Inspiration,” Archives of Physical
Medicine and Rehabilitation, vol. 39, no. 7, pp. 426–430, 1958.
 A. J. Murphy, G. H. Koepke, E. M. Smith, and D. G. Dick-
inson, “Sequence of action of the diaphragm and intercostal
Medicine and Rehabilitation, vol. 40, pp. 337–342, 1959.
 M. Mizuno and N. H. Secher, “Histochemical characteristics
of human expiratory and inspiratory intercostal muscles,”
Journal of Applied Physiology, vol. 67, no. 2, pp. 592–598, 1989.
 M. Mizuno, N. H. Secher, and B. Saltin, “Fibre types, capillary
supply and enzyme activities in human intercostal muscles,”
Clinical Physiology, vol. 5, no. 2, pp. 121–135, 1985.
 L. D. Calvert, T. M. McKeever, W. J. Kinnear, and J. R. Britton,
“Trends in survival from muscular dystrophy in England
and Wales and impact on respiratory services,” Respiratory
Medicine, vol. 100, no. 6, pp. 1058–1063, 2006.
 A. E. Emery, “The muscular dystrophies,” Lancet, vol. 359, no.
9307, pp. 687–695, 2002.
European Respiratory Journal, vol. 10, no. 10, pp. 2404–2410,
 M. G. Klein, H. Cheng, L. F. Santana, Y. H. Jiang, W. J. Lederer,
and M. F. Schneider, “Two mechanisms of quantized calcium
release in skeletal muscle,” Nature, vol. 379, no. 6564, pp. 455–
 L. Kovacs, E. Rios, and M. F. Schneider, “Calcium transients
and intramembrane charge movement in skeletal muscle
fibres,” Nature, vol. 279, no. 5712, pp. 391–396, 1979.
 S. L. Carroll, M. G. Klein, and M. F. Schneider, “Calcium
transients in intact rat skeletal muscle fibers in agarose gel,”
American Journal of Physiology, vol. 269, no. 1, pp. C28–C34,
 S. L. Carroll, M. G. Klein, and M. F. Schneider, “Decay of
calcium transients after electrical stimulation in rat fast- and
slow-twitch skeletal muscle fibres,” Journal of Physiology, vol.
501, no. 3, pp. 573–588, 1997.
 Y. Liu, S. L. Carroll, M. G. Klein, and M. F. Schneider,
“Calcium transients and calcium homeostasis in adult mouse
fast-twitch skeletal muscle fibers in culture,” American Journal
of Physiology, vol. 272, no. 6, pp. C1919–C1927, 1997.
 Y. Liu, Z. Cseresny´ es, W. R. Randall, and M. F. Schneider,
“Activity-dependent nuclear translocation and intranuclear
distribution of NFATc in adult skeletal muscle fibers,” Journal
of Cell Biology, vol. 155, no. 1, pp. 27–39, 2001.
 B. L. Prosser, N. T. Wright, E. O. Hern˜ andez-Ochoa et al.,
“S100A1 binds to the calmodulin-binding site of ryanodine
coupling,” Journal of Biological Chemistry, vol. 283, no. 8, pp.
 L. D. Brown, G. G. Rodney, E. Hern´ andez-Ochoa, C. W. Ward,
and M. F. Schneider, “Ca2+sparks and T tubule reorganization
in dedifferentiating adult mouse skeletal muscle fibers,” Amer-
ican Journal of Physiology. Cell Physiology, vol. 292, no. 3, pp.
 L. D. Brown and M. F. Schneider, “Delayed dedifferentiation
fibers in vitro,” In Vitro Cellular and Developmental Biology,
vol. 38, no. 7, pp. 411–422, 2002.
 T. Ploug, B. van Deurs, H. Ai, S. W. Cushman, and E. Ralston,
“Analysis of GLUT4 distribution in whole skeletal muscle
fibers: identification of distinct storage compartments that are
recruited by insulin and muscle contractions,” Journal of Cell
Biology, vol. 142, no. 6, pp. 1429–1446, 1998.
 G. R. Crabtree and E. N. Olson, “NFAT signaling: chore-
ographing the social lives of cells,” Cell, vol. 109, no. 2,
supplement 1, pp. S67–S79, 2002.
 A. Rao, C. Luo, and P. G. Hogan, “Transcription factors of
the NFAT family: regulation and function,” Annual Review of
Immunology, vol. 15, pp. 707–747, 1997.
 S. M. Cardoso, P. Mutch, P. J. Scotting, and P. M. Wigmore,
“Gene transfer into intact fetal skeletal muscle grown in vitro,”
Muscle and Nerve, vol. 30, no. 1, pp. 87–94, 2004.
 S. G. Cull-Candy, R. Miledi, and O. D. Uchitel, “Denervation
during organ culture,” Journal of Physiology, vol. 333, pp. 227–
 R. L. Ruff and D. Whittlesey, “Na+current densities and
voltage dependence in human intercostal muscle fibres,”
Journal of Physiology, vol. 458, pp. 85–97, 1992.
 L. Ziskind-Conhaim and M. J. Dennis, “Development of rat
neuromuscular junctions in organ culture,” Developmental
Biology, vol. 85, no. 1, pp. 243–251, 1981.
 L. Ziskind-Conhaim, I. Geffen, and Z. W. Hall, “Redistribu-
tion of acetylcholine receptors on developing rat myotubes,”
Journal of Neuroscience, vol. 4, no. 9, pp. 2346–2349, 1984.
 K. Yamanouchi, T. Hosoyama, Y. Murakami, S. Nakano, and
M. Nishihara, “Satellite cell differentiation in goat skeletal
muscle single fiber culture,” Journal of Reproduction and
Development, vol. 55, no. 3, pp. 252–255, 2009.