Contractile Output Research Articles

Without visual feedback voluntary torque is overestimated during muscle potentiation despite similar motor unit firing rate and perception of exertion.

The purpose was to assess whether visual feedback of torque contributes to motor unit (MU) firing rate reduction observed during post-activation potentiation (PAP) of skeletal muscle. From 15 participants 23 MUs were recorded with intramuscular fine-wire electrodes from the tibialis anterior during isometric dorsiflexion contractions at 20% of maximum, with and without both PAP and visual feedback of torque. A 5s maximal voluntary contraction (MVC) was used to induce PAP, and evoked twitch responses were assessed before and after. After the MVC twitch torque was 188% of baseline (p<0.001). Without visual feedback of torque and with participants targeting 20% MVC, torque, MU firing rates and rating of perceived exertion (RPE) were 22.8 ± 5.3 %MVC, 14.3 ± 2.6 Hz, 1.79 ± 0.93 a.u., respectively. Inducing PAP without feedback but targeting 20% MVC torque was overestimated by 50% (p<0.001) despite similar firing rates and RPE as baseline (both p≥0.3). With visual feedback, torque was not overestimated during PAP (p=0.14), however, rates and RPE were lower (13 and 20%, respectively) than baseline (both p≤0.008). Therefore, no compensatory modifications in MU output occurred despite muscle potentiation. This indicates lower voluntary drive, reflected additionally by reduced RPE, was responsible for the reduced firing rates so that torque did not exceed the required task, compared to modified peripheral feedback. During PAP, the motoneuron is not sensitive to alterations in the active state of the muscle unit per se, but rather compensatory adjustments to optimize contractile output are due to reductions in descending input.

Anillin tunes contractility and regulates barrier function during Rho flare-mediated tight junction remodeling.

To preserve barrier function, cell-cell junctions must dynamically remodel during cell shape changes. We have previously described a rapid tight junction repair pathway characterized by local, transient activation of RhoA, termed 'Rho flares,' which repair leaks in tight junctions via promoting local actomyosin-mediated junction remodeling. In this pathway, junction elongation is a mechanical trigger that initiates RhoA activation through an influx of intracellular calcium and recruitment of p115RhoGEF. However, mechanisms that tune the level of RhoA activation and Myosin II contractility during the process remain uncharacterized. Here, we show that the scaffolding protein Anillin localizes to Rho flares and regulates RhoA activity and actomyosin contraction at flares. Knocking down Anillin results in Rho flares with increased intensity but shorter duration. These changes in active RhoA dynamics weaken downstream F-actin and Myosin II accumulation at the site of Rho flares, resulting in decreased junction contraction. Consequently, tight junction breaks are not reinforced following Rho flares. We show that Anillin-driven RhoA regulation is necessary for successfully repairing tight junction leaks and protecting junctions from repeated barrier damage. Together, these results uncover a novel regulatory role for Anillin during tight junction repair and barrier function maintenance.

Differential effects of stimulation frequency on isometric and concentric isotonic contractile function in human quadriceps.

Electrically evoked contractions are used to assess the relationship between frequency input and contractile output to characterize inherent muscle function, and these have been done mostly with isometric contractions (i.e., no joint rotation). The purpose was to compare the electrically stimulated frequency and contractile function relationship during isometric (i.e., torque) with isotonic (i.e., concentric torque, angular velocity, and mechanical power) contractions. The knee extensors of 16 (5 female) young recreationally active participants were stimulated (∼1-2.5 s) at 14 frequencies from 1 to 100 Hz. This was done during four conditions, which were isometric and isotonic at loads of 0 (unloaded), 7.5%, and 15% isometric maximal voluntary contraction (MVC), and repeated on separate days. Comparisons across contractile parameters were made as a % of 100 Hz. Independent of the load, the mechanical power-frequency relationship was rightward shifted compared with isometric torque-frequency, concentric torque-frequency, and velocity-frequency relationships (all P ≤ 0.04). With increasing load (0%-15% MVC), the isotonic concentric torque-frequency relationship was shifted leftward systematically from 15 to 30 Hz (all P ≤ 0.04). Conversely, the same changes in load caused a rightward shift in the velocity-frequency relationship from 1 to 40 Hz (all P ≤ 0.03). Velocity was leftward shifted of concentric torque in the unloaded isotonic condition from 10 to 25 Hz (all P ≤ 0.03), but concentric torque was leftward shifted of velocity at 15% MVC isotonic condition from 10 to 50 Hz (all P ≤ 0.03). Therefore, isometric torque is not a surrogate to evaluate dynamic contractile function. Interpretations of evoked contractile function differ depending on contraction type, load, and frequency, which should be considered relative to the specific task.NEW & NOTEWORTHY In whole human muscle, we showed that the electrically stimulated power-frequency relationship was rightward shifted of the stimulated isometric torque-frequency relationship independent of isotonic load, indicating that higher stimulation frequencies are needed to achieve tetanus. Therefore, interpretations of evoked contractile function differ depending on contraction type (isometric vs. dynamic), load, and frequency. And thus, isometric measures may not be appropriate as a surrogate assessment when evaluating dynamic isotonic contractile function.

Abstract P1104: Mantarray 3D Engineered Muscle Tissue Platform Demonstrates Clinically-relevant Disease Stratification Of An In Vitro Human Duchenne Muscular Dystrophy Model

Accurately modeling disease in vitro is vital for the development of new treatment strategies and therapeutics. For cardiac diseases, direct assessment of contractile output is the “ultimate phenotype” and a reliable metric to study overall tissue function, as other ‘proxy’ measurements or biomarkers are not good predictors of muscle strength. Human 3D engineered heart tissues (EHTs) from induced pluripotent stem cells hold great potential for modeling contractile function. However, the bioengineering strategies required to generate these predictive models present limitations for many investigators. Here, we have developed an instrument platform that utilizes 3D EHTs in conjunction with a label-free, magnetic sensing array (Mantarray). The platform enables facile and reproducible fabrication of 3D EHTs using virtually any cell source and is coupled with parallel direct measurement of contractility. This approach enables clinically relevant functional measurements of muscle, stratification of healthy vs. diseased muscle phenotypes, and facilitates therapeutic modality-agnostic compound safety and efficacy screening. We present a 3D model of Duchenne muscular dystrophy (DMD) that utilizes EHTs formed from an isogenic pair of healthy and diseased cells. DMD tissues display functional deficits across numerous metrics of contractility, including force and relaxation kinetics, though beat rate remains unchanged. EHTs remain functional over months in culture and provide a large experimental window to not only study therapeutic effect, but also disease phenotypes that may present at later stages of development and maturity. We show both acute and chronic effects of compounds in EHTs, including a drug (BMS-986094) that failed clinical trials due to unanticipated cardiotoxicity. These data demonstrate a first-and-only commercial platform that integrates individual, well-based control of electrical stimulation across a 24-well plate of tissues to pace muscle constructs, model force-frequency, and enable exercise regimens or damage protocols. Stimulation is coupled with automated assessment of muscle contraction, providing an inclusive, medium-throughput platform for disease modeling and therapeutic discovery.

Reproducibility of drug-induced effects on the contractility of an engineered heart tissue derived from human pluripotent stem cells.

Introduction: Engineered heart tissues (EHTs) are three-dimensional culture platforms with cardiomyocytes differentiated from human pluripotent stem cells (hPSCs) and were designed for assaying cardiac contractility. For drug development applications, EHTs must have a stable function and provide reproducible results. We investigated these properties with EHTs made with different tissue casting batches and lines of differentiated hPSC-cardiomyocytes and analyzed them at different times after being fabricated. Methods: A video-optical assay was used for measuring EHT contractile outputs, and these results were compared with results from motion traction analysis of beating hPSC-cardiomyocytes cultured as monolayers in two-dimensional cultures. The reproducibility of induced contractile variations was tested using compounds with known mechanistic cardiac effects (isoproterenol, EMD-57033, omecamtiv mecarbil, verapamil, ranolazine, and mavacamten), or known to be clinically cardiotoxic (doxorubicin, sunitinib). These drug-induced variations were characterized at different electrical pacing rates and variations in intracellular calcium transients were also assessed in EHTs. Results: To ensure reproducibility in experiments, we established EHT quality control criteria based on excitation-contraction coupling and contractile sensitivity to extracellular calcium concentration. In summary, a baseline contractile force of 0.2mN and excitation-contraction coupling of EHTs were used as quality control criteria to select suitable EHTs for analysis. Overall, drug-induced contractile responses were similar between monolayers and EHTs, where a close relationship was observed between contractile output and calcium kinetics. Contractile variations at multiple time points after adding cardiotoxic compounds were also detectable in EHTs. Discussion: Reproducibility of drug-induced effects in EHTs between experiments and relative to published work on these cellular models was generally observed. Future applications for EHTs may require additional mechanistic criteria related to drug effects and cardiac functional outputs to be measured in regard to specific contexts of use.

Isometric Force-Frequency and Dynamic Power-Frequency Relationships in Human Knee Extensors

Under voluntary control the neuromuscular system grades contractile output of recruited motor units by modulating discharge frequency. Electrically evoked (i.e., involuntary) contractions have been used to assess relationships between frequency input and contractile output. During an isometric contraction (i.e., no joint rotation) it is well-established that the force-frequency relationship is sigmoidal. However, there are few data describing the relationship between evoked input and dynamic (i.e., joint rotation) contractile parameters. Thus, the purpose was to examine the relationships between stimulated frequencies and dynamic power (i.e., product of concentric torque and angular velocity) during an isotonic contraction in comparison to an isometric force-frequency relationship. Knee extensors of 7 healthy young adult males were tested in a Cybex dynamometer. The right knee was at 90° and hip at 110°. Seat belts secured to the dynamometer were strapped across the hips and shoulders, and a non-elastic strap was secured across the thigh to avoid extraneous movement. Two stimulation electrodes, which consisted of aluminum wrapped in a conductive gel-soaked cloth were secured transversely to the proximal and distal portion of the knee extensors. For dynamic contractions the dynamometer was set to the “isotonic mode” and range of motion was 60°. The muscles were stimulated at 1, 5, 7.5, 10, 12, 15, 17, 20, 25, 30, 40, 50, 75 and 100 Hz. Besides the 1 Hz (i.e., single pulse) all frequencies during isometric contractions were 2s in duration. During isotonic contractions with the load set to 7.5% maximal voluntary contraction (MVC), pulses were programmed to terminate once range of motion was complete or until full range of motion was unachievable. Electrical current for all frequencies was determined from that required at 100 Hz to produce 50% isometric MVC force. Stimulation was done in ascending order with 20s rest between. The order between contraction modes was randomized and a 5-minute rest was given between modes. Results are presented as a percentage of relative peak force or peak power for 100 Hz during isometric or isotonic contractions, respectively. Both isometric force-frequency and dynamic power-frequency relationships were sigmoidal. During stimulation of increasing frequencies peak power was lower than isometric force by ~82%, ~47%, ~43%, ~38%, ~32%, ~25%, ~30%, ~23%, ~17%, ~12%, ~8% at ascending frequencies from 1 Hz to 40Hz, respectively. From 50 Hz to 100 Hz there were minimal differences (0-4%). These data indicate for the same stimulation frequencies the power-frequency relationship of a moderately loaded isotonic contraction is shifted rightward relative to an isometric force-frequency relationship. Therefore, isometric force-frequency and dynamic power-frequency relationships are not equivalent, and assessments of both relationships may be required to adequately characterize contractile function depending on the task. Supported by NSERC. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Calcaneal tendon stiffness is not associated with dynamic time-dependent contractile output.

The ability to rapidly generate muscular torque and velocity is important in specialized activities and daily tasks of living. Tendon stiffness is one factor in the neuromuscular system that influences musculoskeletal torque transmission. Previous studies have reported weak-to-moderate correlations between tendon stiffness and rate of torque development (RTD). However, these correlations have been reported only for isometric contractions, which may not be relevant to contractions involving joint rotation (i.e., dynamic). The purpose was to investigate the effect of calcaneal tendon stiffness on the dynamic RTD and rate of velocity development (RVD) in plantar flexor muscles. Young adult males (n=13) and females (n=2) performed prone isometric- and isotonic-mode maximal voluntary plantar flexion contractions (MVC). Ultrasound imaging was used to quantify tendon morphological characteristics to estimate Young's elastic modulus (YM). Maximal voluntary and electrically evoked (300 Hz) isometric- and isotonic-mode (at 10% and 40% MVC loads) contractions were evaluated for RTD and RVD through a 25° ankle joint range of motion. YM was correlated with isometric RTD, but only for evoked contractions (RTD0-50 ms: r=0.54, p=0.02, RTD0-200 ms: r=0.62, p=0.01). Conversely, YM was not correlated with dynamic RTD (voluntary: r = -0.07-0.41, p=0.06-0.40, evoked: r = -0.2-0.3, p=0.14-0.24) nor RVD (voluntary: r = -0.08-0.24, p=0.27-0.40, evoked: r=0.12-0.3, p=0.14-0.34). These correlations would indicate that calcaneal tendon stiffness is an important factor for rapid isometric torque development, but less so for isotonic contractions. The determinants of dynamic contractile rates are more complex and warrant further study.

Stretch-activated nuclear YAP localization in cardiomyocytes.

Yes Associated Protein (YAP) is a mechanosensitive transcriptional activator regulating a variety of cell fates, including proliferation and cell growth. YAP is activated (localized to the nucleus) in both physiological development (e.g., organ growth mediated by cell proliferation) and pathological heart diseases (e.g., organ growth mediated by cell enlargement). Cardiomyocytes (CMs) generate the mechanical work that powers each heartbeat, and each contraction shortens the CM and strains the nuclei. Mechanical forces and nuclear deformation are considered putative drivers of YAP activation. However, the mechanoregulation of YAP's dual roles in organ growth - through proliferation during development and via hypertrophic enlargement in adulthood - remain unknown. Both contractile output and strain increase as the heart works harder under increased load (e.g., with exercise, disease, aging) and over time can lead to remodeling characteristic of hypertrophic cardiomyopathy. To mimic this additional loading, we first patterned extracellular matrix (Matrigel) single-cell rectangles of 7:1 aspect ratio on silicon substrate of a previously published, microfabricated, silicone pneumatic cell-stretching device (Hart, CAMB, 2021). Then, we then seeded human induced pluripotent stem cell-derived CMs (hiPSC-CM) on these patterns to elicit an elongated, anisotropic subcellular structure. We applied 15% stretch strain in static and cyclic, at 1 Hz frequency, for 10 mins to hiPSC-CM aligned in perpendicular or in parallel to the direction of stretch. Finally, we fixed and immunostained with YAP antibodies to quantify YAP localization. We quantified the ratio of nuclear/non-nuclear YAP in hiPSC-CM and found an upregulation of nuclear YAP in parallel, 97±10% in static and 42±5% in cyclic, and no significant change in regulation in perpendicular. Ultimately, these experiments will help us understand how mechanical cues and directionality of forces drive nuclear YAP, and the role of YAP signaling in driving hypertrophic hiPSC-CM remodeling.

Prolonged loss of force and power following fatiguing contractions in rat soleus muscles. Is low-frequency fatigue an issue during dynamic contractions?

Low-frequency fatigue (LFF) is defined by a relatively larger deficit in isometric force elicited by low-frequency electrical stimulation compared with high-frequency stimulation. However, the effects of LFF on power during dynamic contractions elicited at low and high frequencies have not been thoroughly characterized. In the current study, rat soleus muscles underwent fatiguing either concentric, eccentric, or isometric contractions. Before and 1 h after the fatiguing contractions, a series of brief isometric and dynamic contractions elicited at 20 and 80 Hz stimulation to establish force-velocity relationships. Maximal force (Fmax), velocity (Vmax), and power (Pmax) were assessed for each frequency. Sarcoplasmic reticulum (SR) Ca2+ release and reuptake rates were assessed pre- and postfatigue. Prolonged fatigue was observed as a loss of Fmax and Pmax in muscles fatigued by concentric or eccentric, but not by isometric contractions. When quantified as a decrease in the ratio between 20 Hz and 80 Hz contractile output, LFF was more pronounced for isometric force than for power (-21% vs. -16% for concentrically fatigued muscles, P = 0.003; 29 vs. 13% for eccentrically fatigued muscles, P < 0.001). No changes in SR Ca2+ release or reuptake rates were observed. We conclude that LFF is less pronounced when expressed in terms of power deficits than when expressed in terms of force deficits, and that LFF, therefore, likely affects performance to a lesser degree during fast concentric contractions than during static or slow contractions.

Abstract P1131: Modeling Contractile Diseases Using Scalable 3D Engineered Heart Tissues For Drug Discovery

Model systems that accurately recapitulate healthy and diseased function in a dish are critical for the development of novel therapeutics. For cardiac diseases, direct assessment of contractile output constitutes the most reliable metric with which to assess overall tissue function, as other ‘proxy’ measurements are poor predictors of muscle strength. 3D engineered muscle tissues (EMTs) derived from iPSCs hold great potential for modeling contractile function. Here, we have developed a platform and device that utilizes 3D EMTs in conjunction with a label-free magnetic sensing array. The platform enables facile and reproducible fabrication of 3D EMTs using virtually any cell source and is coupled with a highly parallel direct measurement of contractile strength. This approach enables the stratification of healthy and diseased phenotypes and facilitates compound safety and efficacy screening for evaluation of a drug’s effect on contractile output. We will present data from a drug (BMS-986094) that failed clinical trials due to unanticipated cardiotoxicity. We go on to show both the acute and chronic effects of doxorubicin in cardiac EMTs. Contractile force decreased in a dose dependent-like manner when the drug was applied continuously. Interestingly, a single 1uM bolus induced a transient effect that could be washed out over time. A repeat bolus, however, irreversibly abolished contraction, suggesting that repeat dosing may have a cardiotoxic effect on the muscle. These data demonstrate a first-and-only commercial platform for high-throughput assessment of 3D cardiac muscle contraction with potential for widespread adoption within the drug development field.

Allosteric destabilization of the super-relaxed state of cardiac myosin by hypertrophic cardiomyopathy-causing mutations

Hypertrophic cardiomyopathy (HCM), a leading cause of heart failure and sudden death, results from mutations in sarcomeric proteins. ∼35% of causative mutations affect β-cardiac myosin. It is now recognized that a large fraction of these may be affecting the conformational dynamics of myosin, and not its catalytic activity. Studies over the past decade have shifted the focus of HCM research to the evolutionarily conserved ‘super-relaxed’ (SRX) state of myosin, which is an important modulator of cardiac contractile output and energy consumption.

High-throughput, real-time monitoring of engineered skeletal muscle function using magnetic sensing

Engineered muscle tissues represent powerful tools for examining tissue level contractile properties of skeletal muscle. However, limitations in the throughput associated with standard analysis methods limit their utility for longitudinal study, high throughput drug screens, and disease modeling. Here we present a method for integrating 3D engineered skeletal muscles with a magnetic sensing system to facilitate non-invasive, longitudinal analysis of developing contraction kinetics. Using this platform, we show that engineered skeletal muscle tissues derived from both induced pluripotent stem cell and primary sources undergo improvements in contractile output over time in culture. We demonstrate how magnetic sensing of contractility can be employed for simultaneous assessment of multiple tissues subjected to different doses of known skeletal muscle inotropes as well as the stratification of healthy versus diseased functional profiles in normal and dystrophic muscle cells. Based on these data, this combined culture system and magnet-based contractility platform greatly broadens the potential for 3D engineered skeletal muscle tissues to impact the translation of novel therapies from the lab to the clinic.

Myosin Sequestration Regulates Sarcomere Function, Cardiomyocyte Energetics, and Metabolism, Informing the Pathogenesis of Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is caused by pathogenic variants in sarcomere protein genes that evoke hypercontractility, poor relaxation, and increased energy consumption by the heart and increased patient risks for arrhythmias and heart failure. Recent studies show that pathogenic missense variants in myosin, the molecular motor of the sarcomere, are clustered in residues that participate in dynamic conformational states of sarcomere proteins. We hypothesized that these conformations are essential to adapt contractile output for energy conservation and that pathophysiology of HCM results from destabilization of these conformations. We assayed myosin ATP binding to define the proportion of myosins in the super relaxed state (SRX) conformation or the disordered relaxed state (DRX) conformation in healthy rodent and human hearts, at baseline and in response to reduced hemodynamic demands of hibernation or pathogenic HCM variants. To determine the relationships between myosin conformations, sarcomere function, and cell biology, we assessed contractility, relaxation, and cardiomyocyte morphology and metabolism, with and without an allosteric modulator of myosin ATPase activity. We then tested whether the positions of myosin variants of unknown clinical significance that were identified in patients with HCM, predicted functional consequences and associations with heart failure and arrhythmias. Myosins undergo physiological shifts between the SRX conformation that maximizes energy conservation and the DRX conformation that enables cross-bridge formation with greater ATP consumption. Systemic hemodynamic requirements, pharmacological modulators of myosin, and pathogenic myosin missense mutations influenced the proportions of these conformations. Hibernation increased the proportion of myosins in the SRX conformation, whereas pathogenic variants destabilized these and increased the proportion of myosins in the DRX conformation, which enhanced cardiomyocyte contractility, but impaired relaxation and evoked hypertrophic remodeling with increased energetic stress. Using structural locations to stratify variants of unknown clinical significance, we showed that the variants that destabilized myosin conformations were associated with higher rates of heart failure and arrhythmias in patients with HCM. Myosin conformations establish work-energy equipoise that is essential for life-long cellular homeostasis and heart function. Destabilization of myosin energy-conserving states promotes contractile abnormalities, morphological and metabolic remodeling, and adverse clinical outcomes in patients with HCM. Therapeutic restabilization corrects cellular contractile and metabolic phenotypes and may limit these adverse clinical outcomes in patients with HCM.

A multiscale active structural model of the arterial wall accounting for smooth muscle dynamics.

Arterial wall dynamics arise from the synergy of passive mechano-elastic properties of the vascular tissue and the active contractile behaviour of smooth muscle cells (SMCs) that form the media layer of vessels. We have developed a computational framework that incorporates both these components to account for vascular responses to mechanical and pharmacological stimuli. To validate the proposed framework and demonstrate its potential for testing hypotheses on the pathogenesis of vascular disease, we have employed a number of pharmacological probes that modulate the arterial wall contractile machinery by selectively inhibiting a range of intracellular signalling pathways. Experimental probes used on ring segments from the rabbit central ear artery are: phenylephrine, a selective α1-adrenergic receptor agonist that induces vasoconstriction; cyclopiazonic acid (CPA), a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase; and ryanodine, a diterpenoid that modulates Ca2+ release from the sarcoplasmic reticulum. These interventions were able to delineate the role of membrane versus intracellular signalling, previously identified as main factors in smooth muscle contraction and the generation of vessel tone. Each SMC was modelled by a system of nonlinear differential equations that account for intracellular ionic signalling, and in particular Ca2+ dynamics. Cytosolic Ca2+ concentrations formed the catalytic input to a cross-bridge kinetics model. Contractile output from these cellular components forms the input to the finite-element model of the arterial rings under isometric conditions that reproduces the experimental conditions. The model does not account for the role of the endothelium, as the nitric oxide production was suppressed by the action of L-NAME, and also due to the absence of shear stress on the arterial ring, as the experimental set-up did not involve flow. Simulations generated by the integrated model closely matched experimental observations qualitatively, as well as quantitatively within a range of physiological parametric values. The model also illustrated how increased intercellular coupling led to smooth muscle coordination and the genesis of vascular tone.

Myosin phosphorylation improves contractile economy of mouse fast skeletal muscle during staircase potentiation

Phosphorylation of the myosin regulatory light chain (RLC) by skeletal myosin light chain kinase (skMLCK) potentiates rodent fast twitch muscle but is an ATP-requiring process. Our objective was to investigate the effect of skMLCK-catalyzed RLC phosphorylation on the energetic cost of contraction and the contractile economy (ratio of mechanical output to metabolic input) of mouse fast twitch muscle in vitro (25°C). To this end, extensor digitorum longus (EDL) muscles from wild-type (WT) and from skMLCK-devoid (skMLCK-/-) mice were subjected to repetitive low-frequency stimulation (10 Hz for 15 s) to produce staircase potentiation of isometric twitch force, after which muscles were quick frozen for determination of high-energy phosphate consumption (HEPC). During stimulation, WT muscles displayed significant potentiation of isometric twitch force while skMLCK-/- muscles did not (i.e. 23% versus 5% change, respectively). Consistent with this, RLC phosphorylation was increased ∼3.5-fold from the unstimulated control value in WT but not in skMLCK-/- muscles. Despite these differences, the HEPC of WT muscles was not greater than that of skMLCK-/- muscles. As a result of the increased contractile output relative to HEPC, the calculated contractile economy of WT muscles was greater than that of skMLCK-/- muscles. Thus, our results suggest that skMLCK-catalyzed phosphorylation of the myosin RLC increases the contractile economy of WT mouse EDL muscle compared with skMLCK-/- muscles without RLC phosphorylation.

Simulation of cell-substrate traction force dynamics in response to soluble factors

Finite element (FE) simulations of contractile responses of vascular muscular thin films (vMTFs) and endothelial cells resting on an array of microposts under stimulation of soluble factors were conducted in comparison with experimental measurements reported in the literature. Two types of constitutive models were employed in the simulations, i.e. smooth muscle cell type and non-smooth muscle cell type. The time histories of the effects of soluble factors were obtained via calibration against experimental measurements of contractile responses of tissues or cells. The numerical results for vMTFs with micropatterned tissues suggest that the radius of curvature of vMTFs under stimulation of soluble factors is sensitive to width of the micropatterned tissue, i.e. the radius of curvature increases as the tissue width decreases. However, as the tissue response is essentially isometric, the time history of the maximum principal stress of the micropatterned tissues is not sensitive to tissue width. Good agreement has been achieved for predictions of the vasoconstrictor endothelin-1-induced contraction stress between the FE numerical simulation and the experiment-based approach of Alford (Integr Biol 3:1063-1070, 2011) for the vMTFs with 40, 60, 80 and 100 [Formula: see text] width patterns. This may suggest the contraction stress is weakly sensitive to the tissue width for these patterns. However, for 20 [Formula: see text] width tissue patterning, the numerical simulation result for contraction stress is less than the average value of experimental measurements, which may suggest the thinner and more elongated spindle-like cells within the 20 [Formula: see text] width tissue patterning have higher contractile output. The constitutive model for non-smooth muscle cells was used to simulate the contractile response of the endothelial cells. The substrate was treated as an effective continuum. For agonists such as lysophosphatidic acid and vascular endothelial growth factor, the deformation of the cell diminishes from edge to centre and the central part of the cell is essentially under isometric state. Numerical studies demonstrated the scenarios that cell polarity can be triggered via manipulation of the effective stiffness and Possion's ratio of the substrate.

Valve interstitial cell shape modulates cell contractility independent of cell phenotype

Valve interstitial cells are dispersed throughout the heart valve and play an important role in maintaining its integrity, function, and phenotype. While prior studies have detailed the role of external mechanical and biological factors in the function of the interstitial cell, the role of cell shape in regulating contractile function, in the context of normal and diseased phenotypes, is not well understood. Thus, the aim of this study was to elucidate the link between cell shape, phenotype, and acute functional contractile output. Valve interstitial cell monolayers with defined cellular shapes were engineered via constraining cells to micropatterned protein lines (10, 20, 40, 60 or 80µm wide). Samples were cultured in either normal or osteogenic medium. Cellular shape and architecture were quantified via fluorescent imaging techniques. Cellular contractility was quantified using a valve thin film assay and phenotype analyzed via western blotting, zymography, and qRT-PCR. In all pattern widths, cells were highly aligned, with maximum cell and nuclear elongation occurring for the 10μm pattern width. Cellular contractility was highest for the most elongated cells, but was also increased in cells on the widest pattern (80μm) that also had increased CX43 expression, suggesting a role for both elongated shape and increased cell-cell contact in regulating contractility. Cells cultured in osteogenic medium had greater expression of smooth muscle markers and correspondingly increased contractile stress responses. Cell phenotype did not significantly correlate with altered cell shape, suggesting that cellular shape plays a significant role in the regulation of valve contractile function independent of phenotype.

Bio-inspired micropatterned hydrogel to direct and deconstruct hierarchical processing of geometry-force signals by human mesenchymal stem cells during smooth muscle cell differentiation

Micropatterned biomaterial-based hydrogel platforms allow the recapitulation of in vivo-like microstructural and biochemical features that are critical physiological regulators of stem cell development. Herein, we report the use of muscle mimicking geometries patterned on polyacrylamide hydrogels as an effective strategy to induce smooth muscle cell (SMC) differentiation of human mesenchymal stem cells (hMSCs). hMSCs were systemically coerced to elongate with varying aspect ratios (AR) (that is, 1:1, 5:1, 10:1 and 15:1) at a fixed projection area of ~7000 μm2. The results showed engineered cellular anisotropy with an intermediate AR 5:1 and AR 10:1, promoting the expression of alpha smooth muscle actin (α-SMA) and enhancement of contractile output. Further mechanistic studies indicated that a threshold cell traction force of ~3.5 μN was required for SMC differentiation. Beyond the critical cytoskeleton tension, hMSCs respond to higher intracellular architectural cues such as the stress fiber (SF) alignment, SF subtype expression and diphosphorylated myosin regulatory light-chain activity to promote the expression and incorporation of α-SMA to the SF scaffold. These findings underscore the importance of exploiting biomimetic geometrical cues as an effective strategy to guide hMSC differentiation and are expected to guide the rational design of advanced tissue-engineered vascular grafts. Muscle-mimicking geometries patterned on hydrogel facilitate the acquisition of a smooth-muscle-cell-like phenotype from human stem cells. Adult mesenchymal stem cells (MSCs) are a valuable source of cells for regenerative medicine. Now, researchers in Singapore and China have examined the complex interdependence of engineered cell shape and human transforming growth factor beta-1 treatment to direct smooth muscle cells differentiation of MSCs. They discovered that geometries resembling those of human smooth muscle cells provide the optimal shape for the expression and recruitment of the mechano-sensitive protein α-smooth muscle actin to the filamentous actin cytoskeleton. Based on these results, the researchers proposed an elegant model to describe the decision-making process that human MSCs take during differentiation into smooth muscle cells. The findings highlight the importance of using biomimetic cues for guiding differentiation of human MSCs. Inspired by the intrinsic morphology of smooth muscle cells (SMCs), a micropatterned hydrogel is developed to direct and define the boundary conditions for efficient SMC differentiation of human mesenchymal stem cells (hMSCs). The results show that in conjunction with TGF-β1 treatment, muscle-mimicking shapes with intermediate aspect ratios ranging from 5:1 to 10:1 exert the strongest pro-SMC differentiation effects in a structural–contractile force-dependent manner. These findings are expected to provide critical insights and design rules for vascular-related engineered tissue grafts.

Slow motor units in female rat soleus are slower and weaker than their male counterparts.

The aim of the study was to investigate sex-related differences in contractile properties, parameters of action potentials, and mechanisms of force regulation of motor units in the rat soleus muscle, which is a frequent experimental model in animal research. It was revealed that the mean mass of the muscle in males was bigger than in females, by approximately 80%. However, the relation of the muscle mass to the body mass was not significantly different. These results correspond to approximately twice as much tetanic force per motor unit in male rats, and higher maximal contractile output, reflected by the force-time area per stimulus pulse. On the other hand, no differences were observed with respect to twitch forces of motor units. Thus the twitch-to-tetanus ratio was significantly higher in females. Additionally, the contraction and the half-relaxation times were significantly longer in female motor units, which might be due to differences in muscle architecture. The force-frequency curve in males was shifted rightwards with respect to females, indicating that the same relative level of tetanic force could be achieved at considerably lower stimulation frequency in females. The analysis of motor unit action potentials revealed about four times higher amplitudes in male rats, whereas the time parameters of action potentials were similar. The motor units in male and female rat soleus are considerably different and these observations should be taken in the consideration in various experiments on the muscle.

Smooth muscle architecture within cell-dense vascular tissues influences functional contractility.

The role of vascular smooth muscle architecture in the function of healthy and dysfunctional vessels is poorly understood. We aimed at determining the relationship between vascular smooth muscle architecture and contractile output using engineered vascular tissues. We utilized microcontact printing and a microfluidic cell seeding technique to provide three different initial seeding conditions, with the aim of influencing the cellular architecture within the tissue. Cells seeded in each condition formed confluent and aligned tissues but within the tissues, the cellular architecture varied. Tissues with a more elongated cellular architecture had significantly elevated basal stress and produced more contractile stress in response to endothelin-1 stimulation. We also found a correlation between the contractile phenotype marker expression and the cellular architecture, contrary to our previous findings in non-confluent tissues. Taken with previous results, these data suggest that within cell-dense vascular tissues, smooth muscle contractility is strongly influenced by cell and tissue architectures.