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Development of a novel
multiphysical approach for the
characterization of mechanical
properties of musculotendinous
tissues
Malek Kammoun1, Redouane Terni1, Vincent Dupres2, Philippe Pouletaut1, Sandra Même3,
William Même3, Frederic Szeremeta3, Jessem Landoulsi
4, Jean-Marc Constans5,
Frank Lafont2, Malayannan Subramaniam6, John R. Hawse6 & Sabine F. Bensamoun1
At present, there is a lack of well-validated protocols that allow for the analysis of the mechanical
properties of muscle and tendon tissues. Further, there are no reports regarding characterization of
mouse skeletal muscle and tendon mechanical properties in vivo using elastography thereby limiting
the ability to monitor changes in these tissues during disease progression or response to therapy.
Therefore, we sought to develop novel protocols for the characterization of mechanical properties in
musculotendinous tissues using atomic force microscopy (AFM) and ultrasound elastography. Given
that TIEG1 knockout (KO) mice exhibit well characterized defects in the mechanical properties of
skeletal muscle and tendon tissue, we have chosen to use this model system in the present study. Using
TIEG1 knockout and wild-type mice, we have devised an AFM protocol that does not rely on the use of
glue or chemical agents for muscle and tendon ber immobilization during acquisition of transversal
cartographies of elasticity and topography. Additionally, since AFM cannot be employed on live
animals, we have also developed an ultrasound elastography protocol using a new linear transducer,
SLH20-6 (resolution: 38 µm, footprint: 2.38 cm), to characterize the musculotendinous system in vivo.
This protocol allows for the identication of changes in muscle and tendon elasticities. Such innovative
technological approaches have no equivalent to date, promise to accelerate our understanding of
musculotendinous mechanical properties and have numerous research and clinical applications.
Musculotendinous tissue is complex and consists of well-organized hierarchical structures that inuence its func-
tion and mechanical properties. At present, the mechanical properties of muscle tissue are characterized in vitro
using stretching tests (at dierent velocities) or contractile tests (using calcium) to measure the passive and active
properties of the muscle1–3. However, few studies have performed transversal tests, such as indentation, to ana-
lyze the transversal elasticity of the tissue4,5. Atomic Force Microscopy (AFM) is a perfectly suited technique for
carrying out such tests. Indeed, in the last two decades, AFM has emerged as a ubiquitous technique utilized in
biological research under near-physiological conditions from single molecules to living cells6–10. In addition to
its outstanding imaging capabilities, force-distance curve based modes are widely used to probe the mechanical
properties of biological samples11–13. Using AFM, the viscoelastic behavior of skeletal muscle cells (myoblasts)14
and dystrophic muscle15 have been characterized using the Young’s modulus to quantify the functional benet
1Alliance Sorbonne Universités, Université de Technologie de Compiègne, Biomechanics and Bioengineering
Laboratory, UMR CNRS 7338, Compiègne, France. 2Université Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille,
U1019 - UMR 8204 - CIIL - Center for Infection and Immunity of Lille, F-59000, Lille, France. 3Centre de Biophysique
Moléculaire, CNRS UPR4301, Orléans, France. 4Sorbonne Université, Laboratoire de Réactivité de Surface UMR
7197, Paris, France. 5Institut Faire Faces, EA Chimère 7516 UPJV, CHU Amiens Imagerie Médicale, Amiens, France.
6Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA. Correspondence and
requests for materials should be addressed to S.F.B. (email: sabine.bensamoun@utc.fr)
Received: 17 September 2018
Accepted: 3 May 2019
Published: xx xx xxxx
OPEN
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aer gene therapy. At the level of muscle bers, AFM has been used on xed tissue samples from Mdx, or Col6a1
KO mice to analyze the mechanical properties of the sarcolemma16. However, it is dicult to compare the elastic
values measured with AFM to other studies due to the experimental conditions such as preparation and xation
of the samples, type of cantilever employed and mathematical models used to extract the Young’s modulus from
the force-distance curves which are specic to each protocol17. For the rst time, we have devised a novel AFM
protocol with the ability to be performed on intact living muscle and tendon bers that can be used for numerous
research applications across the eld. We have also applied the most recent AFM data acquisition modes, more
specially the Quantitative Imaging mode (QImode), in this new protocol allowing for the generation of high res-
olution elasticity maps. is technique highlights the distribution of the mechanical properties along the bers
and reveals the striated structure of the tissue.
However, AFM has limitations in that it is unsuitable for application on live animals. us, non-invasive
imaging techniques such as elastography (magnetic resonance imaging (MRI) or ultrasound (US)) have begun
to be used for in vivo estimation of the mechanical properties of mouse or rat muscle18,19. Elastography is based
on the propagation of shear waves using an external driver placed on the muscle. Previous studies have imple-
mented needle drivers implanted in the hindlimb of mice to acquire magnetic resonance elastography (MRE)
based acquisitions for assessment of the mechanical anisotropic ratio of the shear storage moduli18. Recently,
Nelissen et al.20 have developed a MRE-based approach to analyze skeletal muscle damage in rats. With regard to
US elastography, a recent study19 has used the Aixplorer machine to show correlations between elastic properties
and pathological characteristics of spinal cord injuries in rats. For ultrasound elastography, the driver is a probe
placed on the tissue which generates localized acoustic radiation force to produce waves that propagate transver-
sally and provide a two-dimensional real-time quantitative map of the elasticity of muscle. In the present report,
we have utilized these technologies to devise a non-invasive US elastography protocol that allows for the charac-
terization ofmuscle and tendon tissues mechanical properties in live animals. Ultrasound elastography is a more
practical and available technique compared to MRE which requires the use of a MRI machine. Additionally, we
have developed this protocol using a novel clinical probe that could allow for the use of this technique for clinical
applications.
Typically, elastography and AFM protocols for assessment of skeletal muscle and tendon tissues are developed
independently and therefore have substantial dierences between them regarding machine settings, data analysis
protocols etc. One goal of the present report was to develop a protocol that was identical for analysis of both skel-
etal muscle and tendon and that could therefore be universally applied to both tissues for mice.
In addition, we have also validated and correlated the elastic results with other analyses including MRI tex-
ture analysis (MRI-TA) and Transmission Electron Microscopy (TEM). For all of our studies, we have utilized
wild-type (WT) and TIEG1 KO mice as our model system given their well-characterized biological and structural
defects throughout the musculotendinous system. Such innovative technological approaches have no equivalent
to date and promise to accelerate our understanding of musculotendinous mechanical properties.
Results
Atomic force microscopy (AFM) analysis of the elastic properties of musculotendinous tis-
sues. AFM experiments were performed on skeletal muscle and tendon bers to generate elasticity maps. e
sarcomeric structure of muscle bers was observed with this technique demonstrating its capability to measure
elasticity of single muscle bers (Fig.1A–D). e Young’s modulus was signicantly decreased for TIEG1 KO
muscle bers isolated from both the soleus and the EDL muscles compared to WT controls. is decrease in the
Young’s modulus is represented by less intense color mapping for the TIEG1 KO bers (Fig.1). ese analyses
also revealed that slow twitch bers isolated from WT soleus muscle have greater elasticity than fast twitch bers
isolated from WT EDL muscle (Fig.2A). Conversely, TIEG1 KO tendon bers exhibited a signicant increase
in the Young’s modulus compared to WT tendon bers (Figs1E,F and 2B). e Young’s modulus of the WT ten-
don (53.2 kPa) and WT EDL (46.5 kPa) were nearly identical indicating a similar range of the transversal elastic
modulus for these two tissues. Finally, these results indicate that the impact of TIEG1 expression on elasticity is
tissue specic.
Transmission electron microscopy (TEM) analysis. In order to further understand the basis for the
defects in the elastic properties of skeletal muscle tissues derived from TIEG1 KO mice via AFM, we performed
TEM studies. Longitudinal sections of WT and TIEG1 KO soleus and EDL muscles revealed disorganization of
the muscle structure in the absence of TIEG1 expression (Fig.3). Specically, smaller sarcomere lengths (1.5 µm
for KO and 2.5 µm for WT) and the near complete disappearance of the I band were observed.
Evaluation of the musculotendinous elastic and textural properties using ultrasound elastogra-
phy. Given that AFM and TEM evaluation of tissues is performed on ex vivo tissues, we sought to evaluate the
utility of ultrasound elastography of skeletal muscle and tendon tissues in live animals. From the B-mode image
(Figs4A and 5A), no signicant dierence of echo intensity mean or coecient of variation were found between
WT and TIEG1 KO tissues. Similar results were found for the contrast measured within the muscle region of
interest (ROI) (TIEG1 KO: 54.5 ± 18.2% vs WT: 50.6 ± 14.1%) and the tendon ROI (TIEG1 KO: 40.9 ± 14.0% vs
WT: 36.0 ± 9.7%). However, the TIEG1 KO and WT ROIs were well discriminated by texture analysis as high-
lighted in the PCA images (Fig.6A,B).
From the elastography images, no signicant dierence in mean stiness, coecient of variation or con-
trast was observed. More specically, no signicant dierence in the stiness was detected between TIEG1
KO (36.1 ± 10.3 kPa) and WT (44.4 ± 13.4 kPa) muscle nor between TIEG1 KO (157.3 ± 56.3 kPa) and WT
(161.1 ± 54.1 kPa) tendon (Fig.6C,D). However, we noted signicant stiness dierences between WT muscle
(44.4 ± 13.4 kPa) and WT tendon (161.1 ± 54.1 kPa) with higher values observed in the tendon tissue.
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Hierarchical ascending classication (HAC) of skeletal muscles. Comparison of the Hierarchical
Ascending Classications (HAC) for all muscles (tibialis anterior, plantaris, gastrocnemius lateralis, gastrocne-
mius medialis, soleus and extensor digitorum longus) analyzed via MRI revealed two distinct groups of texture
proles which clustered as a function of mouse genotype (Fig.7). Dierences between Class I (CI) and Class
II (CII) demonstrate a genotype eect on the dierent muscles. ese results indicate dierent texture proles
between WT and TIEG1 KO muscles. e global values for the tibialis anterior, plantaris, gastrocnemius lateralis,
gastrocnemius medialis, soleus, and EDL were found to be 73% (WT vs. TIEG1), 62.5% (WT vs. TIEG1), 81.2%
(WT vs. TIEG1), 59% (WT vs. TIEG1), 76% (WT vs. TIEG1) and 70% (WT vs. TIEG1), respectively.
Discussion
Muscle bers are composed of dierent compartments (myobril, extra sarcomeric cytoskeleton and sarco-
lemma) which ideally would be characterized in longitudinal and transverse directions in order to comprehen-
sively characterize the entire ber. However, with the use of existing technology and protocols, measurements in
the transversal direction are oen neglected despite the fact that important information could be obtained from
Figure 1. Elasticity maps (15 µm× 15 µm) obtained from the AFM protocol for slow (A,B soleus: Sol) and fast
(C,D EDL) twitch muscle bers and tendon bers (E,F) of wild-type (WT) and TIEG1 knockout (KO) mice.
e muscle and tendon cartographies are represented by dierent colors which are representative of lower
(blue/green) and higher (red/orange) elasticities.
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such analyses for structural proteins located perpendicular to the ber axis21. For instance, desminopathy is a
muscle disease related to the defect of an intermediate lament (called desmin), which is aligned in a transverse
manner through the Z-disks, known to play a critical role in the maintenance of structural integrity and mechan-
ical properties22.
e AFM technique, which is primarily used to image biomolecular and cellular systems, is currently poorly
utilized for analysis of mechanical properties in brous tissues. Past studies have employed AFM on tissue cry-
osections4 or following tissue xation5 at resolutions of the whole tendon (diameter units: millimeter) or at the
tendon bril level (diameter units: nanometer). For the rst time, we report the development of a novel protocol
for the use of AFM on living tendon tissues at the resolution of single tendon bers (diameter units: micrometer).
From an experimental standpoint, a challenge with such analyses is to immobilize the muscle ber during
the indentation test without using glue or chemical agents16,23 which could alter the mechanical properties of the
tissue. e acquisition of the force curve during the indentation is also a key point of this technique. Indeed, the
curve of the force, which is recorded for each pixel (128 × 128) of the cartography, must be validated according to
the artifact of ber movement leading to incorrect interpretation of the Young’s modulus. Another novel aspect
Figure 2. Quantication of Young’s modulus obtained via AFM for wild-type (WT) and TIEG1 knockout (KO)
soleus (SOL) and EDL muscle bers (A) and tendon (B) bers. ***p < 0.001 between indicated groups.
Figure 3. Transmission Electronic Microscopy (TEM) acquisition of soleus (Sol) and extensor digitorum
longus (EDL) muscles isolated from wild-type (WT) and TIEG1 knockout (KO) mice. Longitudinal sections
revealed a disorganized ultrastructure in both TIEG1 KO Sol and EDL muscles (B–D) compared to WT
littermates (A–C).
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of this study was to generate high resolution elasticity maps of single muscle and tendon bers through the use of
the QI mode of AFM. is allows for fast data acquisitions that are required to generate high resolution maps that
highlight the spatial distribution of the musculotendinous mechanical properties.
e maps generated from these studies have revealed the regular periodic structure of the muscle ber, with
smaller sarcomeres (1.5 µm) observed in the TIEG1 KO muscle, and the collagenous parallel laments within
the tendon bers. ese structural observations are in agreement with TEM acquisitions performed on WT and
TIEG1 KO muscle which revealed a disorganization of the muscle structure in both TIEG1 KO soleus and EDL
muscles compared to WT littermates. Specically, smaller sarcomere lengths (1.5 µm for KO and 2.5 µm for WT)
and disappearance of the I band where observed in both slow and fast twitch TIEG1 KO skeletal muscles. ese
results demonstrate that the eciency of the AFM protocols (experimental and acquisition) are representative of
the main structures of muscle and tendon bers with regard to the elastic cartography allowing for the avoidance
of topographic acquisition that is currently performed with AFM.
Regardless of mouse genotype, slow skeletal muscle bers (soleus) exhibited higher elasticity compared to fast
bers (EDL). ese results most likely relate to the oxidative and glycolytic ber composition of these specic
skeletal muscles. In addition, the lower elasticity as detected by AFM in the TIEG1 knockout soleus and EDL
bers relative to the WT bers may be reective of the disorganized structure. e disorganization of TIEG1 KO
skeletal muscle bers likely leads to a decreased resistance under the tip of the AFM probe. e control (WT)
values are of interest for numerical modelling, which need referent data to simulate the true mechanical behavior
of biological samples. We also observed higher elasticity for the Z-disk region of the muscle structure compared
to the M-band and this observation was in agreement with Ogneva’s study21 for rat muscle bers.
In comparison with muscle bers, the transversal elasticity of WT tendon bers is within the same range as
those of the WT muscle bers. Interestingly, deletion of the TIEG1 gene increased the transversal elasticity of the
tendon bers compared to WT control bers. Previous TEM studies on tendon tissue along the longitudinal axis of
the tendon ber did not reveal any structural changes between the dark and light bands24. However, Gumez et al.25
reported dierent X-ray diraction patterns between WT and TIEG1 KO tendon bers demonstrating that lack
of TIEG1 expression results in a disorganization of the collagenous structure and a signicant distortion of the
triple helix network26.
It is interesting to note that deletion of TIEG1 has opposite eects on the elasticity of muscle vs. tendon bers.
Given the intimate association of these two tissues with one another, it is possible that these dierences are due
Figure 4. Representative B-mode image of the mouse hindlimb (A) and the corresponding elastogram (B)
showing the elasticity of the dierent tissues present within the hindlimb. e region of interest (ROI) analyzed
for quantication of the Young’s modulus (E)is indicated (B).
Figure 5. Representative B-mode image of the mouse hindlimb (A) and the corresponding elastogram (B)
showing the elasticity of the dierent tissues present within the hindlimb. e region of interest (ROI) for
measurement of the Young’s modulus (E)of the Achilles tendon is indicated (B).
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to compensation of one tissue for another. However, it is also possible that these eects are due to tissue specic
dierences exerted by this transcription factor. TIEG1 is known to mediate the down-stream eects of many
cytokines and hormones including TGFβ, BMP, EGF, and estrogen among others27–30. It is therefore possible that
Figure 6. Principal component analysis of texture parameters from the B-mode acquisitions of muscle (A)
and tendon (B) according to mouse genotype (1: TIEG1 knockout (KO) vs 2: wild-type (WT)). Signicant
dierences for both tissues between the two genotypes are apparent. Quantication of muscle stiness (C) and
tendon stiness (D) according to mouse genotype.
Figure 7. Hierarchical ascending classication (HAC) of the six muscles as a function of mouse genotype
(wild-type (WT) vs TIEG1 knockout (KO)). Dierences between Class I (CI) and Class II (CII) demonstrate a
genotype eect on the dierent muscles.
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loss of TIEG1 expression dierentially impacts these signaling pathways in these two tissues. It is also possible
that other transcriptional co-regulators and/or transcription factors that are essential for muscle and tendon
development/function are dierentially impacted by loss of TIEG1 expression. Additional studies are needed to
identify the precise mechanisms by which loss of TIEG1 expression results in opposite eects with regard to the
elasticity of muscle and tendon bers.
To overcome the necessity to sacrice mice, recent elastography imaging techniques (MRI, US) have enabled
the measurement of mechanical properties in live animals. While magnetic resonance elastography has been
used on muscles of mdx (Duchene) mice18, US elastography has never been performed on mouse muscle or
tendon. In the present study, US elastography was performed with the Aixplorer machine which is clinically used
for human tissues including muscle31 and sciatic nerve32 among others. Here, we have applied this technique to
characterize the elastic properties of the musculotendinous system in a non-invasive manner. We have utilized
a high frequency probe (SLH20-6) that has been recently adopted in clinical practice to image the functionality
of supercial musculotendinous tissues. It should be noted that ultrasound imaging of mouse tissue is usually
performed with homemade constructor probes. Using the SLH20-6 probe, muscle ber structure and Achilles
tendon were accurately analyzed with the use of the B-mode image (resolution 38 µm). Moreover, texture anal-
ysis revealed signicant dierences as a function of mouse genotype for both tissues, demonstrating that US
elastography (Aixplorer) has the capability of identifying structural defects in muscle and tendon tissues. We
have validated these ndings with MRI texture analysis (MRI-TA) which also indicated signicant structural
changes between TIEG1 KO and WT muscle bers. In comparison with the US technique, MRI has the ability
to analyze the structure of all individual muscles within the hindlimb, while US can only characterize groups
of muscles33–35. Interestingly, the hierarchical ascending classication (HAC) revealed that deletion of TIEG1
aected all muscle-texture proles regardless of muscle type (oxidative: soleus, glycolytic: EDL, mix: gastrocne-
mius, plantaris, tibialis).
While US was able to detect structural changes as a function of genotype, the elastography acquisitions were
not able to dierentiate between WT and TIEG1 KO mice with regard to the elastic properties. is result indi-
cates that US elastography, based on the analysis of shear waves, is not suciently sensitive to detect dierences in
elasticity. e development of higher frequency probes could allow for better spatial resolution and may allow for
better resolution for detection of dierences in elastic properties. erefore, US elastography performed on the
musculotendinous system cannot currently replace in vitro tests. However, this technique was able to dierentiate
the elasticity between tendon and muscle tissues regardless of mouse genotype. is dierentiation was most
likely related to the increased stiness of the Achilles tendon compared to skeletal muscle.
It should be noted that the protocols developed in the present study have some limitations. Concerning
AFM experiments, it remains dicult to compare the elastic modulus values with the ones obtained from sim-
ilar experiments. is is primarily due to the numerous experimental conditions (type of AFM tip, calibration
method, maximal indentation force, acquisition mode, mathematical models used to extract the Young’s modulus
from the force-distance curves, etc …) which inuence the measurement of the elastic properties. Nevertheless,
this problem is being increasingly recognized, and some improvements have recently been proposed with the
introduction of the Standardized Nanomechanical AFM Procedure (SNAP)17. We are perfectly aware of these
limitations and for this reason we apply a series of precautions to ensure that our results are comparable from one
experiment to another. is implies keeping the same parameters for the whole study, not changing the laser posi-
tion and using the same type of cantilever (and where possible, the same cantilever) and the same mathematical
model for the analysis.
As an extension of the results presented here, future studies will focus on developing the AFM protocol for
analysis of muscle bers under active conditions by altering calcium concentrations during the mechanical test.
Furthermore, it is of interest to use this technique on tissues undergoing various degrees of stretching to provide
a more dynamic measure of their functional properties. As for the US elastography protocol, the clinical probe
utilized in this study did not allow for dierentiating the elastic properties as a function of mouse genotype. To
improve upon this limitation, it will be necessary to design a specic US probe with higher frequency that is
compatible with the Aixplorer machine allowing for a better characterization of the mechanical properties of
rodent so tissues (muscle and tendon). In the future, it will be also of interest to adapt the present elastography
protocols for other tissues.
In conclusion, we have developed a novel multiphysical approach that enables a better understanding of the
structural and mechanical properties of so living tissues including muscle and tendon. ese novel applications
will be useful in both research and clinical applications for the evaluation of normal and diseased states. Finally,
through the use of these mouse models, we have further implicated important roles for TIEG1 in the musculo-
tendinous system and have demonstrated that loss of TIEG1 expression reduces the elastic properties of muscle
bers while improving the transversal elastic properties of tendon bers.
Methods
Mice and study design. e generation of TIEG1 KO mice has been previously described36 and initial char-
acterization of TIEG1 expression and function in skeletal muscle and tendon were previously reported24,25,37–41.
For the studies presented here, 3 month old littermate female animals from heterozygous breeding pairs were
used. All mice were maintained in a temperature controlled room (22 ± 2 °C) with light/dark cycle of 12 hours.
Animals had free access to water and were fed with standard laboratory chow ad libitum. is study was carried
out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of
the National Institutes of Health. e protocol was approved by the French ministry of higher education, research
and innovation (Permit Number: DUO-4776) and the Mayo Clinic Institutional Animal Care and Use Committee
(Permit Number: A9615).
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Atomic force microscopy (AFM) measurements. All experiments were performed on a commer-
cial AFM (NanoWizard 3, JPK Instruments, Berlin, Germany) combined with an inverted optical microscope
(AxioObserver.Z1, Zeiss) driven by JPK NanoWizard soware 6.0 and Zen Blue 2012. Data were acquired in
Quantitative Imaging mode (QImode) using PFQNM-LC cantilevers with a nominal spring constant of 0.08 N/m
and a 3.5 µm high conical tip with 8 nm tip radius (Bruker, Santa Barbara, CA, USA). Cantilever spring constant
and sensitivity were calibrated before each experiment using the thermal uctuation method of the JPK soware,
yielding spring constant values ranging from 0.019 to 0.025 N/m.
Control (NWT_Tendon = 7, NWT_Soleus = 7, NWT_EDL = 7) and TIEG1 KO (NKO_Tendon = 7, NKO_Soleus = 7,
NKO_EDL = 7) bers were extracted from slow (soleus) and fast (EDL) muscles39 as well as tail tendon24. Perfect
immobilization of the sample is a prerequisite during each AFM experiment. is is particularly the case in exper-
iments conducted on bers where the slightest movement (vertical or lateral) is likely to generate artifacts during
the acquisition of force curves, which will result in making the elasticity maps unusable. e absence of move-
ment during transverse indentations is therefore a fundamental element of our experimental protocol. Fibers
were immobilized on plexiglass and submersed in a drop of PBS. Bright eld imaging was used to identify the
area of interest on bers. A visual control of the immobility of the ber was performed using the optical micro-
scope coupled with the AFM instrument. is ensured that only Quantitative Imaging (QI) les were recorded
in a static condition. Subsequently, the AFM tip was engaged in the central portion of the ber (to ensure a near
plane contact surface to avoid curvature eects leading to artifacts) to scan an area of 15 µm × 15 µm. e AFM
tip was scanned on three areas along each muscle and tendon ber by applying a force trigger of 2 nN with a 3 µm
Z ramp (i.e. the range between the two extreme positions of the AFM tip), at 80 µm/s. Compared to conventional
Force-Volume (FV) mode, QI mode uses a specic movement algorithm that enables precise control of the verti-
cal force at every pixel of the image without applying any lateral force that could damage so biological samples.
Combined with the new generation of short cantilevers, QI mode allows fast tip displacements, thus reducing the
time required to capture elasticity maps. Hence, we were able to perform 128 × 128 pixel force mapping scans in
less than 15 minutes while more than 5 hours would be needed for the same resolution with FV.
Data analysis. e AFM data analysis was performed using in-house pyAF (python Atomic Force) soware,
version 1.5.1. An algorithm based on the tting of the baseline was used to detect the point of contact on the
force curve. A linear t was made using the baseline of the approach curve. A noise threshold is used to shi the
t along the force axis. e noise parameter can be manually adjusted by the user to optimize the detection of
the point of contact. e intersection between this shied t and the force curve denes the position of the point
of contact. Subsequently, the elasticity was deduced from the Young’s modulus which was determined from the
indentation portion of the curve using the elastic contact model for conical indenters (named Sneddon model42):
π
α
δ=
−
F
E
v
2tan
1
22
where F is the measured force, E the elastic modulus, α the half opening angle of the tip, δ the indentation, and ν
the sample’s Poisson’s ratio, that was set to 0.5.
Elasticity maps. Arrays of force curves in the x and y planes (typically 128 curves × 128 curves) were recorded on
areas of a given size (15 µm × 15 µm). From these force mapping scans, the Young’s modulus was then estimated for
each force curve and displayed as colored pixels that reect the magnitude of ber stiness (elasticity maps).
Box plot. Box plots were generated by pooling all of the data used to create elasticity maps for each ber
type (tendon, muscle) (elastic modulus deduced from each individual force curve) as a function of mouse geno-
type. Unpaired t-test with Welch’s correction (which assume no equal SDs) was performed to compare elasticity
between mouse genotypes for muscle (soleus, EDL) and tendon bers (***p-value < 0.0001). Bottom and top of
the boxes represent the rst and third quartiles. e line within the box represents the second quartile, i.e. the
median. e end of the whiskers represents the 9th and the 91st percentiles.
Transmission electron microscopy (TEM). Prior to processing for TEM, the soleus (N = 7) and the EDL
(N = 7) muscles were dissected from WT and TIEG1 KO mice with both extremities pinned to avoid muscle
contraction and to maintain original length. Muscles were then immediately placed in xative [1% (vol/vol)
glutaraldehyde and 4% (vol/vol) paraformaldehyde in 0.1 M phosphate buer, pH 7.2]43 and incubated over-
night at 4 °C. Subsequently, two pieces (1 mm × 2 mm) of muscle were dissected from the middle of the tissue
and rinsed for 30 min in three changes of 0.1 M phosphate buer, pH 7.2, followed by a 1 h secondary xation in
phosphate-buered 1% OsO4 and 30 min in 1% uranyl acetate at room temperature. Following dehydration in a
series of ethanol washes, tissue was embedded in EMbed 812 resin (EMS, Hateld PA) and polymerized at 60 °C
for 18h44. Ultrathin (90 nm) sections were cut using a Leica UC7 ultramicrotome (Bualo Grove, IL) and placed on
200 mesh copper grids and stained with lead citrate. Five micrographs of each specimen were randomly captured
across the muscle tissue using a JEOL 1400Plus TEM (Peabody, MA), operating at 80 kV with a magnication
of 42000×. ImageJ 1.46/Java 8 soware (National Institute of Health, Bethesda, MD, United States)45 was used to
manually quantify the sarcomere length.
Shear wave elastography imaging. Mice (NWT = 7, NKO = 7) were anesthetized with 1.5% isourane
and a mixture of O2/air (1:1) at an output of 0.7 L/min and placed in a prone position. Landmarks were dened
to orient the mice in a reproducible manner with the paw bent at a xed angle (Fig.8). Tendon and muscle
in the right hindlimb were imaged by the same operator with an ultrasound machine (Aixplorer MultiwaveTM
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System, Supersonic Imagine, Aix-en-Provence, France) using the SLH 20-6 linear transducer probe having the
specic characteristics: resolution: 38 µm, 2.38 cm footprint, 192 composite elements, eective bandwidth from
6 to 20 MHz.
For muscle imaging, the probe was placed on the surface of the hindlimb, which was shaved, parallel to
the Achilles tendon. A thin layer of water-soluble transmission gel was applied for optimal acoustic coupling.
To ensure that the probe was reproducibly placed, a visual control was performed using the B-mode image
where the bone and the heel, considered as referent tissues, were identied. Subsequently, the same box size
(10 mm× 10 mm) was dened for all acquisitions according to the length of the muscle and bone.
For tendon imaging, the probe was shied to clearly visualize the Achilles tendon on the B-mode image. Bone
and heel were also used as referent tissues and a box (10 mm× 10 mm) was placed in the same area for each
mouse. e Aixplorer MultiwaveTM System generates two types of waves that propagate within the tissue: a com-
pression wave that creates a high-quality B-mode image showing the anatomical structure within the hindlimb
(skin, muscle, tendon, bone), and a shear wave that provides a quantitative color-coded map of tissue elasticity
(Figs7 and 8). e setting parameters for B-mode image were:
==
μ
sp
ulse duration 600
Frequency
number _half_cycle
2
, gain
50%, pulse repetition frequency: 20 kHz and spatial resolution: 38 µm. To obtain mapping of elasticity with the
shear wave elastography (SWE) sequence the setting parameters were: musculoskeletal preset, resolution mode
enabled, tissue tuner at 1540 m/s, gain at 50%, dynamic range at 60 dB and the spatial resolution is 0.9 mm. e
SWE mode is based on the generation of localized acoustic radiation force by the probe in the tissue; the locally
excited tissue then produces waves which propagate transversally. In addition, the range of elasticity (Young’s
modulus) was set between 0 kPa and 180 kPa, which corresponds to a shear wave velocity range of 0–7.7 m/s.
Image analysis. In the B-mode image for US analysis (anatomical), a region of interest (ROI) was manually
drawn within the hindlimb muscle group (lateralis and medialis gastrocnemius, soleus, EDL: Extensor Digitorum
Longus, plantaris and tibialis) and within the surface Achilles tendon. ImageJ soware (NIH, Bethesda, USA) was
used to create the ROI. e ROI was then superimposed on B-mode images and elasticity images. Subsequently,
the ROI Manager Tool of ImageJ was used to measure the following parameters: mean and standard deviation
for echo intensity and stiness, contrast (ratio of the dierence (maximum - minimum) to the sum (maxi-
mum + minimum)) and coecient of variation (ratio of standard deviation to mean).
e ROI of B-mode images were also analyzed for texture analysis using Mazda 4.6 soware45, which includes
several texture-analysis methods. In this study, gray-level histogram parameters and co-occurrence parameters
were used. Principal Component Analyses (PCA) were performed to identify the most signicant texture param-
eters that were able to discriminate KO muscle/tendon from WT muscle/tendon.
Statistical analysis. e SystatTM V11 soware package (Systat Soware Inc., CA, USA) was used to per-
form all statistical analyses. Non parametric two-sample Kolmogorov-Smirnov tests were performed in order to
compare the elasticity values and the B-mode values between WT and TIEG1 KO mice. e statistical analysis
was considered signicant for P < 0.05.
Figure 8. Model indicating the orientation and placement of the ultrasound probe on the mouse hindlimb.
is image was drawn by Frank M. Corl from the department of Biomedical and Scientic Visualization, Mayo
Clinic. Used with permission of Mayo Foundation for Medical Education and Research, all rights reserved.
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Magnetic resonance imaging (MRI). Selection of skeletal muscles. MRI experiments were performed on
10 TIEG1 KO and 10 WT mice. For each mouse, muscles known to have dierent ber types46,47 (slow, mixed,
fast) were selected, namely tibialis anterior, plantaris, gastrocnemius lateralis, gastrocnemius medialis, soleus,
and EDL muscles.
MRI acquisition. Prior to MRI analysis, mice were anesthetized with 1.5% isourane and a mixture of O2/air
(1:1) at an output of 0.7 L/min. Respiration was monitored during the entire experiment, and body tempera-
ture was maintained at 37 °C using a warm-water circulation system. MRI was performed on a 9.4T horizon-
tal ultra-shielded superconducting magnet dedicated to small-animal imaging (94/20 USR Bruker Biospec,
Wissembourg, France) and was equipped with a 950 mT/m gradient set. A loop gap coil (10 mm inner diameter)
was used for both proton transmission and reception. Axial images of the TIEG1 KO and WT hindlimb muscles
were obtained using a gradient echo (Flash) sequence with the following parameters: TE/TR = 6 ms/252 ms, ip
angle = 20°, FOV size = 2 × 2 cm, matrix size = 256×256, bandwidth = 50 kHz, slice thickness = 570μm, to dis-
play 78 µm× 78 μm in plane resolution for a duration of 1 min (one accumulation).
Texture analyses. Acquired MR images were transferred to an external computer for data processing. ROIs
were manually drawn as large as possible within the selected muscles and subsequently analyzed using Mazda
software48 (Mazda 4.6, MRI analysis software, ©1998–2007 by P.M.Szczypinski), which includes several
texture-analyses methods. In this study, the greylevel histograms, composed of various parameters including
mean, standard deviation, skewness, kurtosis and dierent percentiles were used. In addition, a co-occurrence
matrix based on the following parameters: contrast, correlation, entropy, homogeneity, energy and run length
matrix with run length and grey level non uniformity, long and short run emphasis, wasapplied.
Data analyses. PCA (principalcomponent analyse) were performed to identify the most signicant texture
parameters able to discriminate TIEG1 KO muscles from the WT muscles with a condence level of 0.95. A
two-class (WT/TIEG1 KO) Hierarchical Ascending Classication (HAC) was performed with the most relevant
texture parameters. is was performed for all muscles: tibialis anterior, plantaris, gastrocnemius lateralis, gas-
trocnemius medialis, soleus and extensor digitarum longus. e global value (true positive + true negative/total
number of mice) was calculated for each HAC. is parameter represents the number of mice that are grouped
into WT or TIEG1 KO by means of their texture proles. A value superior to 60% is considered good.
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Acknowledgements
is work was carried out and funded in the framework of the Labex MS2T. It was supported by the French
Government, through the program “Investments for the future” managed by the National Agency for Research
(Reference ANR-11-IDEX-0004-02). is work was also supported by the National Institutes of Health (R01
DE14036 to JRH and MS). We would also like to thank Dr. omas Spelsberg for his excellent mentorship.
We thank the Institut Pasteur Core for experimental and technical support (ANR 10-EQPX-04-01 & FEDER
12,001,407 to FL). We acknowledge Frank Corl as the artist, who has created the illustration (Figure 8), as a work-
for-hire for Mayo Foundation for Medical Education and Research.
Author Contributions
S.F.B., M.K., J.R.H. and M.S. conceived the project. V.D., M.K., R.T., S.F.B., M.S., J.R.H., S.M., W.M., F.S., J.L.
and P.P. designed and performed the experiments. V.D., M.K., R.T., S.F.B., M.S., J.R.H., S.M., W.M., J.L. and P.P.
analyzed and interpreted data. V.D., M.K., R.T., S.F.B., M.S., J.R.H., S.M., W.M., F.L., J.M.C., J.L. and P.P. wrote and
revised the manuscript. All authors have read and approved the nal manuscript.
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