# Age-related changes in longitudinal and transversal elastic properties of murine skeletal muscle fibers

Vincent Dupres, Philippe Pouletaut, Mashhour Chakouch, Alexandra Josse, John R Hawse, Sabine F Bensamoun

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## To cite this version:

Vincent Dupres, Philippe Pouletaut, Mashhour Chakouch, Alexandra Josse, John R Hawse, et al. Age-related changes in longitudinal and transversal elastic properties of murine skeletal muscle fibers. Innovation and Research in BioMedical engineering, 2025, 46 (5), pp.100908. 10.1016/j.irbm.2025.100908. hal-05191767

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<figure><img src="image_2.png" /></figure>
Age-related changes in longitudinal and transversal elastic properties of murine
skeletal muscle fibers

Vincent Dupres¹, Philippe Pouletaut², Mashhour Chakouch², Alexandra Josse², John R. Hawse³, Sabine F. Bensamoun²

¹ Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 – UMR9017 - CIIL
- Center for Infection and Immunity of Lille, F-59000 Lille, France

²Université de technologie de Compiègne, CNRS, BMBI (Biomechanics and Bioengineering), Compiègne, France

³Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA

Corresponding author:

Sabine

F.

Bensamoun

Université de technologie de Compiègne, Centre de recherche Royallieu, CS 60 319, 60 203
Compiègne Cedex, France

Tel: (33) 03 44 23 43 90

E-mail address: sabine.bensamoun@utc.fr
## Highlights

* Longitudinal stresses for soleus and EDL muscle fibers decreased with age in mice

* Accentuated decreases in longitudinal stresses were observed for EDL muscle fiber

* Transversal stiffness revealed no changes for soleus or EDL fibers with age in mice

* AFM tests may be used for quantification of transversal stiffness of the endomysium

## Graphical abstract

<figure><img src="image_3.png" /></figure>
**Abstract**

*Purpose:* Aging causes notable effects on skeletal muscle structure and function which accumulate over time to negatively impact quality of life in the elderly. To better understand how aging impacts the mechanical properties of muscle as a function of age, a series of studies were conducted, using mice as a model system, to assess responses to longitudinal and transversal stresses at a single muscle fiber resolution.

*Methods:* Muscle fibers isolated from primarily slow-twitch fibred muscle (soleus) and a primarily fast-twitch fibred muscle (extensor digitorum longus, EDL) were extracted from young (3-month, N=5) and old (21-month, N=5) animals. Passive mechanical tests (ramp, relaxation) were performed in the longitudinal direction at 50% strain, and Atomic Force Microscopy (AFM) tests were applied transversally. Longitudinal stresses (dynamic (σ<sub>d</sub>), instantaneous (σ<sub>i_Ramp</sub>) and static (σ<sub>s_Relax</sub>)) and the Young's modulus were measured in both directions.

*Results:* For both types of fibers, longitudinal stresses were shown to be significantly decreased (*p* < 0.001) in aged mice, with accentuated decreases observed for the EDL. Transversal AFM tests were used to elucidate the cartographies of elasticity and revealed no global differences between young and old animals for either fiber type. Regardless of age, decreased mechanical properties were universally observed for **EDL fibers** compared to **soleus fibers**.

*Conclusion:* These findings highlight the potential of AFM to quantify transversal stiffness of the endomysium and have begun to better delineate the impacts of aging on the mechanical characteristics of skeletal muscle at single fiber resolution.

**Keywords:** Age, Endomysium, Longitudinal passive test, Muscle fiber, Transversal AFM test
# 1. Introduction

Aging itself is not defined as a disease, but rather a normal biological process that is a primary risk factor for many chronic diseases and conditions. As with other tissues, aging is known to modify the structural, biochemical, cellular and functional properties of skeletal muscle [1]. Among these changes, a progressive loss of muscle mass and strength, referred to as sarcopenia, is particularly significant. Sarcopenia contributes substantially to decreased quality of life [2], functional decline, and increased risks of falls or injuries in the elderly population [3]. Importantly, muscle strength is a strong predictor of longevity and functional independence in older adults. Skeletal muscle can be described as a “nested” tissue where several similar elements (fascicule, fiber), but of decreasing size, are contained within one another to form a large structure that works in unison with its individual parts [4]. Whole primal muscles, as well as the fascicles and individual muscle fiber, are encased with collagen-rich connective tissue called the epimysium, perimysium and endomysium respectively [5].

With aging, muscle undergoes profound changes in composition and mechanical properties. Hill et al. [6] demonstrated an increase in mouse body mass, soleus and EDL muscle mass, atrophy of the EDL, and a greater loss of muscle quality in the soleus compared to EDL in aged female animals. One of the most notable alterations is the increase in collagen cross-linking, which contributes to a progressive stiffening of the intramuscular connective tissue. These cross-links, both enzymatic (lysyl-oxidase derived) and non-enzymatic (advanced glycation end-products), accumulate especially in slow-twitch postural muscles [7-8]. They reduce extracellular matrix (ECM) turnover, alter passive mechanical properties, and persist even in decellularized matrices from aged muscle, highlighting their long-lasting impact on tissue structure and regenerative potential. Also, aging is associated with increased type I collagen and reduced levels of hyaluronan and elastic fibers, further contributing to the decline in tissue elasticity and adaptability [9-11].
Although research characterizing the skeletal muscle ECM in the context of aging remains limited, several studies reported structural and molecular alterations. In the epimysium, collagen fibers become stiffer in old rats, despite no changes in ultrastructure or fibril size [12]. In the perimysium and endomysium, collagen shows reduced tortuosity and increased alignment, leading to greater stiffness and altered anisotropy [13]. These structural changes are supported by transcriptomic data showing increased expression of ECM remodeling and fibrillar collagen genes with age [14].

To further explore the effects of aging on mechanical properties of muscle, longitudinal mechanical test, such as stretch tests applied in the direction of the muscle fiber, have been performed at the macroscopic scale (i.e on the whole muscle). Specifically, such mechanical tests have revealed increased whole muscle stiffness with age [15]. Moreover, impaired lateral transmission of force with age has also been reported, and were concluded to be caused by structural changes in the epimysium [16] with collagen accumulation [5]. A recent study by our group demonstrated scale-related changes in the elastic properties of murine skeletal muscle [17]. Finally, at the microscopic scale, longitudinal tests performed on single skeletal muscle fibers have been used as a biomarker of human muscle aging [18] and sarcopenia [19].

To complement the above studies and to develop a more complete characterization of the functional behavior of skeletal muscle at the macro-micro scales, transversal muscle stiffness assessments are expected to provide additional information. However, such studies are extremely rare. Of the few reported in the literature, Lopez et al. [20] used biaxial testing, and demonstrated in the transverse direction a marked increase in muscle stiffness of whole dystrophin-deficient skeletal muscles when compared with control mice. This increase, closely associated with ECM expansion and fibrosis, highlights the significant role of ECM remodeling in transverse mechanical resistance, and supports the relevance of evaluating transversal force transmission of Duchenne muscular dystrophy. In addition, Stearns-Reider et al. [21], using
AFM on decellularized gastrocnemius muscles from young and aged mice, showed that aging induces significant ECM stiffening along with increased fiber alignment. These age-related mechanical alterations highlight the importance of assessing transverse forces to better understand the impact of aging on the muscle microenvironment. AFM was also applied to single rat muscle fibers [22] and cryosection of mdx (muscular dystrophy) mice skeletal muscle [23], confirming its sensitivity in detecting sub-sarcomeric mechanical behavior [22] and abnormal fiber resistance in the case of muscle diseases such as Duchenne myopathy [23]. However, these assessments have not yet been conducted in the context of normal aging. Here, we report a series of findings describing the impact of aging on both longitudinal and transversal mechanical properties of muscle fibers from primarily slow-twitch muscle (soleus) and a primarily fast-twitch muscle (extensor digitorum longus, EDL).

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## 2. Materials and methods

### 2.1. Animal husbandry and muscle fiber isolation

All mice utilized throughout these studies were littermates generated from an in-house breeding colony of congenic C57BL/6 animals. Mice were maintained at 22 ± 2 °C with a light/dark cycle of 12 hours, with free access to food and water. Young (3 months of age, N = 5) and old (21 months of age, N = 5) female mice were employed for analysis. Only female mice were used given our ongoing assessment of this sex as it relates to the biological roles of Klf10 in mediating the biomechanical properties of skeletal muscle that we have published extensively on including [24], from which additional tissue was repurposed for the experiments described here. Then, soleus (N<sub>3-month</sub> = 5, N<sub>21-month</sub> = 5) and EDL (N<sub>3-month</sub> = 5, N<sub>21-month</sub> = 5) muscles were dissected from the left hindlimbs and permeabilized at 4°C using a relaxing solution with different percentages of glycerol (12.5%, 25%, and 50%). Muscle fiber bundles were stored in 50% glycerol relaxing solution at -20°C for 2 weeks. All protocols were approved
76 by the French ethics committee (CREMEAP; Permit Number: APAFIS #8905-
77 2021011109249708).

78 2.2. *Passive mechanical tests performed in the longitudinal direction*

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**Soleus fibers and EDL fibers** were isolated from young (N<sub>soleus</sub> = 17, N<sub>EDL</sub> = 16) and old (N<sub>soleus</sub> = 19, N<sub>EDL</sub> = 18) skinned muscles. First, individual fibers were placed in a small bath (Fig. 1A) filled with a relaxing solution (70 mM potassium propionate, 8 mM magnesium acetate, 5 mM EGTA, 7 mM ATP, 6 mM Imidazole, 10 mM PMSF, 50 mg.L<sup>-1</sup>, Trypsin inhibitor, 4 mg.L<sup>-1</sup> Leupeptine, pH = 7.1) [25]. The extremities were connected to a force transducer (5 mN) on one end and to a motor (Model 1400A; Aurora Scientific, Aurora, Ontario, Canada) on the other end. The fiber length, diameter (φ), and the distance between the successive sarcomeres were measured using a camera (UI-1220LE). Each fiber was set at a sarcomere length of 2.4 µm and a slack length of L<sub>s</sub> ≈ 2 mm, under which diameter measurements were acquired. Subsequently, each fiber underwent two preconditioning tests, based on Kammoun's study [26] applying quasi static stretches, followed by two passive mechanical tests (ramp stretch, stress-relaxation).

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The first passive mechanical test was a ramp stretch (Fig. 2) whereby the fibers were elongated to 150 % L<sub>s</sub> (about 3 mm) with a velocity of 0.033 L<sub>s</sub>.s<sup>-1</sup> (i.e about 0.066 mm.s<sup>-1</sup>) and released at the same velocity, allowing for the measurement of the instantaneous force (F<sub>i_Ramp</sub>) reached at the end of the stretch. From the linear region of the loading curve (between 60 and 80 % of the applied stretch stress-strain curve), the Young's modulus (E) was determined [27].

The second passive mechanical test was a relaxation test in which fibers were rapidly elongated to 150 % L<sub>s</sub> (about 3 mm) at a high velocity of 3.3 L<sub>s</sub>.s<sup>-1</sup> (i.e about 6.6 mm.s<sup>-1</sup>) and released to their original slack length after a 60 s hold. The dynamic force (F<sub>d</sub>), corresponding to the maximal force value, reached at the end of the stretch, the half time of relaxation (τ<sub>1/2</sub>),
and the static force ($F_{s\_Relax}$) measured at the end of the test, before the fiber was released, were determined.

Forces were divided by the anatomical cross-sectional area of the fiber to obtain dynamic ($\sigma_d$), instantaneous ($\sigma_{i\_Ramp}$) and static ($\sigma_{s\_Relax}$) stresses.

## 2.3. *Passive mechanical tests performed in the transversal direction*

Additional soleus fibers (N<sub>3 months</sub> = 4, N<sub>21 months</sub> = 4) and EDL fibers (N<sub>3-month</sub> = 4, N<sub>21-month</sub> = 4) were extracted from the skinned muscles and placed inside the Atomic Force Microscopy (AFM) machine.

All experiments were performed using a commercial Nanowizard III AFM (JPK Instruments, Berlin, Germany) coupled to an inverted optical microscope (AxioObserver.Z1, Zeiss) driven by JPK NanoWizard software 6.4 and Zen Blue 2012. Data were acquired in Quantitative Imaging mode (QI) using PFQNM-LC cantilevers with a nominal spring constant of 0.08 N/m and a 3.5 µm high conical tip with 8 nm tip radius (Bruker, Santa Barbara, CA, USA). Cantilever spring constant and sensitivity were calibrated before each experiment using the thermal fluctuation method of the JPK software, yielding spring constant values ranging from 0.031 to 0.047 N.m<sup>-1</sup>.

Importantly, we ensured perfect immobilization of the sample which is a prerequisite for AFM experiments. This is particularly the case in experiments conducted on muscle fibers where the slightest of movements (vertical or lateral) can generate artifacts during the acquisition of force curves and thus compromise elasticity maps. If we observed any concerns regarding potential fiber movement during an experiment, all resulting data was systematically discarded from the analysis. Fibers were immobilized on a plexiglass support and submersed in a drop of PBS. Bright field imaging was used to identify the area of interest on each fiber.
visual control of the immobility of the fiber was performed using the optical microscope coupled with the AFM instrument (Fig. 1B). This ensured that 1) only Quantitative Imaging (QI) files were recorded in a static condition and 2) the selected region resulted in a structure of successive sarcomeres without any coating of extracellular matrix. Subsequently, the AFM tip was positioned to engage the central portion of the fiber (to ensure a near plane contact surface to avoid curvature effects leading to artifacts) and was set to scan an area of 15 µm x 15 µm through 128 x 128 pixels maps. Moreover, a thresholding was applied on this area to identify the extracellular matrix (endomysium). Force-distance curves were then collected in QI on three areas along each muscle fiber by applying a force trigger of 2 nN with a Z ramp length of 3 µm (i.e. the range between the two extreme positions of the AFM tip), at 200 µm.s⁻¹.

Force-distance curves were analyzed using in-house pyAF (python Atomic Force) software, version 2.0.0. An algorithm based on the fitting of the baseline was used to detect the point of contact on the force curve. A linear fit was made using the baseline of the approach curve. A noise threshold was used to shift the fit along the force axis. The intersection between this shifted fit and the force curve defined the position of the point of contact. Subsequently, the elasticity of fibers was deduced from the Young's modulus which was determined from the indentation portion of the curve using the elastic contact model for conical indenters (named Sneddon model [28]):

$$F = \frac{2E \tan\alpha}{\pi(1 - v^2)} \delta^2$$

where F is the measured force, E the elastic modulus, α the half opening angle of the tip, δ the indentation, and v the sample's Poisson's ratio (set to 0.5, treating fibers as an incompressible material).
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Arrays of force curves in the x and y planes (typically 128 curves x 128 curves) were recorded on areas of a given size (15 µm x 15 µm). From these force mapping scans, the Young's modulus was then estimated for each force curve and displayed as colored pixels that reflect the magnitude of fiber stiffness (elasticity maps).

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## 2.4. Statistical analysis

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From data of the longitudinal tests, box plots were generated by pooling data for each muscle fiber type (soleus vs EDL) (elastic modulus deduced from each individual force curve) and each age. Two-sample Wilcoxon-Mann-Whitney's tests were performed to compare elasticity between ages for soleus/EDL muscle fibers. For all experiments, a *P* value < 0.05 was considered significant.

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# 3. Results

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From a morphological standpoint, the mean diameter (φ) of single skeletal muscle fibers isolated from both the soleus and EDL was increased in aged (Δφ_Soleus = 32.3%, Δφ EDL = 49.7%) mice compared to young animals (Supplementary material, Table 1). The mechanical properties of both **soleus and EDL** muscle fibers performed in the longitudinal direction exhibited significant decreases in the ramp and relaxation tests (Fig. 2) with corresponding decreases in passive stress and Young's modulus (σᵢ_Ramp, E, σd, σs_Relax) as a function of age (Fig. 3). Also, the mean half relaxation time (τ₁/₂) was elevated in aged (Δτ₁/₂_Soleus = 126.7%, Δτ₁/₂ EDL = 151.9%) fibers isolated from both the soleus and EDL muscles (Supplementary material, Table 1). Moreover, the decrease in mechanical parameters as a function of age was more pronounced for **EDL muscle** fibers (from 83.1 % to 87.3 %) compared to **soleus muscle** fibers (from 70.5 % to 78.8 %). Interestingly, both the soleus and EDL fibers isolated from aged muscles exhibited more homogeneous values regarding these mechanical properties compared

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171 to fibers from young mice. When comparing fibers isolated from mice of the same age group,
172 old **EDL fibers** showed a trend towards decreases stresses, and the Young's modulus, compared
173 to old **soleus fibers** (Supplementary material, Table 1). For fibers isolated from young mice, the
174 opposite variation was found when comparing **soleus fibers** to **EDL fibers**.

175 The transversal Young's modulus measured for **soleus fibers** showed no difference as a
176 function of age (E<sub>T_3-month</sub> = 133.8 ± 55.5 kPa vs E<sub>T_21-month</sub> = 141.8 ± 59.1 kPa) (Fig. 4A-B;
177 Fig. 5A). However, a different mechanical response was found for the **EDL fiber** which
178 exhibited an increase in the transversal stiffness with age (E<sub>T_3-month</sub> = 18.5 ± 7.6 kPa vs E<sub>T_21-
179 month</sub> = 49.4 ± 19.7 kPa) (Fig. 4C-D; Fig. 5B). Interestingly, lower mechanical values were found
180 for **EDL fibers** compared to **soleus fibers** regardless of age. This result was clearly reflected by
181 the blue color distribution (Fig. 4C-D) for the EDL fibers compared to the warmer colors (from
182 green to red) for the soleus fibers (Fig. 4 A-B). Of note, a high standard deviation was found
183 due to the non-homogeneous distribution of elastic data represented by the different
184 components present within the muscle fibers (Z disks, myofibrils).

185 AFM tests were performed in the presence of endomysium tissue located around both types
186 of muscle fibers isolated from young and old mice. An example of topographic images (Fig. 6,
187 top row) obtained on a single endomysium tissue, was clearly represented with a lighter
188 structure and identified as a white region after thresholding (Fig. 6, bottom row). Based on this
189 selected image, it is assumed that the transversal stiffness of the endomysium was higher for
190 the **soleus fiber** (E<sub>T_3-month</sub> = 977 ± 457 kPa vs E<sub>T_21-month</sub> = 608 ± 249 kPa) compared to the
191 **EDL fiber** (E<sub>T_3-month</sub> = 125 ± 20 kPa vs E<sub>T_21-month</sub> = 136 ± 28 kPa) regardless of age. The
192 corresponding cartography of elasticity (Fig. 6, middle row) illustrated the potential of the AFM
193 test to differentiate the elastic properties of the endomysium and the muscle fiber.

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## 4. Discussion

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Due to medical advances, the human population as a whole continues to age [29] and a better understanding of the impact of age on the mechanical properties of many tissues, and particularly skeletal muscle tissue, is becoming increasingly more important for the assessment of muscle quality for a variety of indications. Previous experimental and numerical studies have characterized skeletal muscle fibers from a mechanical standpoint, however nearly all such studies have only considered assessment of mechanical properties in the longitudinal direction of the myofibrils, an orientation in which titin filaments run [30]. However, muscle fiber is also composed of important transversal component such as the Z discs and desmin filaments which are involved in maintaining the structural integrity of myofibrils. Thus, in order to have a comprehensive understanding of the structural effect of aging on skeletal muscle, the longitudinal and transversal mechanical properties should be characterized. Therefore, we sought to provide novel mechanical information in which we have compared both longitudinal and transversal mechanical tests on soleus and EDL muscle fibers isolated from the same mice among two different age groups (young: 3 months; and old: 21 months).

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The longitudinal stretch tests were commonly performed at the microscopic scale due to the accessibility of the MTS device compared to the AFM technology which is currently primarily applied to the study of the mechanical properties of epithelial cells, often in relation to pathologies such as cancer. Other points that hinder the assessment of transversal tests relate to difficulties in immobilizing the muscle fibers during the test and their subsequent characterization only under passive conditions. To tackle these limitations, improvements could be made with regard to: 1) the design of a specific device to better stabilize the fiber; 2) the measurement of stiffness at different sarcomeric lengths; and 3) the quantification of the contractile behavior of the transversal components by performing the indentation after the steady state of contraction [31].
In the present study, age-related changes in the longitudinal mechanical properties of skeletal
muscle fibers were observed. Indeed, a significant increase in fiber diameter, including the thin
thickness of ECM (endomysium), between 3 month and 21 month old animals was found for
both **soleus and EDL fibers** which was correlated with a significant decrease in the passive
mechanical properties (static/dynamic/instantaneous stresses and Young's modulus). In
addition, increases in the half relaxation time for the aged fibers were in agreement with Lim's
study [19]. An analysis of the size and amount of titin and desmin isoforms could be
investigated to support these mechanical findings [32]. Further, it is possible that the larger
percent decrease observed for the **EDL fiber** between 3 month and 21 month old animals
compared to fibers isolated from the soleus may be due to a diminished solicitation of the active
fibers, and/or a modification of the endomysium composition. Similar decreases in passive
mechanical properties between 4 month and 12 month old **rat soleus fibers** have also been
reported [33] using a similar experimental methodology. It is expected that this result would be
further accentuated as animals continue to age. In addition, Lim's study [34] showed a higher
passive force and viscoelastic properties for old human muscle fibers. This conclusion was
obtained from successive relaxation tests applying a slow velocity (0.1 µm.s⁻¹) and a sarcomere
length increment of 0.2 µm. The present study applied a highest velocity (66 µm.s⁻¹) and a
higher single stretch length (1 mm) of the mouse fiber. Thus, as stated in the literature, Binder-
Markey et al. [35] showed that the comparison of data between studies was difficult due to the
variability of the experimental protocols [36], the species (Human [18], rabbit [25], rat [15],
mice [17], frog [37]) and the age [38]. Concerning the comparison of the types of fibers at the
same age, the present study is in agreement with Lim's study [34] which showed no significant
difference for all of the mechanical parameters assessed between human fiber types. Taken
together, these results indicate similar passive state-related outcomes due to age regardless of
fiber type or species.
The present study primarily investigated the passive mechanical properties of single fibers to expound upon our understanding of how the aging process impacts these variables. A limitation of the present work relates to the lack of contractile analysis. However, numerous studies have previously reported such active tests with comparable protocols, because well-established concentrations of calcium are used, leading the possibility to use existing data from the literature [19]. To complement the longitudinal passive/active tests, here we additionally provide transversal passive mechanical properties to further understand the impact of the aging process. Interestingly, EDL fibers exhibited lower transversal stiffness compared to soleus fibers, regardless of age, revealing different structural organization as a function of fiber type which conferred different transversal mechanical stability. This additional information was not revealed with the longitudinal stretching tests implicating the necessity to perform such assays in the transversal direction. In addition, the similar transversal properties observed for the soleus fiber at both age groups indicate that the primary effect of aging on soleus fibers impacts longitudinal mechanical properties. Of note, the AFM tests were applied at a small depth of indentation (between 15 nm and 45 nm) above the dystrophin which is linked to the glycoprotein complex within the sarcolemma [20], and a larger indentation would be necessary to confirm this surface result. It is difficult to compare the transversal data with the literature due to vast differences in experimental protocols and especially since the various AFM-based studies are generally carried out using different tips and models which also vary according to the geometry of the tip. The small strain applied with the AFM test compared to the high strain used during the stretching tests renders the comparison of data between the two directions not possible.

During the AFM experiments performed on the surface of the fibers, it was also possible to realize indentation tests on some remaining pieces of endomysium left after the skinning. In the literature, there is a lack of AFM-based muscle ECM mechanics compared to longitudinal stiffness of each layer of ECM, due to technical and experimental challenges. The preliminary
272 AFM results revealed a higher stiffness for the extracellular matrix (ECM) surrounding the
273 soleus fibers compared to the EDL fibers. Nevertheless, this result cannot be related to the ECM
274 longitudinal stiffness [39], or the effect of age, given the results obtained from the present study.
275 These preliminary results pave the way to a possible experimental quantification of the ECM
276 stiffness surrounding the muscle fiber. In perspective, immunohistochemistry or biochemical
277 characterization of the skinned fiber samples would allow to have further information about the
278 types of fiber (I, IIa), and the content of collagen I and IV in function of age.

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280 **5. Conclusion**

281 This study has provided complementary longitudinal and transversal mechanical assessment
282 of skeletal muscle fibers as a function of both age and fiber type (soleus vs EDL) and these data
283 have allowed for a better understanding of the effects of aging skeletal muscle mechanical
284 properties. In addition, these experimental data could be implemented in the development of a
285 more biofidelic numerical model of the muscle at different scales [39].

286

287 **Acknowledgements**

288 This work was founded by CNRS internal resources. We wish to thank the Mayo Foundation
289 for the support of the experimental work.

290

291
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439 **Figure legends**

440

441 **Fig. 1.** Experimental set up (A) for the application of passive mechanical tests (ramp and relaxation) in the longitudinal direction of the muscle fiber (i.e along the myofibril), and 442 experimental set up (B) coupling an optical microscope, to visualize the muscle fiber, with the 443 AFM tip to apply a transversal force. 444

445 **Fig. 2.** Representative stress vs time curves obtained for a ramp test and a relaxation test 446 with a zoom of the initial stress, for a single fiber, isolated from a representative 3-month and 447 21-month animal, extracted from soleus (A) and extensor digitorum longus (EDL) (B). 448

449 **Fig. 3.** Representation of the boxplots showing the instantaneous stress (σᵢ_Ramp), static stress (σs_Relax), dynamic stress (σd) and Young's modulus (E) obtained with both longitudinal tests (ramp, relaxation) performed on **soleus and extensor digitorum longus (EDL) muscle fibers** at different ages (3-month vs 21-month). Variations in mean passive stress and Young's modulus with age are indicated with an arrow and expressed as a percentage. ***: p < 0.001 (Mann-Whitney's test). 450 451 452 453

454 **Fig. 4.** AFM cartographies of transversal Young's modulus (E_T) obtained on **soleus and extensor digitorum longus (EDL) fibers** isolated from 3-month and 21-month old animals. Fiber structure with the successive sarcomeres (S) is represented in A. 455 456

457 **Fig. 5.** Bivariate histograms of transversal Young's modulus (E_T), obtained with AFM test 458 of selected fibers from the soleus (A) and extensor digitorum longus (B) muscles of 3-month- 459 old and 21-month-old mice. The mean value is represented by a vertical dashed line. 460

460 **Fig. 6.** Representative cartographies of topography (top row), transversal Young's modulus 461 E_T (middle row), and thresholded images of E_T (bottom row) obtained on a soleus and extensor 462 digitorum longus (EDL) fibers isolated from a 3-month and 21-month old mouse. 463
<figure><img src="image_4.png" /><figcaption>Fig. 1</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Fig. 2</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Fig. 3</figcaption></figure>

<figure><img src="image_7.png" /></figure>
<figure><img src="image_8.png" /><figcaption>Fig. 4</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>Fig. 5</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Fig. 6</figcaption></figure>
## Supplementary Material

479

**Table 1.** Values of the passive mechanical parameters (fiber diameter: φ, instantaneous stress: σᵢ_Ramp, Young's modulus: E, static stress: σₛ_Relax, dynamic stress: σ₉ and half relaxation time) obtained with the ramp and relaxation tests for the young (3-month) and old (21-month) soleus fibers and extensor digitorum longus (EDL) fibers. Data are presented as mean and standard error of the mean, with stiffness variation (%) of 21-month values in comparison with 3-month values. *: p < 0.05. **: p < 0.01. ***: p < 0.001 (Wilcoxon-Mann-Whitney's test).

<table><thead><tr><th rowspan="2"></th><th colspan="2">Soleus</th><th colspan="2">EDL</th></tr><tr><th>3-month<br>(n = 17)</th><th>21-month<br>(n = 19)</th><th>3-month<br>(n = 16)</th><th>21-month<br>(n = 18)</th></tr></thead><tbody><tr><td>φ (μm)</td><td>33.8 ± 1.3</td><td>44.8 ± 1.4***</td><td>32.9 ± 1.4</td><td>49.2 ± 2.7***</td></tr><tr><td>Δφ (%)</td><td></td><td>32.3</td><td></td><td>49.7</td></tr><tr><td>σᵢ_Ramp<br>(kPa)</td><td>212.6 ± 20.4</td><td>45.1 ± 4.1***</td><td>286.9 ± 34.0</td><td>36.4 ± 3.8***</td></tr><tr><td>Δσᵢ_Ramp<br>(%)</td><td></td><td>- 78.8</td><td></td><td>- 87.3</td></tr><tr><td>E (kPa)</td><td>602.7 ± 60.6</td><td>132.1 ± 11.2***</td><td>863.4 ± 126.6</td><td>106.9 ± 11.2***</td></tr><tr><td>ΔE (%)</td><td></td><td>- 78.1</td><td></td><td>- 87.6</td></tr><tr><td>σₛ_Relax<br>(kPa)</td><td>108.9 ± 11.0</td><td>27.0 ± 2.0***</td><td>147.8 ± 19.9</td><td>20.9 ± 2.0***</td></tr><tr><td>Δσₛ_Relax<br>(%)</td><td></td><td>- 75.2</td><td></td><td>- 85.8</td></tr><tr><td>σ₉ (kPa)</td><td>305.6 ± 26.1</td><td>90.1 ± 6.4***</td><td>396.9 ± 47.1</td><td>67.3 ± 7.1***</td></tr><tr><td>Δσ₉ (%)</td><td></td><td>- 70.5</td><td></td><td>- 83.1</td></tr><tr><td>τ₁/₂ (ms)</td><td>64.9 ± 2.2</td><td>147.1 ± 23.1***</td><td>74.2 ± 7.6</td><td>186.9 ± 39.6**</td></tr><tr><td>Δ τ₁/₂ (%)</td><td></td><td>126.7</td><td></td><td>151.9</td></tr></tbody></table>

486