Vol.:(0123456789) Pflügers Archiv - European Journal of Physiology (2025) 477:729–739 https://doi.org/10.1007/s00424-025-03075-7 RESEARCH Reproducibility of smooth muscle mechanical properties in consecutive stretch and activation protocols Simon Kiem1  · Stefan Papenkort1  · Mischa Borsdorf1  · Markus Böl2  · Tobias Siebert1,3 Received: 25 June 2024 / Revised: 31 December 2024 / Accepted: 4 March 2025 / Published online: 22 March 2025 © The Author(s) 2025 Abstract Mechanical organ models are crucial for understanding organ function and clinical applications. These models rely on input data regarding smooth muscle properties, typically gathered from experiments involving stimulations at different muscle lengths. However, reproducibility of these experimental results is a major challenge due to rapid changes in active and pas- sive smooth muscle properties during the measurement period. Usually, preconditioning of the tissue is employed to ensure reproducible behavior in subsequent experiments, but this process itself alters the tissue’s mechanical properties. To address this issue, three protocols (P1, P2, P3) without preconditioning were developed and compared to preserve the initial mechani- cal properties of smooth muscle tissue. Each protocol included five repetitive experimental cycles with stimulations at a long muscle length, varying in the number of stimulations at a short muscle length (P1: 0, P2: 1, P3: 2 stimulations). Results showed that P2 and P3 successfully reproduced the initial active force at a long length over five cycles, but failed to maintain the initial passive forces. Conversely, P1 was most effective in maintaining constant passive forces over the cycles. These findings are supported by existing adaptation models. Active force changes are primarily due to the addition or removal of contractile units in the contractile apparatus, while passive force changes mainly result from actin polymerization induced by contractions, leading to cytoskeletal stiffening. This study introduces a new method for obtaining reproducible smooth muscle parameters, offering a foundation for future research to replicate the mechanical properties of smooth muscle tissue without preconditioning. Keywords Adaptation · Urinary bladder · Stimulation · Stress–strain-relationship · Biological soft tissue Introduction Organ models are crucial for understanding organ function [29] and for clinical applications, such as predicting surgical procedures and disease effects [11]. For a realistic represen- tation of organ function, these mechanical models require accurate material parameters. Typically, the determination of material parameters as input data for mechanical mus- cle models requires the execution of multiple experiments involving muscle stimulation at varying lengths [6, 20, 32, 35]. Determining these parameters is particularly difficult for smooth muscle (SM) tissue due to the rapid changes in active and passive properties caused by cellular adaptation [46]. Furthermore, the reproducibility of experimental test results is a pressing challenge in current SM research [44]. The aim of this study is to reproduce the initial mechanical properties of SM tissue after stretch- and activation-induced changes in muscle structural properties using appropriate experimental protocols. Microstructurally, SM lacks the striation pattern seen in skeletal muscle due to the absence of parallel myofibrils and Z-discs separating sarcomeres [1, 38, 43]. SM cells (SMC) are spindle-shaped with a central nucleus and are several hundred micrometers long and 5–6 µm wide when fully relaxed [1]. An SM sarcomere is composed of thick (myosin) and thin (actin) filaments, as well as so-called dense bodies that anchor the actin filaments. Additionally, the dense bod- ies serve as attachment points for the intermediate filaments, which form an elastic cytoskeleton within the cell. During * Simon Kiem simon.kiem@inspo.uni-stuttgart.de 1 Department of Sport and Movement Science, University of Stuttgart, Stuttgart, Germany 2 Institute of Mechanics and Adaptronics, Technische Universität Braunschweig, Brunswick, Germany 3 Stuttgart Center for Simulation Science, University of Stuttgart, Stuttgart, Germany http://crossmark.crossref.org/dialog/?doi=10.1007/s00424-025-03075-7&domain=pdf http://orcid.org/0009-0009-2075-8242 http://orcid.org/0000-0003-0134-3147 http://orcid.org/0000-0002-0045-8981 http://orcid.org/0000-0003-1185-6309 http://orcid.org/0000-0003-4090-5480 730 Pflügers Archiv - European Journal of Physiology (2025) 477:729–739 contraction, myosin heads attach to actin, followed by the force-generating power stroke [1, 9, 33]. A number of studies have demonstrated that activation [15, 19, 45], passive stretch amplitude [47], and resting time [48] result in changes in active force within minutes to hours. Wang et al. [48] observed that passive stretching and short- ening of rabbit tracheal SM over a 24-h period resulted in changes in active force and the optimal length (Lopt) shifted as a consequence. Starting from an initial in situ length, pas- sive stretching of tissue strips shifted Lopt to longer lengths, whereas passive shortening shifted Lopt to shorter lengths. In addition, the active force increased when multiple isometric contractions were performed at the same length. The authors proposed that these changes in Lopt are the result of an adap- tive response by the SM tissue by adjusting the number of contractile units in series to maintain an optimal overlap of filament pairs [22, 48]. Increased SM forces are possible by increasing the number of contractile units in parallel [19]. The idea of a malleable myofilament lattice in an SMC, whereby sarcomeres are added and removed, is supported by several sources [4, 19, 22]. In SMCs, there are pools of free myosin monomers as well as filamentous myosin integrated into sarcomeres [8]. These pools are under constant remod- eling, e.g., in response to activations [8]. Myosin monomers are added to the contractile units to adapt to longer lengths and removed from them to adapt to shorter lengths [4, 8, 46]. Smolensky et al. [37] showed that when the SM tis- sue is stimulated at long lengths, an increase in birefrin- gence occurs within seconds, indicating an increased mass of thick filaments (myosin). During relaxation and shorten- ing, myosin filaments partially dissociate and become free myosin monomers again. These observations indicate that the contractile apparatus in SMCs undergoes remodeling to maintain optimal overlap of contractile elements, ensur- ing the necessary contractile properties after large length changes [8, 34, 46]. In addition to the observed changes in active forces, Speich et  al. [40] reported changes in the passive force–length relationship and a reduction in the maximum passive force of the rabbit detrusor muscle for repeated stretches combined with intermediate contractions at differ- ent lengths. Additionally, repeated stretches resulted in an increase in slack length [5] and changes in the curvature of the force-strain relationship [27, 31]. Interestingly, inducing an isometric contraction at short lengths appears to restore the passive forces [5, 40] and the slack length [5]. Preconditioning is a widely used method to adjust the adaptation and reproducibility of measured mechani- cal parameters during experiments. This method is used to ensure that the SM tissue reaches an “equilib- rium state” where passive stress-stretch curves or active force responses are stable and repeatable, allowing the determination of the tissue’s mechanical properties [27]. Typically, SM tissue strips are cyclically stretched approximately 10 times during preconditioning [7, 14, 21, 28, 49]. This results in a reduction of passive forces and hysteresis, as well as an increase in resting length [27, 44]. Thus, the muscle parameters obtained after precon- ditioning do not correspond to the initial tissue response. Therefore, there is a gap in the literature regarding repro- ducible mechanical parameters without preconditioning that characterizes the initial mechanical state. Materials and methods Tissue handling and preparation This study was exempted from ethical committee review in accordance with national regulations (German Animal Welfare Act), as urinary bladders from nine healthy, female domestic pigs (Sus scrofa domestica, age: ≈ 6 months, mass: ≈ 100 kg) were obtained from a slaughterhouse immediately after animal sacrifice. During transport to the laboratory, the bladder tissue was maintained in a modified Krebs solu- tion (25 mM NaHCO3; 1.2 mM NaH2PO4; 2.4 mM MgSO4; 5.9 mM KCl; 2.5 mM CaCl2; 117 mM NaCl; and 11 mM C6H12O6) [24] at a constant temperature of 4°C [26]. During the experiments, the solution was maintained at a tempera- ture of 37 °C and was aerated with 95% O2 and 5% CO2 (pH 7.4) [3]. The experiments were completed within 14 h on each day of testing. The bladder was cut open along the ligamentum vesicae medianum, and three strips were excised in longitudinal direction from the body region of each blad- der. This resulted in a total of 27 tissue strips (N = 27) with a mean slack length (LS) of 10.4 ± 1.7 mm and a mean cross- sectional area (CSA) of 18.9 ± 4.5 mm2. After the measure- ments, the muscle tissue of the tissue strip was first separated from the serosa and then weighed. We determined the CSA as where m is the sample mass, � = 1.05 g/cm3 is the SM den- sity [18], and LS is the sample slack length. For the purpose of tissue testing, a setup was used that consisted of a muscle lever system (305C-LR, Aurora Sci- entific, Canada) and a real-time software package (610A Dynamic Muscle Analysis, Aurora Scientific, Canada) for data acquisition. The length and force signals from the lever were recorded at 100 Hz using an analog-to-digital A/D interface (604A, Aurora Scientific, Canada). The tis- sue strips were sewn into 3D-printed mounting parts and then attached vertically to the muscle lever (Fig. 1). While the strips were fixed in the setup, they were held at a very CSA = m �L S 731Pflügers Archiv - European Journal of Physiology (2025) 477:729–739 short length (clearly sagging) to prevent any pull on the tis- sue. The slack length (LS) was then adjusted by manually stretching the strip until a force of 10 mN was reached. The exact LS was then measured with a digital sliding caliper. The respective protocol was then initiated. Tensile testing protocols In total, three different protocols have been developed (Fig. 2). All three protocols share the same basic structure, starting from LS (corresponding to λ = 1). At the beginning, a length ramp was induced, stretching the strip to a stretch of λ = 2 at a strain rate of 1/180 [− /s]. The strip was then held at this length for 1 min. Subsequently, an isometric contraction (Fig. 2, first red bar from the left) was induced for 20 s, followed by a 40 s pause. Subsequently, the strip was shortened back to LS at a rate of 1/10 [− /s]. The three protocols differ in the number of stimulations at LS. For protocol 1 (P1), the strip was maintained at λ = 1 for 10:20 min until the next length ramp started (Fig. 2a). Therefore, no stimulation was induced at the short length. This cycle was repeated five times (designated C1–C5). The second protocol (P2 – Fig. 2b) included an additional iso- metric contraction at λ = 1 (Fig. 2b, second red bar from the left), timed precisely halfway between the contractions at λ = 2. The third protocol (P3 – Fig. 2c) included a third isometric contraction, again at λ = 1 (Fig. 2c, third red bar from the left). The protocols were designed with a minimum pause of at least 5 min between contractions to allow the tissue to fully recover [3, 30, 42, 48]. Muscle contractions during the protocols were induced by electrical stimulations (900 mA, 100 Hz, 5 ms) accord- ing to van Mastrigt and Glerum [24] for 20 s. From each bladder, the three strips were each assigned to one protocol, resulting in nine strips (n = 9) per protocol. The order of the assigned protocols was randomized for each bladder to avoid systematic errors in the results due to the storage time of the tissue strips. Analyzed mechanical parameters To assess the adaptation of SM tissue to the different pro- tocols (P1, P2, P3), three parameters were determined. The first parameter was the maximum active force (Fact) gen- erated during the isometric contractions at λ = 2. Fact was calculated by subtracting the passive force during the stress relaxation just before the start of stimulation (Frel) from the maximum total force during stimulation (Ftot) (Fig. 3). The second parameter observed was the maximum passive force (Fpass) at the end of the lengthening ramp (Fig. 3). The final parameter was the curvature of the normalized passive stress–strain response during the lengthening ramps. For this purpose, a circle was fitted to the stress–strain curve of each ramp using least-squares fitting. Therefore, the end- points of the circle fit had to align with the endpoints of the stress–strain curve. The curvature was then calculated as the inverse of the radius of the circle. Because for the curvature calculation process, the stress–strain curves were normal- ized with respect to stress and strain, the resulting curvature values are dimensionless and are therefore presented without units. Data processing and statistics Smoothing of the raw data and further data analysis were conducted using Matlab (version R2023a Update 2, The MathWorks Inc., Natick, MA, USA). The data presented in this study are presented as mean ± standard deviation. For the statistical analysis, the parameters were normalized with respect to the first cycle (C1). An ANOVA was conducted to test for significant differences between the protocols at C5. Post-hoc analyses were conducted using the Bonferroni correction. To test for differences within each protocol, a regression analysis was conducted. First, a regression line was constructed for each protocol from the mean values for Fig. 1 The illustration shows the experimental setup of the Aurora Scientific muscle lever system. To the right is a detailed image of a sutured SM strip. The upper end of the strip is connected to the mus- cle lever by a braided fishing line, which is used to induce stretch and record force. Muscle contractions are induced by electrical stimula- tion via platinum electrodes placed laterally along the strip. The tis- sue strip shown here is ~ 18 mm long. The graphic is for demonstra- tion of the experimental setup and the strip shown is not included in the sample size. Figure adapted from Borsdorf [2] 732 Pflügers Archiv - European Journal of Physiology (2025) 477:729–739 the cycles 1–5 (Fig. 3). T-tests were then used to test the null hypothesis for the slope of the linear regression line, namely whether the slope differed significantly from a slope of zero. All analyses were conducted with a significance level of p < 0.05. The ANOVA and the t-tests were conducted using SPSS 23 (version 29.0.1.1, IBM Corp., Armonk, NY, USA), while the regression analysis was conducted with Matlab. Results For P1, the active forces increased with each cycle (Fig. 4). The mean active stress during C1 was 35.0 ± 24.4 kPa for P1, 34.5 ± 17.3 kPa for P2, and 38.9 ± 17.4 kPa for P3. Regres- sion analysis revealed a statistically significant increase in slope for P1, with p < 0.001. For both P2 and P3, no sig- nificant trend in slope was observed. When comparing the values at C5, the results of the ANOVA showed a significant difference in active force between P1 and P2 (p = 0.005) and between P1 and P3 (p < 0.001). For P2 and P3, the passive forces increased with each cycle (Fig. 5). The mean nominal passive stress during C1 was 5.6 ± 3.1 kPa for P1, 4.2 ± 1.6 kPa for P2, and 4.6 ± 2.1 kPa for P3. Regression analysis showed a significant increase in slope for both P2 (p = 0.002) and P3 (p = 0.001). However, no signifi- cant trend in slope was observed for P1 (p > 0.05). The ANOVA showed no significant difference between the protocols at C5. For all three protocols, there is an increase in curvature from C1 to C5 (Fig. 6). An increase in curvature in this case is equivalent to an increase in stiffness at the end of the lengthening ramp. The mean absolute value at C1 was 0.5 ± 0.3 [ −] for P1, 0.4 ± 0.2 [ −] for P2, and 0.4 ± 0.2 [ −] for P3. Regression analysis showed a significant increase in slope for all three protocols (P1: p = 0.01; P2: p = 0.001; P3: p = 0.002). The ANOVA showed no significant differences between the protocols at C5. Fig. 2 Schematic representation of the stretch-time curves of the three developed adaptation protocols. The sub-figure in a shows the full protocol that each tissue strip was subjected to (cycles C1 to C5). The first (C1) of the five repetitive cycles for each protocol is shown for each protocol: a Protocol 1 (P1), b Protocol 2 (P2), and c Proto- col 3 (P3). The red bars indicate the isometric stimulation times (20 s each). LS corresponds to λ = 1 733Pflügers Archiv - European Journal of Physiology (2025) 477:729–739 Fig. 3 Exemplary force–time curves of lengthening ramps from λ = 1 to λ = 2 (see Fig.  2a) and subsequent isometric contractions for the repetitive 5 cycles. Note that the force responses of the 5 cycles are superimposed. The first cycle (C1) is shown in black, while the fol- lowing cycles C2 to C5 are shown in various shades of blue. Fact is the force difference between Ftot (total force) and Frel (force value after the relaxation phase of the tissue strip just before the induced contrac- tion). Fpass is the passive force at the end of the lengthening ramp. The sub-figure shows a schematic example of how the circle segment (dotted line) was fitted using the start and endpoints (blue dots) of the ramp’s stress-stretch curve as anchor points. In this case, the circle was fitted to C1 (black line) Fig. 4 Change in active forces at λ = 2 over 5 cycles for each of the three protocols (n = 9). The data have been normalized to the value of the first cycle. The asterisk symbol (*) indicates a statistically significant trend (p < 0.05) in the regression analysis within each protocol for the slope of C1–C5. The number sign symbol (#) shows a statistically significant dif- ference between the protocols with regard to the values at C5 734 Pflügers Archiv - European Journal of Physiology (2025) 477:729–739 Fig. 5 Changes in passive forces at λ = 2 over 5 cycles for each of the three protocols (n = 9). The data have been normalized to the value of the first cycle. The asterisk symbol (*) indicates a statistically signifi- cant difference (p < 0.05) in the regression analysis within each proto- col for the slope from C1 to C5 Fig. 6 Change in curvature of the stress–strain curves over 5 cycles for each of the three protocols (n = 9). The data were normalized to the value of the first cycle. The asterisk symbol (*) indicates a statisti- cally significant difference (p < 0.05) in the regression analysis within each protocol for the slope of C1 to C5 735Pflügers Archiv - European Journal of Physiology (2025) 477:729–739 Discussion The aim of this study was to find a method to reproduce the active and passive force response in SM tissue con- stantly over five repeated cycles of muscle stimulation at long lengths, thus reversing the induced SM adaptation. Three protocols were developed for this purpose, differing in the number of additional stimulations at a short muscle length (P1: 0, P2: 1, P3: 2 stimulations). The results show that the lengthening ramp and the sub- sequent isometric contraction induced an adaptation in Fact. This is evidenced by the significant increase (68%) in Fact (p < 0.05) in P1 over the course of the five cycles. The repro- duction of Fact was achieved in both P2 and P3, with the regression lines showing no significant change in Fact over five cycles (p > 0.05 in both cases). Although the passive forces in the final cycle did not differ between the three pro- tocols, only P1 showed no significant change in Fpass over the five cycles. Consequently, a change in the passive forces was induced by P2 and P3, and the design of P1 appears to be the most suitable for keeping Fpass constant over five cycles. None of the protocols were able to reproduce the curvature of the stress–strain response over five cycles. Comparison with literature A comparison with the existing literature is difficult due to the lack of comparable protocols in previous studies. Nevertheless, a qualitative comparison with selected stud- ies on muscle adaptivity is presented here. Wang et al. [48] showed an increase in Fact with multiple contractions of the same length. This is consistent with the results obtained for protocol 1 (Fig. 4). Speich et al. [40] reported an increase in Fpass after inducing contractions at short lengths on previously stretched tissue. This observa- tion is consistent with our results for protocols P2 and P3 (Fig. 4), where stimulations were also performed at short muscle lengths. Furthermore, our results for increasing the curvature of stress–strain curves with increasing number of passive stretches (Fig. 6) align with the results of previous studies on rabbit detrusor muscle [39] and porcine blad- der [27]. Both studies demonstrate a significant increase in curvature from the first to the second cycle, which persists with each additional cycle, although to a lesser extent. The change in curvature appears to depend mainly on passive lengthening, as there was no discernible effect of the num- ber of activations (P1, P2, P3) on the slope of the regression line (Fig. 6). An increase in curvature is associated with a decrease in the area under the stress–strain curve and thus a loss of stored energy in the passive elements [39]. Accord- ing to Speich et al. [39], the tissue reaches a viscoelastic steady state after approximately seven to eight repeated stretch ramps. This explains the continuous increase in cur- vature observed during the five ramps in our data. Both Gazzola et al. [12] and Lee-Gosselin et al. [23] reported a clear relationship between SM length and force development. A 30% increase in airway SM length resulted in an increase in total force (Ftot) of up to 117.8% [23]. In our results, Fact for P1 increased by an average of ~ 60% after 5 cycles at the same strip length (λ = 2), while Fpass remained unchanged. The influence of different strip lengths on the relative changes in muscle force during repeated activations at short lengths remains to be investigated. Adaptivity mechanisms Kuo et al. [22] and Bossé et al. [4] propose that changes in SM length lead to alterations in the contractile appara- tus, which in turn affect the active and passive forces of the tissue. According to this hypothesis, in order to adapt to longer lengths, contractile units (sarcomeres) are added Fig. 7 Schematic representation of the model proposed by Wang et al. [46] for the reversible adaptation processes within the contrac- tile apparatus of SMCs. The gray square represents a contractile unit. When a SM is first stretched (initial state) to λ = 2, the myosin and actin filaments do not have an optimal overlap. By inducing contrac- tions to the muscle tissue at the longer length, additional contractile units are added to the contractile apparatus to restore the optimal overlap of the myosin and actin filaments. With the protocols P2 and P3 (Fig.  2), this process is reversible, with contractile units being built out. Adapted from Kuo et al. [22] and Wang et al. [46] 736 Pflügers Archiv - European Journal of Physiology (2025) 477:729–739 to the contractile apparatus. Conversely, to adapt to shorter lengths, contractile units are removed from the array (Fig. 7). This enables the SM tissue to maintain an optimal overlap of actin and myosin filaments for cross-bridge cycling at dif- ferent lengths, which explains the shift in the force–length relationship after length adaptation [19, 48]. The underly- ing mechanism for these structural changes remains largely unknown. However, Chitano et al. [8] found evidence that myosin monomers are recruited into the filament lattice dur- ing length adaptation. The authors describe two myosin pools within the SMC: the monomeric myosin (non-muscle myo- sin) and the filamentous myosin (SM myosin). The process of length adaptation now alters the equilibrium between the two pools. This model provides an explanation for the observed changes in Fact for the three different protocols. For P1, each ramp, in combination with the isometric contraction at λ = 2, induces the installation of additional myosin filaments in series and in parallel [19, 22]. Fact could be increased by a better overlap of myofilaments, longer myosin filaments, or more contractile units in parallel [19, 41]. Shortening the tissue to λ = 1, followed by an isometric contraction, results in the removal of thick filaments (or impedes the previous induced installation of more myosin). This does not result in an increase in Fact with each cycle; rather, it maintains the same level (see P2). The results of this study (Fig. 3) indicate that increasing the number of contractions at the shortened length from one (at P2) to two (at P3) does not result in any additional effect on the removal of thick filaments. While the malleability of the actomyosin complex appears to influence the development of active forces, there is evidence in the literature that the cytoskeleton, with its integrated actin filaments, also plays an important role in the development of mechanical tension in the SMC. As with myosin, there are pools of free globular actin as well as fila- mentous actin [13, 52, 53]. Gazzola et al. [13] showed that SM activation alters the ratio of these two actin pools, as is the case with myosin. Other studies have provided evi- dence that not only activation [25] but also passive stretch [10] induces processes that alter the cytoskeleton of SMCs, including the actin filament lattice. Both stimuli activate a cascade of molecular processes via the so-called adhesion complexes, resulting in the polymerization of actin filaments and corresponding remodeling of the cytoskeleton in the peripheral region (e.g., cortical actin filaments) of the cell (Fig. 8) [16, 17, 36, 53]. The formation of this cortical actin- cytoskeleton structure in response to stretch or activation increases the stiffness of the cell. This remodeling is benefi- cial for the transmission of cross-bridge forces to the outside of the cell and ultimately results in increased stiffness and passive forces throughout the SM tissue [17]. The onset of cytoskeletal remodeling processes towards increased stiffness seems to be independent of the direc- tion of mechanical stretch and the length of the contractions. Fig. 8 Schematic illustration of actin-specific adaptation processes in SMCs in response to contractile and mechanical stimuli. Both conditions initiate a molecular cascade via the membrane adhesion complexes, which ultimately lead to actin polymerization at the cortical region of the cell. Subsequently, the polymerized actin is utilized for cytoskeletal rearrangements, which ultimately result in cell stiffening. Adapted from Gunst and Zhang [17] 737Pflügers Archiv - European Journal of Physiology (2025) 477:729–739 In the case of protocols P2 and P3, it can be reasonably concluded that with each additional contraction, the corti- cal polymerization of actin filaments was induced, thereby increasing the stiffness of the cell or muscle tissue. This is reflected in the increasing Fpass for P2 and P3 (Fig. 5). Since P3 contains one additional contraction compared to P2, it is evident that P3 has the highest Fpass after five cycles (Fig. 5). As proposed by Yamin and Morgan [50], depolymerization is likely to occur during relaxation to reverse the cytoskeletal stiffening that has occurred during the contraction phase. However, this effect has been little studied. Limitations The protocols presented in this study do not resemble the physiological behavior of bladder tissue. Physiologically, SMCs are relaxed during the filling phase and contractions occur only at long lengths, when micturition is induced in a full bladder [1]. The stimulation duration of 20 s is within the physiological micturition duration, which is 21 ± 13 s for all mammals over 3 kg body weight [51]. The proto- cols presented here are able to control muscle adaptation by introducing rest periods and short-length activation. In future studies, these steps can be incorporated into experi- mental protocols on SM tissue where adaptation processes would otherwise alter mechanical properties. Conclusion The aim of this study was to develop experimental protocols to reproduce the initial mechanical properties of SM tissue by inducing and reversing adaptation processes through the application of stretches and contractions of different lengths. The protocols developed allowed the reproduction of an initial active force at long lengths by adding contractions at short lengths (P2 and P3). However, the same protocols failed to reproduce the initial passive forces. Conversely, the passive forces increased with each cycle. Furthermore, the increase in curvature over five cycles indicates the loss of stored energy in the passive cell elements. The observed behavior of both active and passive forces can be explained by existing theories in the literature for the adaptation of SM tissues. Active force is primarily influenced by the addition or removal of contractile units to and from the contractile apparatus, whereas changes in passive force are mainly due to the contraction-induced actin polymerization and conse- quent cytoskeletal stiffening. The question of actin depo- lymerization and subsequent recovery of the initial passive forces remains open for further investigation. Author contributions TS, MaB, SK, MiB and SP conceived and designed the experiments. SK performed the experiments. SK, MiB and SP analyzed the data, interpreted the results, and SK drafted the first version of the manuscript. SK and SP prepared the figures. MaB, TS, SK, SP and MiB edited and revised the manuscript. All authors approved the final version of the manuscript. Funding Open Access funding enabled and organized by Projekt DEAL. This research was funded by the Deutsche Forschungsgemein- schaft (DFG) under the grant number 514952469. Data availability The datasets generated during and/or analysed dur- ing the current study are available from the corresponding author on reasonable request. Declarations Ethics approval The study was exempted from ethical committee review according to the National Regulations (German Animal Wel- fare Act) as porcine bladder smooth muscle tissue was obtained from a slaughterhouse immediately after animal sacrifice. Competing interests The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References 1. Andersson KE, Arner A (2004) Urinary bladder contraction and relaxation: physiology and pathophysiology. Physiol Rev 84:935– 986. https:// doi. org/ 10. 1152/ physr ev. 00038. 2003 2. Borsdorf M (2022) Biomechanical and microstructural characteri- zation of porcine smooth muscle properties [Doctoral dissertation, University of Stuttgart, Germany]. University of Stuttgart Reposi- tory http:// elib. uni- stutt gart. de/ handle/ 11682/ 12206 3. Borsdorf M, Tomalka A, Stutzig N, Morales-Orcajo E, Böl M, Siebert T (2019) Locational and directional dependencies of smooth muscle properties in pig urinary bladder. Front Physiol 10:1–12. https:// doi. org/ 10. 3389/ fphys. 2019. 00063 4. Bossé Y, Sobieszek A, Paré PD, Seow CY (2008) Length adap- tation of airway smooth muscle. Proc Am Thorac Soc 5:62–67. https:// doi. org/ 10. 1513/ pats. 200705- 056VS 5. Bossé Y, Solomon D, Chin LYM, Lian K, Paré PD, Seow CY (2010) Increase in passive stiffness at reduced airway smooth muscle length: potential impact on airway responsiveness. Am J Physiol - Lung Cell Mol Physiol 298:277–287. https:// doi. org/ 10. 1152/ ajplu ng. 00275. 2009 6. Brandstaeter S, Fuchs SL, Aydin RC, Cyron CJ (2019) Mechanics of the stomach: a review of an emerging field of biomechanics. GAMM Mitteilungen 42:1–17. https:// doi. org/ 10. 1002/ gamm. 20190 0001 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1152/physrev.00038.2003 http://elib.uni-stuttgart.de/handle/11682/12206 https://doi.org/10.3389/fphys.2019.00063 https://doi.org/10.1513/pats.200705-056VS https://doi.org/10.1152/ajplung.00275.2009 https://doi.org/10.1152/ajplung.00275.2009 https://doi.org/10.1002/gamm.201900001 https://doi.org/10.1002/gamm.201900001 738 Pflügers Archiv - European Journal of Physiology (2025) 477:729–739 7. Chen J, Drzewiecki BA, Merryman WD, Pope JC (2013) Murine bladder wall biomechanics following partial bladder obstruction. J Biomech 46:2752–2755. https:// doi. org/ 10. 1016/j. jbiom ech. 2013. 07. 022 8. Chitano P, Wang L, Tin GYY, Ikebe M, Paré PD, Seow CY (2017) Smooth muscle function and myosin polymerization. J Cell Sci 130:2468–2480. https:// doi. org/ 10. 1242/ jcs. 202812 9. Cooke R (1997) Actomyosin interaction in striated muscle. Physiol Rev 77:671–697. https:// doi. org/ 10. 1152/ physr ev. 1997. 77.3. 671 10. Deng L, Fairbank NJ, Fabry B, Smith PG, Maksym GN (2004) Localized mechanical stress induces time-dependent actin cytoskeletal remodeling and stiffening in cultured airway smooth muscle cells. Am J Physiol - Cell Physiol 287:440–448. https:// doi. org/ 10. 1152/ ajpce ll. 00374. 2003 11. Fontanella CG, Salmaso C, Toniolo I, de Cesare N, Rubini A, De Benedictis GM, Carniel EL (2019) Computational Models for the Mechanical Investigation of Stomach Tissues and Struc- ture. Ann Biomed Eng 47:1237–1249. https:// doi. org/ 10. 1007/ s10439- 019- 02229-w 12. Gazzola M, Henry C, Couture C, Marsolais D, King GG, Fredberg JJ, Bossé Y (2016) Smooth muscle in human bronchi is disposed to resist airway distension. Respir Physiol Neurobiol 229:51–58. https:// doi. org/ 10. 1016/j. resp. 2016. 04. 005 13. Gazzola M, Henry C, Lortie K, Khadangi F, Park CY, Fredberg JJ, Bossé Y (2020) Airway smooth muscle tone increases actin filamentogenesis and contractile capacity. Am J Physiol - Lung Cell Mol Physiol 318:L442–L451. https:// doi. org/ 10. 1152/ AJPLU NG. 00205. 2019 14. Gilbert TW, Wognum S, Joyce EM, Freytes DO, Sacks MS, Bady- lak SF (2008) Collagen fiber alignment and biaxial mechanical behavior of porcine urinary bladder derived extracellular matrix. Biomaterials 29:4775–4782. https:// doi. org/ 10. 1016/j. bioma teria ls. 2008. 08. 022 15. Griffiths DJ, van Mastrigt R, van Duyl WA, Coolsaet BLRA (1979) Active mechanical properties of the smooth muscle of the urinary bladder. Med Biol Eng Comput 17:281–290. https:// doi. org/ 10. 1007/ BF024 43812 16. Gunst SJ, Fredberg JJ (2003) The first three minutes: Smooth muscle contraction, cytoskeletal events, and soft glasses. J Appl Physiol 95:413–425. https:// doi. org/ 10. 1152/ jappl physi ol. 00277. 2003 17. Gunst SJ, Zhang W (2008) Actin cytoskeletal dynamics in smooth muscle: a new paradigm for the regulation of smooth muscle con- traction. Am J Physiol - Cell Physiol 295:C576–C587. https:// doi. org/ 10. 1152/ ajpce ll. 00253. 2008 18. Herlihy JT, Murphy RA (1973) Length-tension relationship of smooth muscle of the hog carotid artery. Circ Res 33:275–283. https:// doi. org/ 10. 1161/ 01. RES. 33.3. 275 19. Herrera AM, McParland BE, Bienkowska A, Tait R, Paré PD, Seow CY (2005) “Sarcomeres” of smooth muscle: Functional characteristics and ultrastructural evidence. J Cell Sci 118:2381– 2392. https:// doi. org/ 10. 1242/ jcs. 02368 20. Klemm L, Seydewitz R, Borsdorf M, Siebert T, Böl M (2020) On a coupled electro-chemomechanical model of gastric smooth muscle contraction. Acta Biomater 109:163–181. https:// doi. org/ 10. 1016/j. actbio. 2020. 04. 007 21. Korossis S, Bolland F, Southgate J, Ingham E, Fisher J (2009) Regional biomechanical and histological characterisation of the passive porcine urinary bladder: Implications for augmentation and tissue engineering strategies. Biomaterials 30:266–275. https:// doi. org/ 10. 1016/j. bioma teria ls. 2008. 09. 034 22. Kuo K-H, Herrera AM, Wang L, Paré PD, Ford LE, Stephens NL, Seow CY (2003) Structure-function correlation in airway smooth muscle adapted to different lengths. Am J Physiol - Cell Physiol 285:C384–C390. https:// doi. org/ 10. 1152/ ajpce ll. 00095. 2003 23. Lee-Gosselin A, Pascoe CD, Couture C, Paré PD, Bossé Y (2013) Does the length dependency of airway smooth muscle force con- tribute to airway hyperresponsiveness? J Appl Physiol 115:1304– 1315. https:// doi. org/ 10. 1152/ jappl physi ol. 01480. 2012 24. van Mastrigt R, Glerum JJ (1985) Electrical stimulation of smooth muscle strips from the urinary bladder of the pig. J Biomed Eng 7:2–8. https:// doi. org/ 10. 1016/ 0141- 5425(85) 90002-0 25. Mehta D, Gunst SJ (1999) Actin polymerization stimulated by contractile activation regulates force development in canine tra- cheal smooth muscle. J Physiol 519:829–840. https:// doi. org/ 10. 1111/j. 1469- 7793. 1999. 0829n.x 26. Menzel R, Böl M, Siebert T (2017) Importance of contrac- tion history on muscle force of porcine urinary bladder smooth muscle. Int Urol Nephrol 49:205–214. https:// doi. org/ 10. 1007/ s11255- 016- 1482-x 27. Morales-Orcajo E, Siebert T, Böl M (2018) Location-dependent correlation between tissue structure and the mechanical behaviour of the urinary bladder. Acta Biomater 75:263–278. https:// doi. org/ 10. 1016/j. actbio. 2018. 05. 014 28. Nagatomi J, Gloeckner DC, Chancellor MB, DeGroat WC, Sacks MS (2004) Changes in the biaxial viscoelastic response of the urinary bladder following spinal cord injury. Ann Biomed Eng 32:1409–1419. https:// doi. org/ 10. 1114/B: ABME. 00000 42228. 89106. 48 29. Papenkort S, Borsdorf M, Böl M, Siebert T (2023) A geometry model of the porcine stomach featuring mucosa and muscle layer thicknesses. J Mech Behav Biomed Mater 142:105801. https:// doi. org/ 10. 1016/j. jmbbm. 2023. 105801 30. Pratusevich VR, Seow CY, Ford LE (1995) Plasticity in canine airway smooth muscle. J Gen Physiol 105:73–94. https:// doi. org/ 10. 1085/ jgp. 105.1. 73 31. Ratz PH, Speich JE (2010) Evidence that actomyosin cross bridges contribute to “passive” tension in detrusor smooth muscle. Am J Physiol - Ren Physiol 298:1424–1435. https:// doi. org/ 10. 1152/ ajpre nal. 00635. 2009 32. Schenk P, Papenkort S, Böl M, Siebert T, Grassme R, Rode C (2020) A simple geometrical model accounting for 3D mus- cle architectural changes across muscle lengths. J Biomech 103:109694. https:// doi. org/ 10. 1016/j. jbiom ech. 2020. 109694 33. Schmidt RF, Lang F, Thews G (2005) Physiologie des Menschen, 29th edn. Springer Medizin Verlag, Heidelberg 34. Seow CY (2005) Myosin filament assembly in an ever-changing myofilament lattice of smooth muscle. Am J Physiol - Cell Physiol 289:C1363–C1368. https:// doi. org/ 10. 1152/ ajpce ll. 00329. 2005 35. Seydewitz R, Menzel R, Siebert T, Böl M (2017) Three-dimen- sional mechano-electrochemical model for smooth muscle con- traction of the urinary bladder. J Mech Behav Biomed Mater 75:128–146. https:// doi. org/ 10. 1016/j. jmbbm. 2017. 03. 034 36. Smith BA, Tolloczko B, Martin JG, Grütter P (2005) Probing the viscoelastic behavior of cultured airway smooth muscle cells with atomic force microscopy: stiffening induced by contractile agonist. Biophys J 88:2994–3007. https:// doi. org/ 10. 1529/ bioph ysj. 104. 046649 37. Smolensky AV, Ragozzino J, Gilbert SH, Seow CY, Ford LE (2005) Length-dependent filament formation assessed from bire- fringence increases during activation of porcine tracheal muscle. J Physiol 563:517–527. https:// doi. org/ 10. 1113/ jphys iol. 2004. 079822 38. Sobieszek IJ, Sobieszek A (2022) Myosin assembly of smooth muscle: from ribbons and side polarity to a row polar helical model. J Muscle Res Cell Motil 43:113–133. https:// doi. org/ 10. 1007/ s10974- 022- 09622-4 39. Speich JE, Borgsmiller L, Call C, Mohr R, Ratz PH (2005) ROK- induced cross-link formation stiffens passive muscle: Reversible strain-induced stress softening in rabbit detrusor. Am J Physiol https://doi.org/10.1016/j.jbiomech.2013.07.022 https://doi.org/10.1016/j.jbiomech.2013.07.022 https://doi.org/10.1242/jcs.202812 https://doi.org/10.1152/physrev.1997.77.3.671 https://doi.org/10.1152/ajpcell.00374.2003 https://doi.org/10.1152/ajpcell.00374.2003 https://doi.org/10.1007/s10439-019-02229-w https://doi.org/10.1007/s10439-019-02229-w https://doi.org/10.1016/j.resp.2016.04.005 https://doi.org/10.1152/AJPLUNG.00205.2019 https://doi.org/10.1152/AJPLUNG.00205.2019 https://doi.org/10.1016/j.biomaterials.2008.08.022 https://doi.org/10.1016/j.biomaterials.2008.08.022 https://doi.org/10.1007/BF02443812 https://doi.org/10.1007/BF02443812 https://doi.org/10.1152/japplphysiol.00277.2003 https://doi.org/10.1152/japplphysiol.00277.2003 https://doi.org/10.1152/ajpcell.00253.2008 https://doi.org/10.1152/ajpcell.00253.2008 https://doi.org/10.1161/01.RES.33.3.275 https://doi.org/10.1242/jcs.02368 https://doi.org/10.1016/j.actbio.2020.04.007 https://doi.org/10.1016/j.actbio.2020.04.007 https://doi.org/10.1016/j.biomaterials.2008.09.034 https://doi.org/10.1152/ajpcell.00095.2003 https://doi.org/10.1152/japplphysiol.01480.2012 https://doi.org/10.1016/0141-5425(85)90002-0 https://doi.org/10.1111/j.1469-7793.1999.0829n.x https://doi.org/10.1111/j.1469-7793.1999.0829n.x https://doi.org/10.1007/s11255-016-1482-x https://doi.org/10.1007/s11255-016-1482-x https://doi.org/10.1016/j.actbio.2018.05.014 https://doi.org/10.1016/j.actbio.2018.05.014 https://doi.org/10.1114/B:ABME.0000042228.89106.48 https://doi.org/10.1114/B:ABME.0000042228.89106.48 https://doi.org/10.1016/j.jmbbm.2023.105801 https://doi.org/10.1016/j.jmbbm.2023.105801 https://doi.org/10.1085/jgp.105.1.73 https://doi.org/10.1085/jgp.105.1.73 https://doi.org/10.1152/ajprenal.00635.2009 https://doi.org/10.1152/ajprenal.00635.2009 https://doi.org/10.1016/j.jbiomech.2020.109694 https://doi.org/10.1152/ajpcell.00329.2005 https://doi.org/10.1016/j.jmbbm.2017.03.034 https://doi.org/10.1529/biophysj.104.046649 https://doi.org/10.1529/biophysj.104.046649 https://doi.org/10.1113/jphysiol.2004.079822 https://doi.org/10.1113/jphysiol.2004.079822 https://doi.org/10.1007/s10974-022-09622-4 https://doi.org/10.1007/s10974-022-09622-4 739Pflügers Archiv - European Journal of Physiology (2025) 477:729–739 - Cell Physiol 289:12–21. https:// doi. org/ 10. 1152/ ajpce ll. 00418. 2004 40. Speich JE, Dosier C, Borgsmiller L, Quintero K, Koo HP, Ratz PH (2007) Adjustable passive length-tension curve in rabbit detrusor smooth muscle. J Appl Physiol 102:1746–1755. https:// doi. org/ 10. 1152/ jappl physi ol. 00548. 2006 41. Squire JM (2009) Muscle myosin filaments : cores, crowns and couplings. Biophys Rev 1:149–160. https:// doi. org/ 10. 1007/ s12551- 009- 0017-4 42. Tomalka A, Borsdorf M, Böl M, Siebert T (2017) Porcine stomach smooth muscle force depends on history-effects. Front Physiol 8:289206. https:// doi. org/ 10. 3389/ fphys. 2017. 00802 43. Tomalka A, Heim M, Klotz A, Rode C, Siebert T (2022) Ultra- structural and kinetic evidence support that thick filaments slide through the Z-disc. J R Soc Interface 19. https:// doi. org/ 10. 1098/ rsif. 2022. 0642 44. Trostorf R, Morales-Orcajo E, Siebert T, Böl M (2021) Location- and layer-dependent biomechanical and microstructural character- isation of the porcine urinary bladder wall. J Mech Behav Biomed Mater 115:104275. https:// doi. org/ 10. 1016/j. jmbbm. 2020. 104275 45. Uvelius B (1976) Isometric and isotonic length-tension relations and variations in cell length in longitudinal smooth muscle from rabbit urinary bladder. Acta Physiol Scand 97:1–12. https:// doi. org/ 10. 1111/j. 1748- 1716. 1976. tb102 30.x 46. Wang L, Chitano P, Seow CY (2021) Filament evanescence of myosin II and smooth muscle function. J Gen Physiol 153:1–12. https:// doi. org/ 10. 1085/ JGP. 20201 2781 47. Wang L, Paré PD, Seow CY (2000) Effects of length oscillation on the subsequent force development in swine tracheal smooth muscle. J Appl Physiol 88:2246–2250. https:// doi. org/ 10. 1152/ jappl. 2000. 88.6. 2246 48. Wang L, Paré PD, Seow CY (2001) Plasticity in skeletal, cardiac, and smooth muscle selected contribution: effect of chronic passive length change on airway smooth muscle length-tension relation- ship. J Appl Physiol 90:734–740 49. Wognum S, Schmidt DE, Sacks MS (2009) On the mechanical role of de novo synthesized elastin in the urinary bladder wall. J Biomech Eng 131. https:// doi. org/ 10. 1115/1. 40001 82 50. Yamin R, Morgan KG (2012) Deciphering actin cytoskel- etal function in the contractile vascular smooth muscle cell. J Physiol 590:4145–4154. https:// doi. org/ 10. 1113/ jphys iol. 2012. 232306 51. Yang PJ, Pham JC, Choo J, Hu DL (2013) Law of Urination: all mammals empty their bladders over the same duration. arXiv Prepr arXiv 1310.3737. https:// doi. org/ 10. 48550/ arXiv. 13103 737 52. Zhang W, Gunst SJ (2008) Interactions of airway smooth mus- cle cells with their tissue matrix implications for contraction. Proc Am Thorac Soc 5:32–39. https:// doi. org/ 10. 1513/ pats. 200704- 048VS 53. Zhang W, Wu Y, Du L, Tang DD, Gunst SJ (2005) Activation of the Arp2/3 complex by N-WASp is required for actin polym- erization and contraction in smooth muscle. Am J Physiol - Cell Physiol 288:1145–1160. https:// doi. org/ 10. 1152/ ajpce ll. 00387. 2004 Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. https://doi.org/10.1152/ajpcell.00418.2004 https://doi.org/10.1152/ajpcell.00418.2004 https://doi.org/10.1152/japplphysiol.00548.2006 https://doi.org/10.1152/japplphysiol.00548.2006 https://doi.org/10.1007/s12551-009-0017-4 https://doi.org/10.1007/s12551-009-0017-4 https://doi.org/10.3389/fphys.2017.00802 https://doi.org/10.1098/rsif.2022.0642 https://doi.org/10.1098/rsif.2022.0642 https://doi.org/10.1016/j.jmbbm.2020.104275 https://doi.org/10.1111/j.1748-1716.1976.tb10230.x https://doi.org/10.1111/j.1748-1716.1976.tb10230.x https://doi.org/10.1085/JGP.202012781 https://doi.org/10.1152/jappl.2000.88.6.2246 https://doi.org/10.1152/jappl.2000.88.6.2246 https://doi.org/10.1115/1.4000182 https://doi.org/10.1113/jphysiol.2012.232306 https://doi.org/10.1113/jphysiol.2012.232306 https://doi.org/10.48550/arXiv.13103737 https://doi.org/10.1513/pats.200704-048VS https://doi.org/10.1513/pats.200704-048VS https://doi.org/10.1152/ajpcell.00387.2004 https://doi.org/10.1152/ajpcell.00387.2004 Reproducibility of smooth muscle mechanical properties in consecutive stretch and activation protocols Abstract Introduction Materials and methods Tissue handling and preparation Tensile testing protocols Analyzed mechanical parameters Data processing and statistics Results Discussion Comparison with literature Adaptivity mechanisms Limitations Conclusion References