Pflügers Archiv - European Journal of Physiology (2018) 470:1503–1519

https://doi.org/10.1007/s00424-018-2167-3

MUSCLE PHYSIOLOGY

Sarcopenia in a mice model of chronic liver disease: role

of the ubiquitin–proteasome system and oxidative stress
Fabián Campos 1,2 & Johanna Abrigo 1,2 & Francisco Aguirre 1,2 & Bruno Garcés 1,2 & Marco Arrese 3 & Saul Karpen 4 &
Daniel Cabrera 3,5 & Marcelo E. Andía 6,7 & Felipe Simon 1,2 & Claudio Cabello-Verrugio 1,2

Received: 13 December 2017 / Revised: 6 June 2018 / Accepted: 11 June 2018 / Published online: 20 June 2018
# Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
Sarcopenia is the loss of muscle mass and strength produced by aging or secondary to chronic diseases such as chronic
liver disease (CLD). Although not all types of sarcopenia involve the same features, the most common are decreased
fiber diameter and myosin heavy chain (MHC) levels, increased activity of ubiquitin–proteasome system (UPS) and
reactive oxygen species (ROS). In this study, we aim to characterize the development of sarcopenia secondary to CLD
induced by the hepatotoxin 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). For this purpose, four-months-old male
C57BL6 mice were fed with normal diet or DDC supplemented diet for 6 weeks. Functional tests to evaluate muscle
strength, mobility, and motor skills were performed in alive mice. The muscle strength in isolated gastrocnemius was
also assayed via electrophysiological measurements. Morphometric measures of fibers’ diameter, total and ubiquitinated
protein levels of myosin heavy chain (MHC), E3 ubiquitin ligases, ROS, and oxidation-dependent modified proteins in
gastrocnemius tissue were also determined. Our results demonstrated that mice fed the DDC diet developed muscle
wasting as evidenced by a loss of muscle mass and decreased muscle strength. The muscles of mice fed with DDC diet
have a decreased diameter of fibers and MHC levels, also as increased MuRF-1 and atrogin-1 protein levels, ROS levels,
and oxidation-modified protein levels. Additionally, control and DDC mice have the same food and water intake as well
as mobility. Our results demonstrate mice with CLD develop sarcopenia involving decreased levels of myofibrillar
proteins, increased UPS, and oxidative stress, but not for impaired caloric intake or immobility.

Keywords Sarcopenia . Chronic liver diseases . Hepatotoxin . UPS . Oxidative stress

F.C. and J.A. contributed equally to the present work.

Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00424-018-2167-3) contains supplementary
material, which is available to authorized users.

* Claudio Cabello-Verrugio 4
Department of Pediatrics, Emory University School of Medicine,
claudio.cabello@unab.cl Atlanta, GA, USA

1 5
Laboratorio de Patologías Musculares, Fragilidad y Envejecimiento, Facultad de Salud, Departamento de Ciencias Químicas y Biológicas,
Departamento de Ciencias Biológicas, Facultad de Ciencias de la Universidad Bernardo O Higgins, Santiago, Chile
Vida, Universidad Andres Bello, Avenida República 239,
8370146 Santiago, Chile 6
Biomedical Imaging Center, Pontificia Universidad Católica de
2
Millennium Institute on Immunology and Immunotherapy, Chile, Santiago, Chile
Santiago, Chile
3 7
Departamento de Gastroenterología, Facultad de Medicina, Radiology Department, School of Medicine, Pontificia Universidad
Pontificia Universidad Católica de Chile, Santiago, Chile Católica de Chile, Santiago, Chile
1504 Pflugers Arch - Eur J Physiol (2018) 470:1503–1519

Introduction and outcomes after liver transplantation, as well as being re-

sponsible for the development of additional health complica-
Skeletal muscle is one of the most abundant tissues in the tions [22, 34, 52, 71]. The few, but highly relevant, studies
body and makes the generation of force, movement, and relating CLD and sarcopenia confirm the negative impacts of
breathing possible. Since skeletal muscle is exposed to many sarcopenia. One of these studies found sarcopenia is an inde-
different internal and external stimuli, it has high plasticity, pendent predictor of mortality in cirrhosis patients [52].
evidenced by changes in muscle mass. Skeletal muscle atro- Moreover, a retrospective analysis in CLD patients found
phy can be defined as the loss of muscle mass with a concom- the loss of muscle mass to be an independent predictor of
itant decrease in function and force generation [12]. Among mortality for patients awaiting liver transplantation [22, 74].
the main factors altering muscle mass are aging, disuse, and Thus, sarcopenia associated with CLD is gaining relevance in
several chronic diseases such as cancer, cardiac, renal, and hepatology, with clinicians trying to incorporate this condition
liver pathologies. Sarcopenia is defined as a complex syn- as a new parameter for consideration in liver transplants [21,
drome characterized by loss of body weight, muscle mass, 22]. However, the mechanisms underlying sarcopenia in CLD
and strength which is not only developed during aging but are not yet fully understood. In this study, we evaluated the
also by chronic illness or inflammatory conditions [29]. mechanism involved in muscle weakness in CLD, using a
During sarcopenia, the major degraded skeletal muscle pro- previously characterized murine model of xenobiotic-
teins are myofibrillar proteins such as myosin heavy chain induced cholangiopathy that produces hepatobiliary injury
(MHC) which is a key component in the process of muscle and biliary fibrosis by intake of the hepatotoxin 3,5-
contraction [14, 37]. One of the main mechanisms involved in diethoxycarbonyl-1,4-dihydrocollidine (DDC) [32].
this catabolic process is the ubiquitin–proteasome system Our results indicate mice fed a DDC supplemented diet for
(UPS) [10, 67]. Overactivation of the UPS during muscle 6 weeks developed muscle wasting, evidenced by loss of mus-
wasting is characterized by a muscle-specific increase of type cle mass and decreased muscle strength. Muscles of these
E3 ubiquitin ligases, atrogin-1 (Muscle Atrophy F-box, sarcopenic mice have a decreased diameter of muscle fibers,
MAFbx), and MuRF-1 (Muscle-specific RING-finger protein reduced MHC, and increased UPS components and ROS
1) [67]. Oxidative stress is also present in skeletal muscle levels. Furthermore, mice fed either normal or DDC diet have
wasting [63, 69]. The main oxidant types are reactive oxygen the same food and water intake as well as motor skills and
species (ROS). Oxidative stress has been functionally linked mobility.
to muscle wasting through UPS activation, where increased Our data showed mice with CLD induced by a hepatotoxin
ROS activates proteasome-dependent protein degradation develop sarcopenia which involves decreased levels of myo-
through the expression of atrogin-1 and MuRF-1 [1, 63, 66]. fibrillar proteins, increased UPS activity, and oxidative stress.
Another consequence of oxidative stress is oxidative injury Notably, we also demonstrated muscle weakness is not pro-
generated in the cell, as evidenced by protein and lipid oxida- duced by differential caloric intake or immobility.
tion [5, 62, 68].
Among the causes of muscle wasting are chronic liver dis-
eases (CLD) [16, 40]. CLDs are a group of liver dysfunctions Methods
resulting in severe injury characterized by hepatocellular al-
terations and fibrosis, which progress to cirrhosis [8]. Several Animals Male C57BL/6J (16 weeks old) strain of mice was
types of liver damage are classified as CLDs, including used. The animals were randomized and separated into exper-
fibrosing cholangiopathies, a group of cholestatic liver dis- imental groups, and three independent experiments were per-
eases affecting biliary ducts responsible for transporting and formed. Two experimental groups (five to seven animals/
modifying bile [44]. Xenobiotic-induced cholangiopathies group) were designed: normal diet (Control) and diet supple-
produce biliary disease that may progress to vanishing bile mented with 0.1% (1 mg/kg) of 5-diethoxycarbonyl-1,4-
duct syndrome, biliary fibrosis, and cirrhosis [46]. CLD re- dihydrocollidine (DDC) [32]. The mice were fed with
sults in cirrhosis and, usually, in end-stage liver disease requir- Control or DDC diet for 6 weeks. At the end of the experi-
ing a liver transplant to extend survival [78]. None of these ment, the animals were euthanized under anesthesia and the
consequences is reversible [8]. There are few effective thera- gastrocnemius (GA) muscles were dissected, removed, sepa-
peutic options for treating CLD etiology, the consequence of rated from plantaris, weighed and rapidly frozen, and stored at
which is an increased incidence of cirrhosis [22, 36]. The most − 80 °C until processing. All applicable international, nation-
recognized clinical complications of CLD include ascites, en- al, and/or institutional guidelines for the care and use of ani-
cephalopathy, portal hypertension, renal dysfunction, hepato- mals were followed. All procedures performed in studies in-
cellular carcinoma, and malnutrition [6, 8, 28]. However, and volving animals were in accordance with the ethical standards
despite being less studied, sarcopenia is the most common and with the formal approval of the Animal Ethics Committee
complication and adversely affects survival, quality of life, at the Universidad Andrés Bello institution.
Pflugers Arch - Eur J Physiol (2018) 470:1503–1519 1505

Liver histopathology Liver sections from the right lobe of all mice were coated with non-toxic blue paint. The animals were
mouse livers were routinely fixed in 10% formalin and em- then allowed to walk along a 38-cm-long, 5.2-cm-wide run-
bedded in paraffin. Hematoxylin and eosin was then carried way (with 5-cm-high walls) into an enclosed box. All mice
out according to the standard procedures [15]. had three training runs and were then given one run per week.
A fresh sheet of white paper was placed on the floor of the
Biochemical determinations Serum alanine aminotransferase runway for each run. The footprint patterns were analyzed for
(ALT) was quantified using the Kovalent kit (Río de Janeiro, three step parameters (all measured in centimeters): (1) stride
Brazil) following manufacturer instructions, as described pre- length was measured as the average distance of forward move-
viously [15]. ment between each stride. (2) Stance length was measured as
the average distance between left and right hind footprints, (3)
Contractile properties After treatment, the mice were anes- sway length of the posterior base was measured as the average
thetized, the GA muscles were removed, and the muscle distance between the left and right hind footprints and left, and
contractile properties were measured as previously de- these values were determined by measuring the perpendicular
scribed [47]. The maximum isometric tetanic force was distance of a given step to a line connecting its opposite pre-
determined. The muscle mass and the optimum muscle ceding and proceeding steps. For each step parameter, three
length (Lo) were used to calculate the specific net force values were measured from each run, excluding footprints
[force normalized per total muscle fiber cross-sectional ar- made at the beginning and end of the run where the animal
ea (CSA), mN/mm2] [13, 53, 54]. was initiating and finishing movement, respectively. The
mean value of each set of three values was used in subsequent
Weightlifting test At the end of the treatment, the mice were analysis.
subjected to a measurement of muscle strength by a
weightlifting test as previously described [25]. Briefly, the Locomotor activity test Spontaneous locomotor activity was
apparatus consisted of a series of chain links of increasing measured in open-field chambers measuring 50 cm long,
length attached to a ball of tangled fine wire. The number of 30 cm wide, and 20 cm high, in a room with lighting and
links ranged from one to seven with total weights between noise-free and was used to compare the mobility of mice fed
15.5 and 54.1 g. Before performing the test and prior to treat- with a normal (Control) or DDC diet for 6 weeks. The mice
ments, the mice were subjected to training (once per day for were placed in the arena and allowed to freely move for 5 min
2 weeks). To perform the test, the mouse grasps (with its while recording with a camera above. The images are ana-
forepaws) the different weights and a score was assigned. lyzed by a made-home automated tracking system. Traveled
The final score was calculated as the summation of the product distance was calculated from the locomotor activity data.
between the link weight and the time held. The average of After each test session, the equipment was cleaned with 70%
three measures from each mouse was normalized by the body ethanol to remove animal odors.
weight [47].
Water and food intakes The animals were separated in indi-
Running test Mice were subjected to perform exercises on a vidual cages 2 weeks previous to the start of the experi-
treadmill for 15 min at 20 cm/s on a treadmill, divided in three ment and kept during the complete period of treatment
sessions of 5 min with a rest of 5 min. The test was recorded using like a bed paper pelletized that allow major absorp-
and the video analyzed to calculate the mice’s permanence tion of fluids. Food and water were weekly administered in
time in three zones of the treadmill, accordingly to scheme fixed amounts (200 ml of water and 200 g of ground food),
of Fig. 2a. and the intakes were measured at 0, 3, and 6 weeks by
difference between the remainder and the initial food and
Rotarod test Before starting the experiment, the mice were water for each mouse [4].
accustomed to the rotarod for 1 week, at different rotation
speeds in 5-min-long sessions. The rotarod test was performed Muscle fiber’s diameter determination and quantification
before starting the treatment with normal or DDC diet, and Cryosections (7 μm) of the GA were stained with Alexa-
also during weeks 1, 3, 4, 5, and 6 of the treatments. To Fluor® 594 tagged WGA (Life Technologies™, USA) ac-
perform the test, mice were placed in the rotarod, with an cording to standard procedures. Fiber sizes were determined
initial rotation speed of 5 rpm. The speed was gradually in- by WGA staining and the Image J software (NIH, USA), as
creased from 5 to 35 rpm over a time of 5 min. The time (in previously described [19, 50]. In brief, fibers were manually
seconds) that mice spend on the rotarod was recorded. selected, and the minimal Feret diameter of each fiber was
computed by the software. Myofiber cross-sectional area
Footprint test The footprint test was used to measure the gait (CSA) was determined and measured regarding to the fiber
of mice. To obtain footprints, the hind- and forefeet of the types, as previously reported [49].
1506 Pflugers Arch - Eur J Physiol (2018) 470:1503–1519

Determination of fiber types For determining of fiber types in anti-atrogin-1 (1:500; Santa Cruz Biotechnology, USA), rab-
the GA muscles, immunofluorescence analysis of MHC ex- bit anti-MuRF-1 (1:500; Santa Cruz Biotechnology, USA),
pression was performed with primary antibodies against the mouse anti-tubulin (1:5000; Santa Cruz Biotechnology,
specific types of myosin [11]. For this, cryosections of GA USA), and mouse anti-β-actin (Abcam, USA; 1:2000). All
were rinsed once with PBS, then incubated 30 min with immunoreactions were visualized by enhanced chemilumi-
mouse-to-mouse blocking reagent (1:100 in PBS), then incu- nescence (Thermo Scientific, USA).
bated with a blocking solution (PBS, 10% goat serum, 1:100
mouse-to-mouse blocking reagent) for 1 h. After of this incu- Determination of ubiquitinated MHC protein levels Total pro-
bation, a mix of antibodies were used: MHC-I (BA-D5, 1:10), tein extracts were immuno-precipitated with mouse anti-MHC
MHC-IIA (SC-71, 1:300), and MHC-IIB (BF-F3, 1:50), in (1:100; MF-20, Developmental Studies, Hybridoma Bank,
blocking solution for 2 h. All the primary antibodies were University of Iowa, USA). The antibody/antigen complex
purchased from the Developmental Studies Hybridoma was then pulled out of the sample using protein G-coupled
Bank (University of Iowa). Then, the sections were rinsed agarose beads to isolate MHC. The immuno-precipitated ex-
three times for 5 min with PBS. A mix of secondary antibodies tract was separated in SDS-PAGE and transferred to PVDF
(Invitrogen, USA) were incubated for 1 h (1:250; Alexa membranes that were incubated with the antibodies for ubiq-
IgG2b, A21140; Alexa IgM, A21426; Alexa IgG1, uitin (1:1000; Santa Cruz Biotechnology, USA), MHC
A21121). The slides were washed in PBS three times and (1:3000; MF-20, Developmental Studies, Hybridoma Bank,
were mounted with a fluorescent mounting medium (Dako University of Iowa, USA), and β-actin (1:5000; Merck,
Corporation, USA). Finally, the slides were visualized with a USA). All immunoreactions were visualized by enhanced
Motic BA310 fluorescence microscopy. Fiber types were de- chemiluminescence (Thermo Scientific, USA) that were ac-
termined within the entire muscle/cross-section (red, IIB; quired using the Fotodyne FOTO/Analyst Luminary
green, IIA; blue, I; black or no stained, IIX and mixed) and Workstation Systems (Fotodyne, Inc., USA). Densitometry
then counted. The quantification of fiber type was expressed analysis was determined by scanning immunoreactive bands,
as the percentages that were individually calculated in each and intensity values were obtained for further normalization
image and further plotted. against the control group.

Measurement of the ROS Fresh-frozen GA muscles were Determination of carbonylated protein levels Carbonylation
cryosectioned to 7 μm and placed on glass slides. The sections modification in GA muscle was assessed by immunoblot de-
were washed twice with ice-cold HBSS (Hank’s Balanced Salt tection of protein carbonyl groups, using the OxyBlot assay
Solution) and incubated with the cell permeate dye following the indications of the supplier (OxyBlot Protein
dichlorodihydrofluorescein (H2-DCF-DA) (5 μM, Oxidation Detection Kit, Millipore, S7150). Briefly, 30 μg
Invitrogen, USA) for 15–30 min in the dark at room temper- of total proteins was subjected to SDS-PAGE and transferred
ature. Then, the sections were rinsed twice with HBSS and to PVDF membranes, which were incubated with the antibod-
once PBS, and then were fixed with 4% paraformaldehyde for ies provided in the kit. All immunoreactions were visualized
10 min and rinsed with PBS. Further, cryosections were incu- by enhanced chemiluminescence (Thermo Scientific, USA),
bated with 1 μg/ml Hoechst 33258 in PBS for 10 min for which were acquired using the Fotodyne FOTO/Analyst
nuclear staining. After rinsing, the sections were mounted Luminary Workstation Systems (Fotodyne, Inc., USA).
with fluorescent mounting medium (Dako Corporation, CA), Densitometry analysis was determined by scanning immuno-
viewed and photographed using the Motic BA310 reactive bands, and intensity values were obtained for further
epifluorescence microscope (Motic, Hong Kong) to 488 nm. normalization against the control group.
Muscle sections were immediately analyzed and the ROS
production was measured by the increases in DCF fluores- Quantitative real-time PCR analysis RNA was isolated from
cence, as an indicator of ROS production. The ROS levels liver samples using the SV Total RNA Isolation System
were quantified by the analysis of DCF fluorescence intensity (Promega, Madison, WI) and then quantified spectrophoto-
in the pictures using the software Image J [2]. metrically in a NanoDrop ND-1000 (NanoDrop
Technologies, Wilmington, DE). cDNA synthesis was per-
Immunoblot analysis GA muscles were homogenized in Tris– formed starting with 1 μg of total RNA (Improm II system;
EDTA buffer with a cocktail of protease inhibitors and 1 mM Promega, Madison, WI) according to the manufacturer’s
PMSF. Proteins were subjected to SDS-PAGE, transferred guidelines. We measured the hepatic expression of Col1A1.
onto PDVF membranes (Millipore, USA), and probed with All probes were obtained from Applied Biosystems (Foster
mouse anti-MHC (1:3000; MF-20, Developmental Studies, City, CA). The relative amounts of all mRNAs were calculat-
Hybridoma Bank, University of Iowa, USA), goat anti-4- ed using the comparative threshold cycles (dCT) method and
hydroxynonenal (4HNE) (1:1000; Merck, USA), mouse normalized to 18S RNA as a housekeeping gene.
Pflugers Arch - Eur J Physiol (2018) 470:1503–1519 1507

Magnetic resonance imaging (MRI) protocol In vivo MR im- DDC hepatotoxin for 6 weeks, a model in which there is
aging was performed using a 1 T Bruker ICON MR scanner considerable hepatic dysfunction evidenced by histological
(Bruker, MA, USA). Briefly, anesthetized mice were imaged alterations in the third week (ESM-1A): increased liver
in the prone position. Following a 3D gradient echo scout weight (ESM-1B), ALT activity (ESM-1C), and Col1A1
scan, an ECG gated, T1-Flash cine images for cardiac function expression (ESM-1D) [32].
analysis were acquired (imaging parameters—FOV, 25 × Several functional tests with live mice were performed on
25 mm; matrix acquisition, 116 × 116 × 6; in-plane resolution, chow and DDC-supplemented diet fed mice along 6 weeks.
0.21 × 0.21 mm). Muscle quantification was performed from Figure 1a indicates mice with chronic liver damage caused by
T1w abdominal MR (imaging parameters—FOV, 30 × consuming the DDC diet experienced a decrease in their mus-
30 mm; matrix acquisition, 256 × 256 × 12; in-plane resolu- cle’s ability to support weight beginning in the second week
tion, 0.12 × 0.12 mm) [51]. after receiving the DDC diet when a weightlifting test was
performed. At the end of the experiment (week 6), muscle
MRI analysis Cardiac function was studied calculating the end- strength fell until it reached 40% of the value obtained from
systolic and end-diastolic left ventricle volume and the ejec- the normal diet control group. Another functional test con-
tion fraction using OsiriX (OsiriX Foundation, Geneva, ducted was on exercise performance using the rotarod test.
Switzerland). Psoas muscle volume was calculated by manu- Figure 1b demonstrates that the riding time for the control
ally segmentation in the T1w images using OsiriX. mice fed with the normal diet remained unchanged during
the 6 weeks of treatment. However, mice fed with the DDC
Cell cultures The skeletal muscle cell line C2C12 obtained diet showed a decline in the riding time beginning in the third
from the American Type Culture Collection was grown in week of treatment and reaching the minimal value at the end
DMEM 10% fetal bovine serum and differentiated into of the sixth week (25% of the control group).
myotubes until day 5 with DMEM 2% horse serum [3].

Cell viability (MTT assays) Cytotoxicity was determined by the

MTT assay. C2C12 myotubes were incubated with or without
several concentrations of DDC (between 0 and 400 μg/ml) in
DMEM 2% horse serum for 24 h. At the end of experiment,
10 μl of MTT solution (5 mg/ml, pH 7.5) was added. After 1 h,
blue formazan crystals were resolved with 100 μl of DMSO.
Absorbance was measured at 595 nm. Cell viability (percent of
control) was calculated relative to untreated control.

Atrophic effect in vitro C2C12 myotubes were incubated with

DDC (10 or 25 μg/ml) or serum from mice fed with control or
DDC-supplemented diets (10% final) for 72 h. The MHC
protein levels were determined by Western blot as previously
described [19].

Statistics For statistical analysis, we used a t test to compare

two groups. To compare three or more groups, we used one or
two-way analysis of variance (ANOVA) with a post hoc
Bonferroni multiple-comparison test (Prisma). A difference
was considered statistically significant at P < 0.05.

Fig. 1 Mice with chronic liver damage induced by a DDC-supplemented

Results diet develop muscular weakness. C57BL/6J male mice were fed a
standard chow (Control) or DDC-supplemented diet (DDC) for
6 weeks. Each week, mice were subjected to the following: a A
DDC-fed mice exhibit liver damage, muscle weakness weightlifting test to determine limb muscle strength. Values represent
and loss of muscle functionality the score normalized per body weight reached by the mice with each
weight and correspond to the mean ± SEM (five animals per group,
three independent experiments; *P < 0.05 vs. Control, two-way
We evaluated the effects of chronic liver damage on ANOVA). b A rotarod test. The values correspond to the time (s). The
strength and muscle function. For this purpose, hepatic values correspond to the mean ± SEM (five animals per group, three
injury was induced in mice by a diet supplemented with independent experiments; *P < 0.05 vs. Control, two-way ANOVA)
1508 Pflugers Arch - Eur J Physiol (2018) 470:1503–1519

Then we evaluated exercise performance on a treadmill by Thus, these results reveal CLD that caused by the DDC diet
measuring how much time mice spent running over the front, impaired exercise performance in mice.
middle, or bottom of treadmill’s sections. These measures To determine whether the changes observed in the func-
were categorized as follows: front section was categorized as tional tests for living mice can be extrapolated to a specific
high performance (HP), the middle section was referred to as deficiency in skeletal muscle, we evaluated the muscle
medium performance (MP), and the last portion was catego- strength in isolated gastrocnemius tissue through electro-
rized as low performance (LP), as displayed in Fig. 2a. The physiological assays of tetanic force. Figure 3a reflects
results indicate at the beginning of the experiment (week 0), muscles from mice fed the DDC diet had a lower muscle
both groups of mice (Control or DDC) have a similar perma- force in most of the assessed frequency ranges. Figure 3b
nence time in treadmill’s distinct zones, with a higher propor- shows gastrocnemius from mice fed the DDC diet present-
tion in the HP (Fig. 2b). However, after 3 weeks of treatment, ed a decrease in maximal tetanic force (381 mN/mm2)
mice fed the DDC diet spent a shorter length of time in the HP compared to the control group (548 mN/mm 2). Similar
zone (3.86 vs. 84.6% of control mice) and more time in the LP results were obtained by evaluating the maximal tetanic
zone (87.2 vs. 2.3% of control mice), indicating an impair- force in the tibialis anterior muscle (ESM-2).
ment in physical performance (Fig. 2c). This situation was To rule out the possibility that mice fed the DDC diet had a
accentuated in the sixth week after treatment where mice fed problem with the motricity of their paws, we conducted a
the DDC diet only reached 1.5% of permanence time in the footprint test. ESM-3A displays the scheme of this test and
HP zone (control mice 92.3%), while they spent 94.1% of the distances measured. Results presented in ESM-3B, C, and
permanence time in LP zone (control mice 1.8%) (Fig. 2d). D suggest there were no differences between mice fed the

Fig. 2 Chronic liver damage

induced by DDC-supplemented
diet impairs the ability to perform
physical exercise. a Treadmill
scheme in which is illustrated the
treadmill and the three zones used
to calculate exercise performance:
high (HP), medium (MP), and
low (LP) performance stones of
the treadmill. Each experiment
was recorded by video and further
analyzed for the permanence time
in each zone. Briefly, C57BL/6J
male mice were trained to run on a
treadmill before the beginning of
the experiment with a standard
chow (Control) or DDC-
supplemented diet (DDC) for
6 weeks. In weeks 0 (b), 3 (c), and
6 (d) after the mice were fed the
differential diet, mice were
subjected to the running test to
measure exercise performance.
The permanence time (s) was
calculated from observing video
footage and expressed as the
percentage of time spent in each
of the three zones. The values
correspond to the mean ± SEM
(five animals per group, three
independent experiments; *P <
0.05 vs. Control, two-way
ANOVA)
Pflugers Arch - Eur J Physiol (2018) 470:1503–1519 1509

Fig. 4 Mice with chronic liver disease have a decreased body and
gastrocnemius (GA) weight. a C57BL/6J male mice were fed a
standard chow (Control) or DDC-supplemented diet (DDC) for 6 weeks.
a At 0, 3, and 6 weeks, body weight was measured. The value is
expressed in grams. Values represent the mean ± SEM of three
independent experiments. In each experiment, five mice were used for
Fig. 3 Gastrocnemius (GA) tissue from mice with chronic liver disease
each experimental condition (*P < 0.05 vs. Control, two-way ANOVA).
has decreased isometric force. C57BL/6J male mice were fed a standard
b At the end of experiment (week 6), GA tissue weight was measured.
chow (Control) or DDC-supplemented diet (DDC) for 6 weeks. At the
The values represent the ratio between GA tissue and body weight (mg/g)
end of the experiment, GA muscles were excised. a A curve of force
and are the mean ± SEM of three independent experiments. In each
versus frequency was determined (*P < 0.05 vs. Control, two-way
experiment, five mice were used for each experimental condition. (*P
ANOVA). b Maximal isometric strength (mN/mm2) was evaluated.
< 0.05 vs. Control, t test)
Values represent the mean ± SEM of three independent experiments. In
each experiment, five mice were used for each experimental condition
(*P < 0.05 vs. Control, t test)
end of the experiment (Fig. 4b). Interestingly, mice from the
control and DDC groups did not have differences among them
control or DDC diet in the stride, stance, and sway length of or at the time of treatment regarding food and water intake
their hind paws, respectively, suggesting motor skills were not (ESM-5A and B, respectively).
altered in mice fed the DDC diet. Additionally, we evaluated Overall, these results suggest chronic liver damage induced
the locomotor activity of mice using an open field test. by the intake of a DDC-supplemented diet produced muscle
Tracings of the distance walked by mice fed either the normal weakness evidenced by decreased exercise performance and
(ESM-4A) or DDC diet (ESM-4B). Quantification of this data isometric tetanic force which would not be explained by
determines the mobility of both experimental groups is similar changes in motor skills, immobility, or food or water intake.
(ESM-4C).
We also evaluated several other parameters such as body Mice fed the DDC diet have a decrease in the myofiber
weight, muscle mass, and food and water intake. Figure 4a diameter of gastrocnemius (GA) muscles
demonstrates mice fed the DDC diet experienced a decline in
body weight evidenced in the third and sixth weeks of treat- To evaluate the diameter of muscle fibers, a WGA stain was
ment, reaching 17.1 g compared to the control group’s 26.8 g performed on GA muscle. Figure 5a shows GA tissue from
in the sixth week. Mice from the DDC group indicated the mice fed the DDC diet showed a decrease in muscle fiber
ratio between the mass of gastrocnemius tissue and body diameter. Quantification of the Feret’s diameter revealed GA
weight was lower (5.1) than the control group’s (5.9) at the tissue from the control group has a normal distribution curve
1510 Pflugers Arch - Eur J Physiol (2018) 470:1503–1519

with a peak in the range of 41–45 μm, whereas the fibers’ size 13% in control mice to 29% in DDC mice) (Fig. 6b). The
distribution from mice fed the DDC diet was clearly displaced same also reflects there is an increase in the number of type
to the left and experienced a peak in the range of 31–35 μm, I fibers (from 0% in control mice to 10% in DDC mice). In
indicating a smaller diameter than the control group (Fig. 5b). addition, we evaluated the CSA of the different fibers’ types.
These data suggest GA tissue from mice fed the DDC diet has GA from mice fed with DDC diet show a decrease of CSA in
a decrease in myofiber diameter. To evaluate muscle atrophy IIB and IIX fibers, and an increase in IIA fibers compared to
in another muscle, we measured the volume of psoas by MRI mice fed with control diet (Fig. 6c). Normally, in GA, the CSA
analysis. Our data showed that mice fed with DDC for 3 weeks of IIB is bigger than IIA, so this transition in the fiber types
present a decrease in the volume of psoas compared to control (IIB to IIA) could explain the displacement of fiber diameter
mice (EMS-6A). Interestingly, the cardiac function evaluated toward minor size (Fig. 5b). Our results suggest that in this
by MRI analysis was unchanged between mice fed with con- case it is not the reason because CSA of IIA is increased in
trol and DDC diet (EMS-6B). mice fed with DDC diet. However, since GA in normal con-
ditions is a muscle that is mainly composed for IIB fibers, the
Gastrocnemius (GA) from mice with chronic liver decrease in its proportion and CSA can explain the displace-
damage presents transition in the fiber type ment of fiber diameter toward minor size (Fig. 5b).

The most abundant fiber type in GA tissue is IIB [1]. When Gastrocnemius (GA) tissue from mice with chronic
atrophied, there is a transition from IIB fibers to IIA and I liver damage has decreased myosin heavy chain
fibers as well as hybrid fibers IIB/IIX, IIX, IIX/IIA (Fig. 6a) protein levels but higher levels of ubiquitination than
[38, 57]. In mice fed the DDC-supplemented diet, GA tissue control mice
exhibits a clear decrease in IIB fibers (from 63% in control
mice to 27% in DDC mice) also as an increase in IIX (from One of the main targets altered in skeletal muscle atrophy is
9% in control mice to 14% in DDC mice) and IIA fibers (from the sarcomeric proteins, specifically MHC. Therefore, we

Fig. 5 Gastrocnemius (GA)

tissue from mice with chronic
liver disease have decreased fiber
diameters. C57BL/6J male mice
were fed a standard chow
(Control) or DDC-supplemented
diet (DDC) for 6 weeks. At the
end of the experiment, GA
muscles were excised. a Muscle
cross-sections were stained with
WGA to delimit muscle fiber
sarcolemma. b Minimal Feret
diameters were determined in TA
cross-sections from experiments
depicted in (a). Fiber diameters
were grouped from 0 to 80 μm
and values expressed as the
percentage of the total fibers
quantified. Counted images are
representative of three
independent experiments, using
five mice for each experimental
condition. Values correspond to
the mean ± SEM. (*P < 0.05 vs.
Control, two-way ANOVA)
Pflugers Arch - Eur J Physiol (2018) 470:1503–1519 1511

Fig. 6 Mice with chronic liver damage induced by DDC-supplemented the percentage of specific fiber types relative to the total fibers counted
diet have fiber type transitions in the gastrocnemius (GA) muscle. per field. Values represent the mean ± SEM of three independent
C57BL/6J male mice were fed a standard chow (Control) or DDC- experiments. In each experiment, five mice were used for each
supplemented diet (DDC) for 6 weeks. a At the end of the experiment, experimental condition (*P < 0.05 vs. Control, two-way ANOVA). c
mice were sacrificed, and the GA muscles were analyzed to determine Analysis of CSA for each type of fiber. Images from GA obtained in
fiber type through the immunoflourescence detection of myosin heavy the experiments for (a) were used for the quantitative analysis. Values
chain isoforms (IIA, IIB, I). Images obtained at ×40 magnification reveal represent the mean ± SEM (μm2) of three independent experiments. In
fiber types were determined within the entire muscle/cross-section (red, each experiment, five mice were used for each experimental condition
IIB; green, IIA; blue, I; black or not stained, IIX; and mixed, IIB/X, X/ (*P < 0.05 vs. Control, t test)
IIA, IIA/I). b Quantitative analysis of the fiber type. Graph representing

evaluated MHC levels using a Western blot analysis. Skeletal muscle from mice with chronic liver disease
Figure 7a demonstrates the decrease in MHC protein levels exhibits an increase of MuRF-1 and atrogin-1
in GA tissue from mice fed the DDC diet compared to the expression
control group. This decline is 0.58-fold compared to control
group (Fig. 7b). To assess the UPS’s involvement, we evaluated the protein
The main pathway involved in altered MHC protein levels levels of atrogin-1 and MuRF-1, two key enzymes involved
is the UPS, which ubiquitinates MHC before its degradation. in the process of skeletal muscle atrophy. Figure 8a and c
To evaluate this, MHC was immunoprecipitated with an MHC illustrates GA muscles from mice fed the DDC diet had in-
antibody and then immunodetected with ubiquitin antibody creases in both atrogin-1 and MuRF-1. These increases are
using the Western blot method. Figure 7c shows GA tissue’s 2.0-fold for atrogin-1 and 4.4-fold for MuRF-1, compared to
total MHC protein levels decreased in mice from the DDC the control group’s GA tissue (Fig. 8b and d, respectively).
group as presented in Fig. 7a, but MHC ubiquitination levels These results suggest GA tissue from mice with CLD
are 2.4-fold higher in GA tissue from mice fed the DDC diet has a UPS more active to degrade muscle proteins than
compared to the control diet (Fig. 7c, d). control mice.
1512 Pflugers Arch - Eur J Physiol (2018) 470:1503–1519

Fig. 7 Mice with chronic liver damage induced by DDC-supplemented Ubiquitinated-MHC protein levels through immunoprecipitation with
diet have decreased myosin heavy chain (MHC) protein levels in the anti-MHC and further Western blot with anti-ubiquitin. The upper panel
gastrocnemius (GA) muscle. C57BL/6J male mice were fed a standard illustrates the immunodetection of MHC and tubulin in the input. The
chow (Control) or DDC-supplemented diet (DDC) for 6 weeks. At the lower panel shows the detection of ubiquitinated-MHC levels in the
end of the experiment, mice were sacrificed, and the GA muscles were immunoprecipitated. Tubulin is depicted as a negative control.
excised and homogenized to evaluate a MHC protein levels through Molecular weight markers are shown in kilodaltons. d Quantitative
Western blot analysis. Tubulin levels were used as the loading control. analysis of the experiments from (c). The levels of ubiquitinated MHC
Molecular weight markers are depicted in kilodaltons. b Quantitative were normalized to total MHC and expressed relative to control. Values
analysis of the experiments from (a). Values represent the mean ± SEM represent the mean ± SEM of three independent experiments. In each
of three independent experiments. In each experiment, five mice were experiment, five mice were used for each experimental condition (*P <
used for each experimental condition (*P < 0.05 vs. Control, t test). c 0.05 vs. Control, t test)

Gastrocnemius (GA) tissue from mice with chronic oxidative stress by protein modifications such as carbonyla-
liver damage presents oxidative stress tion and 4-HNE. Figure 9c and d show GA tissue from mice
fed the DDC diet has higher levels of carbonylated proteins
To assess the involvement of oxidative stress, we evaluated than mice fed the normal diet (2.2-fold vs. control mice).
the ROS levels in a slice of GA muscle. A DCF probe was Moreover, Fig. 9e and f reflects that GA tissue from the
used to detect and quantify ROS (Fig. 9a). The GA tissue of DDC group has greater 4-HNE reactivity compared to the
mice fed the DDC diet presented a higher ROS reactivity (an control group (2.0-fold vs. control mice).
8.3-fold increase compared to mice fed the control diet) All these results suggest GA tissue from mice with CLD
(Fig. 9b). We subsequently evaluated consequences of developed oxidative stress.
Pflugers Arch - Eur J Physiol (2018) 470:1503–1519 1513

Fig. 8 Gastrocnemius (GA) tissue from mice with chronic liver disease levels were used as the loading control. Molecular weight markers are
develop increased atrogin-1 and MuRF-1 protein levels. C57BL/6J male shown in kilodaltons. Quantitative analysis of the atrogin-1 (b) and
mice were treated with standard chow (Control) or DDC-supplemented MuRF-1 (d). Values represent the mean ± SEM of three independent
diet (DDC) for 6 weeks. At the end of the treatment, mice were sacrificed, experiments. In each experiment, five mice were used for each
and the GA muscles were excised and homogenized to evaluate of experimental condition (*P < 0.05 vs. Control, t test)
atrogin-1 (a) and MuRF-1 (c) protein levels by Western blot. Tubulin

Hepatotoxin DDC did not directly induce an atrophic Discussion

effect in skeletal muscle cells
In this paper, we demonstrated that a murine model of CLD
A possible explanation for the atrophic effect observed in induced by the DDC hepatotoxin showed an evident muscular
GA is that DDC directly affect skeletal muscle. To eval- weakness with decreased fiber diameter and MHC total pro-
uate this, we incubated C2C12 myotubes with different tein levels. Among the mechanisms involved in this atrophic
concentrations of DDC. First, we determined that effect, we demonstrated that UPS is overactivated by detecting
myotubes maintained the viability between 1 and 50 μg/ increased levels of ubiquitinated MHC, atrogin-1, and MuRF-
ml of DDC and decreased in 100, 200, and 400 μg/ml of 1, and we demonstrated important evidence of oxidative stress
DDC (EMS-7A). In the same figure, it can be observed by the increased ROS as well as carbonylated- and 4-HNE-
that serum from mice fed with DDC diet, but not with modified protein levels.
control diet, decreased the MHC levels. Interestingly, This model of CLD [32, 46] has not been previously char-
DDC increased the HO-1 expression in C2C12 myotubes acterized for muscular weakness. To address this gap in the
(EMS-7B), an effect that has been reported in other cell research literature, several approaches with live mice were
types [56]. The same figure shows that serum from mice performed in which the DDC group always exhibited lower
fed with control or DDC diet did not change the HO-1 muscular strength or performance than control mice, which is
expression from basal levels. consistent with clinical observations in patients with CLD [21,
Further, we evaluated the effect of DDC and serum 40, 52, 59]. Furthermore, these results agree with data regard-
from mice fed with DDC on the MHC protein levels. ing isometric and tetanic force measured in isolated gastroc-
Thus, myotubes incubated with 1 and 50 μg/ml of DDC nemius tissue, in which the DDC group exhibited decreased
did not present changes in the MHC protein levels muscle strength.
(Fig. 10a, b). Among the factors associated with mortality in patients
These results suggest that DDC did not directly induce with CLD are poor physical fitness and endurance [27].
an atrophic effect in skeletal muscle cells, and that there is Regarding these factors, we observed that basal mobility
a soluble factor in the serum from mice fed with DDC that was unchanged by the DDC diet, while mice in the DDC
induces skeletal muscle atrophy. group demonstrated lesser ability to perform an involuntary
1514 Pflugers Arch - Eur J Physiol (2018) 470:1503–1519

Fig. 9 Gastrocnemius (GA) tissue from mice with chronic liver disease loading control. Molecular weight markers are shown in kilodaltons. d
has an increase of ROS and protein oxidation levels. C57BL/6J male Quantitative analysis based in the densitometry of the bands. Values
mice were treated with standard chow (Control) or DDC-supplemented represent the mean ± SEM of three independent experiments. In each
diet (DDC) for 6 weeks. At the end of the treatment, mice were sacrificed, experiment, five mice were used for each experimental condition (*P <
and the GA muscles were excised. a Cryosections obtained from the GA 0.05 vs. Control, t test). e Levels of oxidation-dependent 4HNE protein
tissue were incubated with a DCF probe for ROS detection through adducts were detected by Western blot using anti-4HNE antibody. β-
fluorescence microscopy. b Quantification of ROS levels from Actin levels were used as the loading control. Molecular weight
experiments demonstrated in (a). The values are expressed as the fold markers are shown in kilodaltons. f Quantitative analysis based in the
of induction of the DCF probe intensity (*P < 0.05 vs. Control, t test). c densitometry of the bands. Values represent the mean ± SEM of three
Levels of oxidation-dependent carbonylated protein levels were detected independent experiments. In each experiment, five mice were used for
by Western blot using Oxyblot kit. β-Actin levels were used as the each experimental condition (*P < 0.05 vs. Control, t test)

exercise test on a treadmill. Similar results were obtained with suggesting caloric and liquid intake did not cause the DDC
the rotarod test. Interestingly, our data suggest that in the DDC mice’s decreased body and muscle weight. Despite these data,
model of CLD, immobilization does not contribute to muscle we must acknowledge nutrient absorption problems could im-
weakness. Thus, our data reveal there were no significant dif- pact body and muscle weight.
ferences in mobility or motor skills test scores between the In addition, our results suggest that the muscle wasting can
experimental and control groups, allowing us to assert immo- be observed in mice at early stages after DDC treatment (from
bility or motor problems did not cause the observed muscle 2 to 3 weeks in different muscles, e.g., gastrocnemius, tibialis
weakness. The measures of water and food intake were not anterior, and psoas). In our hands, DDC treatment did not
significantly different between the control and DDC mice, show change in the cardiac function at early stages, despite
Pflugers Arch - Eur J Physiol (2018) 470:1503–1519 1515

Fig. 10 Serum from mice fed with DDC-supplemented diet contain a determined by Western blot analysis. Tubulin levels were used as a
factor, different to DDC itself, produces muscle atrophy in vitro. C2C12 loading control. Molecular weights are indicated in kilodaltons. b
myoblasts were differentiated into myotubes for 5 days. a Myotubes were Quantitative analysis for MHC levels was conducted, with values
incubated with DDC (1 and 50 μg/ml) or serum derived from mice fed normalized to tubulin levels and plotted as fold of induction relative to
with control (Serum Ctrl) or DDC diet (Serum DDC) at the final control (*P < 0.05 vs. Control, one-way ANOVA)
concentration of 10% v/v. After of 72 h of incubation, MHC levels were

it has been previously reported [26] which can be explained by Thus, in further studies, NAC treatment could be a useful tool
the different experimental approaches to measure the cardiac to elucidate if oxidative stress is responsible for MuRF-1 and
parameters. Moreover, we cannot discard that alterations of atrogin-1 expression induction.
cardiac function induced by DDC later stages could contribute Interestingly, our evidence suggests there is oxidative stress
to muscle function in alive mice. In this line, the diminution in in the skeletal muscle DDC-fed mice. In addition to the ROS
isometric muscle strength in isolated muscle is a parameter increase, we detected an increment of the protein modifica-
which is independent of cardiac function. tions dependent on oxidative stress such as 4-HNE and car-
Our data indicate that DDC-treated mice have lesser mus- bonylation [5, 42]. These findings are relevant because they
cle strength than control, which was evident when mice were not only detected an early event such as ROS increases but
subjected to an exercise challenge. Patients with cirrhosis also also determined the oxidative stress-induced cellular damage
exhibit decreased physical activity [77]. However, most of by the modification of proteins, which could not be the only
these patients also present other comorbidities that affect the result of cell damage because it has been DNA and cell mem-
physical activity like obesity [70] which is absent in our mod- branes can be also be altered [62, 68]. Regarding the carbon-
el. Thus, other experiments must be done in order to evaluate ylation of protein, there is evidence that in several models of
the contribution of those factors in the physical activity of muscle atrophy such as disuse, aging, or sarcopenia in cancer,
cirrhotic patients or experimental models of CLD. sepsis, or COPD, there is an increase of carbonylated proteins
Regarding mechanisms involved in muscle atrophy, we dependent on oxidative stress [5, 61]. Several studies have
evaluated the involvement of UPS and oxidative stress. Our demonstrated carbonylation and most posttranslational oxida-
data confirm muscle-specific E3 ligases MuRF-1 and atrogin- tive modifications may result in loss of protein function as
1 are increased in DDC mice. Moreover, these data are con- well as accelerated protein degradation by the proteasome
sistent with the decreased levels of total MHC and increased [17, 20, 31, 48, 58].
levels of ubiquitinated MHC, a step prior to degrading MHC Myofibrillar proteins such as actin and myosin are among
by proteasome. Thus, we can suggest the decrease in MHC the main targets [30]. Thus, further experiments must be per-
levels caused by CLD can be explained, at least, for an in- formed to study possible MHC carbonylation and the effect on
crease in its ubiquitination by MuRF-1 which is increased in its degradation by proteasome in muscles from mice with
muscle tissue from mice fed the DDC diet. Moreover, we CLD. In other models of muscle wasting such as disuse mus-
detected an increase in ROS levels which could be responsible cle atrophy, oxidative stress may contribute to the muscle
for the increase in MuRF-1 levels. Thus, we can speculate that dysfunction by the increment of protein catabolism through
in CLD, skeletal muscle increments ROS production concom- activation of calpains, caspase-3, and UPS [60]. Oxidative
itantly with the MuRF-1 expression and the ubiquitination and stress can trigger the activation of calpains responsible for
degradation of MHC. However, it is not possible to disregard myofilament disassembly as well as caspase-3, which can
the involvement of a synthesis mechanism which could be degrade actin and MHC [60, 61].
decreased in CLD. Previously, we have demonstrated that Another underlying mechanism have to be involved re-
ROS can induce the increase of the E3 ubiquitin-ligases garding skeletal muscle atrophy induction since food intake
MuRF-1 and atrogin-1, which can be avoided by N- and mobility were not affected in DDC-fed mice. In this line, it
acetylcysteine (NAC) treatment [2]. NAC is an antioxidant is plausible to think that unknown molecules can be released
agent widely used to reduce ROS content and oxidative stress. from the liver into the blood, reaching the muscles and acting
1516 Pflugers Arch - Eur J Physiol (2018) 470:1503–1519

endocrine. In this context, when the liver is damaged, there are (21161353). F.C. thanks Universidad Andrés Bello-Dirección de
Investigación for providing a PhD Scholarship.
inflammatory processes that are activated and several soluble
molecules can be released into the blood [75, 76], events that
Author’s contribution J.A., F.C., F.A., and B.G. were responsible for
have been also shown in mice fed with DDC diet [32, 33]. carrying out the experiments as well as analyzing and interpreting the
These molecules can reach the muscle by the circulation. data. D.C., S.K., and M.A. were responsible for collecting and reporting
Interestingly, some of these factors, such as tumor necrosis liver-related data. MEA was responsible for carrying out the MRI analy-
sis. C.C.V. and F.S. were involved in drafting the manuscript for publica-
factor alpha (TNF-α) [9, 72, 80] and interleukin-6 (IL-6) [7, tion. C.C.V. was responsible for conceiving all the experiments and was
35, 55], have been described as muscle wasting inducers in involved in analyzing the data, preparing it for publication, and drafting
other types of chronic diseases. Indeed, CLD can develop a the manuscript.
condition denominated hyperammonemia, which is character-
ized by an increase in the ammonia concentration in the blood Compliance with ethical standards
due to a disruption in the urea cycle [24]. This ammonia in-
crement can damage other organs, mainly the brain [41]. Conflict of interest The authors declare that they have no conflict of
interest.
Recent studies have also involved this condition with muscle
wasting in patients with cirrhosis [23]. The muscle expresses
ammonia transporters [73], and growing evidence have shown
that ammonia could induce the transcription of myostatin in References
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