Trends inOpinion
A perspective on muscle phenotyping in
musculoskeletal research
Endocrinology & MetabolismInes Foessl1,7,*, Cheryl L. Ackert-Bicknell2,7, Erika Kague3,7, Faidra Laskou4,7, Franz Jakob5,
David Karasik6, Barbara Obermayer-Pietsch1, and Co-authors8Nerea Alonso, Åshild Bjørnerem, Maria Luisa Brandi, Björn Busse, Ângelo Calado, Alper Han Cebi,
Maria Christou, Kathleen M. Curran, Jannie Dahl Hald, Maria Donatella Semeraro, Eleni Douni,
Emma L. Duncan, Ivan Duran, Melissa M. Formosa, Yankel Gabet, Samuel Ghatan, Artemis Gkitakou,
Eva Maria Hassler, Wolfgang Högler, Terhi J. Heino, Gretl Hendrickx, Patricia Khashayar, Douglas P. Kiel,
Fjorda Koromani, Bente Langdahl, Philippe Lopes, Outi Mäkitie, Antonio Maurizi, Carolina Medina-Gomez,
Evangelia Ntzani, Claes Ohlsson, Vid Prijatelj, Raquel Rabionet, Sjur Reppe, Fernando Rivadeneira,
Gennady Roshchupkin, Neha Sharma, Kent Søe, Unnur Styrkarsdottir, Pavel Szulc, Anna Teti, Jon Tobias,
Amina Valjevac, Jeroen van de Peppel, Bram van der Eerden, Bert van Rietbergen, Tatjana Zekic, and
M. Carola ZillikensHighlights
There is a close relationship between
muscle loss (sarcopenia) and bone loss
(osteoporosis), integrated in the term
‘osteosarcopenia’; however, detailed
musclephenotyping is under-represented
in musculoskeletal studies.
Muscle phenotyping in both humans
and animal models, such as mice
and zebrafish, requires validation and
agreement.
Parameters ofmuscle strength and func-
tion should be measured in health and
disease while considering age and sexMusculoskeletal research should synergistically investigate bone and muscle
to inform approaches for maintaining mobility and to avoid bone fractures. The
relationship between sarcopenia and osteoporosis, integrated in the term
‘osteosarcopenia’, is underscored by the close association shown between
these two conditions in many studies, whereby one entity emerges as a predictor
of the other. In a recent workshop of Working Group (WG) 2 of the EU Coopera-
tion in Science and Technology (COST) Action ‘Genomics of MusculoSkeletal
traits Translational Network’ (GEMSTONE) consortium (CA18139), muscle char-
acterization was highlighted as being important, but currently under-
recognized in the musculoskeletal field. Here, we summarize the opinions of
the Consortium and research questions around translational and clinical musculo-
skeletal research, discussingmuscle phenotyping in human experimental research
and in two animal models: zebrafish and mouse.differences.
Animal models, such as mice and
zebrafish, add valuable information
and detail on muscle development and
function, but need careful assessment
to provide translatable insights.
1Division of Endocrinology and
Diabetology, Department of Internal
Medicine, Medical University of Graz,
Graz, Austria
2Colorado Program for Musculoskeletal
Research, Department of Orthopedics,
University of Colorado, Aurora, CO, USA
3Centre for Genomic and Experimental
Medicine, Institute of Genetics and
Cancer, University of Edinburgh,
Edinburgh, UKThe global impact of musculoskeletal health
Musculoskeletal disorders affect physical function andmetabolic homeostasis, thereby impacting
several other organ systems [1]. Their ultimate importance is captured by the ‘Global Burden of
Disease’ study [2], which ranked five musculoskeletal conditions (low back pain, neck pain,
falls, osteoarthritis and ‘other musculoskeletal’ diseases) among the top 11 leading causes of
disability.
Osteoporosis (see Glossary) is the systemic deterioration of bone tissue, characterized by low
bonemass andmicroarchitectural impairment, causing an increased fracture risk. Corresponding
with low bone mass and impaired bone architecture in osteoporosis, sarcopenia has been
defined as an age-associated loss of muscle mass, strength, and/or function, and is a major
contributor to falls, a key event portending worseningmusculoskeletal health and the subsequent
injuries [3,4]; sarcopenia is also a predictor of fracture risk in both men and women, and478 Trends in Endocrinology & Metabolism, June 2024, Vol. 35, No. 6 https://doi.org/10.1016/j.tem.2024.01.004
Crown Copyright © 2024 Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
4MRC Lifecourse Epidemiology Centre,
Southampton, UK
5Bernhard-Heine-Centrum für
Bewegungsforschung und Lehrstuhl für
Funktionswerkstoffe der Medizin und der
Zahnheilkunde, Würzburg, Germany
6Azrieli Faculty of Medicine, Bar-Ilan
University, Ramat Gan, Israel
7These authors contributed equally.
8Co-authors are listed in the back matter
and their affiliations are available in the
supplemental information online.
*Correspondence:
Ines.foessl@medunigraz.at (I. Foessl).
Trends in Endocrinology &Metabolismquadriceps weakness predicts mortality after a fragility fracture [5,6]. The close correlation be-
tween osteoporosis and sarcopenia is reflected in the term ‘osteosarcopenia’. While the age-
related deterioration of the musculoskeletal systemmight not be completely avoidable, identifica-
tion of at-risk groups, timely diagnoses, and initiation of appropriate prophylaxis and treatment
can prevent rapid deterioration [7]. Therefore, musculoskeletal research should investigate
bone and muscle characteristics synergistically to generate comprehensive directives for main-
taining mobility and avoiding fractures.
The COST Action GEMSTONE (CA18139) is an EU initiative that aims to make knowledge, data,
and technologies available to a wider range of researchers, to help generate additional discoveries
in musculoskeletal science. Of the six WGs in GEMSTONE, members of WG2 (phenotyping) re-
cently summarized and discussed developments and open questions in skeletal phenotyping [8],
giving a comprehensive review of techniques available for clinical and basic research applications in
bone tissue. In a recent workshop, detailed muscle characterization was highlighted as an impor-
tant but often neglected part of phenotyping. The Consortium members presented techniques of
muscle phenotyping and engaged in discussions regarding potential knowledge gaps, both topics
that may be valuable to skeletal experts.Box 1. Methods of muscle phenotyping
Human phenotyping
Physical measurements include muscle volume and function using the surrogate parameters mass, strength and, perfor-
mance [10].
Muscle mass assessment includes imaging techniques, such as computed tomography (CT), magnetic resonance
imaging (MRI), and dual X-ray absorptiometry (DXA). The appendicular lean mass measured by DXA was part of the initial
definition of sarcopenia. Bioelectrical impedance analysis based on electric current attenuation and calculations based on
anthropometric measurements can approximate muscle mass.
Muscle strength in clinical settings is most commonly assessed by hand grip. Impaired results are better associated with
poor mobility and clinical outcomes compared with muscle mass alone [107]. Knee flexion and extension can be assessed
with variable techniques with and without the use of equipment. Peak expiratory flow in people without lung disorders can
determine the strength of the respiratory muscles, but data on its use as a sarcopenia surrogate are limited.
Physical performance can be measured with a variety of tests. Most commonly used are variations of speed tests for gait,
stair climb, and combinations such as the ‘Short Physical Performance Battery’ (SPPB).
Muscle biopsies are not common in clinical use, but can provide invaluable information, usually done and considered
tolerable at the vastus lateralis (largest muscle in the thigh). Muscle biopsy analysis can provide details of morphological
characteristics, gene transcription and expression, and metabolism, allowing identification of the mechanisms of skeletal
muscle loss/sarcopenia [108–111].
Mouse phenotyping
Besides biological and histological analyses of muscles in the mouse, in analogy to humans, muscle mass can be
assessed by imaging usingmicro-CT, MRI, and DXA. Muscle strength as theminimal force required to oppose the forelimb
of a mouse and whole-limb grip can be measured with a grip strength meter. The four-limb hang test is designed to
measure endurance as the time until grip strength exhausts. Exercise capacity and endurance can be tested by treadmill
exhaustion tests [112,113].
Fish phenotyping
Among the strengths of the zebrafish model for microscopy techniques is the transparency of the larvae. Muscle function
via locomotion assay analyzes larval swimming parameters, such as speed, distance traveled, and number of movements.
Commonly performed in larvae 5 days post fertilization, tests include assessing evoked responses by metal touch or
acoustic stimuli and tests assessing swimming within a time frame [114]. The force generated during muscle contraction
can be measured from live larvae subjected to an electrical stimulus, but is technically challenging [115].
Trends in Endocrinology & Metabolism, June 2024, Vol. 35, No. 6 479
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Glossary
Altricial: in bird and mammal biology,
describes the dependence of newly
hatched or born young on adults for
food and protection.
Axial musculature: muscles that
originate on the axial skeleton (i.e.,
bones in the head, neck, and core of the
body).
Bonemineral density (BMD): amount
of mineral in bone tissue, defined as
mass per volume.
Dual X-ray absorptiometry (DXA):
X-ray based method to measure bone
density, considered the gold standard
for osteodensiometry. Capable of
whole-body scans, DXA can also be
used to measure body composition and
fat/lean mass.
Fibro-adipogenic precursors
(FAPs): of mesenchymal stromal cell
with stem cell potential. They are
resident between muscle cells and are
important for muscle regeneration and
homeostasis.
Inbredmouse strains: individuals with
at least 20 generations of inbreeding
(mating brother × sister or offspring ×
parent). Individuals of inbred strains can
be considered genetically identical for
practical purposes.
Myoblast: precursor cells with single
nuclei that fuse to form muscle fibers.
Myofibers: fibrous muscle cells.
Myosin: group of motor proteins
abundant in the sarcomere region of
muscle. They ATP-dependently mediate
actin-based motility.
Myostatin: member of the TGF-βIn this review, we present narrative expert statements on muscle phenotyping in human experi-
mental muscular research. While many insights into human muscular function and its changes
upon aging can be studied from biopsies and by functional assessment of the muscle (Box 1),
model organisms are essential to study the function of genes and their products via genetic
modifications. As a result, two animal models, zebrafish and mouse, are also discussed.
Aging muscle and sarcopenia in humans
Sarcopenia is a heritable trait influenced by lifestyle factors [9]. In the original definition, appendicular
lean mass measured by dual X-ray absorptiometry (DXA)was used alongside grip strength and
gait speed, andmany studies rely on DXA to define sarcopenia.While thesemeasures are predictors
of adverse health events, such as falls, mobility limitation, and mortality, there is nowmore emphasis
on muscle strength (dynapenia), as opposed to muscle mass, as the primary criterion for defining
sarcopenia. However, general consensus on the definitions and assessment of sarcopenia is yet
to be reached [4,10].
Human muscle changes in sarcopenia
Humanmuscle comprises various fibers (Table 1) and specialized cell types [4], and is structurally
and functionally adaptive to neural stimulation, loading, availability of substrates, and hormonal
signals, among others [11]. With age, declining blood flow to skeletal muscles limits the diffusion
of substrates, oxygen, hormones, and nutrients in the tissue, thereby contributing to sarcopenia
and reduced aerobic capacity [12,13].
Comparing older patients who are immobile with those who are mobile can provide valuable
insights into physiological versus pathological muscle aging. Sedentary octogenarians display a
very high abundance of hybrid fibers [14,15], which are transitory intermediates between different
fiber types that are observed during skeletal muscle development, adaptation to exercise,
and aging [16]. Muscle deterioration is accompanied by fiber deformation [17], variations in the
number of myonuclei [18], and imbalance in protein synthesis by muscle cells [19]. For example,
the synthesis of myosin heavy chain declines with age [20], as do myofiber number and size
[21]. Type II fast myofibers responsible for strength are typically more affected compared with
type I slow fibers. Gait speed is strongly influenced by muscle strength and, thus, by the age-Table 1. Main types of human muscle fiber and their characteristics
Characteristic Type I Type IIA Type IIB
Slow Fast Fast
Function Endurance Speed Speed
Endurance High Intermediate Low
Power Low High High
Speed Low High High
Fatigue Low Intermediate High
Myosin-ATPase Low High High
Activity Aerobic Aerobic and anaerobic Aerobic
Capillarization High Intermediate low
Mitochondria High numbers Intermediate numbers Low numbers
Myoglobin High High Low
Color Dark red Red White
Lipid content High Low Low
Creatin phosphate Low High High
480 Trends in Endocrinology & Metabolism, June 2024, Vol. 35, No. 6
superfamily and an important regulator
of muscle mass and function. It acts on
muscle cells and inhibits muscle growth.
Deletion of the gene encoding
myostatin, MSTN, results in extremely
muscular animals.
Osteoporosis: systemic deterioration
of bone tissue, characterized by low
bone mass and microarchitectural
impairment, causing an increased
fracture risk.
Osteosarcopenia: co-occurrence of
osteoporosis and sarcopenia in a
patient.
Phenotype: set of observable
characteristics or traits of an organism.
The phenotype is defined as the result of
the genetic code (genotype) of an
organism and the influence of
environmental factors.
Sarcomere: basic contractile unit of a
myocyte, comprising actin and myosin
filaments.
Trends in Endocrinology &Metabolism
Sarcopenia: degenerative loss of
muscle mass, quality, and strength.
Satellite cells: small multipotent cells
found in mature muscle that reside
between the basement membrane of
the muscle and the muscle fibers, where
they are important for tissue repair.associated reduction of these type II fibers [10,22]. The decline of myofibers with age might be
caused partially bymitochondrial dysfunction driven by decreased neuronal input [23]. Mitochondrial
density is known to varywith physical training and activity. This, alongwith the decrease inmitochon-
drial protein synthesis, may explain increased fatigue and reduced endurance [24–26]. Indeed,
decreased mitochondrial bioenergetic capacity is observed in the transition from physiological to
pathological muscle aging [27].
Satellite cells beneath the basal lamina of skeletal muscle fibers are important for myofibermain-
tenance, and satellite cell numbers decline during aging [28]. However, this loss does not appear
to be causative for sarcopenia in the uninjured muscle, since mice lacking satellite cells show
severe defects in muscle regeneration after injury, but are little affected during normal maintenance
[29,30]. Multipotent human satellite cells were suspected to be driven toward the adipogenic line-
age by adverse stimuli in vitro [31], but this was later refuted since accurate labeling showed them
committed to a myogenic lineage [32]. By contrast, fibro-adipogenic precursors (FAPs), a type
of mesenchymal stromal cell, are necessary for both muscle regeneration and maintaining muscle
homeostasis [33,34]. They respond to proinflammatory signaling and can become senescent cells,
which impair muscle regeneration and support aberrant fibrogenic and adipogenic commitment and
differentiation [35–37]. Adipogenic differentiation of FAPs can be delayed by exercise, whereasmuscle
regeneration can be rescued by senolytic depletion of senescent FAP populations [38]. ‘Fatty infiltra-
tion’ is present in both bone and muscle in osteosarcopenia and is being explored as an indicator
and propagator of disease development. Both intermuscular adipose tissue accumulation (i.e., the ac-
cumulation of adipocytes between the muscle fibers) and intracellular lipid infiltration within the muscle
cells are increased in aging and sarcopenia [39]. Fatty and fibrotic infiltration can be measured by dis-
tinct computed tomography (CT) and magnetic resonance imaging (MRI) approaches, and there is in-
tense ongoing research to determine the extent to which these quantitative measures may serve as
surrogates for impaired muscle function [40]. In addition, resident macrophages are also important
modulators of muscle regeneration currently under investigation [41,42].
Muscle–bone crosstalk
It was long assumed that the interaction between muscle and bone is mainly mechanical, but
evidence for a secretory crosstalk between the two organs is increasing, as comprehensively
reviewed elsewhere [43,44]. Examples of proteins involved in such crosstalk includemyostatin,
a transforming growth factor beta (TGF-β) family member, which negatively regulatesmyoblast
proliferation, thus affectingmuscle mass and strength (althoughwith conflicting results on its age-
related expression) [45–47]. Its downregulation increases muscle mass and osteogenic differen-
tiation of mesenchymal stem cells (MSCs), but its signaling can also lead to inflammation, insulin
resistance, and cancer cachexia, among others [48]. Interleukin (IL) 6 and 7 promote osteoclas-
togenesis [49]. Irisin is mainly secreted from the muscle in response to exercise and its adminis-
tration prevented both bone loss andmuscle atrophy in hind limb-suspendedmice [50]. While the
mechanism involved is not yet fully elucidated, irisin appears to act through integrin receptors
in vitro, blocking osteocyte cell death and stimulating sclerostin expression [51].
Energy metabolism is well linked to muscle and bone. IGF-1 causes muscle hypertrophy and
promotes the differentiation of osteoblasts into osteocytes and, thus, bone formation [52].
Osteocalcin is secreted by bones during endurance exercise and its signaling in myofibers
promotes glucose uptake and the breakdown of glucose into pyruvate [53].
Modeling sarcopenia in mice
As mammals, mice are anatomically similar to humans and their tissues are identical in cellularity,
organization, and function. They have, or can be induced to have, many of the same disorders asTrends in Endocrinology & Metabolism, June 2024, Vol. 35, No. 6 481
Trends in Endocrinology & Metabolism
Figure 1. Animal models of sarcopenia. Figure created with BioRender (www.biorender.com).
Trends in Endocrinology &Metabolismaging humans and, therefore, serve as a model to study age-related processes [54,55]. Only
~1% of human protein-coding genes lack a homolog in mice [56]. Despite the advent of alternative
models, mice remain a preferred choice for molecular studies of muscle aging, pharmacological
tests, and therapeutic interventions designed to slow down or prevent muscle wasting [57,58].
Inbredmouse strains and transgenic mice are valuable to model human aging, including studies
on the musculoskeletal system and sarcopenia in particular (Figure 1) [54,59–61].
What age in human years is my mouse?
Mice progress through the same developmental milestones as humans. They are altricial when
they are born, depending on their mothers for food and protection. A period of rapid postnatal
growth precedes an ordered sexual maturation phase, and a long adulthood with high reproduc-
tive fecundity, followed by reproductive and systemic decline [55]. However, the lengths of each
life phase versus chronological age do not follow a linear relationship between mice and human.
For most inbred strains, median lifespan is just under 3 years [62]. By comparison, the global life
expectancy at birth for humans is currently 71 years for men and 76 years for women (being
slightly higher in industrialized nations) [63]. Data collected over the past 3 months of the natural482 Trends in Endocrinology & Metabolism, June 2024, Vol. 35, No. 6
Trends in Endocrinology &Metabolismlife of a mouse reflect the accumulated pathological burden; thus, it is best to study aging pro-
cesses, such as age-related musculoskeletal decline, from the advent of adulthood to 3 months
before the median lifespan of the strain [64]. Similar to the concept of ‘healthy lifespan’ versus
‘chronological lifespan’ in humans [65], 3 months before the median total lifespan for a given
mouse strain is achieved is considered the end of healthy aging in mice.
In female C57BL/6J (B6) mice, a common laboratory strain [66], sexual maturity occurs relatively
late for inbred strains, at ~5 weeks of age [62]. They can become pregnant as early as 6.8 weeks
of age [67]. Male mice generally produce mature sperm at 5.7 weeks of age, but are not consid-
ered to have completed the pubertal/adolescent phase until ~8.6 weeks of age [68]. The median
lifespan for B6 mice is 901 days for males and 866 days for females [62], diverging from humans,
where women usually outlive men [69]. Assuming puberty at 10 years for girls and 12 years for
boys in humans, extrapolation would suggest that 1 month of healthy adulthood in B6 mice is
operationally equivalent to ~2.6 years of adulthood in humans (averaging for males and females).
Other models use more generalized median lifespans and ages of sexual maturity [70], and may
be used when actual data for a specific mouse strain are missing.
Placing benchmarks around musculoskeletal development and aging highlights why considering
these differences matters when studying musculoskeletal phenotypes in mouse models. Peak
bone mass is reached at ~4 months of age in both males and females of most inbred mouse
strains [71]. In humans, peak bone mineral density (BMD) is reached during the early 20s for
both sexes [72]. Thus, peak BMD in mice largely reflects corresponding human timelines.
Human bone growth is completed between 17 and 22 years of age on average, with epiphyseal
fusion triggered by puberty. By contrast, the long bone epiphyses of mice do not fuse at sexual
maturation: continued femur lengthening in B6 females has been observed up 12 months of age
[71], when the growth plates finally bridge [73]. This would be the equivalent of a human growing
up until their late 30s. In C57BL/6jRj mice, a strain closely related to B6 mice, the mass of major
hind limbmuscles is maintained until 18months of age and then appears to steadily decline, begin-
ning at 22 months of age in males [74], equivalent to ~64 years of age in humans. By contrast,
humans show a slow decline of muscle mass beginning at ~30 years of age until age 60, when
decline accelerates [75]. Therefore, C57BL/6jRj mice might mirror this accelerated decline phase.
Mouse and human physical parameters
For practical purposes, all mice within an inbred strain are genetically identical, allowing for
repeated measures within strains. By using panels of inbred strains, ‘surveys’ across genetic
backgrounds can be conducted allowing for the identification of trends and exploration of rela-
tionships between aging and age-related decline in musculoskeletal health, and highlighting
strain-specific differences that need consideration when interpreting murine musculoskeletal
aging studies. For example, considering measures of muscle mass, in a cross-sectional study
of body composition in 30 inbred strains, lean mass was measured with DXA at 6, 12, and
18 months of age in male and female mice (data set Ackert1, data set number 25046 in the
Mouse Phenome Databasei). The analysis showed no consistent pattern for either gain or loss
of whole-body lean mass. Females of strains such as MRL/MpJ, A/J and DBA/2J consistently
lose lean body mass over time, while other females of 129S1/SvImJ and BALB/cByJ strains con-
sistently increase lean mass, with similar patterns observed in males. These data suggest strong
strain (genetic) differences for the ‘lean mass’ phenotype. Therefore, one needs to consider the
genetic background of the mouse model for the interpretation of studies of age effects on lean
mass. As a further example, considering functional assessments of muscle strength, grip strength
decreases from 12months of age onwards (chronologically ‘equivalent’ to ~38 years in humans) in
male C57BL/6N mice and in an F1 C57BL/6×BALB/c cross [76]. Thus, these strains of mice loseTrends in Endocrinology & Metabolism, June 2024, Vol. 35, No. 6 483
Trends in Endocrinology &Metabolismgrip strength at a slightly older ‘equivalent’ age compared with the peak hand grip strength in
humans, which is at ~30 years [77]. Analysis of data on grip strength in a wider study of 32 different
inbred strains suggests that most male mice lose grip strength between 6 and 12 months of age.
By contrast, female mice show a plateau effect, with grip strength loss not beginning until after
18 months of age (data set Seburn2, data set number 24801 in the Mouse Phenome Databasei).
The phenotypic diversity of grip strength between inbred strains signifies that the trait is highly
genetically determined in mice.
Mice largely share the same major bone and muscle groups as humans. However, there are
specializations of the axial musculature reflecting the need of wild mice to exert quick bursts
of speed and to jump to avoid predators [78]. Muscle fiber excursion and biomechanical properties
of individual muscles used in walking differ down to the molecular level when comparing four-
legged mice with humans [79]. Mice widely express the gene encoding myosin heavy chain IIb
isoform (found in high-force, fast-contracting fibers), whereas, in humans, expression of this
gene was found in only a few muscles as mRNA and was not detected as a protein [80]. During
muscle fiber growth in mice, the addition of myonuclei to fibers precedes myonuclear domain
expansion, whereas, in growing humans, these two processes occur simultaneously [81].
Multiple studies across several tissues have shown that there are substantial differences in gene
regulation between mice and humans, evidenced by a lack of conservation of gene regulation
across cell types and divergences in gene expression patterns in biological pathways [82].
Therefore, it is not surprising that similar issues arise when comparing muscle gene expression
profiles across ages in mice and humans. Although molecular changes affecting the mitochon-
dria, and systemic contributors to aging such as inflammation, do occur in rodents as in humans,
these processes are conserved at the pathway level rather than for individual genes. Furthermore,
age-related changes in muscle gene expression occur progressively in mice, but in two distinct
stages in humans [74], which may contribute to the two phases of muscle loss seen in humans
[75]. A key accelerator of human bone and muscle decline is menopause, which has to be
modeled in mice [83]. Such differences may explain the difficulties translating therapeutic agents
from mice to humans [81].
The above caveats notwithstanding, mice are overall attractive models to study age-related
muscle decline and can be used to model aspects of sarcopenia. Careful consideration of the
genetics is needed because not all inbred strains experience the same age-related changes in
muscle function or mass at the same rate. Chronological age correlated between mice and
humans should not be used to select time points for observations, but instead the model life
stage needs to be appropriately calculated. Lastly, analysis at the pathway level rather than for
individual genes may be key for the translatability of studies between mice and human.
Zebrafish: a model organism for muscle conditions
Zebrafish are small-sized freshwater fish requiring relatively simple and cost-effective mainte-
nance [84,85] with several potential advantages over mammalian models, including their fast
development, transparency, genetic amenability, and the availability of a plethora of reporter
lines (Figure 2). Although the evolutionary divergence of mammals from zebrafish dates to
~450million years ago, zebrafish have the same bone andmuscle cells asmammals. Approximately
80% of human disease-causing genes have at least one copy in zebrafish [86]. Muscle contractile
units corresponding to those in mammals, formed by actin and myosin filaments (sarcomeres),
are found in zebrafish [87]. Specific cell types are also traceable in vivo and can enable the study
of cell interaction dynamics. Zebrafish have extraordinary regenerative capacity in their exoskeleton
and muscles (including in response to heart injuries), serving as models for regeneration; however,484 Trends in Endocrinology & Metabolism, June 2024, Vol. 35, No. 6
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Figure 2. Pros and cons of different animal models for the study of musculoskeletal processes. Figure created
with BioRender (www.biorender.com).
Trends in Endocrinology &Metabolismthis capacity does not halt the development of sarcopenia and osteoporosis during aging [87]. Thus,
although muscle research in zebrafish is lagging behind that in mice, they are an alternative and
promising model system to advance our understanding of the genetics and mechanisms of muscle
conditions and to identify potential therapeutics against age-related sarcopenia.
Embryos and larval states of zebrafish are versatile models in muscle research. They are decreas-
ingly transparent and can be phenotypically assessed in vivo and ex vivo. Larval analysis has been
used in large forward genetic screens aiming to identify genes associated with muscle diseases,
reverse genetics with genetic targeting of specific genes using CRISPR/Cas9, drug screening,
in vivo muscle phenotyping, and in vivo muscle function assessment [88,89]. Muscle fiber mor-
phogenesis can be rapidly analyzed through the anterior–posterior axis of the zebrafish
embryo. Within 24 h post fertilization, all 30 segments of muscles (myotomes) are formed and
the first spontaneous muscle contractions are observed. More differentiated muscle fibers are
located rostrally (toward the head), formed by fused myoblasts, resulting in multinucleated
myofibers, while the posterior developing myotomes harbor developing muscle cells undergoing
elongation and attachment to somite boundaries (myotendinous junctions). When analyzing
zebrafish muscles, the superficial slow-twitch fibers of slower contractions and high endurance,
and the fast-twitch fibers responsible for rapid high intensity contractions, located more internally,
should be considered [90].
Assessments of zebrafish muscle physiology and function
Muscle birefringence, an optical property of muscle, is frequently used for a quick phenotypic
assessment of muscle fibers of zebrafish larvae in vivo and has been intensively used inTrends in Endocrinology & Metabolism, June 2024, Vol. 35, No. 6 485
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Outstanding questions
What could we learn from alternative
model organisms in musculoskeletal
research, such as birds (which have
lightweight yet robust skeletons) or
sloths (which show reduced skeletal
muscle mass despite high strength
and fatigue resistance)?
Can insights (clinical, preclinical, and
‘omic studies) from the bone field be
extrapolated to skeletal muscle
research?
What surrogate parameters should be
used to study musculoskeletal
physiology and disease and how
would these tools be standardized?
Which biomarkers (e.g., laboratory
markers or imaging features) could be
recommended in clinical assessment
and studies?
What would be an appropriate model
of sarcopenia scoring comparable to
FRAX, and what parameters should it
be based on?phenotypic and drug screenings [88,89]. Besides reporter lines to label specific muscle types,
Evans Blue Dye, a vital stain that enters cells with damaged membranes, reveals sarcolemmal
damage in vivo [91]. For ex vivo phenotyping, immunostainings to distinguish slow- (e.g., anti-
F59 antibody) and fast-twitch fibers (e.g., anti-Phalloidin, anti-F20, and anti-F310) are available
[92].Histological sections and electronmicroscopy can also be used for fine and ultra-finemuscle
fiber phenotyping in zebrafish [93].
While most zebrafish muscle research is done in larvae, adult zebrafish phenotyping and disease
modeling comprise an emergent research niche, highly relevant for advancing our understanding
of muscle degeneration during aging. Zebrafish transparency is lost during their transition from
the larval to juvenile stage (during approximately the first month of life). Therefore, the assessment
of zebrafish muscles in 3D can be done ex vivo using enhanced contrast micro-CT (μCT) [94]. As
in larvae, slow muscle fibers are located externally and fast fibers, representing most zebrafish
muscles, are located more internally. Swimming behavior analysis is used as surrogate of muscle
function [94].
Zebrafish show a natural age-related reduction in muscle mass, along with reduced swimming
performance with increasing age, indicative of a decline in muscle function [95–97]. In older
zebrafish, skeletal muscle degeneration is accompanied by increased senescence-associated
β-galactosidase activity, mitochondrial dysfunction, oxidized protein accumulation, as well as
spinal curvature (kyphosis) [98,99]. Similar to mammals, fish have slow and fast muscle fibers;
however, adults are able to restore muscles via hypertrophy [100]. Aged zebrafish still show a re-
duced fiber cross-sectional area, although it is unknown whether this is specific to a fiber type.
Given that the average lifespan of zebrafish is 2–3 years or more, waiting for natural aging for
experimentation is not feasible. Modeling of muscle mass reduction can be achieved by
genetic manipulations and environmental changes. Recently, a sarcopenia phenotype was
induced through a high-fat diet [101]. In addition, mutant models that display premature aging
phenotypes are commonly used to study the biology of sarcopenia.
Although fewer in zebrafish than in mice, various models of skeletal muscle aging have been
reported in recent years with the development of CRISPR/Cas9 technology [102]. In addition,
an age-related muscle phenotype has been identified in zebrafish with mutations in celsr1a
(a non-classical cadherin involved in maintenance of progenitor cells) and tert (telomerase reverse
transcriptase involved in maintenance of telomere length) [103,104]. An age-related sarcopenia
phenotype in zebrafish might be mitigated by a prophylactic exercise regime, whereby an 8-week
swimming exercise regime in zebrafish was sufficient to restore muscle mass in 21-month-old fish
[87]. Exercise training improves swimming performance in young (8–12 months) and middle-aged
(15–20months) fish, but not old fish (25–30months) [98]. This might suggest that, as in the humans,
exercise alone is not sufficient to treat sarcopenia.
Concluding remarks and future perspectives
Clinical muscle phenotyping in human health and disease has significantly progressed over the
past few years, although many questions remain (see Outstanding questions) [40]. There is a
need for an agreement on specific and reproducible measurement parameters in health and
disease, while considering age and sex differences. Such standardization is crucial not only
for enhancing clinical assessments, but also for promoting data exchange and facilitating
research endeavors. Drawing analogies from research on bone disease, the concept of a muscle-
equivalent to the FRAX (a fracture risk assessment tool) score for osteoporosis has been pro-
posed [105], emphasizing the importance of similar initiatives for muscle health. Additionally,
there is a need for the development or refinement of questionnaires, laboratory biomarkers,486 Trends in Endocrinology & Metabolism, June 2024, Vol. 35, No. 6
Trends in Endocrinology &Metabolismand imaging tools, and consolidation of diagnostic criteria for various musculoskeletal dis-
eases [106].
The interaction of preclinical and clinical scientists is a key goal for the GEMSTONE COST action.
Many further aspects emerged following the writing of this review, including the complexity of
measuring interactions between muscles, bones, joints, and tendons both during movement
and at rest, and the need for better definitions of human disease phenotypes. Finally, we antici-
pate that, in addition to advances in clinical and preclinical muscle phenotyping, human aspects,
such as social interactions, movement, and behavioral patterns and their feedback to the muscu-
loskeletal function, will be a focus for future research.
Co-authors
Nerea Alonso, Åshild Bjørnerem, Maria Luisa Brandi, Björn Busse, Ângelo Calado, Alper Han
Cebi, Maria Christou, Kathleen M. Curran, Jannie Dahl Hald, Maria Donatella Semeraro, Eleni
Douni, Emma L Duncan, Ivan Duran, MelissaM Formosa, Yankel Gabet, Samuel Ghatan, Artemis
Gkitakou, Eva Maria Hassler, Wolfgang Högler, Terhi J. Heino, Gretl Hendrickx, Patricia
Khashayar, Douglas P. Kiel, Fjorda Koromani, Bente Langdahl, Philippe Lopes, Outi Mäkitie,
Antonio Maurizi, Carolina Medina-Gomez, Evangelia Ntzani, Claes Ohlsson, Vid Prijatelj, Raquel
Rabionet, Sjur Reppe, Fernando Rivadeneira, Gennady Roshchupkin, Neha Sharma, Kent Søe,
Unnur Styrkarsdottir, Pavel Szulc, Anna Teti, Jon Tobias, Amina Valjevac, Jeroen van de Peppel,
Bram van der Eerden, Bert van Rietbergen, Tatjana Zekic, and M. Carola Zillikens. Affiliations are
available in the supplemental information online.
Acknowledgments
This publication is based upon work from the COST Action GEMSTONE (CA18139), a funding agency for research and in-
novation networks (www.cost.eu). The Origins of Bone and Cartilage Disease Programme analyzed the skeletal phenotypes
of knockout mice generated by the International Mouse Phenotyping Consortium (IMPC) and was funded by a Wellcome
Trust Strategic Award (101123). EK is funded by Versus Arthritis (Career Development Award, 23115).
Declaration of interests
No interests are declared.
Resources
ihttps://phenome.jax.org
Supplemental information
Supplemental information associated with this article can be found online at https://doi.org/10.1016/j.tem.2024.01.004.
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