Higher Contractile Force Research Articles

Dual-Responsive MXene-Functionalized Wool Yarn Artificial Muscles.

Fiber-based artificial muscles are promising for smart textiles capable of sensing, interacting, and adapting to environmental stimuli. However, the application of current artificial muscle-based textiles in wearable and engineering fields has largely remained a constraint due to the limited deformation, restrictive stimulation, and uncomfortable. Here, dual-responsive yarn muscles with high contractile actuation force are fabricated by incorporating a very small fraction (<1wt.%) of Ti3C2Tx MXene/cellulose nanofibers (CNF) composites into self-plied and twisted wool yarns. They can lift and lower a load exceeding 3400 times their own weight when stimulated by moisture and photothermal. Furthermore, the yarn muscles are coiled homochirally or heterochirally to produce spring-like muscles, which generated over 550% elongation or 83% contraction under the photothermal stimulation. The actuation mechanism, involving photothermal/moisture-mechanical energy conversion, is clarified by a combination of experiments and finite element simulations. Specifically, MXene/CNF composites serve as both photothermal and hygroscopic agents to accelerate water evaporation under near-infrared (NIR) light and moisture absorption from ambient air. Due to their low-cost facile fabrication, large scalable dimensions, and robust strength coupled with dual responsiveness, these soft actuators are attractive for intelligent textiles and devices such as self-adaptive textiles, soft robotics, and wearable information encryption.

Abstract 737: Arrays of 3D ECM-embedded fibroblast clusters for characterization of ECM remodeling by primary cancer associated fibroblasts and for evaluation of drugs attenuating fibrotic ECM remodeling

Abstract Chronic fibrosis can lead to organ failure and is also associated with cancer, where the tumor is interpreted as a chronic wound, and the fibrotic tumor environment drives disease progression. Fibrosis occurs in an inflammatory environment where macrophages are activated, and fibroblasts transdifferentiate into myofibroblasts. Activated macrophages stimulate the myofibroblasts to produce excessive amounts of collagen-rich ECM. Changes in proteolytic activity and high contractile forces that myofibroblasts apply onto their tissue environment, further lead to extensive remodeling of the healthy tissue matrix into a scar matrix. Similar activity is displayed by cancer-associated fibroblasts (CAFs) in tumors. Intervening with the excessive ECM remodeling by activated fibroblasts represents a candidate therapeutic strategy to normalize the architecture of fibrotic and malignant tissues. We developed a screening platform using an assay based on automated image guided injection of clusters of fibroblasts in wells of multi-well plates preloaded with a collagen-rich ECM network. The identical x-y-z position, spacing, and size of the ECM-embedded fibroblast clusters in each well facilitates automated real time confocal microscopy and quantitative image analysis algorithms. We use this setup for automated quantitative analysis of ECM remodeling activity of CAFs obtained from early-stage colorectal cancer patients. We find that CAFs from these early-stage lesions display increased ECM remodeling capacity as compared to patient matched normal fibroblasts. We then use the same setup to screen a series of compounds for their ability to attenuate ECM remodeling triggered by TGFb-activated primary human fibroblasts. We detect known and novel pathways, and we identify small molecule inhibitors with a promising efficacy versus toxicity profile. Lastly, we detect a previously unknown concentration-dependent side effect of ALK5 inhibitors that leads to the identification of a novel profibrotic pathway and indicates that caution is warranted for the clinical application of ALK5 inhibitors. Citation Format: Erik H. J. Danen. Arrays of 3D ECM-embedded fibroblast clusters for characterization of ECM remodeling by primary cancer associated fibroblasts and for evaluation of drugs attenuating fibrotic ECM remodeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 737.

Human induced pluripotent stem cell-derived cardiac muscle rings for biohybrid self-beating actuator.

Cardiac muscle, a subtype of striated muscle composing our heart, has garnered attention as a source of autonomously driven actuators due to its inherent capability for spontaneous contraction. However, conventional cardiac biohybrid robots have utilized planar (2D) cardiac tissue consisting of a thin monolayer of cardiac myotubes with a thickness of 3-5 μm, which can generate a limited contractile force per unit footprint. In this study, 3D cardiac muscle rings were proposed as robotic actuator units. These units not only exhibit higher contractile force per unit footprint compared to their 2D counterparts due to their increased height, but they can also be integrated into desired 3D configurations. We fabricated cardiac muscle rings from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), evaluated their driving characteristics, and verified the actuation effects by integrating them with artificial components. After the 10th day from culture, the cardiac muscle rings exhibited rhythmic spontaneous contraction and increased contractile force in response to stretching stimuli. Furthermore, after constructing a centimeter-sized biohybrid self-beating actuator with an antagonistic pair structure of cardiac muscle rings, the periodic antagonistic beating motion at its tail portion was confirmed. We believe that 3D cardiac muscle rings, possessing high contractile force and capable of being positioned within limited 3D space, can be used as potent biohybrid robotic actuators.

The impact of life-long strength versus endurance training on muscle fiber morphology and phenotype composition in older men.

Aging is typically associated with decreased muscle strength and rate of force development (RFD), partly explained by motor unit remodeling due to denervation, and subsequent loss of fast-twitch type II myofibers. Exercise is commonly advocated to counteract this detrimental loss. However, it is unclear how life-long strength versus endurance training may differentially affect markers of denervation and reinnervation of skeletal myofibers and, in turn, affect the proportion and morphology of fast-twitch type II musculature. Thus, we compared fiber type distribution, fiber type grouping, and the prevalence of atrophic myofibers (≤1,494 µm2) in strength-trained (OS) versus endurance-trained (OE) master athletes and compared the results to recreationally active older adults (all >70 yr, OC) and young habitually active references (<30 yr, YC). Immunofluorescent stainings were performed on biopsy samples from vastus lateralis, along with leg press maximal strength and RFD measurements. OS demonstrated similar type II fiber distribution (OS: 52.0 ± 16.4%; YC: 51.1 ± 14.4%), fiber type grouping, maximal strength (OS: 170.0 ± 18.9 kg, YC: 151.0 ± 24.4 kg), and RFD (OS: 3,993 ± 894 N·s-1, YC: 3,470 ± 1,394 N·s-1) as young, and absence of atrophic myofibers (OS: 0.2 ± 0.7%; YC: 0.1 ± 0.4%). In contrast, OE and OC exhibited more atrophic fibers (OE: 1.2 ± 1.0%; OC: 1.1 ± 1.4%), more grouped fibers, and smaller proportion of type II fibers (OE: 39.3 ± 11.9%; OC: 35.0 ± 12.4%) than OS and YC (all P < 0.05). In conclusion, strength-trained master athletes were characterized by similar muscle morphology as young, which was not the case for recreationally active or endurance-trained old. These results indicate that strength training may preserve type II fibers with advancing age in older men, likely as a result of chronic use of high contractile force generation.NEW & NOTEWORTHY Aging is associated with loss of fast-twitch type II myofibers, motor unit remodeling, and grouping of myofibers. This study reveals, for the first time, that strength training preserves neural innervation of type II fibers, resulting in similar myofiber type distribution and grouping in life-long strength-trained master athletes as young moderately active adults. In contrast, life-long endurance-trained master athletes and recreationally active old adults demonstrated higher proportion of type I fibers accompanied by more marked grouping of type I myofibers, and more atrophic fibers compared with strength-trained master athletes and young individuals. Thus, strength training should be utilized as a training modality for preservation of fast-twitch musculature, maximal muscle strength, and rapid force capacity (RFD) with advancing age.

Application of Noncircular Pulleys to Straight-Fiber-Type Pneumatic Artificial Muscle Manipulator

This study proposes a method for improving the performance of a manipulator driven by pneumatic artificial muscles. Although the straight-fiber-type pneumatic artificial muscle (SF-PAM), a kind of pneumatic artificial muscle, is lightweight and exhibits high contractile force and contraction percentage, its contractile force decreases as contraction increases. To compensate for the decrease in the SF-PAM contractile force, we developed a noncircular pulley and integrated it into the manipulator driven by a wire pulley mechanism. Because this noncircular pulley is designed in accordance with the output characteristics of SF-PAM, the contraction force of SF-PAM can be converted into manipulator torque efficiently. In addition, the radius of the noncircular pulley is expressed as a function, which can be incorporated into a numerical model for the manipulator’s controller. Subsequently, simulation and experimentation to verify the proposed method showed that, when using the same actuator, the manipulator with a noncircular pulley can optimize both output torque and range of motion better than that with a conventional circular pulley. However, a few differences between simulation results and experimental results were observed. These differences were caused by SF-PAM stretching which was not considered in the model. This drawback can be overcome by improving the SF-PAM and the numerical model in future studies. We believe that this study will provide designers of robots that coexist with humans with a high degree of freedom.

Engineered Human Muscle Tissue from Multilayered Aligned Myofiber Sheets for Studies of Muscle Physiology and Predicting Drug Response.

In preclinical drug testing, human muscle tissue models are critical to understanding the complex physiology, including drug effects in the human body. This study reports that a multilayering approach to cell sheet-based engineering produces an engineered human muscle tissue with sufficient contractile force suitable for measurement. A thermoresponsive micropatterned substrate regulates the biomimetic alignment of myofiber structures enabling the harvest of the aligned myofibers as a single cell sheet. The functional muscle tissue is produced by layering multiple myofiber sheets on a fibrin-based gel. This gel environment promotes myofiber maturation, provides the tissue an elastic platform for contraction, and allows the attachment of a measurement device. Since this multilayering approach is effective in enhancing the contractile ability of the muscle tissue, this muscle tissue generates a significantly high contractile force that can be measured quantitatively. The multilayered muscle tissue shows unidirectional contraction from electrical and chemical stimulation. In addition, their physiological responses to representative drugs can be determined quantitatively in real time by changes in contractile force and fatigue resistance. These physiological properties indicate that the engineered muscle tissue can become a promising tissue model for preclinical in vitro studies in muscle physiology and drug discovery.

BODY WEIGHT INFLUENCES MUSCULOSKELETAL ADAPTATION TO LONG-TERM VOLUNTARY WHEEL RUNNING DURING AGING

Abstract Frailty is a key hallmark of aging and exercise has been shown to delay aging effects. This study was initiated based on the hypothesis that voluntary wheel running (VWR) starting at 12 mo until 18 or 22 mo of age would benefit the female murine musculoskeletal system. Based on the final body weight, the mice were separated into high (HBW) and low body weight (LBW) subgroups. Beneficial effects of VWR were observed on soleus muscle mass and contractile force at both ages, although HBW led to greater increases at 22 mo. VWR increased fiber cross-sectional area by 20%, leading to more type I and fewer IIA fibers in soleus. HBW mice were resistant to age-related decline in Extensor digitorum longus (EDL) mass and contractile force. EDL in 18 mo HBW also showed 15% higher contractile force following VWR while muscle from 18 &amp; 22 mo LBW responded to VWR with greater osteocyte protective factor secretion. Skeletal adaptation to VWR was also dependent on body weight, with HBW showing higher femoral cortical thickness and area under sedentary conditions. VWR maintained osteocyte dendrite number in HBW. VWR increased periosteal and endosteal circumferences in HBW, suggesting compensation for loss of material strength. Consistent with this, VWR maintained higher bone mechanical properties in 18mo LBW. In summary, VWR alters musculoskeletal parameters depending on body weight with HBW contributing to more muscle mass and strength to prevent sarcopenia while bone retains better mechanical properties in LBW but HBW contributes structural modification to prevent osteopenia.

Miniature coiled artificial muscle for wireless soft medical devices.

Wireless small-scale soft-bodied devices are capable of precise operation inside confined internal spaces, enabling various minimally invasive medical applications. However, such potential is constrained by the small output force and low work capacity of the current miniature soft actuators. To address this challenge, we report a small-scale soft actuator that harnesses the synergetic interactions between the coiled artificial muscle and radio frequency–magnetic heating. This wirelessly controlled actuator exhibits a large output force (~3.1 N) and high work capacity (3.5 J/g). Combining this actuator with different mechanical designs, its tensile and torsional behaviors can be engineered into different functional devices, such as a suture device, a pair of scissors, a driller, and a clamper. In addition, by assuming a spatially varying magnetization profile, a multilinked coiled muscle can have both magnetic field–induced bending and high contractile force. Such an approach could be used in various future untethered miniature medical devices.

The desmin mutation R349P increases contractility and fragility of stem cell-generated muscle micro-tissues.

Desminopathies comprise hereditary myopathies and cardiomyopathies caused by mutations in the intermediate filament protein desmin that lead to severe and often lethal degeneration of striated muscle tissue. Animal and single cell studies hinted that this degeneration process is associated with massive ultrastructural defects correlating with increased susceptibility of the muscle to acute mechanical stress. The underlying mechanism of mechanical susceptibility, and how muscle degeneration develops over time, however, has remained elusive. Here, we investigated the effect of a desmin mutation on the formation, differentiation, and contractile function of in vitro-engineered three-dimensional micro-tissues grown from muscle stem cells (satellite cells) isolated from heterozygous R349P desmin knock-in mice. Micro-tissues grown from desmin-mutated cells exhibited spontaneous unsynchronised contractions, higher contractile forces in response to electrical stimulation, and faster force recovery compared with tissues grown from wild-type cells. Within 1 week of culture, the majority of R349P desmin-mutated tissues disintegrated, whereas wild-type tissues remained intact over at least three weeks. Moreover, under tetanic stimulation lasting less than 5 s, desmin-mutated tissues partially or completely ruptured, whereas wild-type tissues did not display signs of damage. Our results demonstrate that the progressive degeneration of desmin-mutated micro-tissues is closely linked to extracellular matrix fibre breakage associated with increased contractile forces and unevenly distributed tensile stress. This suggests that the age-related degeneration of skeletal and cardiac muscle in patients suffering from desminopathies may be similarly exacerbated by mechanical damage from high-intensity muscle contractions. We conclude that micro-tissues may provide a valuable tool for studying the organization of myocytes and the pathogenic mechanisms of myopathies.

Quantitative Analysis of Myofibroblast Contraction by Traction Force Microscopy.

Myofibroblasts play important roles in physiological processes such as wound healing and tissue repair. While high contractile forces generated by the actomyosin network enable myofibroblasts to physically contract the wound and bring together injured tissue, prolonged and elevated levels of contraction also drive the progression of fibrosis and cancer. However, quantitative mapping of these forces has been difficult due to their extremely low magnitude ranging from 100pN/μm2 to 2nN/μm2. Here, we provide a protocol to measure cellular forces exerted on two-dimensional compliant elastic hydrogels. We describe thefabrication of polyacrylamide hydrogels labeled with fluorescent fiducial markers, functionalization of substrates with ECM proteins, setting up the experiment, and imaging procedures. We demonstrate the application of this technique for quantitative analysis of traction forces exerted by myofibroblasts.

Bioinspired high-power-density strong contractile hydrogel by programmable elastic recoil.

Stimuli-responsive hydrogels have large deformability but-when applied as actuators, smart switch, and artificial muscles-suffer from low work density due to low deliverable forces (~2 kPa) and speed through the osmotic pressure-driven actuation. Inspired by the energy conversion mechanism of many creatures during jumping, we designed an elastic-driven strong contractile hydrogel through storing and releasing elastic potential energy in polymer network. It can generate high contractile force (40 kPa) rapidly at ultrahigh work density (15.3 kJ/m3), outperforming current hydrogels (~0.01 kJ/m3) and even biological muscles (~8 kJ/m3). This demonstrated elastic energy storing and releasing method endows hydrogels with elasticity-plasticity switchability, multi-stable deformability in fully reversible and programmable manners, and anisotropic or isotropic deformation. With the high power density and programmability via this customizable modular design, these hydrogels demonstrated potential for broad applications in artificial muscles, contractile wound dressing, and high-power actuators.

Increased Platelet Force Is Associated with Decreased Bleeding Severity in Pediatric Immune Thrombocytopenia

Background: Immune Thrombocytopenia Purpura (ITP) can result in severely low platelet counts and while most of the 4,000 new cases of pediatric ITP diagnosed each year self-resolve, 10% of these patients have major bleeding episodes. The platelet count remains the mainstay method for predicting hemorrhage and unfortunately has only proven to be loosely correlated to bleeding severity. Consequently, there currently exists no biomarker that accurately and reliably predicts which patients need immediate medical treatment, all of which have side effects, and which patients only require monitoring. To that end, we leveraged our Platelet Contraction Cytometer (PCC), a versatile system that measures platelet contraction forces at the single-cell level and at high-throughput, to study platelets of patients with ITP prospectively. Buchanan bleeding scores were used to distinguish patients with severe symptoms from asymptomatic patients. With 49 patients, we observed two significant findings: 1) Tracking both single platelet force measurements and platelet count enables stratifying patients into having major, minor, or no risk for bleeding. Accordingly, patients in the major risk category have a combined low platelet force and low platelet count. 2) Longitudinal studies showed that when major risk patients had increased platelet force or higher platelet counts, this was associated with the alleviation of major symptoms. Thus, when utilized together, platelet force and platelet count can more accurately predict bleeding severity in pediatric ITP patients. Platelet Contraction Cytometry: Our PCC utilizes a large array of fibrinogen "microdot" pairs patterned on a flat hydrogel of known stiffness. Thrombin-activated platelets adhere to these microdots, spread to the neighboring microdot, and contract, pulling the microdots closer together. As platelet force is directly proportional to the microdot displacement, only a single microscopy measurement is necessary to determine the force applied by each single platelet (Fig 1). As such, hundreds of platelets are measured in a controlled mechanical and biochemical environment (Myers et al, 2017) Results: With our cohort of 49 ITP patients, we observed that low average single cell platelet contraction forces highly correlate with severe bleeding symptoms (Buchannan scores 2-4). Using a regression tree, we found that an average force of 26nN best separates symptomatic from asymptomatic patients with a diagnostic sensitivity of 100% and specificity of 86.8% (AUC 0.97, 95% CI: 0.9361-1). However, platelets from some asymptomatic patients exerted forces of less than 26nN as well. In a more thorough examination, we found that majority of those asymptomatic patients with low platelet forces had platelet counts &gt; 40,000/uL. Although our data shows that platelet count alone is a poor predictor of severe bleeding symptoms (AUC 0.8, 95% CI: 0.6757-0.9189), when a platelet count &lt;40k/uL is coupled alongside our criterion of average contraction force &lt;26 nN, we find that the specificity of our system increases to 94.7% and thus platelet count and platelet force synergistically predict which patients are symptomatic with high accuracy. Using a logistic regression model, we also found that with each 1nN decrease in average contraction force, the odds of the patient bleeding increases 2.1 (95% CI: 1.1-4.0) times that of being asymptomatic and when this decrease in contraction force is increased 5nN, the odds of a patient bleeding is 44.4 (95% CI: 1.8-1066.5) times that of being asymptomatic (Fig 2A-B). Moreover, when tracking individual patients (n=4) over time, we demonstrate that whenever a patient's blood sample is associated with increased single platelet average contraction force &gt;26 nN or platelet count &gt;40k/uL, bleeding symptoms are alleviated. Conversely, whenever a patient exhibited high platelet contractile force and low platelet count, the onset of bleeding symptoms correlated with a decrease in platelet forces. Unlike bulk or micro-clot assays, our assay functions as a "stress test" of single platelets by placing them in conditions that maximize contractility, and in both cases, the reduction in the subpopulation of highly contractile platelets is correlated with bleeding severity. As such, our work suggests that single platelet forces could be used as a diagnostic biomarker to assess bleeding risk in patients with ITP regardless of count (Fig 2C-D). Disclosures Bennett: Novartis: Research Funding. Lam:Sanguina, LLC: Equity Ownership.

Skeletal Muscle Constructs Engineered from Human Embryonic Stem Cell Derived Myogenic Progenitors Exhibit Enhanced Contractile Forces When Differentiated in a Medium Containing EGM-2 Supplements.

Three-dimensional (3D) skeletal muscle constructs engineered from myogenic progenitors derived from human pluripotent stem cells (hPSCs) have a wide range of applications, but to date, such constructs generate lower specific tetanic force than adult human muscles. Methods enhancing functional muscle differentiation and force generation of these constructs are highly desirable. The finding of this study is that addition of the supplements in the endothelial cell growth medium-2 (EGM-2) to the myogenic differentiation medium can substantially enhance contractile force generation. For constructs differentiated for 4 weeks, addition of the EGM-2 supplements in the first 2 weeks leads to tenfold and sevenfold increases in twitch and tetanic forces, respectively. The specific tetanic force generated by these constructs is 33 mN mm-2 , which is significantly higher than previously reported. These constructs show wider myotubes and higher gene expression levels for all skeletal muscle-specific myosin heavy chain isoforms, suggesting that a more mature differentiation stage of the cells underlies the greater contractile force generation. The constructs exposed to these supplements for 4 weeks do not generate as high contractile forces, suggesting that prolonged treatment is not beneficial. These results suggest that temporal conditioning with the EGM-2 supplements assists functional development of hPSC-derived skeletal muscle constructs.

(Invited) High-Throughput Single Cell Nanomechanical Measurements

Microsystems have the potential to make an enormous contribution to biomedical and clinical settings. Fully realizing the capabilities of this established technology lies in designing new robust microsystems capable of answering clinically relevant problems. Here, I discuss the creation of the platelet contraction cytometer, a tool that has led to important insights into our understanding of the biomechanical process of clotting and may even represent a new type of diagnostic based on biophysics. Platelets constantly circulate in the bloodstream and respond to injury to prevent excessive blood loss. At a wound, platelets will aggregate on exposed collagen and undergo muscle-like contraction with nascent fibrin polymers to mechanically stabilize the clot and stem hemorrhage. During clot contraction, platelets can dramatically shrink the fibrin mesh and increase the clot stiffness by an order of magnitude. Previous studies have established that the clot stiffness is linked to disease, as overly softened and stiffened clots are associated with bleeding and thrombotic disorders, respectively. However, defining the reasons that clot mechanical properties change in disease has remained difficult, as clots are inherently complex and heterogeneous, and all existing bulk measurements depend on contributions from fibrin stiffness, fibrin architecture, red blood cell incorporation, and platelet contraction. Moreover, changes to the microenvironmental stiffness, biochemical concentration, and shear are known to alter platelet force or behavior at the single cell level. To that end, we developed a “platelet contraction cytometer (PCC)” that simultaneously measures the contractile force of hundreds of individual adherent platelets across the physiological range of mechanical microenvironments within a clot. Within the PCC, a single platelet attaches, spreads, and applies contractile force to a pair of fibrinogen microdots that are attached to a moveable, spring-like, surface. Since the applied platelet contractile force is directly proportional to the microdot displacement, the force may be calculated from a single fluorescent image of the platelet. As this device is microfabricated, thousands of microdots may be created on a single device to enable high-throughput measurements that are performed in a highly controlled mechanical, biochemical, and shear environment. Using this device, we have defined the range of forces that platelets apply within a clot and shown that mechanical and biochemical cues can synergistically combine to create highly contractile platelets. Moreover, we have been able to probe the pathways involved in contraction and shown which pathways control how platelets apply force in response to local stiffness. This device has even enabled new comparative studies that examine how platelet force varies between humans and other animal species, and may lead to new biological insights into how contractile force is generated and regulated. In addition to enabling mechanistic studies, we have collected data showing that platelet contraction cytometry may represent a new class diagnostic that is based on biophysics. In particular, our studies have focused on immune thrombocytopenia (ITP), a disease which is characterized by low platelet counts in the absence of any other cause. Approximately 10% of these patients will have major bleeding issues, but no existing biomarker can determine which patients are at risk for bleeding and whether the disease will self-resolve or become chronic. In a small cohort of patients, we have found that all patients with bleeding and/or bruising symptoms also have significantly lower platelet contractile forces. By defining an average force cutoff value of 26nN, we found that low forces identified bleeding in ITP with 100% sensitivity and 89.4% specificity. Importantly, since the PCC measures single platelets in high throughput, it also provides information on the distribution of forces applied by platelets and has helped to identify unique subpopulations of platelets in ITP. Healthy individuals have a normal distribution of contractile forces with a single peak, while ITP patients tend to have a bimodal distribution with two prominent peaks, one with a lower contractile force and one with a higher contractile force. Surprisingly, patients with bleeding symptoms are missing the highly contractile peak and suggests that the lack of highly contractile platelets may contribute to bleeding. Taken together, this work highlights the incredible potential for microsystems in solving clinical challenges. Designing a device capable of measuring single platelet forces led to new insights into the basic biophysics of platelets and clotting and may also represent a new type of diagnostic.

Isometric-based test improves EMG-threshold determination in boys vs. men.

Children have been hypothesized to utilize higher-threshold (type-II) motor units (MUs) to a lesser extent than adults. Two recent studies, using a cycling-based EMG-threshold (EMGTh) protocol, supported the hypothesis, showing children's EMGTh intensities to be higher than adults'. Conclusions, however, were hampered by children's low EMGTh detection rates. Insufficiently high contractile forces at exhaustion were postulated as the reason for non-detection, predominantly in children. An intermittent isometric contraction test (IICT) protocol facilitates higher contractile forces prior to exhaustion and was shown effective in EMGTh testing of adults. Determine whether an IICT protocol would enhance EMGTh detection in children, and consequently increase the magnitude of the previously observed child-adult EMGTh differences. 18 boys and 21 men completed one-repetition-maximum (1RM) isometric knee-extension test. The IICT protocol followed, commencing at 25%1RM and comprising five isometric contractions per load, incremented by ~ 3%1RM to exhaustion. Vastus lateralis surface EMG was recorded and EMGTh, expressed as %1RM, was defined as the onset of the EMG-response's steeper segment. EMGTh was detected in 88.9% of boys and 95.2% of men, and occurred at higher relative intensities in boys (56.4 ± 9.2%1RM) than in men (46.0 ± 6.8%1RM). This 10.4% difference was 57% greater than the corresponding, previously reported cycling-based age-related difference. With the boys' detection rate nearly on par with the men's, the IICT protocol appears to overcome much of the intensity limitation of cycling-based protocols and provide a more sensitive EMGTh detection tool, thus extending the previously observed boys‒men difference. This difference adds supports to the notion of children's more limited type-II MU recruitment capacity.

Therapeutic potential of AAV9-S15D-RLC gene delivery in humanized MYL2 mouse model of HCM.

Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disorder characterized by ventricular hypertrophy, myofibrillar disarray, and fibrosis, and is primarily caused by mutations in sarcomeric genes. With no definitive cure for HCM, there is an urgent need for the development of novel preventive and reparative therapies. This study is focused on aspartic acid-to-valine (D166V) mutation in the myosin regulatory light chain, RLC (MYL2 gene), associated with a malignant form of HCM. Since myosin RLC phosphorylation is critical for normal cardiac function, we aimed to exploit this post-translational modification via phosphomimetic-RLC gene therapy. We hypothesized that mimicking/modulating cardiac RLC phosphorylation in non-phosphorylatable D166V myocardium would improve heart function of HCM-D166V mice. Adeno-associated virus, serotype-9 (AAV9) was used to deliver phosphomimetic human RLC variant with serine-to-aspartic acid substitution at Ser15-RLC phosphorylation site (S15D-RLC) into the hearts of humanized HCM-D166V mice. Improvement of heart function was monitored by echocardiography, invasive hemodynamics (PV-loops) and muscle contractile mechanics. A significant increase in cardiac output and stroke work and a decrease in relaxation constant, Tau, shown to be prolonged in HCM mice, were observed in AAV- vs. PBS-injected HCM mice. Strain analysis showed enhanced myocardial longitudinal shortening in AAV-treated vs. control mice. In addition, increased maximal contractile force was observed in skinned papillary muscles from AAV-injected HCM hearts. Our data suggest that myosin RLC phosphorylation may have important translational implications for the treatment of RLCmutations-induced HCM and possibly play a role in other disease settings accompanied by depressed Ser15-RLC phosphorylation. KEY MESSAGES: HCM-D166V mice show decreased RLC phosphorylation and decompensated function. AAV9-S15D-RLC gene therapy in HCM-D166V mice, but not in WT-RLC, results in improved heart performance. Global longitudinal strain analysis shows enhanced contractility in AAV vs controls. Increased systolic and diastolic function is paralleled by higher contractile force. Phosphomimic S15D-RLC has a therapeutic potential for HCM.

Analysis of Contractility and Invasion Potential of Two Canine Mammary Tumor Cell Lines.

Cancer cells are surrounded by a mechanically and biochemically distinct microenvironment that undergoes dynamic changes throughout the neoplastic progression. During this progression, some cancer cells acquire abnormal characteristics that potentiate their escape from the primary tumor site, to establish secondary tumors in distant organs. Recent studies with several human cancer cell lines have shown that the altered physical properties of tumor cells, such as their ability to apply high traction forces to the surroundings, are directly linked with their potential to invade and metastasize. To test the hypothetical interconnection between actomyosin-mediated traction forces and invasion potential within 3D-microenvironment, we utilized two canine mammary tumor cell lines with different contractile properties. These cell lines, canine mammary tumor (CMT)-U27 and CMT-U309, were found to have distinct expression patterns of lineage-specific markers and organization of actin-based structures. In particular, CMT-U309 carcinoma cells were typified by thick contractile actomyosin bundles that exerted high forces to their environment, as measured by traction force microscopy. These high contractile forces also correlated with the prominent invasiveness of the CMT-U309 cell line. Furthermore, we found high contractility and 3D-invasion potential to be dependent on the activity of 5′AMP-activated protein kinase (AMPK), as blocking AMPK signaling was found to reverse both of these features. Taken together, our findings implicate that actomyosin forces correlate with the invasion potential of the studied cell lines.

Why is cytoskeletal contraction required for cardiac fusion before but not after looping begins?

Cytoskeletal contraction is crucial to numerous morphogenetic processes, but its role in early heart development is poorly understood. Studies in chick embryos have shown that inhibiting myosin-II-based contraction prior to Hamburger–Hamilton (HH) stage 10 (33 h incubation) impedes fusion of the mesodermal heart fields that create the primitive heart tube (HT), as well as the ensuing process of cardiac looping. If contraction is inhibited at or after looping begins at HH10, however, fusion and looping proceed relatively normally. To explore the mechanisms behind this seemingly fundamental change in behavior, we measured spatiotemporal distributions of tissue stiffness, stress, and strain around the anterior intestinal portal (AIP), the opening to the foregut where contraction and cardiac fusion occur. The results indicate that stiffness and tangential tension decreased bilaterally along the AIP with distance from the embryonic midline. The gradients in stiffness and tension, as well as strain rate, increased to peaks at HH9 (30 h) and decreased afterward. Exposure to the myosin II inhibitor blebbistatin reduced these effects, suggesting that they are mainly generated by active cytoskeletal contraction, and finite-element modeling indicates that the measured mechanical gradients are consistent with a relatively uniform contraction of the endodermal layer in conjunction with constraints imposed by the attached mesoderm. Taken together, our results suggest that, before HH10, endodermal contraction pulls the bilateral heart fields toward the midline where they fuse to create the HT. By HH10, however, the fusion process is far enough along to enable apposing cardiac progenitor cells to keep ‘zipping’ together during looping without the need for continued high contractile forces. These findings should shed new light on a perplexing question in early heart development.

Utilization and control of bioactuators across multiple length scales

In this review, we summarize the recent developments in the emerging field of bioactuators across a multitude of length scales. First, we discuss the use and control of biomolecules as nanoscale actuators. Molecular motors, such as DNA, kinesin, myosin, and F1-ATPase, have been shown to exert forces in the range between 1 pN to 45 pN. Second, we discuss the use and control of single and small clusters of cells to power microscale devices. Microorganisms, such as flagellated bacteria, protozoa, and algae, can naturally swim at speeds between 20 μm s(-1) to 2 mm s(-1) and produce thrust forces between 0.3 pN to 200 pN. Individual and clustered mammalian cells, such as cardiac and skeletal cells, can produce even higher contractile forces between 80 nN to 3.5 μN. Finally, we discuss the use and control of 2D- and 3D-assembled muscle tissues and muscle tissue explants as bioactuators to power devices. Depending on the size, composition, and organization of these hierarchical tissue constructs, contractile forces have been demonstrated to produce between 25 μN to 1.18 mN.

Treatment of Stress Urinary Incontinence by Cinnamaldehyde, the Major Constituent of the Chinese Medicinal Herb Ramulus Cinnamomi

Stress urinary incontinence (SUI) is a common disorder in middle-aged women and the elderly population. Although surgical treatment of SUI has progressed, pharmacological therapies remain unelucidated. We screened potential herbal medicines against SUI with an ex vivo organ bath assay. Ramulus Cinnamomi and its major constituent cinnamaldehyde cause a high contractile force of the urethra and a low contractile force of blood vessels. Cinnamaldehyde dose-dependently reduced lipopolysaccharide-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells. In the vaginal distension- (VD-) induced SUI model in mice, cinnamaldehyde significantly reversed the VD-induced SUI physical signs and reduced blood pressure. Cinnamaldehyde may offer therapeutic potential against SUI without the possible side effect of hypertension. The modulation of several SUI-related proteins including myosin, iNOS, survival motor neuron (SMN) protein, and superoxide dismutase 3 (SOD3) may play some crucial roles in the therapeutic approach against SUI. This information may offer clues to the pathogenesis of SUI and open additional avenues for potential therapy strategies.