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Skeletal Muscle Resident Progenitor Cells Coexpress Mesenchymal and Myogenic Markers and Are Not Affected by Chronic Heart Failure-Induced Dysregulations

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Background and Purpose . In heart failure (HF), metabolic alterations induce skeletal muscle wasting and decrease of exercise capacity and quality of life. The activation of skeletal muscle regeneration potential is a prospective strategy to reduce muscle wasting; therefore, the aim of this project was to determine if functional properties of skeletal muscle mesenchymal progenitor cells (SM-MPC) were affected by HF-induced functional and metabolic dysregulations. Methods . Gastrocnemius muscle biopsy samples were obtained from 3 healthy donors (HD) and 12 HF patients to purify mRNA for further analysis and to isolate SM-MPC. Cells were expanded in vitro and characterized by immunocytochemistry and flow cytometry for expression of mesenchymal (CD105/CD73/CD166/CD146/CD140b/CD140a/VIM) and myogenic (Myf5/CD56/MyoG) markers. Cells were induced to differentiate and were then analyzed by immunostaining and Q-PCR to verify the efficiency of differentiation. The expression of genes that control muscle metabolism and development was compared for HD/HF patients in both muscle biopsy and in vitro-differentiated myotubes. Results . The upregulation of MYH3/MYH8/Myf6 detected in HF skeletal muscle along with metabolic alterations indicates chronic pathological activation of the muscle developmental program. SM-MPC isolated from HD and HF patients represented a mixed population that coexpresses both mesenchymal and myogenic markers and differs from AD-MMSC, BM-MMSC, and IMF-MSC. The functional properties of SM-MPC did not differ between HD and HF patients. Conclusion . In the present work, we demonstrate that the metabolic and functional alterations we detected in skeletal muscle from HF patients do not dramatically affect the functional properties of purified and expanded in vitro SM-MPC. We speculate that skeletal muscle progenitor cells are protected by their niche and under beneficial circumstances could contribute to muscle restoration and prevention and treatment of muscle wasting. The potential new therapeutic strategies of HF-induced skeletal muscle wasting should be targeted on both activation of SM-MPC regeneration potential and improvement of skeletal muscle metabolic status to provide a favorable environment for SM-MPC-driven muscle restoration.
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Research Article
Skeletal Muscle Resident Progenitor Cells Coexpress
Mesenchymal and Myogenic Markers and Are Not Affected by
Chronic Heart Failure-Induced Dysregulations
R. I. Dmitrieva ,
1
T. A. Lelyavina,
2
M. Y. Komarova,
3
V. L. Galenko,
2
O. A. Ivanova,
4
P. A. Tikanova,
5
N. V. Khromova,
1
A. S. Golovkin ,
1
M. A. Bortsova,
2
A. Sergushichev,
4
M. Yu. Sitnikova,
2
and A. A. Kostareva
1
1
Institute of Molecular Biology and Genetics, National Almazov Medical Research Centre, Saint Petersburg, Russia
2
Heart Failure Department, National Almazov Medical Research Centre, Saint Petersburg, Russia
3
Peter the Great St. Petersburg Polytechnic University, Saint Petersburg, Russia
4
ITMO University, Saint Petersburg, Russia
5
Saint Petersburg State University, Saint Petersburg, Russia
Correspondence should be addressed to R. I. Dmitrieva; renata.i.dmitrieva@gmail.com
Received 4 July 2018; Revised 6 October 2018; Accepted 7 November 2018; Published 3 January 2019
Guest Editor: Zhaoping Ding
Copyright © 2019 R. I. Dmitrieva et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background and Purpose. In heart failure (HF), metabolic alterations induce skeletal muscle wasting and decrease of exercise
capacity and quality of life. The activation of skeletal muscle regeneration potential is a prospective strategy to reduce muscle
wasting; therefore, the aim of this project was to determine if functional properties of skeletal muscle mesenchymal progenitor
cells (SM-MPC) were aected by HF-induced functional and metabolic dysregulations. Methods. Gastrocnemius muscle biopsy
samples were obtained from 3 healthy donors (HD) and 12 HF patients to purify mRNA for further analysis and to isolate
SM-MPC. Cells were expanded in vitro and characterized by immunocytochemistry and ow cytometry for expression of
mesenchymal (CD105/CD73/CD166/CD146/CD140b/CD140a/VIM) and myogenic (Myf5/CD56/MyoG) markers. Cells were
induced to dierentiate and were then analyzed by immunostaining and Q-PCR to verify the eciency of dierentiation. The
expression of genes that control muscle metabolism and development was compared for HD/HF patients in both muscle
biopsy and in vitro-dierentiated myotubes. Results. The upregulation of MYH3/MYH8/Myf6 detected in HF skeletal muscle
along with metabolic alterations indicates chronic pathological activation of the muscle developmental program. SM-MPC
isolated from HD and HF patients represented a mixed population that coexpresses both mesenchymal and myogenic markers
and diers from AD-MMSC, BM-MMSC, and IMF-MSC. The functional properties of SM-MPC did not dier between HD
and HF patients. Conclusion. In the present work, we demonstrate that the metabolic and functional alterations we detected in
skeletal muscle from HF patients do not dramatically aect the functional properties of puried and expanded in vitro
SM-MPC. We speculate that skeletal muscle progenitor cells are protected by their niche and under benecial
circumstances could contribute to muscle restoration and prevention and treatment of muscle wasting. The potential new
therapeutic strategies of HF-induced skeletal muscle wasting should be targeted on both activation of SM-MPC regeneration
potential and improvement of skeletal muscle metabolic status to provide a favorable environment for SM-MPC-driven
muscle restoration.
1. Introduction
In heart failure (HF), functional and metabolic alterations
are detected not only in cardiac muscle [1, 2] but also in
skeletal muscle tissue. Oxidative stress, systemic inamma-
tion, chronic hypoxia, and decreased fatty acid oxidation
coupled with mitochondrial dysfunction are the factors
contributing to HF-induced muscle damage that include
Hindawi
Stem Cells International
Volume 2019, Article ID 5690345, 11 pages
https://doi.org/10.1155/2019/5690345
a shift in ber type, induction of atrophy, development of
insulin resistance, dysregulation of lipid metabolism, and
ectopic fat depositions in the skeletal muscles. Additionally,
chronic activation of adrenergic and natriuretic peptide
systems in HF results in sustained lipolysis in adipocytes
resulting in the accumulation of toxic and neutral lipid
species in adipose and skeletal muscle that also contrib-
utes to skeletal muscle damage [39]. Impairments in
skeletal muscle stem cell function have also been sug-
gested as an important factor causing the loss of muscle
mass with increasing age [10] and could similarly be con-
sidered as a factor contributing to HF-induced skeletal
muscle wasting.
The development of preventive and therapeutic strategies
against muscle wasting disorders remains an unresolved
challenge. By now, exercise training, either alone or in
combination with nutritional support, is the most proven
strategy to reduce skeletal muscle wasting in HF patients
and is recommended by treatment guidelines [7, 11]. Conse-
quently, the activation of skeletal muscle developmental,
growth, and regeneration potential is an essential mechanism
to treat/prevent skeletal muscle wasting. Thus, the skeletal
muscle progenitor cells that contribute to skeletal muscle
regeneration and growth might be a prospective therapeutic
target, and the analysis of the functional properties of skeletal
muscle stem cells derived from heart failure patients has
become a crucial issue.
Identication and characterization of myogenic progeni-
tors in postnatal tissues are important for the evaluation of
regeneration potential. In our recent work [12], we have
demonstrated that bone marrow multipotent mesenchymal
stromal cells (BM-MMSC) derived from heart failure
patients are aected by heart failure in multiple ways: (1) in
HF-derived cultures, we detected the upregulation of genes
that control regeneration and brosis, including the Tgf-β
pathway, synthesis of ECM, remodeling enzymes, and
adhesion molecules; (2) during in vitro expansion, BM-
MMSC from HF patients demonstrated early development
of replicative senescence and decrease of proliferative
activity; and (3) altered dierentiation potential was also
observed in HF-derived samples. However, when culturing
conditions were modied, we have achieved the predomi-
nant purication and expansion of the highly proliferative
nonprobrotic CD146+/SMAαfraction that proves the
potential ecacy of HF-derived BM-MMSC in regeneration
processes [12].
Multipotent mesenchymal stromal cells are tissue-
committed progenitors that preferentially contribute to
the regeneration of certain types of tissue. For skeletal
muscle, the role of nonsatellite resident myogenic progen-
itors including multipotent mesenchymal stromal cells in
tissue regeneration was also reported [1317]. In the cur-
rent work, we sought to investigate whether or not HF-
induced metabolic dysregulations aect the functional
properties of resident skeletal muscle mesenchymal progen-
itor cells (SM-MPC) in order to determine if these cells
could respond suciently to therapeutic interventions
aimed at activating muscle regeneration and growth,
including physical rehabilitation programs focused on the
stabilization of muscle metabolism and prevention of skeletal
muscle wasting.
2. Methods
2.1. Study Design and Ethical Issues. The current work is part
of a complex ongoing project focused at evaluating the
eciency of aerobic physical training and developing person-
alized physical rehabilitation programs for heart failure
patients. The rst results that demonstrate the physiological
response to aerobic physical training were published recently
[18, 19]. During this project, the skeletal muscle biopsies
should be taken from a selected group of patients enrolled
in the program before and after a course of exercise training
in order to examine the regenerative potential of muscle
progenitor cells in HF patients as presented in this work;
RNA-Seq analysis will be employed to reveal the global
response of skeletal muscle to exercise training and deter-
mine potential specic targets when sample collection will
be completed. In the current work, the rst portion of the
biopsy samples was used. All samples were collected under
the agreement of the Institutional Ethics Committee at the
Almazov National Medical Research Centre. All patients
and donors entering the program agreed to and signed an
institutional review board-approved statement of informed
consent. The study was conducted in compliance with cur-
rent Good Clinical Practice standards and in accordance with
the principles under the Declaration of Helsinki (1989).
2.2. Human Subjects and Gastrocnemius Muscle Biopsy
Samples. Only male donors were recruited into this study.
A total of 3 healthy adult donors (HD) and 12 chronic heart
failure patients (HF) were enrolled. HF patients have the
following characteristics: NYHA II-III functional class, age
54 ±12.5 years, body mass index (BMI) 26.5 ±6.4 kg/m
2
,
and left ventricle ejection fraction (LV EF) 26.4 ±1.4%. The
NYHA II : III patient ratio is 67% : 33%.
Inclusion criteria for the study are as follows: age 1865
years, LV EF <40% (Simpson), stable CHF NYHA II-III
functional class, informed consent signing, ability to perform
CPET, and optimal drug and electrophysiological therapy
(ICD, CRT-D).
Exclusion criteria for the study are as follows: myocardial
infarction and myocardial revascularization less than 3
months, stroke and CRT-D implantation less than 6 months,
expressed cognitive impairment, any chronic disease decom-
pensation, and high-gradation ventricular arrhythmias with
no implanted cardioverter-debrillator (ICD).
Gastrocnemius muscle biopsy samples were collected
from each donor/patient at baseline and after 36 months
of follow-up. Biopsy samples were divided into two portions.
One was immediately transferred to liquid nitrogen for
further mRNA purication. The second portion was used
immediately for skeletal muscle progenitor cell purication.
Since our study was limited by the small number of HD
enrolled into the project, we demonstrate the consistent
response of HD-derived cells to stimulation of myogenic
dierentiation and similarities in immunophenotypes in
supplemental Figure S1.
2 Stem Cells International
2.3. Purication and Separation of Skeletal Muscle
Mesenchymal Progenitor Cells (SM-MPC) and Mesenchymal
Stromal Cells from Intermuscular Fat (IMF-MSC). SM-MPC
were isolated enzymatically according to the protocols
described previously [20, 21] with minor changes. In brief,
isolated muscles were placed into an enzyme solution,
mechanically disrupted with scissors, and digested for
60 min at 37
°
Cin5mLltered 0.1% collagenase I (C0130,
Sigma-Aldrich, Germany). To remove collagenase and cell
debris after digestion, the cell suspension was centrifuged
for 5 min at 1000 × g and the supernatant was discarded. To
release the stem cells from the bers, the pellet was resus-
pended using sterile pipette tips in 2.5 mL of washing media
(DMEM supplemented with 10% horse serum (HS) (Gibco,
USA)). After the resuspension, the bers were allowed to
settle for 5 min and then the supernatant containing stem
cells was transferred to a fresh tube. To increase the yield, this
step was repeated twice. The double-collected supernatant
was ltered through a 40 μm nylon cell strainer and centri-
fuged for 10 min at 1000 × g in order to discard debris. Then,
the resultant supernatant was discarded and the pellet of cells
was placed in a proliferation media (DMEM supplemented
with 10% FCS) on cell culture dishes and cultured until
80% conuence.
IMF-MSC samples were obtained from intermuscular
adipose tissue located in biopsy material. IMF-MSC cultures
were prepared as described in [22]. The separated sample of
adipose tissue was washed with phosphate-buered saline
(PBS) and suspended in an equal volume of DMEM supple-
mented with 0.1% collagenase type III, prewarmed to 37
°
C.
The tissue was placed in a shaking water bath at 37
°
C with
continuous agitation for 30 min and centrifuged for 5 min
at 300 ×g at room temperature; then, the tissue sample was
resuspended in culture media (DMEM supplemented with
10% FBC) and plated in a culture dish for expansion.
Bone marrow mesenchymal multipotent stromal cells
(BM-MMSC) and subcutaneous adipose mesenchymal mul-
tipotent stromal cells (AD-MMSC) were collected, character-
ized, and saved as in our previous projects [22]. For this
project, they were obtained from the biobank of the Almazov
National Medical Research Centre, cultured as IMF-MSC,
and used as control samples where appropriate.
2.4. Dierentiation Protocols. Fusion of some cells without
external stimuli usually was observed in subconuent
SM-MPC cultures and served as a reliable indicator, after
which we induced skeletal muscle dierentiation. To induce
dierentiation, the proliferation media was removed and
replaced with dierentiation media that was renewed after
every other day. The DMEM media was supplemented with
2% of horse serum. Cultures were taken for experiments at
day ve and day seven after induction when myotubes were
clearly visualized. Adipose tissue dierentiation was stimu-
lated as described earlier [22] by replacing the culture media
with adipocyte induction medium composed of culture
medium supplemented with 1 μM insulin, 1 μM dexametha-
sone, and 0.5 μM 3-isobutyl-1-methylxanthine. Dierenti-
ated adipocytes were xed and stained with Oil Red O at
day 9 after induction.
2.5. Immunocytochemistry. The nature of the isolated cells
was conrmed by immunocytochemical staining. Cells
seeded onto cover glasses were xed in 4% paraformaldehyde
for 10 min at 4
°
C and then permeabilized with 0.02% Triton
X-100 for 5 min. Nonspecic binding was blocked by incu-
bation in 15% FCS for 30 min, followed by one-hour incu-
bation with the following primary antibodies: anti-MyoG
(R&D Systems, USA), anti-MYF5 (R&D Systems, USA),
anti-vimentin (Sigma-Aldrich, USA), anti-CD146 (Sigma-
Aldrich, USA), anti-desmin (D33, Dako, Denmark), myo-
sin heavy chain (MF20, MAB4470, R&D Systems, USA),
anti-myosin (skeletal fast; human MYH1/MYH2) (M4276,
Sigma-Aldrich, USA), anti-myosin (skeletal slow; human
MYH7) (M8421, Sigma-Aldrich, USA), and anti-myogenin
(MAB6686, R&D Systems, USA). The secondary antibodies
conjugated with Alexa Fluor 546/Alexa-488 (Molecular
Probes, USA) were applied for 45 min at room tempera-
ture. Nuclei were counterstained with DAPI (Molecular
Probes, USA).
2.6. Flow Cytometry Analysis. The immunophenotype of
stem cells was evaluated by ow cytometry analysis per-
formed on CytoFLEX (Beckman Coulter). Сells were resus-
pended in 100 μL of PBS containing 1% of bovine serum
albumin (Sigma-Aldrich, Saint Louis, MO, USA) and
incubated for 20 min at 20
°
C in the dark with the following
monoclonal antibodies (Ab): anti-CD56 PC7 (Beckman
Coulter, USA, A21692), anti-CD146 PE (Beckman Coulter,
USA, A07483), anti-CD166 PE (Beckman Coulter, USA,
A22361), anti-CD73 PE (BD Pharmingen, USA, 550257),
anti-CD105 APC (R&D Systems, USA, FAB1097A-100),
anti-CD45 PC5 (Beckman Coulter, USA, A07785), anti-
PDGFRβAPC (BD Pharmingen, USA, FAB1263A), and
anti-CD140a PE (BioLegend, USA, 323506). Data were
analyzed using the CytExpert 2.0 (Beckman Coulter).
2.7. Cell Sorting. All sorting procedures were performed on a
BD FACSAriaIII (Becton Dickinson, USA) ow cytometer
using BD FACSDiva (Becton Dickinson, USA) software.
Flow calibration was performed using Acudrop Beads (BD
FACS) with following stabilization for at least 25 minutes.
The nozzle size was 100 mm, and the sheath pressure was
set at 17 psi. During sorting, the ow rate was restricted to
<800 events/sec to ensure minimal contamination. Addition-
ally, a 4-way puritysort option was used and is sucient to
gain a 99% pure sample. Before sorting commenced, appro-
priate settings were determined for all parameters.
Cell cultures were stained with antiCD56 PE (Beckman
Coulter, USA, A07788) monoclonal antibodies according to
the manufacturers protocols. Primarily cells were detected
in logarithmical scales in forward scattering (FS) and side
scattering (SC). Sorting was performed according to the
electronic gating strategy. Two target cell populations
(CD56+ and CD56-, respectively) were collected in 15 mL
falcon tubes containing PBS supplemented with 2% of FBS.
Sorting eciency was controlled using additional ow
cytometry analysis of sorted samples. Detected purity was
no less than 95%.
3Stem Cells International
2.8. RNA Isolation, cDNA Synthesis, and Q-PCR. Sequences
for Q-PCR primers can be found in supplemental Table S1.
Total RNA was isolated using the ExtractRNA reagent
(Evrogen, cat. no. BC032, Russia). cDNA was synthesized
from 500 ng of total RNA using a Moloney Murine
Leukemia Virus Reverse Transcriptase MMLV RT kit
(Evrogen, SK021, Russia). A quantitative evaluation of gene
expression was performed with qPCR mix-HS SYBR+ROX
(Evrogen, cat. no. PK156, Russia). Q-PCR data are presented
as arbitrary units of mRNA expression normalized to
GAPDH expression and to expression levels in the
reference sample.
2.9. Statistical Methods. Statistical analysis was performed
using GraphPad Prism 7 software. All data were analyzed
with at least three biological replicates and presented as
mean ±SEM. See gure legends for details for each spe-
cic experiment.
3. Results
3.1. Pathological Upregulation of Genes That Regulate
Developmental/Regeneration Program and Metabolism
Detected in Skeletal Muscle from HF Patients. In order to
detect markers of HF-induced functional and metabolic
alterations in skeletal muscles, the expression analysis was
performed in HD- and HF-derived biopsy samples for
markers and regulators of skeletal muscle development,
maturation and function (Myf6, Myh3,Myh8, Myh1, Myh4,
Myh9, Myh10, Myh7, TNNI2, and TTNC1), and energy
metabolism, including the expression of genes that regulate
lipids and glucose handling (Pgc1a, HIF1a, GLUT1, GLUT4,
aP2, PLIN2, PLIN3, PPARg, ATGL, SCD1, GOS, CGI58,
CD36, NPRA, NPRB, and NPRC). A few genes from this
panel demonstrated signicantly altered expression in
HF-derived samples (Figure 1(a)).
We have found that the balance between expression
levels of slow oxidative skeletal muscle ber MHC isoform
MYH7 and fast glycolytic isoform MYH1 was notably down-
regulated in HF muscle (Figure 1(b)), along with the down-
regulation of the expression of Pgc1a (Figure 1(c)) that
indicates an impairment in the regulation of the transcrip-
tional program for mitochondrial biogenesis and oxidative
metabolism in HF [23]. Furthermore, the expression levels
of both developmental myosins, embryonic (MYH3) and
neonatal (MYH8), were substantially upregulated in HF-
derived skeletal muscle biopsies, which along with the
upregulation of myogenic regulatory factor Myf6 detected
in HF-derived samples (Figures 1(d)1(f)) may indicate the
chronic shift to developmental program and pathological
stimulation of muscle regeneration in HF [24, 25].
We have also detected in HF skeletal muscle the alter-
ations in the expression of the NPRA/B-to-NPRC ratio
(Figures 1(g)1(i)) that controls the biological activity of
the natriuretic peptide system (NP) at the target tissue level
[26], including control of sensitivity to insulin and lipid
oxidative capacity through a Pgc1a-dependent pathway [27]
and cardiac progenitor cell proliferation and dierentiation
into cardiomyocytes [28].
3.2. The Characterization of In Vitro Expanded SM-MPC
Cells and Comparison with Mesenchymal Multipotent Cells
from Dierent Sources. Puried and expanded in vitro
populations of SM-MPC derived from HD and HF muscle
biopsies demonstrated a mixed phenotype that may change
dynamically during in vitro expansion. Cells did not dier
signicantly from sample to sample and between HF
patients and HD in both ow cytometry analysis and immu-
nocytochemistry analysis. BM-MMSC, IMF-MSC, and AD-
MMSC were used to demonstrate dierences and similarities
between muscle-derived stem cells and mesenchymal multi-
potent stromal cells from other sources. The representative
images are presented in Figure 2.
Most of the SM-MPC cells were CD73+/CD105+/
CD166+/CD140b+, and about 3040% of the cells in the
sample expressed NCAM/CD56, known as a reliable molec-
ular marker of satellite cells and myoblasts in human skeletal
muscle, as well as myotubes, and muscle bers during devel-
opment and/or regeneration [29] (Figure 2(a)). Interestingly,
we detected two distinct subpopulations of CD140b
dim/bright
cells in some samples, but none of these populations was
CD56+ (Figure 2(b)). Furthermore, practically all SM-MPC
cells demonstrated no CD140a expression and only about
15% of them were CD146
dim
. Markedly, a substantial fraction
of CD146
dim
cells was CD56+ (Figure 2(b)).
BM-MMSC demonstrated, as expected, the CD73+/
CD166+/CD140a+/CD140b+/CD146+ phenotype and, sur-
prisingly, expressed NCAM/CD56. The expression of CD56
on bone marrow-derived MSC is not common, but was
reported previously at both mRNA and protein levels and
was donor-specic with the CD56+ fraction ranging from
24 to 88.5% [30]. In our experiments in all 3 samples, we
detected quite a big fraction of CD56+ cells; however, the
fraction of CD56
bright
cells in these samples was less than
15%, while in SM-MPC samples this fraction was more
than 30% as indicated on Figure 2(a). Interestingly, unlike
SM-MPC, in BM-MMSC samples all CD56+ cells were
CD140a+/CD140b+.
The immunophenotypes of both fat-derived samples,
IMF-MSC and AD-MMSC, were very similar and dier sub-
stantially from the ones of the SM-MPC and BM-MMSC
samples. Virtually all cells in the IMF-MSC and AD-MMSC
samples were CD56 negative and expressed mesenchymal
markers CD73, CD105, CD166, and CD140b (PDGFRb),
but they demonstrated little or no CD140a (PDGFRa) and
CD146 expression.
The immunostaining analysis also revealed the coexpres-
sion of mesenchymal markers and markers of myogenic cells
in SM-MPC (Figure 2(c)). Virtually all cells were expanded
in vitro; however, the cells were not stimulated to dierenti-
ate into cells expressing early myogenic regulatory factor
Myf5 [25], and some cells, presumably those that will
undergo spontaneous fusion, expressed myogenin (MYOG)
that regulates the fusion of myocytes and the formation of
myotubes [25]. Furthermore, in vitro expanded skeletal
muscle-derived stem cells expressed vimentin (Figure 2(c)),
known to be expressed not only in mesenchymal cells [31]
but also in myoblasts and in myotubes during early stages
of embryonic development [32, 33]. The promyogenic nature
4 Stem Cells International
of cells that express the melanoma cell adhesion molecule
(MCAM, or CD146) was demonstrated recently [16], and
we also have detected the CD146+ population in our samples
(Figure 2(c)). We did Myf5 and MyoG immunostaining
simultaneously for the same cultures that were stained for
vimentin, and these data in combination with FACS analysis
provide evidence of the coexpression of mesenchymal lineage
cell markers with myogenic markers.
3.3. Both CD56+ and CD56- Fractions of SM-MPC
Demonstrate Myogenic Potential. In order to determine if
both major subpopulations of SM-MPC (CD56-/CD56+)
possess the myogenic potential and may therefore be impor-
tant for the maintenance of skeletal muscle regeneration,
we employed FACS sorting to separate these subpopula-
tions from SM-MPC; puried CD56-/CD56+ samples were
expanded in vitro and induced to dierentiate into adipose
and muscle tissue (Figure 3). IMF-MSC samples were used
as a control.
As we expected, IMF-MSC samples did not respond to
the stimulation of myogenesis but were dierentiated actively
into adipocytes (Figure 3(b)). On the contrary, both subpop-
ulations of SM-MPC did not dierentiate into adipocytes
under adipogenic stimuli but demonstrated the ability to dif-
ferentiate into myotubes (Figure 3(c)). The fusion coecient
was slightly but not signicantly higher in the CD56+
subpopulation (30 ± 3% vs 24 ± 35) (Figure 3(d)), which
conrmed the potential signicance of both fractions of
SM-MPC for muscle regeneration/development. Taking
into account all information mentioned above, we choose
to use the whole unsorted HD- and HF-derived SM-MPC
cellular samples in further work.
3.4. Immunohistochemical Analysis and Gene Expression
Analysis during HD- and HF-Derived SM-MPC Dierentiation
In Vitro Did Not Reveal Dierences between Groups. HD-
and HF-derived SM-MPC demonstrated a similar ability
to respond to the stimulation of myogenesis in vitro. We
Fold change (log2)
SCD1
GOS
MYH3
MYH8
MYH1
aP2
HIF1a
CGI58
MYF6
GLUT1
PPARg
CD36
PLIN2
TTNC1
MYH7
NPRB
LXRA
CPT1b
MYH9
GLUT4
MYH10
TNNI2
ATG L
PLIN3
MCAD
ERRA
NPRA
DGAT1
Pgc1a
NPRC
−1.5 5
(a)
HD HF
0
1
2
3
4
5
Relative MYH7/MYH1 mRNA
expression (arbitrary units)
p < 0.01
(b)
HD HF
0
2
4
6p < 0.01
Pgc1c mRNA expression
(arbitrary units)
(c)
HD HF
0
2
4
6
8
10
p < 0.01
MYH3 mRNA expression
(arbitrary units)
(d)
HD HF
0
10
20
30
40
p < 0.02
MYH8 mRNA expression
(arbitray units)
(e)
HD HF
0
5
10
15 p < 0.05
Myf6 mRNA expression
(arbitrary units)
(f)
HD HF
0
5
10
15
NS
NPRA mRNA expression
(arbitrary units)
(g)
HD HF
0
2
4
6
NS
NPRB mRNA expression
(arbitrary units)
(h)
HD HF
0
10
20
30
40
p < 0.05
NPRC mRNA expression
(arbitrary units)
(i)
Figure 1: The expression of genes that regulate skeletal muscle development and metabolism is altered in HF patients. (a) Results of Q-PCR
screening of key regulators and markers of skeletal muscle development and metabolism. Green bars: upregulation in HF; red bars:
downregulation in HF; n=3 (HD) and n=12 (HF). (bi) Results of Q-PCR analysis of mRNA expression for genes that demonstrated
signicant dierences in between HD and HF. n=3(HD) and n=12 (HF).
5Stem Cells International
have done immunocytochemical staining at early and late
steps of dierentiation. At day ve after induction, the
formation of myotubes was observed and immunocyto-
chemical staining detected the expression of myogenin,
CD146, and vimentin. It is known that vimentin is the
most abundant intermediate lamentproteininimmature
myoblasts/muscle progenitors. During the early steps of
muscle development, desmin and vimentin are coexpressed.
Upon further dierentiation into mature muscle cells,
desmin is strongly upregulated, while the expression of
CD56
SM-MPC IMF-MSC AD-MMSC BM-MMSC
41% CD56 0.7% CD56 4% CD56 92%
CD73 88% CD73 45% CD73 96% CD73 100%
CD105 100% CD105 99% CD105 99% CD105 24%
CD166 79% CD166 95% CD166 92% CD166 100%
CD146 16% CD146 8% CD146 1% CD146 75%
CD140a 8% CD140a 3% CD140a 0% CD140a 96%
CD140b 77% CD140b 90% CD140b 92% CD140b 100%
CD45 0% CD45 0% CD45 0% CD45 0%
35% 15%
101
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
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80
60
40
20
0
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80
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0
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80
60
40
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0
100
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0
102103104105106
101102103104105106
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101102103104105106102103104105106102103104105106102103104105106107
101102103104105106102103104105106102103104105106102103104105106107
(a)
CD56 + /CD140b−
CD56 + /CD140a−
CD56 + /CD146b+
CD56
CD140b
CD56
CD140a
CD56
CD146
106
105
104
103
102
101
106
105
104
103
102
101
106
105
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102
101106
105
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103
102
101
106
105
104
103
102
101
(b)
MYOG
MYF5
VIMCD146
DAPI DAPI
DAPI DAPI
(c)
Figure 2: Characterization of SM-MPC, IFM-MSC, BM-MMSC, and AD-MMSC cultures expanded in vitro. (a) Representative histograms
demonstrate the results of a comparative FACS surface marker analysis of SM-MPC, IFM-MSC, BM-MMSC, and AD-MMSC. Green
histograms indicate the stained samples, and grey ones indicate the negative controls. The red ring indicates the CD56
bright
subpopulations
in SM-MPC and BM-MMSC samples (n=35 in each group). (b) Visualization of FACS analysis in SM-MPC samples: CD56+/CD140a-,
CD56+/CD140b-, and CD56+/CD146+ subpopulations are indicated in a rectangle gate; quadrant gates specify the negative/positive
populations. Unstained cells were used as a negative control for each sample. (c) Immunocytological phenotyping of skeletal muscle
precursor cells at early steps of expansion in vitro. Cells were stained for the expression of MYF5 (red; ~100% of positive), MYOG
(red; 25±4% of positive), vimentin (VIM, green; ~100 of positive), or CD146 (green; 20±4.5% of positive). The insert demonstrates
the enlarged region with a CD146+ cell. Nuclei were labelled with DAPI (blue). Scale bars represent 50 μm.
6 Stem Cells International
vimentin completely ceased [34]. In our cultures, we
detected vimentin in progenitor cells (Figure 2(c)) and in
myotubes at the early steps of dierentiation (Figure 4),
while desmin expression was detected in all tested condi-
tions(Figures3and4).
Staining for desmin (Figure 4) and MF20 (data not
shown) at day 5 did not demonstrate a cross-striated pattern
specic for mature skeletal muscle ber (Figure 4). At day
seven of immunocytochemical staining with antibodies
against MF20, slow and fast MyHCs conrmed that dier-
entiated myotubes demonstrate a cross-striated pattern,
similar to that seen in adult muscle bers. Both HD- and
HF-derived cells developed myotubes that were positive
for both slow (MYH7) and fast (MYH1/MYH2) myosins,
and the fractions of nuclei incorporated into myosin-
positive myotubes did not dier signicantly between
HF and HD samples (Figure 5(a)). The fusion coecient
did not dier signicantly between HD and HF samples
(28 ± 4% vs 32 ± 5%) (Figure 5(b)).
The gene expression analysis of markers and regulators of
myogenic dierentiation of SM-MPC derived from HD and
HF patients conrmed the results of immunohistochemistry:
the expression of slow skeletal muscle ber MHC isoform
MYH7 and fast isoform MYH1 did not dier signicantly
between HD- and HF-derived dierentiated myotubes, and
the expression of embryonic (MYH3) and neonatal (MYH8)
myosins did not dier signicantly as well. The expression
of myogenic regulatory factor Myf6, Pgc1a, and of atrial
natriuretic peptide receptor C in dierentiated myotubes also
did not dier signicantly between groups (Figure 5(c)).
4. Discussion
Heart failure is a multiorgan syndrome aecting dierent cell
types, including skeletal muscle. The development of preven-
tive and therapeutic strategies against muscle wasting disor-
ders in HF remains an unresolved challenge, and activation
of developmental/regeneration programs in skeletal muscle
could be considered as a prospective approach. Therefore,
the activation of skeletal muscle progenitor cells would be
benecial for the restoration of skeletal muscle structure
and performance. In this work, we aimed to determine if
HF-induced skeletal muscle alterations aect SM-MPC
developmental/regeneration potential.
IFM-MSC
Stimulation of
myogenesis and adipogenesis
SM-MPC
(a)
IFM-MSC
Adipogenesis Myogenesis
No response
(b)
Adipogenesis Myogenesis
No response
CD56-PC7
CD73-PE
0 10
3
10
2
10
3
10
4
10
5
10
4
10
5
CD56−
CD56+
Desmin
DAPI
DEsmin
DAPI
SM-MPC
(c)
50
40
30
20
10
0CD56−CD56+
Fusion coecient (%)
(d)
Figure 3: Comparative analysis of the functional properties of subpopulations of skeletal muscle progenitor cells derived from muscle tissue
(SM-MPC) and from intermuscular fat (IMF-MSC). (a) The design of the experiment is as follows: SM-MPC and IMF-MSC were puried
from muscle biopsy, expanded in vitro, characterized, and induced to dierentiate. (b) IMF-MSC under appropriate stimulation undergo
adipogenesis but do not respond to promyogenic stimulation. (c) Both CD56+ and CD56- fractions of SM-MPC demonstrate the ability
to dierentiate into myotubes but do not respond to adipogenic stimuli; scale bars represent 100 μm. (d) Fusion coecient is calculated as
a percent of nuclei incorporated in MF20+ myotubes, and it does not dier between the CD56+ and CD56- subpopulations.
HD HDHD
HF HF
HF
DAPI
DAPI DAPI
DAPI
DAPI
DAPI Desmin
Desmin
VIM
VIM
CD146
CD146
MYOG
MYOG
Figure 4: Immunocytological phenotyping of dierentiated myotubes at early steps of myogenesis. At day 5 after stimulation, myotubes
coexpress myogenin (MYOG) and CD146. All cells in culture express vimentin (VIM), and desmin expression is specically associated
with multinucleated myotubes. Scale bars represent 50 μm.
7Stem Cells International
We have detected a number of chronic dysregulations in
the skeletal muscle tissue from our patients. The most
important one was the chronic activation of the develop-
mental program: the expression of mRNA of MYH3/
MYH8 myosins and myogenic regulatory factor Myf6 was
detected in an HF-derived biopsy. These alterations in com-
bination with changes in slow/fast ber composition may
severely inuence skeletal muscle metabolism, structure,
and performance. We suggest that in HF, the regeneration
process is stimulated in response to HF-induced damage:
skeletal muscle progenitor cells progress successfully through
activation, proliferation, dierentiation into myoblasts, and
fusion steps; however, instead of advancing to the ber mat-
uration stage, they get stuckat the developmental phase
presumably due to chronic metabolic alterations observed
in HF skeletal muscle. The reexpression of developmental
myosins in adult skeletal muscle was detected in dierent
pathological conditions that involve muscle degeneration/
regeneration, such as trauma, chronic denervation, muscu-
lar dystrophy, and dierent types of myopathies (reviewed
in [24]); however, to our knowledge none was previously
reported for HF.
The shift from oxidative ber type I to glycolytic ber
type II and reduced oxidative enzyme activities is the best-
described HF-induced metabolic alteration in skeletal muscle
[3, 5, 8, 35, 36]. We have also detected the shift in the expres-
sion ratio between ber type I and ber type II in skeletal
muscle from HF patients, as well as the downregulation of
expression of Pgc1a (Figure 1), which is an important medi-
ator of mitochondrial metabolic properties in skeletal muscle
and is downregulated in various types of atrophying muscle
[37] including skeletal muscle of rats with HF [3841].
Furthermore, HF is a state of chronic activation of adrenergic
and NP systems, which besides their well-documented role in
the cardiovascular system also plays a role in favoring fat
oxidative capacity in human skeletal muscle cells [42] via
the activation of cGMP signaling, induction of PGC1a, and
enhancement of mitochondrial respiration [27]. There are
also recent data showing that the NP system is involved in
the regulation of cardiac progenitor cell proliferation via
NPR-A and dierentiation into cardiomyocytes via NPR-B
[28] contributing to heart development and regeneration.
The switch in expression balance from NPR-A/B to NPRC
detected in our work indicates an increase of NP system
MF20
MF20
Slow
Slow
DAPI
DAPI
DAPI
DAPI
DAPI
DAPI
Fast
Fast
HF HF HF
HD HD HD
(a)
HD HF
0
20
40
60
Fusion coecient (%)
(b)
1
10
100
1000
HD
MYH1
MYH7
MYH3
MYH8
MYH6
NPRA
NPRB
NPRC
Pgc1a
HF
mRNA expression
(arbitrary units, log)
(c)
Figure 5: Dierentiated myotubes do not dier signicantly between HD- and HF-derived skeletal muscle progenitor cells. (a) At day 7 after
stimulation, myotubes were stained for the expression of MyHC with an antibody that recognizes the heavy chain of myosin II (MF20) and
markers of slow MYH7 and fast MYH1/MYH2 bers. Nuclei were labelled with DAPI (blue). Representative images are given for both HF-
and HD-derived samples. Scale bars represent 50 μm. (b) Fusion coecient is calculated as a percent of nuclei incorporated in MF20+
myotubes at day 7 after stimulation, and it does not dier between HD- and HF-derived samples. (c) mRNA expression analysis was
performed for key markers of muscle development and metabolism for both HF- and HD-derived samples.
8 Stem Cells International
activity [9] that could also impact on the upregulation of
developmental signaling in HF skeletal muscle. Together,
we have detected a number of alterations in HF-derived
skeletal muscle that would aect developmental, metabolic,
structural, and functional properties of skeletal muscle.
Next, we puried SM-MPC from HD- and HF-derived
biopsies and investigated if the functional properties of
HF-derived cells were aected by these alterations. The
SM-MPC that we isolated from skeletal muscle biopsy
samples from HD and HF patients represented a mixed
population of cells that express both mesenchymal and
myogenic markers. Samples were characterized by FACS
analysis (Figures 2(a)2(c)) and immunocytochemistry
(Figure 2(c)), and they were also investigated for the abil-
ity to dierentiate in vitro (Figure 3). In order to better
evaluate the myogenic potential of muscle progenitor cells
derived from HD and HF subjects based on obtained
results, we concluded using the whole unsorted samples
in dierentiation experiments in order to retain in cultures
all subpopulations that could possibly contribute to stimu-
lated in vitro myogenesis either via paracrine signaling
mechanisms and/or direct cell-cell interaction.
Indeed, there is a lot of evidence in the literature that
describes dierent subpopulations of skeletal muscle progen-
itor cells that support skeletal muscle development, growth,
and regeneration (reviewed in [13]). The best characterized
myogenic progenitors in postnatal muscle are satellite cells
that are activated in response to injury or stimulation to
growth, which then start to proliferate and generate a pool
of myoblasts able to fuse into newly formed myobers.
CD56 is considered as the most reliable satellite cell surface
marker [38]. However, human satellite cells are not easy to
isolate, purify, and expand in culture: most of the studies with
satellite cells were done on mice, but not on humans [16].
Furthermore, in recent years reports of myogenic cells
distinct from satellite cells have accumulated, and not all
of these cells are reported to be CD56+. For example,
PW1+/Pax7interstitial cells (PICs) that do not express
CD56 but demonstrate bipotential behavior in vitro, gen-
erating both smooth and skeletal muscles, were isolated
and characterized [17]; the coexpression of mesenchymal
(CD90, CD73, CD166, and CD105) and myogenic (CD56)
markers was reported on several cell populations with myo-
genic potential including human embryonic mesodermal
progenitors [43], expanded in vitro muscle-derived primary
cultures [14], and myogenic mesenchymal progenitors
derived from hES and iPSC [29]. It was also shown that sub-
endothelial- (mural) cultured CD146+ cells (also known as
mesenchymal stem cells) puried from various groups of
skeletal muscle were able to spontaneously generate myo-
tubes in vitro and myobrils in vivo, and the expression of
CD146 and CD56 was mutually exclusive in distinct myo-
genic cell subsets, with no coexpression [16]. Importantly,
in this work the authors report that sorted and cultured
CD146+ human muscle-derived cells progressively turn on
the expression of myogenic markers PAX7, PAX3, Myf5,
CD56, desmin, and MyHC, as they were veried by uores-
cent immunocytochemistry [16]. Finally, in some protocols
CD146 is recommended as a positive selection marker in cell
sorting to obtain a human fetal myoblast population [44].
Together, all these previous ndings support observations
made in our work. Firstly, in expanded in vitro adherent
cells puried from gastrocnemius muscle biopsy we also
detected substantial subpopulations of cells that coexpress
mesenchymal and myogenic markers (Figure 2). Secondly,
we have found that not only the sorted CD56+ but also
the sorted CD56- subpopulation demonstrates myogenic
potential (Figure 3). Because of the limited volumes of
samples, we were not able to monitor the dynamics of marker
expression during in vitro expansion of sorted CD56+/CD56-
populations; however, we can speculate that the myogenic
potential of the CD56- subpopulation can be related to the
CD146+ subpopulation of cells and those cells could turn
on the expression of myogenic markers in the course of
expansion as described by Persichini et al. [16]. The data
presented on Figures 2 and 3 support this speculation pretty
well: the coexpression of CD146 and CD56 on certain sub-
populations of expanded in vitro SM-MPC (Figures 2(a)
and 2(c)) and coexpression of myogenic regulatory factor
myogenin (MyoG) with CD146 at early steps of myogenic
dierentiation (Figure 3) conrm the importance of the
CD146+ SM-MPC fraction for myogenic dierentiation.
Also, as indicated on Figure 3, both major fractions of
SM-MPC (CD56+/CD56-) demonstrated a similar ability to
dierentiate into myotubes but not into adipocytes. This is
an important observation: dysregulation of lipid metabolism
in the skeletal muscles of HF patients is a well-described
metabolic disorder [9], and fatty degeneration of skeletal
muscle is often associated with metabolic dysregulation
[45, 46]. In our experiments, neither HD nor HF-derived
SM-MPC demonstrated in vitro adipogenic potential. Our
data t well the observations described by Uezumi et al.
[45] who demonstrated that only the CD140a+ mesenchymal
progenitor population of muscle-derived cells show ecient
adipogenic dierentiation both in vitro and in vivo. In
our work, SM-MPC demonstrated a CD140a- phenotype.
Interestingly, both adipose tissue-derived AD-MMSC and
IMF-MSC, but not BM-MMSC samples that all dierentiate
into adipocytes (Figure 2, our previous data [22]), demon-
strated a CD140a- phenotype.
Interestingly, we have detected myotube formation not
only upon canonical dierentiation conditions (2% horse
serum), but even under adipogenic stimulation (data not
shown). Similar observations were made previously by
others: myogenesis in skeletal muscle-derived progenitor
cells under adipogenic stimulation was mentioned earlier
by Persichini et al. [16] and by Uezumi et al. [45]; those data,
however, did not get much attention and deserve further
investigation. Together, these observations allow us to con-
clude that SM-MSC samples puried from both HD- and
HF-derived skeletal muscle were restricted/committed to
myogenic dierentiation and, in general, do not dier
between healthy donors and heart failure patients.
5. Conclusion
In the present work, we demonstrate that the metabolic and
functional alterations we detected in skeletal muscle from
9Stem Cells International
HF patients do not dramatically aect the functional prop-
erties of puried and expanded in vitro skeletal muscle
mesenchymal progenitor cells (SM-MPC).
These ndings allow us to speculate that skeletal muscle
progenitor cells are quite well protected by their niche from
HF-induced metabolic stress, and they could, under bene-
cial circumstances, contribute to damaged muscle restora-
tion, prevention, and treatment of muscle wasting; the exact
mechanisms behind alterations in the muscle regeneration
program in HF remain to be investigated, and the potential
new therapeutic strategies of HF-induced skeletal muscle
wasting should be targeted on both activation of skeletal
muscle stem cell regeneration potential and improvement
of skeletal muscle metabolic status in order to provide a
favorable environment for SM-MPC-driven improvement
of muscle structure and performance.
Data Availability
The data used to support the ndings of this study are
available from the corresponding author upon request.
Conflicts of Interest
Dmitrieva R.I., Lelyavina T.A., Komarova M.Y., Ivanova
O.A., Galenko V.L., Tikanova P.O., Khromova N.V.,
Golovkin A.S., Bortsova M.A., Sergushichev A., Sitnikova
M.Yu., and Kostareva A.A. declare that they have no
conict of interest.
Acknowledgments
Cell sorting experiments were performed on the FACSAria
III Cell Sorter (Becton, Dickinson and Company, USA) at
the Research Resource Centre Molecular and Cell Tech-
nologies, Saint Petersburg State University; http://www.
biomed.spbu.ru/en/. Work was funded by Russian Science
Foundation grant #16-15-10178 (RD).
Supplementary Materials
Table S1: list of primers used in Q-PCR experiments. Figure
S1: SM-MPC from HD demonstrate the consistent response
to stimulation of myogenic dierentiation and similarities
in immunophenotypes. Upper panel: immunocytological
visualization of dierentiated myotubes in HD-derived sam-
ples: myotubes were stained for expression of MyHC with an
antibody that recognizes the heavy chain of myosin II
(MF20). Nuclei were labelled with DAPI (blue). Lower panel:
histograms demonstrate results of FACS surface marker
analysis of SM-MPC derived from HD. (Supplementary
Materials)
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11Stem Cells International
... Обычно спортивные тренировки стимулируют увеличение массы скелетной мускулатуры за счет активации сателлитных клеток и реализации их потенциала миогенной дифференцировки. Недавно мы показали, что в мышечной ткани пациентов с ХСН хронически активирована программа регенерации мышечной ткани, которая не завершается формированием "взрослой", функциональной мышеч ной ткани; такое состояние является патологическим и было описано ранее для разных типов мышечных дистрофий [3]. Однако в стандартизированных условиях in vitro резидентные стволовые клетки (СК) скелетной мускулатуры пациентов с ХСН активно диф-ференцировались в миотрубки, демонстрируя со храннось регенеративного потенциала мышечной ткани при ХСН [3]. ...
... Недавно мы показали, что в мышечной ткани пациентов с ХСН хронически активирована программа регенерации мышечной ткани, которая не завершается формированием "взрослой", функциональной мышеч ной ткани; такое состояние является патологическим и было описано ранее для разных типов мышечных дистрофий [3]. Однако в стандартизированных условиях in vitro резидентные стволовые клетки (СК) скелетной мускулатуры пациентов с ХСН активно диф-ференцировались в миотрубки, демонстрируя со храннось регенеративного потенциала мышечной ткани при ХСН [3]. Таким образом, мы пришли к заключению, что активация резидентной СК у пациентов с ХСН возможна, и правильно подобранная программа физической реабилитации может способствовать активации регенераторного потенциала СК скелетной мускулатуры, восстановлению мышечной ткани и росту, препятствовать развитию саркопении и увеличивать толерантность к физическим нагрузкам. ...
... Это наблюдение важно и ново: большинство опубликованных работ содержат доказательства, подтверждающие мнение о том, что СК скелетных мышц больных с СН плохо поддерживают рост мышц из-за ряда патологических факторов, включая ингибирование активации и пролиферации сателлитных клеток белком Ang II через его рецептор I типа [12], более низкую плотность капиллярной сети в мышцах [13], устойчивую активацию основных путей деградации белков -протеосомного и лизосомально-аутофагического [14]. В наших последних работах, однако, мы продемонстрировали, что в стандартизированных условиях in vitro СК скелетных мышц и костного мозга, полученные от пациентов с СН, не теряют регенеративного потенциала, что свидетельствует о том, что стабилизация микроокружения in vivo может привести к физиологически значимой активации СК [3,15]. В настоящей работе мы показываем, что этот потенциал может быть стимулирован у пациентов с СН с помощью персонализированного курса тренировок, что приводит к предотвращению мышечного истощения и повышению толерантности к физической нагрузке. ...
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Aim. Heart failure (HF) is accompanied by skeletal muscle atrophy and exercise intolerance. The aim was to study the molecular mechanisms underlying the therapeutic effect of personalized exercise in patients with HF. Material and methods . RNA sequencing obtained from skeletal muscle biopsies before and after a 12-week exercise course was used to identify changes in gene expression and signaling pathways induced by the physical rehabilitation program for patients with HF. Results. We have shown that personalized exercise program in patients with HF stimulates the activation of molecular pathways regulating the differentiation and functioning of skeletal muscles: commitment of muscle progenitor cells; mechanisms regulating the calcium release and sensitivity of myofibrillar contraction, electrical excitability of the muscle membrane, synaptic vesicle proton gradient creation, maintenance of electrochemical gradients of Na ⁺ /K ⁺ . Also, the analysis of differentially expressed genes revealed an increase in the expression of transcription factors MyoD and MEF2, which are responsible for the differentiation of muscle stem cells, and sarcomeric genes MYOM1 , MYOM2 , MYH7 . Along with this, we observed activation of the CYR61 expression — a potential prognostic biomarker for HF patients. Conclusion. Our data show that the beneficial effect of personalized aerobic exercise in patients with HF depends, at least in part, on an improvement in the physiological and biochemical parameters of skeletal muscle.
... Usually, physical training increases the mass of skeletal muscle tissue through the contractility-induced satellite cell activation [6]. In our recent work, we showed that the regeneration potential of bone marrow and skeletal muscle resident stem cells in CHF patients was not severely affected by disease, and under standardized in vitro conditions, these cells maintained proliferation activity [7] and differentiated actively into myotubes [8]. This indicates that exercise-induced activation of the regeneration potential of skeletal muscle stem cells might contribute to muscle tissue restoration and better performance in CHF patients. ...
... Skeletal muscle biopsies were taken twice from a selected group of HF patients enrolled in the personalized exercise training program as indicated: Before and after 12 weeks of training. The portion of the first biopsy was used to evaluate the regeneration potential of HF-derived skeletal muscle progenitor cells as we described recently [8]; the rest was flash frozen and saved for further histology analysis. ...
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Heart failure (HF) is associated with skeletal muscle wasting and exercise intolerance. This study aimed to evaluate the exercise-induced clinical response and histological alterations. One hundred and forty-four HF patients were enrolled. The individual training program was determined as a workload at or close to the lactate threshold (LT1); clinical data were collected before and after 12 weeks/6 months of training. The muscle biopsies from eight patients were taken before and after 12 weeks of training: histology analysis was used to evaluate muscle morphology. Most of the patients demonstrated a positive response after 12 weeks of the physical rehabilitation program in one or several parameters tested, and 30% of those showed improvement in all four of the following parameters: oxygen uptake (VO2) peak, left ventricular ejection fraction (LVEF), exercise tolerance (ET), and quality of life (QOL); the walking speed at LT1 after six months of training showed a significant rise. Along with clinical response, the histological analysis detected a small but significant decrease in both fiber and endomysium thickness after the exercise training course indicating the stabilization of muscle mechanotransduction system. Together, our data show that the beneficial effect of personalized exercise therapy in HF patients depends, at least in part, on the improvement in skeletal muscle physiological and biochemical performance.
... Genes 2020, 11, 1057 2 of 20 mutation-specific manner [6][7][8][9][10].) Namely, if the repairing/regenerating myofibers express the mutant LMNA, they demonstrate the compromised and ineffective regeneration capacity and satellite cells' replicative senescence resulting in higher susceptibility to mechanical damage [11][12][13]. Additionally, LMNA mutations may impact the function of the nuclear envelope as a signaling platform, affecting the dynamics of epigenomic perturbations and gene expression in regenerating skeletal muscle [14,15]. ...
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Laminopathies are a family of monogenic multi-system diseases resulting from mutations in the LMNA gene which include a wide range of neuromuscular disorders. Although lamins are expressed in most types of differentiated cells, LMNA mutations selectively affect only specific tissues by mechanisms that remain largely unknown. We have employed the combination of functional in vitro experiments and transcriptome analysis in order to determine how two LMNA mutations associated with different phenotypes affect skeletal muscle development and metabolism. We used a muscle differentiation model based on C2C12 mouse myoblasts genetically modified with lentivirus constructs bearing wild-type human LMNA (WT-LMNA) or R482L-LMNA/G232E-LMNA mutations, linked to familial partial lipodystrophy of the Dunnigan type and muscular dystrophy phenotype accordingly. We have shown that both G232E/R482L-LMNA mutations cause dysregulation in coordination of pathways that control cell cycle dynamics and muscle differentiation. We have also found that R482/G232E-LMNA mutations induce mitochondrial uncoupling and a decrease in glycolytic activity in differentiated myotubes. Both types of alterations may contribute to mutation-induced muscle tissue pathology.
... Максимальной степени эти изменения могут достигать у больных с сердечной кахексией [3][4][5][6]. Однако наличие нормального регенераторного потенциала в клетках-предшественниках мышечных волокон [6,11] является обоснованием позитивного ответа скелетной мускулатуры на ФР. ...
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Aim . To assess the response of skeletal muscle and myocardium to original aerobic exercise (AE) program in patients with heart failure (HF) with reduced ejection fraction (HFrEF); to assess morphometric changes in skeletal muscle fiber after AE. Material and methods . The study included 100 patients with class III HFrEF (age — 52±5,2 years; body mass index (BMI) — 23,5±2,8 kg/m ² ). At baseline and after 6 months of AE, an echocardiogram, peak oxygen uptake (VO 2peak ), exercise tolerance and quality of life (QOL) were evaluated. Lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity were evaluated in biopsy material of lower leg muscles. Results . After 6 months of AE, the left ventricular ejection fraction (LVEF) increased by 10,5±2,3%, QOL — by 24,8±3,5 points, exercise tolerance — by 9,7±0,5 points, VO2peak — by 5,2±0,5 ml/min/kg (p 1,2,3,4 < 0,05). In 6 patients, the diameter of muscle fiber decreased slightly. The activity of ALP (initially — 0,33±0,09 D) increased by 24,2% (p< 0,05); LDH in glycolytic fibers was initially 0,213±0,08 D, in oxidative fibers — 0,083±0,04, and after 6 months of AE, decreased by 24,4% and 6,0%, respectively (p 1 <0,05, p 2 >0,05). A positive relationship was found between the dynamics of HF class and fiber diameter (r=0,4, p=0,05); an increase in сardiopulmonary exercise test was associated with ALP activity (r=0,5, p=0,05). Conclusion . 1. Dosed aerobic exercise in patients with stable class III HFrEF, normal BMI, based on reaching the lactate threshold, had a positive effect on LVEF, QOL, exercise tolerance and VO 2peak . 2. With exercise training, a decrease in fiber diameter and LDH activity in both oxidative and glycolytic fibers, an increase in ALP activity were revealed. 3. The functional relationship between the increase in exercise tolerance and ALP content in muscle tissue was revealed.
... However, the percentage of human bmMSCs committed to neurogenesis may be limited as only a small proportion (5.9 ± 2.1) of passage 4 bmMSCs expanded in P/ PL were positive for the neural lineage commitment CD56/NCAM phenotypic marker [64,65]. There are conflicting reports on the expression of CD56/NCAM on human bmMSCs with two studies showing that bmMSCs cultured in 10-20% FBS do not express or express very little CD56 [95,96], while another study showed that the fraction of CD56 bright bmMSCs was less than 15% [97]. A study investigating bmMSCs from five healthy females (age 21-31 years old) expanded in human PL and assessed at passages 2 or 3 showed that the proportion of positive cells varied considerably with the percentage of positive cells extending from 23.6 to 88.5% [98]. ...
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Background: Many studies have elegantly shown that murine and rat bone marrow-derived mesenchymal stromal cells (bmMSCs) contribute to muscle regeneration and improve muscle function. Yet, the ability of transplanted human bmMSCs to manifest myogenic potential shows conflicting results. While human adipose- and umbilical cord-derived MSCs can be differentiated into a skeletal muscle phenotype using horse serum (HS), bmMSCs have only been shown to differentiate towards the skeletal muscle lineage using a complex mixture of cytokines followed by transfection with notch intracellular domain. Methods: Since xenogeneic-free growth supplements are increasingly being used in the expansion of bmMSCs in clinical trials, we investigated the effects of human plasma and platelet lysate (P/PL) on the expression of neuromuscular markers and whether P/PL-expanded human bmMSCs could be differentiated towards a skeletal myogenic phenotype. Neuromuscular markers were measured using the highly sensitive droplet digital polymerase chain reaction for measuring the expression of Myf5, MyoD, MyoG, ACTA1, Desmin, GAP-43, and Coronin 1b transcripts, by performing immunofluorescence for the expression of Desmin, GAP-43, and MEF2, and flow cytometry for the expression of CD56/neural cell adhesion molecule (NCAM). Results: Despite that bmMSCs expressed the myogenic regulatory factor (MRF) MEF2 after expansion in P/PL, bmMSCs cultured under such conditions did not express other essential MRFs including Myf5, MyoD, MyoG, or ACTA1 needed for myogenesis. Moreover, HS did not induce myogenesis of bmMSCs and hence did not induce the expression of any of these myogenic markers. P/PL, however, did lead to a significant increase in neurogenic GAP-43, as well as Desmin expression, and resulted in a high baseline expression of the neurogenic gene Coronin 1b which was sustained under further P/PL or HS culture conditions. Fetal bovine serum resulted in equally high levels of GAP-43 and Coronin 1b. Moreover, the proportion of CD56/NCAM-positive bmMSCs cultured in P/PL was 5.9 ± 2.1. Conclusions: These data suggest that P/PL may prime a small portion of bmMSCs towards an early neural precursor cell type. Collectively, this shows that P/PL partially primes the cells towards a neurogenic phenotype, but does not prime adult human bmMSCs towards the skeletal muscle lineage.
... The hSM-MPC were isolated enzymatically according to the protocols described [16]. Briefly, mechanically disrupted paravertebral muscle from 24y old male lumbar disc herniation patients were digested by 0.1% collagenase I (Sigma-Aldrich, Germany) for 60 min at 37°C. ...
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Idiopathic scoliosis (IS) is a disease with unknown etiology characterized by spinal rotation asymmetry. Reports describing the histochemical and pathological analyses of IS patients have shown that necrosis, fibrosis and fatty involution occurred on the apex paraspinal muscles. However, research on the changes in the paraspinal muscles of IS patients compared with those in matched controls is rare; thus, the basic mechanism of how paraspinal muscles are injured in IS patients is still unclear. In this study, we investigated the morphological changes of paraspinal muscles in the control group and IS patients, and the possible mechanisms were examined in vivo and in vitro. Increased myofiber necrosis was found on both sides of the apex paraspinal muscles of IS patients compared with those of the control group, and the number of TUNEL-positive apoptotic cells was also increased. Apoptosis signaling pathways, including pro-apoptosis proteins such as cleaved-caspase 3 and cytochrome c, were markedly upregulated, whereas the anti-apoptotic Bcl-2/Bax was significantly downregulated in IS patients compared with the control group. Moreover, PGC-1α and SOD1 were upregulated in accordance with the increased ROS production in IS patients. The distribution of myofiber types, as well as the mRNA levels of type IIa myofiber marker MYH2 and the important myogenesis regulator MYOG were remarkably changed in IS patients. In addition, C2C12 or human skeletal muscle mesenchymal progenitor cells treated with antimycin A in glucose-free and serum-free culture medium, which can activate oxidative stress and induce apoptosis, showed similar patterns of the changed distribution of myofiber types and downregulation of MYH2 and MYOG. Altogether, our study suggested that the extents of severe muscle injury and accumulated oxidative stress were increased in IS patients compared with the control group, and the abnormal myogenesis was also observed in IS patients. Since elevated oxidative stress can lead to apoptosis and the dysregulation of myogenesis in muscle cells, it may be associated with the pathological changes observed in IS patients and contribute to the development and progression of IS.
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Objective: To summarize the expression and role of CD146 in mesenchymal stem cells (MSCs). Methods: The literature related to CD146 at home and abroad were extensively consulted, and the CD146 expression in MSCs and its function were summarized and analyzed. Results: CD146 is a transmembrane protein that mediates the adhesion of cells to cells and extracellular matrix, and is expressed on the surface of various MSCs. More and more studies have shown that CD146 + MSCs have superior cell properties such as greater proliferation, differentiation, migration, and immune regulation abilities than CD146 - or unsorted MSCs, and the application of CD146 + MSCs in the treatment of specific diseases has also achieved better results. CD146 is also involved in mediating a variety of cellular signaling pathways, but whether it plays the same role in MSCs remains to be demonstrated by further experiments. Conclusion: The utilization of CD146 + MSCs for tissue regeneration will be conducive to improving the therapeutic effect of MSCs.
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Background information: The mechanical properties of cells are essential to maintain their proper functions, and mainly rely on their cytoskeleton. A lot of attention has been paid to actin filaments, demonstrating their central role in the cells mechanical properties, but much less is known about the participation of intermediate filament networks. Indeed the contribution of IFs, such as vimentin, keratins, and lamins, to cell mechanics has only been assessed recently. We study here the involvement of desmin, an intermediate filament specifically expressed in muscle cells, in the rheology of immature muscle cells. Desmin can carry mutations responsible for a class of muscle pathologies named desminopathies. Results: In this study, using 3 types of cell rheometers, we assess the consequences of expressing wild-type or mutated desmin on the rheological properties of single myoblasts. We find that the mechanical properties of the cell cortex are not correlated to the quantity, nor the quality of desmin expressed. On the contrary, the overall cell stiffness increases when the amount of wild-type or mutated desmin polymerized in cytoplasmic networks increases. However, myoblasts become softer when the desmin network is partially depleted by the formation of aggregates induced by the expression of a desmin mutant. Conclusions: We demonstrate that desmin plays a negligible role in the mechanical properties of the cell cortex but is a determinant of the overall cell stiffness. More particularly, desmin participates to the cytoplasm visco-elasticity. Significance: Desminopathies are associated with muscular weaknesses attributed to a disorganization of the structure of striated muscle that impairs the active force generation. The present study evidences for the first time the key role of desmin in the rheological properties of myoblasts, raising the hypothesis that desmin mutations could also alter the passive mechanical properties of muscles, thus participating to the lack of force build up in muscle tissue. This article is protected by copyright. All rights reserved.
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Recent observation identifies subendothelial (mural) cells expressing MCAM, a specific system of clonogenic, self-renewing, osteoprogenitors (a.k.a, “mesenchymal stem cells”) in the microvascular compartment of post-natal human bone marrow (BM). In this study, we used MCAM/CD146, as a marker to localize, isolate and assay subendothelial clonogenic cells from the microvasculature of postnatal human skeletal muscle. We show here that these cells share with their BM counterpart, anatomic position (subendothelial/adventitial) and ex vivo clonogenicity (CFU-Fs). When assayed under the stringent conditions, these cells display a high spontaneous myogenic potential (independent of co-culture with myoblasts or of in vivo fusion with local myoblasts), which is otherwise only attained in cultures of satellite cells. These muscle-derived mural cells activated a myogenic program in culture. Cultured CD146⁺ cells expressed the myogenic factors (Pax7, Pax3 and Myf5), NCAM/CD56, desmin as well as proteins characteristic of more advanced myogenic differentiation, such as myosin heavy chain. In vivo, these cells spontaneously generate myotubes and myofibrils. These data identify the anatomy and phenotype of a novel class of committed myogenic progenitor in human post-natal skeletal muscle of subendothelial cells associated with the abluminal surface of microvascular compartment distinct from satellite cells.
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Sarcopenia (loss of muscle mass and muscle function) is a strong predictor of frailty, disability and mortality in older persons and may also occur in obese subjects. The prevalence of sarcopenia is increased in patients suffering from chronic heart failure. However, there are currently few therapy options. The main intervention is resistance exercise, either alone or in combination with nutritional support, which seems to enhance the beneficial effects of training. Also, testosterone has been shown to increased muscle power and function; however, a possible limitation is the side effects of testosterone. Other investigational drugs include selective androgen receptor modulators, growth hormone, IGF-1, compounds targeting myostatin signaling, which have their own set of side effects. There are abundant prospective targets for improving muscle function in the elderly with or without chronic heart failure, and the continuing development of new treatment strategies and compounds for sarcopenia and cardiac cachexia makes this field an exciting one.
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Purpose of review: Severe exercise intolerance and early fatigue are hallmarks of heart failure patients either with a reduced (HFrEF) or a still preserved (HFpEF) ejection fraction. This review, therefore, will provide a contemporary summary of the alterations currently known to occur in the skeletal muscles of both HFrEF and HFpEF, and provide some further directions that will be required if we want to improve our current understanding of this area. Recent findings: Skeletal muscle alterations are well documented for over 20 years in HFrEF, and during the recent years also data are presented that in HFpEF muscular alterations are present. Alterations are ranging from a shift in fiber type and capillarization to an induction of atrophy and modulation of mitochondrial energy supply. In general, the molecular alterations are more severe in the skeletal muscle of HFrEF when compared to HFpEF. The alterations occurring in the skeletal muscle at the molecular level may contribute to exercise intolerance in HFrEF and HFpEF. Nevertheless, the knowledge of changes in the skeletal muscle of HFpEF is still sparsely available and more studies in this HF cohort are clearly warranted.
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Background: A greater understanding of the different underlying mechanisms between patients with heart failure with reduced (HFrEF) and with preserved (HFpEF) ejection fraction is urgently needed to better direct future treatment. However, although skeletal muscle impairments, potentially mediated by inflammatory cytokines, are common in both HFrEF and HFpEF, the underlying cellular and molecular alterations that exist between groups are yet to be systematically evaluated. The present study, therefore, used established animal models to compare whether alterations in skeletal muscle (limb and respiratory) were different between HFrEF and HFpEF, while further characterizing inflammatory cytokines. Methods and results: Rats were assigned to (1) HFrEF (ligation of the left coronary artery; n=8); (2) HFpEF (high-salt diet; n=10); (3) control (con: no intervention; n=7). Heart failure was confirmed by echocardiography and invasive measures. Soleus tissue in HFrEF, but not in HFpEF, showed a significant increase in markers of (1) muscle atrophy (ie, MuRF1, calpain, and ubiquitin proteasome); (2) oxidative stress (ie, higher nicotinamide adenine dinucleotide phosphate oxidase but lower antioxidative enzyme activities); (3) mitochondrial impairments (ie, a lower succinate dehydrogenase/lactate dehydrogenase ratio and peroxisome proliferator-activated receptor-γ coactivator-1α expression). The diaphragm remained largely unaffected between groups. Plasma concentrations of circulating cytokines were significantly increased in HFrEF for tumor necrosis factor-α, whereas interleukin-1β and interleukin-12 were higher in HFpEF. Conclusions: Our findings suggest, for the first time, that skeletal muscle alterations are exacerbated in HFrEF compared with HFpEF, which predominantly reside in limb, rather than in respiratory, muscle. This disparity may be mediated, in part, by the different circulating inflammatory cytokines that were elevated between HFpEF and HFrEF.
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Skeletal muscle has an extraordinary regenerative capacity due to the activity of tissue-specific muscle stem cells. Consequently, these cells have received the most attention in studies investigating the cellular processes of skeletal muscle regeneration. However, efficient capacity to rebuild this tissue also depends on additional cells in the local milieu, as disrupting their normal contributions often leads to incomplete regeneration. Here, we review these additional cells that contribute to the regenerative process. Understanding the complex interactions between and among these cell populations has the potential to lead to therapies that will help promote normal skeletal muscle regeneration under conditions in which this process is suboptimal.
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Cardiac metabolism is highly adaptive to changes in fuel availability and the energy demand of the heart. This metabolic flexibility is key for the heart to maintain its output during the development and in response to stress. Alterations in substrate preference have been observed in multiple disease states; a clear understanding of their impact on cardiac function in the long term is critical for the development of metabolic therapies. In addition, the contribution of cellular metabolism to growth, survival, and other signalling pathways through the generation of metabolic intermediates has been increasingly noted, adding another layer of complexity to the impact of metabolism on cardiac function. In a quest to understand the complexity of the cardiac metabolic network, genetic tools have been engaged to manipulate cardiac metabolism in a variety of mouse models. The ability to engineer cardiac metabolism in vivo has provided tremendous insights and brought about conceptual innovations. In this review, we will provide an overview of the cardiac metabolic network and highlight alterations observed during cardiac development and pathological hypertrophy. We will focus on consequences of altered substrate preference on cardiac response to chronic stresses through energy providing and non-energy providing pathways.
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Purpose of review: The review is an update on recent research investigating the role of skeletal muscle stem cells (also known as satellite cells) during muscle fiber regeneration and growth in animal and human skeletal muscle, with an emphasis on their role in age-related sarcopenia. Recent findings: Studies indicate clear impairments in satellite cell function with aging, resulting in an impaired muscle fiber regenerative response. The autophagy-mediated switch to an irreversible presenescent state of geriatric satellite cells appears to play a key role in age-related impaired satellite cell function. In addition, inadequate muscle fiber vascularization may be a crucial factor underlying impaired regulation of satellite cells in older adults. Controversy remains on the actual contribution of satellite cells to the development of sarcopenia in later life, this clearly requires further research. Nevertheless, exercise training remains to be a potent intervention strategy, mediated through satellite cells or not, to counteract the ill effects of sarcopenia. Summary: Although important strides are made investigating the importance of satellite cells in the development and/or treatment of sarcopenia, the idea that satellite cell function is a therapeutic target to treat sarcopenia remains controversial.
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Introduction: Atrial and B-type Natriuretic Peptides (NP) are cardiac hormones with potent cardiovascular and metabolic effects. They signal through the NPRA/cGMP system and are inactivated by a clearance receptor NPRC and neutral endopeptidases (NEP). Recombinant ANP and BNP are currently used as drug treatment for acute decompensated congestive heart failure. Recent literature indicate that a defective NP system is linked to obesity and predict the risk of type 2 diabetes (T2D). Areas covered: This article reviews recent epidemiological, clinical and preclinical evidences that NP system deficiency may be causal of obesity and T2D. The molecular mechanisms of the NP pathway in several metabolic target tissues are presented. The therapeutic potential of NP in obesity and T2D is discussed. Expert opinion: Targeting the NP pathway may offer a novel therapeutic avenue for the management of obesity and T2D. The benefit/risk of drugs increasing circulating NP levels by blocking NPRC and NEP, and/or enhancing NPRA signaling should be assessed in obese and type 2 diabetic individuals.