Contractile Force In Response Research Articles

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.

SIRT1 induction in the skeletal muscle of male mice partially preserves limb muscle mass but not contractile force in response to androgen deprivation.

In males, the factors that decrease limb muscle mass and strength in response to androgen deprivation are largely unknown. Sirtuin1 (SIRT1) protein levels are lower in the limb muscle of male mice subjected to androgen deprivation. The present study aimed to assess whether SIRT1 induction preserved limb muscle mass and force production in response to androgen deprivation. Physically mature male mice containing an inducible muscle-specific SIRT1 transgene were subjected to a sham or castration surgery and compared to sham and castrated male mice where the SIRT1 transgene was not induced. SIRT1 induction partially preserved whole-body lean mass, tibialis anterior (TA) mass and triceps surae muscle mass in response to castration. Further analysis of the TA muscle showed that muscle-specific SIRT1 induction partially preserved limb muscle soluble protein content and fibre cross-sectional area. Unilateral AAV9-mediated SIRT1 induction in the TA muscle showed that SIRT1 partially preserved mass by acting directly in the muscle. Despite those positive outcomes to limb muscle morphology, muscle-specific SIRT1 induction did not preserve the force generating capacity of the TA or triceps surae muscles. Interestingly, SIRT1 induction in females did not alter limb muscle mass or limb muscle strength even though females have naturally low androgen levels. SIRT1 also did not alter the androgen-mediated increase in limb muscle mass or strength in females. In all, these data suggest that decreases in SIRT1 protein in the limb muscle of males may partially contribute to the loss of limb muscle mass in response to androgen deprivation. KEY POINTS: SIRT1 induction in skeletal muscle of male mice subjected to androgen deprivation partially preserved limb muscle mass and fibre cross-sectional area. SIRT1 induction in skeletal muscle of male mice subjected to androgen deprivation did not prevent preserve limb muscle force generating capacity. SIRT1 induction in skeletal muscle of females did not alter baseline limb muscle mass, nor did it affect the androgen-mediated increase in limb muscle mass.

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.

Dose-independent threshold illumination for non-invasive time-lapse fluorescence imaging of live cells

Fluorescent microscopy employs monochromatic light for excitation, which can adversely affect the cells being observed. We reported earlier that fibroblasts relax their contractile force in response to green light of typical intensity. Here we show that such effects are independent of extracellular matrix and cell lines. In addition, we establish a threshold intensity that elicits minimal or no adverse effect on cell contractility even for long-time exposure. This threshold intensity is wavelength dependent. We cultured fibroblasts on soft 2D elastic hydrogels embedded with fluorescent beads to trace substrate deformation and cell forces. The beads move toward cell center when cells contract, but they move away when cells relax. We use relaxation/contraction ratio (λr), in addition to traction force, as measures of cell response to red (wavelength, λ=635-650 nm), green (λ=545-580 nm) and blue (λ=455-490 nm) lights with varying intensities. Our results suggest that intensities below 57, 31 and 3.5 W/m2 for red, green and blue lights, respectively, do not perturb force homeostasis. To our knowledge, these intensities are the lowest reported safe thresholds, implying that cell traction is a highly sensitive readout of the effect of light on cells. Most importantly, we find these threshold intensities to be dose-independent; i.e., safe regardless of the energy dosage or time of exposure. Conversely, higher intensities result in widespread force-relaxation in cells with λr > 1. Furthermore, we present a photo-reaction based model that simulates photo-toxicity and predicts threshold intensity for different wavelengths within the visible spectra. In conclusion, we recommend employing illumination intensities below aforementioned wavelength-specific thresholds for time-lapse imaging of cells and tissues in order to avoid light-induced artifacts in experimental observations.

Ageing modulates human dermal fibroblast contractility: Quantification using nano-biomechanical testing

Dermal fibroblasts play a key role in maintaining homoeostasis and functionality of the skin. Their contractility plays a role in changes observed during ageing, especially in processes such as wound healing, inflammation, wrinkling and scar tissue formation as well as structural changes on extracellular matrix. Although alternations in skin physiology and morphology have been previously described, there remains a paucity of information about the influence of chronological ageing on dermal fibroblast contractility. In this study, we applied a novel nano-biomechanical technique on cell-embedded collagen hydrogels in combination with mathematical modelling and numerical simulation to measure contraction forces of normal human dermal fibroblasts (NHDF). We achieved quantitative differentiation of the contractility of cells derived from ‘young’ (< 30 years old) and ‘aged’ (> 60 years old) donors. Transforming growth factor β1 (TGF-β1) was used to stimulate the fibroblasts to assess their contractile potential. NHDF from aged donors exhibited a greater basal contractile force, while in contrast, NHDF from young donors have shown a significantly larger contractile force in response to TGF-β1 treatment. These findings validate our nano-biomechanical measurement technique and provide new insights for considering NHDF contractility in regenerative medicine and as a biomarker of dermal ageing processes.

Functional constipation induces bladder overactivity associated with upregulations of Htr2 and Trpv2 pathways

Bladder and bowel dysfunction (BBD) is a common yet underdiagnosed paediatric entity that describes lower urinary tract symptoms (LUTS) accompanied by abnormal bowel patterns manifested as constipation and/or encopresis. LUTS usually manifest as urgency, urinary frequency, incontinence, and urinary tract infections (UTI). Despite increasing recognition of BBD as a risk factor for long-term urinary tract problems including recurrent UTI, vesicoureteral reflux, and renal scarring, the mechanisms underlying BBD have been unclear, and treatment remains empirical. We investigated how constipation affects the lower urinary tract function using a juvenile murine model of functional constipation. Following four days of functional constipation, animals developed LUTS including urinary frequency and detrusor overactivity evaluated by awake cystometry. Physiological examination of detrusor function in vitro using isolated bladder strips, demonstrated a significant increase in spontaneous contractions without affecting contractile force in response to electrical field stimulation, carbachol, and KCl. A significant upregulation of serotonin receptors, Htr2a and Htr2c, was observed in the bladders from mice with constipation, paralleled with augmented spontaneous contractions after pre-incubation of the bladder strips with 0.5 µM of serotonin. These results suggest that constipation induced detrusor overactivity and increased excitatory serotonin receptor activation in the urinary bladder, which contributes to the development of BBD.

Functional skeletal muscle constructs from transdifferentiated human fibroblasts

Transdifferentiation of human non-muscle cells directly into myogenic cells by forced expression of MyoD represents one route to obtain highly desirable human myogenic cells. However, functional properties of the tissue constructs derived from these transdifferentiated cells have been rarely studied. Here, we report that three-dimensional (3D) tissue constructs engineered with iMyoD-hTERT-NHDFs, normal human dermal fibroblasts transduced with genes encoding human telomerase reverse transcriptase and doxycycline-inducible MyoD, generate detectable contractile forces in response to electrical stimuli upon MyoD expression. Withdrawal of doxycycline in the middle of 3D culture results in 3.05 and 2.28 times increases in twitch and tetanic forces, respectively, suggesting that temporally-controlled MyoD expression benefits functional myogenic differentiation of transdifferentiated myoblast-like cells. Treatment with CHIR99021, a Wnt activator, and DAPT, a Notch inhibitor, leads to further enhanced contractile forces. The ability of these abundant and potentially patient-specific and disease-specific cells to develop into functional skeletal muscle constructs makes them highly valuable for many applications, such as disease modeling.

Cryopreservation of the common carotid artery of the rabbit: Optimization of dimethyl sulfoxide concentration and cooling rate

This paper describes the continuation of studies that demonstrated the suitability of CP-Tes solution as a medium for the introduction and removal of dimethyl sulfoxide in rabbit common carotid arteries and established the kinetics of cryoprotectant permeation in that tissue. In this paper we report the tolerance of rabbit common carotid artery to dimethyl sulfoxide, in concentrations up to 30% (w/w), using a technique of exposure that was designed to control osmotic stress. The maximum concentration achieved without damage was 15% (w/w). Vessels were then equilibrated with 15% dimethyl sulfoxide and cooled to −80 °C at 0.22, 0.69, 2.15, or 9.63 °C/min: they were then transferred to the gas phase of a liquid nitrogen refrigerator {temperature below −160 °C) for storage. Thawing was carried out in a 37 °C water bath. The optimum rate of cooling for these conditions was found to be 0.69 °C/min. The maximal recovery of contractile force in response to 10−6 M norepinephrine was 30–40%; relaxation to acetylcholine (an endothelium-mediated function) was 80% of control, and an estimated 71% of endothelial cells survived with minimal ultrastructural change.

Fabrication of contractile skeletal muscle tissues using directly converted myoblasts from human fibroblasts

Transplantation of stem cell-derived myoblasts is a promising approach for the treatment of skeletal muscle function loss. Myoblasts directly converted from somatic cells that bypass any stem cell intermediary stages can avoid the problem of tumor formation after transplantation. Previously, we reported that co-transduction with the myogenic differentiation 1 (MYOD1) gene and the v-myc avian myelocytomatosis viral oncogene lung carcinoma derived homolog (MYCL) gene efficiently converted human fibroblasts into myoblasts. Although the directly converted myoblasts efficiently fused into multinucleated myotubes invitro and invivo, it is not clear whether they have the contractile ability, which is the most significant phenotype of the muscle. In the present study, we aimed to examine the invitro contractile ability of the myotubes differentiated from the directly converted myoblasts by the overexpression of MYOD1 and MYCL. We fabricated three-dimensional (3D) tissues on a microdevice for force measurement. The 3D culture enhanced the differentiation of the myoblasts into myotubes, which were confirmed by gene expression analysis of skeletal muscle-related genes. The tissues started to generate contractile force in response to electrical stimulation after 4 days of culture, which reached approximately 12μN after 10 days. The addition of IGF-I decreased the contractile force of the 3D tissues, while the use of cryopreserved cells increased it. We confirmed that the tissues fabricated from the cells derived from three different donors generated forces of similar magnitude. Thus, directly converted myoblasts by the overexpression of MYOD1 and MYCL could be a promising cell source for cell therapy.

Vascular Rinsing and Chilling Carcasses: Effects on Quality Attributes and Metabolic Changes in Beef

ObjectivesRinse &amp; Chill® (RC) is a process applied early postmortem that provides the ability to manipulate muscle metabolism and can have a positive impact on meat quality traits. This study aimed to evaluate the effect of RC on pH decline, shear force, sarcomere length and cooking losses on different cull dairy cow carcass grades. Investigate the ability of different substrates to modulate contractile response as an indirect measure of metabolic activity on beef early postmortem.Materials and MethodsFor each carcass grade (lean, LE; light, LI), ten carcasses were conventionally chilled (CC) and twelve carcasses were chilled using RC technology (MPSC Inc.). The RC process involved infusion of a chilled isotonic solution (98.5% water; balance: glucose, phosphates, and maltose) through the vascular system, beginning in the arterial and exiting the venous side of the vasculature. Shear force and cooking losses were measured on Longissimus dorsi steaks aged (7 d). Sarcomere length (SL) was determined by a laser diffraction method. Animal served as the experimental unit and data were analyzed with a PROC MIXED procedure. For contraction measurements, a muscle-fiber bundle from the Sternomandibularis muscles (n = 14) was collected from cull dairy cows in a commercial packing plant, 15 min after bleeding. The muscle bundle was attached to a force transducer (FT-302, iWorx, Dover, NH). Stimulation electrodes were used to elicit a supramaximal electrical stimulus at a frequency of 50 V, 0.1 Hz (HCS-100 stimulator, iWorx). Muscle weight was standardized, and length was adjusted to obtain maximum twitch-tension output. After 3 min of rest in a test solution, 200 stimuli were given, and the contractile response was recorded. Four solutions were tested (A = RC, B = Fructose, C = Sodium phosphate, D = Dipotassium phosphate; substrates added at 1% except fructose 1.5%). Descriptive means for initial peak twitch force, final peak twitch force, percentage decline and percentage half-time decline were calculated to determine the response associated with each solution.ResultsRC reduced (P &lt; 0.05) shear force by 51.9% (6.79 kgf CC) and 55.8% (8.50 kgf CC) for LI and LE cows, respectively. LI cows were more tender than LE for CC (6.79 vs. 8.50 kgf; P &lt; 0.05). RC compared to CC had longer SLs (LE: 1.80 vs. 1.44µ; P &lt; 0.05) and LI (1.80 vs. 1.40µ; P &lt; 0.05). Purge and cooking losses were not affected by chilling method. The contractile responses of the muscle after the exposure to the solutions were slightly different. The average percentage decline of peak twitch force was higher for solution B, followed by solutions A, D, C (54.8%, 53.5%, 48.0%, 43.4%, respectively). Furthermore, the same pattern was observed for the average percentage decline at half time of the test (82.5%, 80.4%, 78.1% and 74.7%, respectively).ConclusionPacking plants that harvest cull dairy cows have the potential to dramatically improve tenderness and thereby merchandize a greater amount of whole muscle cuts as a result of the application of the Rinse &amp; Chill®. This improvement may be associated with accelerating postmortem glycolysis, thereby limiting cold shortening, although enhanced proteolysis may also be involved. Continuous electrical stimulation of isolated muscle-fiber bundles while being soaked in selected test solutions led to decreased and somewhat varied contractile force responses suggesting the potential to modify muscle metabolism.

Altered detrusor contractility and voiding patterns in mice lacking the mechanosensitive TREK-1 channel

BackgroundPreviously published results from our laboratory identified a mechano-gated two-pore domain potassium channel, TREK-1, as a main mechanosensor in the smooth muscle of the human urinary bladder. One of the limitations of in vitro experiments on isolated human detrusor included inability to evaluate in vivo effects of TREK-1 on voiding function, as the channel is also expressed in the nervous system, and may modulate micturition via neural pathways. Therefore, in the present study, we aimed to assess the role of TREK-1 channel in bladder function and voiding patterns in vivo by using TREK-1 knockout (KO) mice.MethodsAdult C57BL/6 J wild-type (WT, N = 32) and TREK-1 KO (N = 33) mice were used in this study. The overall phenotype and bladder function were evaluated by gene and protein expression of TREK-1 channel, in vitro contractile experiments using detrusor strips in response to stretch and pharmacological stimuli, and cystometry in unanesthetized animals.ResultsTREK-1 KO animals had an elevated basal muscle tone and enhanced spontaneous activity in the detrusor without detectable changes in bladder morphology/histology. Stretch applied to isolated detrusor strips increased the amplitude of spontaneous contractions by 109% in the TREK-1 KO group in contrast to a 61% increase in WT mice (p ≤ 0.05 to respective baseline for each group). The detrusor strips from TREK-1 KO mice also generated more contractile force in response to electric field stimulation and high potassium concentration in comparison to WT group (p ≤ 0.05 for both tests). However, cystometric recordings from TREK-1 KO mice revealed a significant increase in the duration of the intermicturition interval, enhanced bladder capacity and increased number of non-voiding contractions in comparison to WT mice.ConclusionsOur results provide evidence that global down-regulation of TREK-1 channels has dual effects on detrusor contractility and micturition patterns in vivo. The observed differences are likely due to expression of TREK-1 channel not only in detrusor myocytes but also in afferent and efferent neural pathways involved in regulation of micturition which may underly the “mixed” voiding phenotype in TREK-1 KO mice.

Cardiomyocyte contractile force changes in response to AGRWE detected by AFM

The cardiac contractile force is an important predictor of healthy and cardiovascular diseases. The changes of cardiomyocyte contractile force in response to American ginseng root water extract (AGRWE) detected by atomic force microscope have not been investigated yet. This study examined the effects of AGRWE on single beating cardiomyocytes extracted from a newborn rat. The same cardiomyocytes were incubated with AGRWE at a concentration of 50 μg/ml for about 30 min, and the cardiomyocytes' contractile force increased from 1.74 ± 1.01 to 3.49 ± 1.53 nN. The mean value of the contractile strain calculated was 3.32 ± 1.55% for the cardiomyocyte before the treatment with AGRWE, while for the cardiomyocyte treated with AGRWE it increased to 4.60 ± 1.35%. The results also showed that the beating rate of the same single beating cardiomyocytes was decreased from 34 ± 11 beats/min (control, n = 10) to 20 ± 9 beats/min. In conclusion, the experimental results have shown clearly that the contractile forces and strain of single beating cardiomyocytes treated with AGRWE are significantly higher than the control group, while the heart rate was decreased. It suggests that ginseng agents are promising candidates in improving cardiac functions for treating heart failure.

Reduction of blood pressure elevation by losartan in spontaneously hypertensive rats through suppression of LARG expression in vascular smooth muscle cells

This study sought to elucidate the mechanism by which losartan inhibits blood pressure (BP) elevation in spontaneously hypertensive rats (SHRs). Four-week-old Wistar-Kyoto (WKY) rats and SHRs were either treated with losartan (20mg/kg/day) for 8 weeks or served as untreated controls. BP was measured by the tail-cuff method. At 12 weeks, isometric contraction of the aortic rings of the rats was evaluated with a force transducer and recorder. The mRNA and protein levels of the target Rho guanine nucleotide exchange factors (RhoGEFs), and the extent of myosin phosphatase target subunit 1 (MYPT-1) phosphorylation in the aorta, were determined using quantitative real-time polymerase chain reaction (qPCR) assay and Western blot analysis. The BP of the four-week-old SHRs did not differ from that of the age-matched WKY rats, whereas the BP of the twelve-week-old control group SHRs was higher than that of the control group WKY rats. Losartan treatment, however, inhibited BP elevation in both rat strains, doing so to a greater extent in the treatment group SHRs. The contractile force in response to angiotensin II of the aortic rings from the SHRs treated with losartan was significantly lower than that of the aortic rings from the non-treated SHRs. The protein expression of leukemia-associated RhoGEF (LARG) was significantly higher in the non-treated SHRs compared to the non-treated WKY rats. The study results showed that the reduction of BP elevation by losartan in SHRs occurs through the suppression of LARG expression and MYPT-1 phosphorylation in vascular smooth muscle cells.

Vascular Rinsing and Chilling Carcasses: Effects on Quality Attributes and Metabolic Changes in Beef

ObjectivesRinse & Chill® (RC) is a process applied early postmortem that provides the ability to manipulate muscle metabolism and can have a positive impact on meat quality traits. This study aimed to evaluate the effect of RC on pH decline, shear force, sarcomere length and cooking losses on different cull dairy cow carcass grades. Investigate the ability of different substrates to modulate contractile response as an indirect measure of metabolic activity on beef early postmortem.Materials and MethodsFor each carcass grade (lean, LE; light, LI), ten carcasses were conventionally chilled (CC) and twelve carcasses were chilled using RC technology (MPSC Inc.). The RC process involved infusion of a chilled isotonic solution (98.5% water; balance: glucose, phosphates, and maltose) through the vascular system, beginning in the arterial and exiting the venous side of the vasculature. Shear force and cooking losses were measured on Longissimus dorsi steaks aged (7 d). Sarcomere length (SL) was determined by a laser diffraction method. Animal served as the experimental unit and data were analyzed with a PROC MIXED procedure. For contraction measurements, a muscle-fiber bundle from the Sternomandibularis muscles (n = 14) was collected from cull dairy cows in a commercial packing plant, 15 min after bleeding. The muscle bundle was attached to a force transducer (FT-302, iWorx, Dover, NH). Stimulation electrodes were used to elicit a supramaximal electrical stimulus at a frequency of 50 V, 0.1 Hz (HCS-100 stimulator, iWorx). Muscle weight was standardized, and length was adjusted to obtain maximum twitch-tension output. After 3 min of rest in a test solution, 200 stimuli were given, and the contractile response was recorded. Four solutions were tested (A = RC, B = Fructose, C = Sodium phosphate, D = Dipotassium phosphate; substrates added at 1% except fructose 1.5%). Descriptive means for initial peak twitch force, final peak twitch force, percentage decline and percentage half-time decline were calculated to determine the response associated with each solution.ResultsRC reduced (P < 0.05) shear force by 51.9% (6.79 kgf CC) and 55.8% (8.50 kgf CC) for LI and LE cows, respectively. LI cows were more tender than LE for CC (6.79 vs. 8.50 kgf; P < 0.05). RC compared to CC had longer SLs (LE: 1.80 vs. 1.44µ; P < 0.05) and LI (1.80 vs. 1.40µ; P < 0.05). Purge and cooking losses were not affected by chilling method. The contractile responses of the muscle after the exposure to the solutions were slightly different. The average percentage decline of peak twitch force was higher for solution B, followed by solutions A, D, C (54.8%, 53.5%, 48.0%, 43.4%, respectively). Furthermore, the same pattern was observed for the average percentage decline at half time of the test (82.5%, 80.4%, 78.1% and 74.7%, respectively).ConclusionPacking plants that harvest cull dairy cows have the potential to dramatically improve tenderness and thereby merchandize a greater amount of whole muscle cuts as a result of the application of the Rinse & Chill®. This improvement may be associated with accelerating postmortem glycolysis, thereby limiting cold shortening, although enhanced proteolysis may also be involved. Continuous electrical stimulation of isolated muscle-fiber bundles while being soaked in selected test solutions led to decreased and somewhat varied contractile force responses suggesting the potential to modify muscle metabolism.

3D printed micro-scale force gauge arrays to improve human cardiac tissue maturation and enable high throughput drug testing

Human induced pluripotent stem cell – derived cardiomyocytes (iPSC-CMs) are regarded as a promising cell source for establishing in-vitro personalized cardiac tissue models and developing therapeutics. However, analyzing cardiac force and drug response using mature human iPSC-CMs in a high-throughput format still remains a great challenge. Here we describe a rapid light-based 3D printing system for fabricating micro-scale force gauge arrays suitable for 24-well and 96-well plates that enable scalable tissue formation and measurement of cardiac force generation in human iPSC-CMs. We demonstrate consistent tissue band formation around the force gauge pillars with aligned sarcomeres. Among the different maturation treatment protocols we explored, 3D aligned cultures on force gauge arrays with in-culture pacing produced the highest expression of mature cardiac marker genes. We further demonstrated the utility of these micro-tissues to develop significantly increased contractile forces in response to treatment with isoproterenol, levosimendan, and omecamtiv mecarbil. Overall, this new 3D printing system allows for high flexibility in force gauge design and can be optimized to achieve miniaturization and promote cardiac tissue maturation with great potential for high-throughput in-vitro drug screening applications. Statement of SignificanceThe application of iPSC-derived cardiac tissues in translatable drug screening is currently limited by the challenges in forming mature cardiac tissue and analyzing cardiac forces in a high-throughput format. We demonstrate the use of a rapid light-based 3D printing system to build a micro-scale force gauge array that enables scalable cardiac tissue formation from iPSC-CMs and measurement of contractile force development. With the capability to provide great flexibility over force gauge design as well as optimization to achieve miniaturization, our 3D printing system serves as a promising tool to build cardiac tissues for high-throughput in-vitro drug screening applications.

MP38-09 THE LACK OF MECHANOSENSITIVE K2P CHANNEL IS ASSOCIATED WITH MIXED VOIDING PHENOTYPE IN MICE

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Basic Research & Pathophysiology II1 Apr 2018MP38-09 THE LACK OF MECHANOSENSITIVE K2P CHANNEL IS ASSOCIATED WITH MIXED VOIDING PHENOTYPE IN MICE Ricardo Pineda, Joseph Hypolite, Sanghee Lee, Alonso Carrasco, Nao Iguchi, Randall B Meacham, and Anna P Malykhina Ricardo PinedaRicardo Pineda More articles by this author , Joseph HypoliteJoseph Hypolite More articles by this author , Sanghee LeeSanghee Lee More articles by this author , Alonso CarrascoAlonso Carrasco More articles by this author , Nao IguchiNao Iguchi More articles by this author , Randall B MeachamRandall B Meacham More articles by this author , and Anna P MalykhinaAnna P Malykhina More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1236AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder capacity is defined by the physiological response of the bladder wall to stretch. We previously reported that compliance of the bladder wall during storage phase of the micturition cycle in the human urinary bladder is regulated by a mechanosensitive TREK-1 channel, a member of the K2P potassium channel family. The use of isolated human tissue limited evaluation of in vivo role of TREK-1 on voiding function as these channels are also expressed in the nervous system and may modulate micturition via neural pathways. Therefore, in this study, we aimed to assess the role of TREK-1 channels in bladder contractility and voiding using TREK-1 knockout (KO) mice. We hypothesized that absence of TREK-1 expression could increase the number of spontaneous contractions in the urinary bladder, and alter its reposne to physiological stretch. We aimed to: 1) study the patterns of TREK-1 expression in visceral organs (kidney, urinary bladder, and colon) in WT and TREK-1 KO mice, and 2) assess the role of TREK-1 channels in bladder contractility and voiding cycle. METHODS Adult C57BL/6J wild-type (WT, N=32) and TREK-1 KO (KO, N=33) mice were used in this study. We performed immunocytochemistry labeling, endpoint RT-PCR, detrusor muscle strip contractility studies, and awake cystometry to evaluate blader function. RESULTS No changes in visceral organ morphology or tissue histology were observed between WT and TREK-1 KO animals. Isometric contractility recordings from TREK-1 KO isolated detrusor strips showed elevated basal muscle tone and enhanced spontaneous activity in response to stretch (109% vs. 61% in WT group, p=0.05). Detrusor strips from TREK-1 KO mice also generated more contractile force in response to electric field and high potassium stimulation. Interestingly, cystometric recordings from TREK-1 KO mice revealed a significant increase in the duration of inter-micturition interval, also expressed enhanced bladder capacity and increased number of non-voiding contractions in comparison to WT mice. CONCLUSIONS Our data suggest that the knockout of TREK-1 channels has dual effects on detrusor contractility and micturition patterns, most likely due to expression of TREK-1 channels on both smooth muscle and neuronal cells. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e503-e504 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Ricardo Pineda More articles by this author Joseph Hypolite More articles by this author Sanghee Lee More articles by this author Alonso Carrasco More articles by this author Nao Iguchi More articles by this author Randall B Meacham More articles by this author Anna P Malykhina More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Exosomal microRNA-21-5p Mediates Mesenchymal Stem Cell Paracrine Effects on Human Cardiac Tissue Contractility.

The promising clinical benefits of delivering human mesenchymal stem cells (hMSCs) for treating heart disease warrant a better understanding of underlying mechanisms of action. hMSC exosomes increase myocardial contractility; however, the exosomal cargo responsible for these effects remains unresolved. This study aims to identify lead cardioactive hMSC exosomal microRNAs to provide a mechanistic basis for optimizing future stem cell-based cardiotherapies. Integrating systems biology and human engineered cardiac tissue (hECT) technologies, partial least squares regression analysis of exosomal microRNA profiling data predicted microRNA-21-5p (miR-21-5p) levels positively correlate with contractile force and calcium handling gene expression responses in hECTs treated with conditioned media from multiple cell types. Furthermore, miR-21-5p levels were significantly elevated in hECTs treated with the exosome-enriched fraction of the hMSC secretome (hMSC-exo) versus untreated controls. This motivated experimentally testing the human-specific role of miR-21-5p in hMSC-exo-mediated increases of cardiac tissue contractility. Treating hECTs with miR-21-5p alone was sufficient to recapitulate effects observed with hMSC-exo on hECT developed force and expression of associated calcium handling genes (eg, SERCA2a and L-type calcium channel). Conversely, knockdown of miR-21-5p in hMSCs significantly diminished exosomal procontractile and associated calcium handling gene expression effects on hECTs. Western blots supported miR-21-5p effects on calcium handling gene expression at the protein level, corresponding to significantly increased calcium transient amplitude and decreased decay time constant in comparison to miR-scramble control. Mechanistically, cotreating with miR-21-5p and LY294002, a PI3K inhibitor, suppressed these effects. Finally, mathematical simulations predicted the translational capacity for miR-21-5p treatment to restore calcium handling in mature ischemic adult human cardiomyocytes. miR-21-5p plays a key role in hMSC-exo-mediated effects on cardiac contractility and calcium handling, likely via PI3K signaling. These findings may open new avenues of research to harness the role of miR-21-5p in optimizing future stem cell-based cardiotherapies.

The reduced myofilament responsiveness to calcium contributes to the negative force-frequency relationship in rat cardiomyocytes: role of reactive oxygen species and p-38 map kinase.

The force-frequency relationship (FFR) is an important intrinsic regulatory mechanism of cardiac contractility. However, a decrease (negative FFR) or no effect (flat FFR) on contractile force in response to an elevation of heart rate is present in the normal rat or in human heart failure. Reactive oxygen species (ROS) can act as intracellular signaling molecules activating diverse kinases as calcium-calmodulin-dependent protein kinase II (CaMKII) and p-38 MAP kinase (p-38K). Our aim was to elucidate the intracellular molecules implicated in the FFR of isolated rat ventricular myocytes. The myocytes were field-stimulated via two-platinum electrodes. Sarcomere length was recorded with a video camera. Ca2+ transients and intracellular pHi were recorded by epifluorescence. Increasing frequency from 0.5 to 3Hz decreased cell shortening without changes in pHi. This negative FFR was changed to positive FFR when the myocytes were pre-incubated with the ROS scavenger MPG, the NADPH oxidase blocker apocynin, or by inhibiting mitochondrial ROS production with 5-HD. Similar results were obtained when the cells were pre-incubated with the CaMKII blocker, KN-93, or the p-38K inhibitor, SB-202190. Consistently, the levels of phosphorylation of p-38K and the oxidation of CaMKII were significantly higher at 2Hz than at 0.5Hz. Despite the presence of positive inotropic effect during stimulation frequency enhancement, Ca2+ transient amplitudes were reduced in MPG- and SB-202190-treated myocytes. In conclusion, our results indicate that the activation of the intracellular pathway involving ROS-CaMKII-p-38K contributes to the negative FFR of rat cardiomyocytes, likely by desensitizing the response of contractile myofilaments to Ca2+.

A resilient formin-derived cortical actin meshwork in the rear drives actomyosin-based motility in 2D confinement.

Cell migration is driven by the establishment of disparity between the cortical properties of the softer front and the more rigid rear allowing front extension and actomyosin-based rear contraction. However, how the cortical actin meshwork in the rear is generated remains elusive. Here we identify the mDia1-like formin A (ForA) from Dictyostelium discoideum that generates a subset of filaments as the basis of a resilient cortical actin sheath in the rear. Mechanical resistance of this actin compartment is accomplished by actin crosslinkers and IQGAP-related proteins, and is mandatory to withstand the increased contractile forces in response to mechanical stress by impeding unproductive blebbing in the rear, allowing efficient cell migration in two-dimensional-confined environments. Consistently, ForA supresses the formation of lateral protrusions, rapidly relocalizes to new prospective ends in repolarizing cells and is required for cortical integrity. Finally, we show that ForA utilizes the phosphoinositide gradients in polarized cells for subcellular targeting.

Bioengineered human myobundles mimic clinical responses of skeletal muscle to drugs.

Existing in vitro models of human skeletal muscle cannot recapitulate the organization and function of native muscle, limiting their use in physiological and pharmacological studies. Here, we demonstrate engineering of electrically and chemically responsive, contractile human muscle tissues ('myobundles') using primary myogenic cells. These biomimetic constructs exhibit aligned architecture, multinucleated and striated myofibers, and a Pax7(+) cell pool. They contract spontaneously and respond to electrical stimuli with twitch and tetanic contractions. Positive correlation between contractile force and GCaMP6-reported calcium responses enables non-invasive tracking of myobundle function and drug response. During culture, myobundles maintain functional acetylcholine receptors and structurally and functionally mature, evidenced by increased myofiber diameter and improved calcium handling and contractile strength. In response to diversely acting drugs, myobundles undergo dose-dependent hypertrophy or toxic myopathy similar to clinical outcomes. Human myobundles provide an enabling platform for predictive drug and toxicology screening and development of novel therapeutics for muscle-related disorders.