Shear sensor mechano-signaling determines tendon stiffness and human jumping performance


 Tendons enable movement by transferring muscle forces to the skeleton, and athletic performances critically rely on mechanically-optimized tendons. How load-bearing structures of tendon sense and adapt to physical demands is an open question of central importance to musculoskeletal medicine and human sports performance. Here, with calcium imaging in tendon explants and primary tendon cells we characterized how tenocytes detect mechanical forces and determined collagen fiber-sliding-induced shear stress as a key stimulus. CRISPR/Cas9 screening in human and rat tenocytes identified PIEZO1 as the crucial shear sensor. In rodents, elevated mechano-signaling increased tendon stiffness and strength both in vitro by pharmacological channel activation and in vivo by a Piezo1 gain-of-function mutation. Strikingly, humans carrying the PIEZO1 gain-of-function E756del mutation revealed a 16% average increase in normalized jumping height, with more effective storage of potential energy released during dynamic jumping maneuvers. We propose that PIEZO1-mediated mechano-signaling regulates tendon stiffness and impacts human athletic performance.


Introduction
Tendons connect muscle to bone and experience mechanical forces among the highest acting in the body (1). During movements with strong body acceleration, such as sprinting and jumping, tendons store and return energy in a catapult-like manner (2) and thereby enable the muscle-tendon unit to generate more power than is possible by the muscle alone (3,4). Interestingly, regular high-load exercising increases the mechanical properties of tendons but with little to no change in tendon thickness (5,6). This is con rmed by sprinters displaying tendons with elevated stiffness and strength compared to endurance runners and non-active individuals (7). Moreover, tendon diseases are conventionally treated with physical therapy that aims to restore the decreased stiffness and impaired performance by speci c application of mechanical stimuli (8,9). However, very little is known about acute cellular dynamics in response to physiological tendon loading and the underlying molecular mechanisms that regulate tendon stiffness.
Mechanotransduction is crucial to a wide variety of physiological processes, such as hearing, touch sensation, regulation of blood ow and pressure, as well as proprioception and breathing (10)(11)(12)(13)(14)(15). These events rely on molecular mechanisms that convert mechanical forces into biological signals using various membrane proteins. In eukaryotes, several ion channels and receptors have been identi ed as mechanosensors (16)(17)(18)(19)(20)(21)(22)(23)(24). Among these, the mechanosensitive ion channel PIEZO1 is responsible for different mechanotransduction processes occurring in the lymphatic, cardiovascular, renal and skeletal systems (25)(26)(27)(28)(29)(30). Genetic mutations in PIEZO1 have revealed the physiological importance of this ion channel in humans. PIEZO1 loss-of-function mutation leads to persistent congenital lymphoedema (25,26), while a PIEZO1 gain-of-function mutation that is common in individuals of African descent has been associated with malaria resistance (31). Although research on mechanically activated ion channels and receptors has made signi cant progress in recent years, the mechanosensors in tendons -one of the most mechanically challenged tissues of the human body -have not been identi ed.
We investigated tendon mechanotransduction by combining calcium (Ca 2+ ) imaging with simultaneous mechanical loading of tendon explants and isolated primary tendon cells. The physiological role of the identi ed molecular mechanism was then studied in mice and humans carrying gain-of-function mutations.

Results
Tenocytes sense tissue stretching by transient intracellular Ca 2+ elevations.
To investigate how tendon cells (tenocytes) detect mechanical forces, we developed a functional imaging system that allows simultaneous uorescence microscopy and tissue stretching of tendon explants from rat tails ( Fig. 1a; see Methods for more details). Using this approach, we performed Ca 2+ imaging in tissue-resident tenocytes labeled with Fluo-4. In an unstretched condition, we detected sparse spontaneous Ca 2+ signals, however, upon stretching from 0-10% strain we observed a tissue-wide Ca 2+ response ( Fig. 1b and Movie S1). By testing different strain rates, we noticed distinct Ca 2+ dynamics.
While at low strain rate each tenocyte exhibited multiple Ca 2+ signals, at high strain rate they displayed only a single Ca 2+ response upon tendon stretching ( Fig. 1c and S1). Additionally, with increasing strain rate higher tissue stretch was required to elicit a Ca 2+ response in 50% of the cells (Fig. 1d). This strain rate dependency can partially be explained by a time lag between stimulus and Ca 2+ signal of 0.77±0.18 s (Fig. 1e). We attributed the remaining differences to inherent viscoelastic properties of the tissue (Fig. S1e) and to potential cell-cell communication processes occurring predominately at low strain rate. The stretch-induced Ca 2+ response was con rmed by two-photon uorescence lifetime imaging (FLIM), with which we also determined absolute Ca 2+ concentrations in tissue-resident tenocytes (Fig. S2) following OGB-1 loading (32). At rest, Ca 2+ levels averaged 43±4 nM, however, upon tissue stretching Ca 2+ levels increased by 18±6 nM reaching on average 61±8 nM ( Fig. 1f and Movie S2). Similar Ca 2+ elevations of 16±9 nM were detected in spontaneous signals, indicating that the stretch-induced Ca 2+ responses were in a physiological range (Fig. 1g). Taken together, we observed a mechanosensitive calcium response in tenocytes that depends both on magnitude and rate of the tissue stretch.
Shear stress triggers calcium signals in isolated tenocytes.
During tissue stretching, collagen bers -the load-bearing elements of the extracellular matrix -slide past each other (33). Tenocytes reside between these bers and are therefore exposed to mechanical shear. Since ber sliding is the predominant mechanism enabling the extension of tendon fascicles (33), we wondered whether shear stress could be the primary mechanical stimulus for tenocytes. We therefore quanti ed the ber sliding by tracking cells from image sequences obtained at low strain rates and by comparing inter-ber displacements (Fig. 2a). And by using a physical model we calculated the resulting shear stress, which ranged between 2 to 6 Pa depending on the cell height (Fig. 2b). Our analysis suggests that shear stress levels may vary across cellular domains of tenocytes, likely being highest around narrow protrusions and lowest around the cell body. To test our prediction, we developed a micro uidic ow chamber that allows simultaneous Ca 2+ imaging and shear stress stimulation of isolated primary tenocytes stained with Fluo-4 ( Fig. 2c; see Methods for more details). Exposing tenocytes to a shear stress of 5 Pa, that occurs during tissue stretching, triggered a prominent Ca 2+ response ( Fig. 2d and Movie S3). The magnitude of shear stress stimulus determines the percentage of responsive cells (Fig. 2e) as well as the amplitude and duration of the Ca 2+ response (Fig. S3).
Interestingly, a Ca 2+ response in about 50% of tenocytes is induced by a shear stress of 3.3 Pa, which falls well within the range of the calculated shear stress that occurs during tissue stretching (Fig. 2b).
Together, this con rms the role of shear stress as a key mechanical stimulus for tenocytes, which show similar responsiveness across anatomical regions (Fig. 2f).
We noticed that Ca 2+ signals typically start at the cell periphery both in isolated and in tissue-resident tenocytes (Fig. 2g). This is in line with our physical model which predicts that narrow regions (i.e. protrusions) experience the highest levels of shear stress. PIEZO1 is required for the shear stress-induced response in tenocytes.
Since mechanotransduction relies on membrane proteins that convert mechanical stimuli into a biological signal (34), we wondered if shear stress in tenocytes activates mechanosensitive membrane channels mediating the Ca 2+ in ux. To test this, we performed mechanical stimulation in Ca 2+ -free medium and observed no overt Ca 2+ response in tissue-resident and isolated tenocytes (Fig. 3a).
However, reperfusion with control medium containing Ca 2+ restored the stimulus-evoked responses (Fig.   3a), suggesting a channel-mediated mechanism.
To identify the responsible ion channel, we focused on candidates associated with mechanosensitive cation channel characteristics that are highly expressed in both mouse tail tendons (35) and human Achilles tendons (36) (Fig. 3b). Using CRISPR/Cas9 genome editing, we generated e cient knockdowns of the selected genes in human primary tenocytes and tested their mechanosensitive response (Fig. 3c,  d). Interestingly, from all examined knockdowns only cells depleted from PIEZO1, known as a shear sensor (12,16,37), showed a signi cant reduction in the shear stress response (Fig. 3e, f). This was further corroborated by analyzing additional CRISPR-guided RNAs targeting different regions of PIEZO1 (Fig. 3g), thereby also precluding the contribution of potential off-target effects. Moreover, we obtained similar results with CRISPR/Cas9-mediated Piezo1 knockdowns in rat tenocytes isolated from tail tendon fascicles ( Fig. S4a-d). Taken together, this con rms that PIEZO1, an abundantly expressed ion channel in tendon tissues (Fig. S4e), is the crucial shear stress sensor in tenocytes. PIEZO1 activity regulates the biomechanical properties of tendons.
To investigate the role of PIEZO1 in tendons, we performed in vitro and in vivo experiments. First, we con rmed that pharmacological activation of PIEZO1 by the speci c agonist Yoda1 (38) triggers a robust Ca 2+ response in tendon-resident tenocytes ( Fig. S4f and Movie S4). Then, we wondered if recurrent pharmacological stimulation of tenocytes in cultured tendon fascicles could alter tissue stiffness. We therefore maintained tendon fascicles from rat tails in our custom bioreactor (39) with minimal loading to preserve tissue integrity (39) and stimulated tenocytes with 5 mM Yoda1 for 30 min every three days for two weeks (mimicking exercise) (Fig. 4a). To characterize changes in biomechanical properties over time, we cut each tendon fascicle in two, tested the rst half at day 0 and the second half after the stimulation paradigm at day 16 (Fig. 4a). By comparing the ramp-to-failures of the two time-points, we noticed that stiffness and strength were signi cantly higher in Yoda1-stimulated fascicles relative to control fascicles ( Fig. 4b). However, fascicle diameter was not affected (Fig. 3b). To verify if PIEZO1-mediated mechanosignaling regulates tendon stiffness in vivo, we analyzed tendons from mice carrying a Piezo1 gain-offunction (Piezo1GOF) mutation equivalent to a human PIEZO1 gain-of-function mutation (R2456H) (31).
These mice express an overactive PIEZO1 that elicits stronger Ca 2+ signals upon channel activation due to a longer inactivation time (31). Remarkably, ramp-to-failure experiments with plantaris tendons (foot exor tendon) revealed an average increase in stiffness of 19% and in strength of 17% in Piezo1GOF mice compared to wild-type littermates, while the macroscale tendon morphology remained unchanged (Fig. 4c, d). These biomechanical differences are particularly prominent at high tendon loading and are very similar to reported changes in tendons of sprinters compared to endurance runners and non-active individuals (7). However, thorough analysis of collagen brils -the load-bearing microstructures (33)from tendons of Piezo1GOF mice revealed no overt differences in bril size distribution compared to their wild-type littermates ( Fig. 4e-h). Hence, elevated mechano-signaling by PIEZO1 regulates tendon stiffness and strength without inducing a hypertrophic collagen production. Similarly, exercise-induced increases in tendon mechanical properties were not associated with changes in collagen bril morphology (40). This likely indicates that increased tendon stiffness and strength in response to mechano-signaling may be induced by a denser collagen cross-linking (41). PIEZO1 activity in uences the jumping performance in humans.
Strong tendons are critical for high physical performance (3) and we wondered if our observed PIEZO1mediated adaptations in tendon stiffness and strength could be relevant for human athletic performance.
In fact, about one out of three individuals of African descent carries a PIEZO1GOF mutation known as E756del which has been associated with malaria resistance and represents the most abundant PIEZO1GOF mutation identi ed to date, particularly common in West African populations (31). Interestingly, athletes of West African descent (including African Americans) excel in sports performances related to sprinting and jumping (42)(43)(44), however, whether the E756del allele is overrepresented in elite athletes is unknown. But could human E756del carriers also present tendon adaptations that associate with athletic performance? We addressed this question in a double-blind study in which we investigated the Achilles tendon of 65 healthy African Americans and assessed their jumping performance.
Genotyping identi ed 20 heterozygous and two homozygous E756del carriers, and 43 non-carrier controls ( Fig. 5a). We observed comparable demographics between E756del carriers and controls, with no differences in age, height, weight, physical activity or sports participation ( Fig. 5b and S5). Ultrasoundbased assessment of the Achilles tendon morphology showed no signi cant differences in tendon crosssectional area and length between E756del carriers and controls (Fig. 5c, d), which is in line with the unchanged tendon morphology observed in Piezo1GOF mice ( Fig. 4d-h). Since the tendon phenotype in Piezo1GOF mice was prominent at high degrees of tendon loading, we speculated that human E756del carriers may show athletic differences in exercises that evoke high tendon loading. We therefore measured the participant's maximal jumping performance in two related jumps, namely a countermovement jump (CMJ) and a drop countermovement jump (DCMJ) (Fig. 5e). The latter differs solely by an initial drop from a height of 20 cm, which leads to greater mechanical loads in the Achilles tendon (45,46). Non-carrier controls demonstrated a similar performance in both jumps (Fig. 5f). Strikingly, E756del carriers performed signi cantly better in DCMJ, which evokes higher tendon loading, compared to CMJ (Fig. 5f). By normalizing the DCMJ-height to the CMJ-height, we assessed the impact of increased tendon loading on jumping performance and found an average performance of 111% in E756del carriers that signi cantly exceeded the average 95% reached by non-carriers (Fig. 5g). Hence, E756del carriers showed a net 16% average increase in normalized jumping performance compared to non-carriers, presumably because of a greater capacity of tendons to store and return elastic energy. Indeed, when converting the jump height into potential energy (i.e. mass x gravitational acceleration x jump height), we found that E756del carriers effectively stored and returned the drop energy (+8.1 J on average), whereas the controls did not (-2.1 J on average) (Fig. 5h). Accounting for the energy return of E756del carriers against the energy dissipation of controls, E756del carriers showed on average a signi cantly increased net energy return of 10.2 J. Thus, the E756del mutation likely in uences sports performances that rely on the power output generated with high tendon loading, such as sprinting and jumping.

Discussion
In tendon mechanotransduction, it has been hypothesized that mechanical forces are converted into biological signals by mechanically activated membrane proteins (47). Isolated tenocytes are sensitive to pipette indentation (48) and show increased frequency of spontaneous calcium signals following a mechanical stimulation (49). However, evidence of acute responses to physiologically relevant mechanical stimuli and the underlying molecular sensing mechanisms have been elusive.
We developed a novel microscope-compatible functional imaging setup to enable direct observation of tenocytes during tissue stretching and harnessed it to characterize tendon mechanotransduction by Ca 2+ imaging. We found a prompt calcium response in tenocytes upon tissue stretching and noticed that at low strain rate less stretch was required to elicit the response. This phenomenon might explain why physical rehabilitation strategies based on slowly performed resistance training show improved outcomes when treating tendinopathies (50,51). Our data suggest that these improvements might arise from an optimal compromise between maximizing cellular stimulation while minimizing tissue strains and loads.
Moreover, we identi ed shear stress as a key mechanical stimulus by determining the shear stress levels that occur during tissue stretching, applying them to isolated primary rat and human tendon cells and nding tight activation limits in vitro and in situ. By combining CRISPR/Cas9 screening with functional shear stress imaging, we found PIEZO1 as the crucial sensor driving shear stress-induced calcium signals.
To investigate the role of this ion channel in tendons, we analyzed the effect of elevated PIEZO1 mechano-signaling in tendons of rodents. In vitro pharmacological PIEZO1 stimulation and in vivo PIEZO1 overactivity both increased tendon stiffness and strength. Surprisingly, these changes seem not to result from a hypertrophic response. Instead, we suspect that a denser collagen cross-linking network causes the phenotypes. This would decrease the relative motions of collagen bers and likely diminish the shear stress within the tissue, counteracting the elevated shear sensor signaling. Such tissue adaptation would imply a tenocyte mechanostat behavior (52), i.e. a feedback mechanism aiming to maintain optimal shear stress stimuli. An adapting cross-link network might also explain why exercise has very little to no effect on tissue-and bril-morphology (40), and why the collagen matrix has a low turnover (53).
The high prevalence of the PIEZO1GOF E756del allele in the African population provides the opportunity to study the function of PIEZO1 in humans (31). The E756del mutation is particularly common in populations of West Africa, likely due to the potential protection it affords against malarial infection (31). Alongside with this potential role, our evidence suggests that the E756del mutation affects the human athletic performance. Speci cally, E756del carriers perform signi cantly better than non-carriers in jumping maneuvers that include high degrees of tendon loading and of energy storage and return (2,45,46). This performance mechanism is presumably enhanced in tendons whose biomechanical characteristics are governed by an overactive PIEZO1, as we observed an increase in energy return in E756del carriers. While the performance is enabled by the muscle-tendon unit, it is worthy to note that skeletal muscle is likely not affected by the E756del mutation due to the very limited Piezo1 expression in muscle tissue (Fig. S4e) (16). The performance difference in humans as well as the tendon phenotype in mice both emerged at high degrees of tendon loading. Concurrently, both Piezo1GOF mice and E756del carriers displayed tendon morphology indistinguishable from wild-type controls. These ndings strongly suggest that E756del carriers present a tendon phenotype similar to the one observed in Piezo1GOF mice.
The phenotypically improved power performance we identi ed in E756del carriers suggests that it might plausibly contribute to the fact that nearly all the top 500 sprint times of the men's 100 m are held by athletes of West African descent (including African Americans) (43), with these athletes reliably excelling in world-class sprinting competitions (42). However, whether the E756del allele is overrepresented in elite athletes is a tantalizing question that remains to be clari ed.
Beyond implications for athletic performance, PIEZO1 may represent a therapeutic target in clinical indications for which physical rehabilitation is currently prescribed. Tendon pathologies are a common human condition, due to the high mechanical demands and the low intrinsic healing capacity (1,8). They account for a substantial part of musculoskeletal diseases, which represent the second leading cause for years lived with disability worldwide (8,54,55). Pharmacological activation of PIEZO1 in diseased mechanically-inferior tendons might trigger tissue reinforcement and accelerate the healing process.  (56). During stretching protocols, fascicles were continually perfused with KHS that was preheated to 29° C and steadily aerated by a gas mixture containing 95% N 2 and 5% CO 2 to maintain 3% O 2 and constant pH levels. Images were acquired with a 10x (N.A. 0.4) objective with excitation set at 488 nm wavelength and 100 ms exposure time. Prior to the stretching protocols, fascicles were preconditioned 5 times to 1.0% initial length L 0 (10mm mounting length from clamp-to-clamp). The cross-sectional area was measured at crimp disappearance and L 0 was de ned at 1MPa tissue stress. Tissue strain was de ned as (L-L 0 )/L 0 in %, with sample length L. Tissue stress was calculated by dividing the force values through the crosssectional area.

Methods
To investigate the cellular response to tissue stretching, single fascicles were stretched at three different strain rates (low 0.01% strain/s, medium 0.1% strain/s, high 1.0% strain/s) from 0 to 10% strain. Baseline activity was investigated at the preload of 1 MPa prior to initiating the stretching protocol. At medium and high strain rate, time lapses of one z-plane were recorded. At low strain rate, series of image stacks (50x2 µm) were acquired and subsequently deconvolved using Huygens Professional 18.10 (Scienti c Volume Imaging) to quantify the micro-mechanical environment (at the cellular/collagen ber level) needed to trigger intracellular calcium signals. To examine the time lag between the mechanical stimulus and the downstream calcium signals, tendon fascicles were subjected to one cycle of 2.7% strain at 30% strain/s. Time lapse images were analyzed with the following steps. Cell movements were tracked with Imaris 7.7 (Bitplane AG, Switzerland) and the exported displacements were further used in a custom software (Matlab R2016a) that divided the images into 6x6 subimages and applied motion correction in each subimage. Calcium events were automati cally detected and measured in the stabilized subimages using CHIPS (Cellular and Hemo dynamic Image Processing Suite) (57). The inter ber sliding was calculated from the relative displacements (in axial direction) of the cells on adjacent bers (e.g. ber u and v): , n and m are the respective cell numbers of the adjacent bers, L 0 is the ber length (58).

Fluorescence Lifetime Imaging (FLIM) of tendon fascicles
Isolated rat tail tendon fascicles were stained with 20 μM cell-permeable OGB-1 (Thermo Fisher Scienti c O6806) for 2 h in KHS at 29° C and 3% O 2 and mounted on our tensile stretching device placed on the stage of a two-photon microscope and continually perfused with KHS during imaging. FLIM was performed on an upright Leica TCS SP8 FLIM two-photon microscope equipped with a tunable (680-1300 nm) 80 MHz infrared laser (Insight DS+ Dual from Spectra Physics) and four non-descanned FLIM enabled hybrid detectors. On the non-descanned detectors the following emission band pass lters were used: 460/50 nm, 525/50 nm, 585/40 nm, 650/50 nm. Data acquired from the channels 525/50 nm and 585/40 nm were used for the FLIM analysis. A 2.6 mm working distance Leica HC IRAPO 25x/1.0 water immersion objective was used for imaging. Time-correlated single-photon counting (TCSPC) was performed using a six channel Picoquant HydraHarp 400 together with the Picoquant Symphotime 64 software package. Femtosecond infrared laser pulses allowed for e cient two-photon uorescence excitation and emission from a thin focal plane in the core of a tendon fascicle (ca. 30-100 μm deep in the tissue). A laser wavelength of 915 nm and a maximum laser power of 12.5 mW measured after the objective were used to avoid any signs of phototoxicity. To avoid photo-induced effects as well as statistical pile-up effects in the TCSPC histograms, the photon count rates on the detectors were always kept below 1% of the excitation rate. For fast sequential imaging an image size of 512 px in width and 80-110 px in height was used with a scanner frequency of 400 Hz.
The calcium calibration buffer kit from Thermo Fisher Scienti c (C3008MP) was used in combination with the cell impermeable Ca 2+ indicator OGB-1 at 1 μM to calibrate the [Ca 2+ ] readout (Fig. S2a).
Temperature and pH were measured and considered by nely adjusting the estimated [Ca 2+ ] using Chris Patton's WEBMAXC program (http://web.stanford.edu/~cpatton/webmaxcS.htm) (32). The TCSPC histograms were tted using a double-exponential tail t within a time gate of 10ns using the Picoquant Symphotime software. From the tail t, we calculated the amplitude weighted average lifetime, which was used throughout the study as a readout for [Ca 2+ ] using a suitable calibration function acquired from tting a nonlinear Hill function to the OGB-1 calibration data (Fig. S2b). ]-landscape. Data analysis and statistics, however, were performed using the raw data, not the ltered pixel maps.

Mathematical model for shear stress prediction in tenocytes
To predict the shear stress experienced by tenocytes during tissue stretching, we applied a numerical model that assumes cell heights (h) between 1-10 μm and that assumes individual tenocytes span the distance between two adjacent collagen bers (59). Shear stress in tenocytes is generated by unilateral collagen ber displacement, which leads to transverse displacement of the cell body. By de nition, the shear stress (τ) arises through the application of a force (F) parallel to the cross-section over a certain surface (A) and is equal to the shear modulus (G) of the material multiplied by the shear strain (γ). The shear strain is de ned as the transverse displacement (Δx) of the material divided by the initial height of the material (h) (Fig. 1i): . The shear modulus of a eukaryotic cell was previously estimated to be around G = 1.5 Pa (60) and the transverse material displacement (Δx) was calculated from the inter ber sliding (s) and the ber length (l ber = 900 μm). This enables the estimation of shear stresses acting on tenocytes resulting from unilateral collagen ber sliding.
Primary human and rat tenocyte cultures Calcium imaging during application of shear stress on isolated tenocytes using ow chambers Custom-made ow chambers were fabricated with the following procedure. A microscope slide was plasma treated, and 3 μl of polydimethylsiloxane (PDMS, Sylgard 184 Silicone Elastomer Kit, Dow Europe) was deposited in its center. Then a silanized PDMS stamp that was molded from the negative of the microgroove pattern (10 μm depth/ridge width/pitch) was placed on top. The assembly was subsequently cured at 70° C for 6 hours before detaching the stamps. PDMS microgrooves were chemically activated using two consecutive treatments of 0 Tenocytes were seeded in the ow chamber at a density of 38'000 cells/cm 2 and incubated at 29° C / 5% CO 2 / 3% O 2 overnight. Staining for 2 h with 1 µM Fluo 4-AM diluted in KHS containing 0.02% pluronic F-127 was performed before placing the ow chamber on the microscope stage and connecting it to a syringe pump (Cetoni, low-pressure module). Next, the ow chambers were ushed for a few minutes at a ow rate of 0.1 ml/min (resulting in negligible shear stress of ca. 0.01 Pa) with KHC that was preheated to 29° C and degassed to 3% O 2 using a gas mixer. Appropriate ow rates resulting in speci c shear stresses on the cell substrate were calculated using established formulas of uid ow in rectangular channels (62). During shear stress experiments, image stacks (5x3 μm) were acquired with the iMic wide eld microscope (10x objective) at a frequency of 1 Hz, a wavelength of 488 nm, and an exposure time of 100 ms.
Image analysis was done with an initial average intensity Z-projection of the image stacks, followed by a segmentation of individual cell bodies performed with a custom ImageJ script based on the uorescence at the baseline, i.e. 30 s interval before application of the shear stress stimulus. The mean uorescence intensity of each segmented cell was normalized to the average intensity measured at the baseline. A calcium signal in a cell was de ned as such when the normalized uorescence intensity (ΔF/F 0 ) exceeded the baseline uorescence intensity by 10 times the standard deviation of the baseline during a 20 s interval following shear stress exposure.

Generation of CRISPR/Cas9-mediated knockdown cells
Single guide RNAs (sgRNAs) against multiple candidate genes were designed with the CRISPRdirect online tool http://crispr.dbcls.jp (63). Only highly speci c target sites were selected, the respective sequences are listed in Table S1. A non-targeting control sgRNA was chosen from the study of Morgens et al. (64) and checked for low targeting potential by BLASTN 2.8.0 search. Target sequences oligos were synthesized with BsmBI restriction site overhangs by Microsynth (Balgach, Switzerland) and then annealed and cloned into the lentiCRISPRv2 transfer plasmid, a gift from Feng Zhang (Addgene plasmid #52961; (65)), following the provided protocol of the Feng Zhang Lab. Lentiviral particles were produced by co-transfection of the lentiCRISPRv2 plasmid, containing the respective gRNA-sequence, with the packaging plasmids pCMV-VSV-G (a gift from Bob Weinberg; Addgene plasmid #8454; (66)) and psPAX2 (a gift from Didier Trono; Addgene plasmid #12260) into HEK293T cells using Lipofectamine 3000 (Thermo Fisher Scienti c L3000008) and following the manufacturer's instructions.
For transduction, human and rat tenocytes were incubated for 24 h with supernatant containing the viral particles and supplemented with 8 µg/ml Polybrene. Subsequently, human and rat cells were selected with 3 µg/ml Puromycin (Gibco A1113803) for 3 days or with 4 µg/ml for 7 days, respectively. The e ciency of the knockouts was tested with quantitative real-time PCR, immuno uorescence and western blotting.

RNA isolation from tissues and cells and quantitative real-time PCR
Freshly isolated tissues were snap frozen in liquid nitrogen and subsequently homogenized with QIAzol lysis reagent (Qiagen 79306) using a cryogenic grinder (SPEXSamplePrep FreezerMill 6870). 1-bromo-3chloropropane (Sigma-Aldrich B9673) was added to the tissue lysates at a 1:4 ratio, and the RNA containing aqueous phase was obtained using Phase Lock Gel -Heavy (LabForce 2302830). In vitro tenocytes were lysed with RLT/bME buffer. Subsequently, RNA from tissue and cell lysates was extracted using the RNeasy micro Kit (Qiagen 74004) following the protocol provided by the manufacturer. Quality and quantity of the RNA was measured with a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scienti c). cDNA was synthesized from 500 ng of total RNA using a High-Capacity cDNA Reverse Transcription Kit with RNAse Inhibitor according to the manufacturer instructions (Applied Biosystems 4374966). Gene expression analysis was performed by quantitative real-time PCR with cDNA corresponding to 10 ng of starting RNA using the PowerUp SYBR Green Master Mix (Thermo Fisher Scienti c A25742). The samples were ampli ed using a StepOnePlus Real-Time PCR System (Applied Biosystems) with the following conditions: 95° C for 10 min followed by 40 PCR cycles of 95° for 15 s and 60° C for 1 min. All experiments were run with technical triplicates. Relative gene expression levels were quanti ed using the 2 -ddCT method with either ANXA5 or GAPDH as reference gene. All primers are listed in Table S2. (Thermo Fisher Scienti c MA5-32876, 1:500) and β-tubulin (MERCK Millipore MAB3408, 1:10'000) were diluted in 5% BSA/TBS-T and incubated overnight at 4° C. Next, the membranes were washed 3 times in TBS-T and incubated with the secondary antibody (anti-mouse, Sigma-Aldrich SAB3701073, 1:20'000) for 1 h at room temperature. Images were taken using UltraScence Pico Ultra Western Substrate (GeneDireX CCH345-B) and the ChemiDoc MP imaging system (Bio-Rad).
Tendon explants cultured in bioreactor and subjected to sham or Yoda1 stimulation Rat tail tendon fascicles were freshly isolated and placed into culture medium (high glucose Dulbecco's Modi ed Eagle's Medium, Sigma Aldrich D6429, supplemented with 1% Penn/Strep, 200 μM ascorbic acid, Wako Chemicals 013-19641, and 1% N-2 supplement, Thermo Fisher Scienti c 7001585). Each fascicle was cut in half, one half was used for mechanical testing at day 0, while the other half was cultured in our custom-made bioreactor at the preload (crimp disappearance, i.e. minimal mechanical load) and mechanically tested at day 16 (39). Distal and proximal samples were randomly distributed between the two days. Diameters were measured at day 0 and day 16 using a 10x objective (Motic AE2000). Cultured fascicles underwent either sham or 5 μM Yoda1 stimulations for 30 min on days 0, 3, 6, 9 and 12 post-isolation. Following the 30 min treatment, fascicles were washed once with medium, then resuspended in medium and incubated at 29° C, 5% CO 2 and 3% O 2 . Ramp-to-failure experiments were performed to assess the biomechanical properties. Samples were preloaded to 0.04 N and preconditioned 5 times to 1% strain. Subsequently, a ramp-to-failure was carried out at 1% strain/s. Fascicle stiffness was calculated in the linear region of the force-strain curves and fascicle strength was determined from the maximal force.

Biomechanical testing and analysis
The biomechanical properties of plantaris tendons from wild-type and Piezo1GOF mice (31) were investigated with ramp-to-failure experiments. Age-matched littermates (20-21 weeks old) were euthanized and stored at -80° C until the day of experiment (approval by the Institutional Animal Care and Use Committees of Scripps Research in accordance with the guidelines established by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)). After thawing, plantaris tendons were carefully isolated and tested in uniaxial tension using a custom clamping technique on a universal testing machine that recorded force-displacement data (Zwick Z010 TN, 20 N load-cell) (67). During testing, tendons were kept in a custom chamber lled with KHS, preloaded to 0.1 N (initial length L 0 corresponding to 0% strain) and preconditioned 5 times to 1% strain (preload reapplied after every cycle). Subsequently, samples were ramped to failure at a constant strain-rate of 1% strain/s. The diameter was measured in microscopic images of the plantaris tendons (4x objective, Motic AE2000). Tendon stiffness was calculated in the linear region of the force-strain curves and tendon strength was determined from the maximal force.

Transmission electron microscopy
Plantaris tendons were freshly isolated from age-matched littermates (29-34 weeks old) euthanized in the middle of the day (normal light/dark cycle) and sequentially xed with 2.5% glutaraldehyde (Sigma-Aldrich G5882) in 0.1 M sodium cacodylate buffer (pH 7.2), with 1% OsO 4 in 0.1 M sodium cacodylate buffer at room temperature and with 1% uranyl acetate in H 2 O at room temperature for at least 1 hour per step. Samples were rinsed 3 times between the xation steps and nally with H 2 O prior to dehydration in an ethanol series and embedding in Epon. Ultrathin (70 nm) sections were post-stained with Reynolds lead citrate and imaged in a FEI Talos 120 at 120 kV using a bottom mounted Ceta camera (CMOS, 4k x 4k pixels) using MAPS software (Thermo Fisher Scienti c). Segmentation of the cross-sectional area of collagen brils was performed with the Trainable Weka Segmentation Fiji plugin (68).

Participants of the human study -activity level and sports participation
Healthy self-reported African American participants (at least 18 years old) were enrolled after approval by the Institutional Review Board of the University of Delaware (ID-1420251-3) and written informed consent.
A clinical evaluation with ultrasound imaging was carried out to ensure that the subjects had no underlying pathologies in their Achilles tendons. Additionally, the Victorian Institute for Sports Assessment -Achilles questionnaire (VISA-A) was applied to con rm that the Achilles tendons were healthy (69). Participants reported their highest level of sports participation (recreational, secondary school, collegiate or professional) and their history of sports participation. The Physical Activity Scale (PAS) was used to assess the subject's reported current physical activity level (70).
Ultrasound-based assessment of the human Achilles tendon morphology B-mode ultrasound imaging (LOGIQ e ultrasound system (GE Healthcare, USA) with wide-band linear array probe (5.0-13.0 MHz)) was used to analyze the Achilles tendon morphology Brie y, Achilles tendon thickness and cross-sectional area were measured at a distance of 2.5 cm from the calcaneal osteotendinous junction (axial direction) in the images acquired parallel and perpendicular to the ber orientation (each 3 repetitions), respectively (71). Achilles tendon length was measured as the distance from the calcaneal osteotendinous junction to the myotendinous junction of the gastrocnemius and soleus muscles using extended eld of view (71). The software OsiriX MD (Pixmeo SARL, Switzerland) was used to analyze the ultrasound images.

Functional performance tests
To investigate the jump heights, we used the MuscleLabÔ (Ergotest Innovation, Norway) light mat measurement system, which creates an infrared light eld 4 mm above the oor and records beam interruptions. Participant height and weight were recorded prior to jumping tests. Jump height was calculated by MuscleLabÔ using participant weight, ground contact time, and ight time. Participants performed two different jump tests. In each test the participants were asked to place their hands behind their back and to jump as high as possible. The rst test was a single leg countermovement jump (CMJ) in which the participants started by standing on the oor on one leg then quickly bent the knee before jumping straight up as high as possible (72). This was repeated three times with each leg. The second test was a single leg drop countermovement jump (DCMJ), in which participants jumped off of a 20 cm high box and then jumped vertically as high as possible (72). For each leg, the average height of three trials was used for analysis.
This amplicon (ca. 200 bp) was sequenced using both primers to identify non-carriers and E756del carriers.

Statistical analysis
For multiple-comparisons, data were analyzed with one-way ANOVA (Tukey's or Dunnett's test). Intergroup comparisons were performed with two-tailed Student's t test or two-tailed Mann-Whitney test. For mouse and human data, n = number of animals or participants, at least n = 4 was used. Analyses using linear mixed effects models (lme4 package in R) were conducted for the biomechanical experiments with mouse tendons (mouse ID as random effect and litter as xed effect, Bonferroni-Holm correction) and for the human jumping data (subject ID as random effect and leg as xed effect). Age, height, weight, highest level of sports participation and activity level of the participants were tested as covariates.