Measurement Of Contractile Force Research Articles

Protein-free media for cardiac differentiation of hPSCs in 2000 mL suspension culture

BackgroundCommonly used media for the differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CMs) contain high concentrations of proteins, in particular albumin, which is prone to quality variations and presents a substantial cost factor, hampering the clinical translation of in vitro-generated cardiomyocytes for heart repair. To overcome these limitations, we have developed chemically defined, entirely protein-free media based on RPMI, supplemented with L-ascorbic acid 2-phosphate (AA-2P) and either the non-ionic surfactant Pluronic F-68 or a specific polyvinyl alcohol (PVA).Methods and ResultsBoth media compositions enable the efficient, directed differentiation of embryonic and induced hPSCs, matching the cell yields and cardiomyocyte purity ranging from 85 to 99% achieved with the widely used protein-based CDM3 medium. The protein-free differentiation approach was readily up-scaled to a 2000 mL process scale in a fully controlled stirred tank bioreactor in suspension culture, producing > 1.3 × 109 cardiomyocytes in a single process run. Transcriptome analysis, flow cytometry, electrophysiology, and contractile force measurements revealed that the mass-produced cardiomyocytes differentiated in protein-free medium exhibit the expected ventricular-like properties equivalent to the well-established characteristics of CDM3-control cells.ConclusionsThis study promotes the robustness and upscaling of the cardiomyogenic differentiation process, substantially reduces media costs, and provides an important step toward the clinical translation of hPSC-CMs for heart regeneration.

Hydrogel-Sheathed hiPSC-Derived Heart Microtissue Enables Anchor-Free Contractile Force Measurement.

In vitro reconstruction of highly mature engineered heart tissues (EHTs) is attempted for the selection of cardiotoxic drugs suitable for individual patients before administration. Mechanical contractile force generated in the EHTs is known to be a critical indicator for evaluating the EHT response. However, measuring contractile force requires anchoring the EHT in a tailored force-sensing cell culture chamber, causing technical difficulties in the stable evaluation of contractile force in long-term culture. This paper proposes a hydrogel-sheathed human induced pluripotent stem cell (hiPSC)-derived heart microtissue (H3 M) that can provide an anchor-free contractile force measurement platform in commonly used multi-well plates. The contractile force associated with tissue formation and drug response is calculated by motion tracking and finite element analysis on the bending angle of the hydrogel sheath. From the experiment of the drug response, H3 M is an excellent drug screening platform with high sensitivity and early testing capability compared to conventionally anchored EHT. This unique platform would be useful and versatile for regenerative therapy and drug discovery research in EHT.

Vasomotion in human arteries and their regulations based on ion channel regulations: 10 years study.

Vasomotion is the oscillation of vascular tone which gives rise to flow motion of blood into an organ. As is well known, spontaneous contractile organs such as heart, GI, and genitourinary tract produce rhythmic contraction. It imposes or removes pressure on their vessels alternatively for exchange of many substances. It was first described over 150 years ago, however the physiological mechanism and pathophysiological implications are not well understood. This study aimed to elucidate underlying mechanisms and physiological function of vasomotion in human arteries. Conventional contractile force measurement, immunohistochemistry, and Western blot analysis were employed to study human left gastric artery (HLGA) and uterine arteries (HUA). RESULTS: Circular muscle of HLGA and/or HUA produced sustained tonic contraction by high K+ (50 mM) which was blocked by 2 µM nifedipine. Stepwise stretch and high K+ produced nerve-independent spontaneous contraction (vasomotion) (around 45% of tested tissues). Vasomotion was also produced by application of BayK8644, 5-HT, prostagrandins, oxytocin. It was blocked by nifedipine (2 µM) and blockers of intracellular Ca2+ stores. Inhibitors of Ca2+ -activated Cl- channels (DIDS and/or niflumic acid) and ATP-sensitive K+ (KATP ) channels inhibited vasomotion reversibly. Metabolic inhibition by sodium cyanide (NaCN) and several neuropeptides also regulated vasomotion in KATP channel-sensitive and -insensitive manner. Finally, we identified TMEM16A Ca2+ -activated Cl- channels and subunits of KATP channels (Kir 6.1/6.2 and sulfonylurea receptor 2B [SUR2B]), and c-Kit positivity by Western blot analysis. We conclude that vasomotion is sensitive to TMEM16A Ca2+ -activated Cl- channels and metabolic changes in human gastric and uterine arteries. Vasomotion might play an important role in the regulation of microcirculation dynamics even in pacemaker-related autonomic contractile organs in humans.

Ryanodine Receptor Staining Identifies Viable Cardiomyocytes in Human and Rabbit Cardiac Tissue Slices.

In terms of preserving multicellularity and myocardial function in vitro, the cultivation of beating myocardial slices is an emerging technique in basic and translational cardiac research. It can be used, for example, for drug screening or to study pathomechanisms. Here, we describe staining for viable cardiomyocytes based on the immunofluorescence of ryanodine receptors (RyRs) in human and rabbit myocardial slices. Biomimetic chambers were used for culture and measurements of contractile force. Fixable fluorophore-conjugated dextran, entering cells with a permeable membrane, was used for death staining. RyRs, nuclei and the extracellular matrix, including the t-system, were additionally stained and analyzed by confocal microscopy and image processing. We found the mutual exclusion of the RyR and dextran signals in cultivated slices. T-System density and nucleus size were reduced in RyR-negative/dextran-positive myocytes. The fraction of RyR-positive myocytes and pixels correlated with the contractile force. In RyR-positive/dextran-positive myocytes, we found irregular RyR clusters and SERCA distribution patterns, confirmed by an altered power spectrum. We conclude that RyR immunofluorescence indicates viable cardiomyocytes in vibratome-cut myocardial slices, facilitating the detection and differential structural analysis of living vs. dead or dying myocytes. We suggest the loss of sarcoplasmic reticulum integrity as an early event during cardiomyocyte death.

Effects of docosahexaenoic acid or arachidonic acid supplementation on the behavior of cardiomyocytes derived from human pluripotent stem cells

<b>Background:</b> Human heart changes its energetic substrates from lactate and glucose to fatty acids during the neonatal period. Noticing the lack of fatty acids in media for the culture of cardiomyocytes derived from human pluripotent stem cells (hiPS-CM), researchers have supplemented mixtures of fatty acids to hiPS-CM and reported the enhancement in the maturation of hiPS-CM. In our previous studies, we separately supplemented two polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA) or arachidonic acid (AA), to rat fetal cardiomyocytes and found that the supplementations upregulated the expressions of mRNAs for cardiomyocyte differentiation, fatty acid metabolism, and cellular adhesion. The enhancement in cellular contractility was attributed to the improvement in intercellular connection rather than a direct enhancement of the contractile force. <b>Methods:</b> This study reports the successive results of the effects of DHA or AA supplementation on hiPS-CM. In addition to the contractile force and mRNA measurements used in the previous study, we further investigated the effect of different cellular aggregations on the contractile force output by means of finite element analysis, measured glucose and fatty acids metabolites, and assessed cTNT and MLC2v expressions through immunofluorecsence evaluation. <b>Results:</b> It showed that the sole supplementation of albumin-conjugated DHA or AA can be taken up by hiPS-CM without other uptake-enhancing factors, and the supplementations may activate the CD36_­ERRγ metabolic pathway. DHA or AA supplementation increased the cellular contractile ratio on collagen gels and AA supplementation stimulated hiPS-CM aggregation to form cellular clusters. The enhancement effect on the hiPS-CM contractile force was modest since the increase in contractile force was not significant. AA supplementation was more effective than DHA supplementation because it significantly upregulated mRNA expressions of <i>P300</i> and <i>CD36</i>. However, finite element analysis showed that the formation of clusters on a collagen gel attenuated the contractile force exerted by the gel on its surroundings. <b>Conclusion:</b> DHA and AA, as having been supplemented in infant formulas, have no direct and significant enhancement effect on the performance of the hiPS-CM when they were supplemented individually, although they were able to enter the cellular metabolic system. The AA supplementation showed some auxiliary effect on the maturation of hiPS-CM, which is worthy of further investigation under the consideration of membrane composition alteration and remodeling of membrane molecules.

Ex vivo Culture and Contractile Force Measurements of Non-human Primate Heart Slices.

Cardiovascular diseases are the leading cause of death and morbidity worldwide. Patient mortality has been successfully reduced by nearly half in the last four decades, mainly due to advances in minimally invasive surgery techniques and interventional cardiology methods. However, a major hurdle is still the translational gap between preclinical findings and the conversion into effective therapies, which is partly due to the use of model systems that fail to recapitulate key aspects of human physiology and disease. Large animal models such as pigs and non-human primates are highly valuable because they closely resemble humans but are costly and time intensive. Here, we provide a method for long-term ex vivo culture of non-human primate (NHP) myocardial tissue that offers a powerful alternative for a wide range of applications including electrophysiology studies, drug screening, and gene function analyses.

Engineered Heart Tissues for Contractile, Structural, and Transcriptional Assessment of Human Pluripotent Stem Cell-Derived Cardiomyocytes in a Three-Dimensional, Auxotonic Environment.

Three-dimensional, humanengineered heart tissue promotes maturation of humaninduced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and provides a useful platform for in vitro cardiac development and disease modeling. This protocol describes the generation of fibrin-based engineered heart tissues (EHTs) containing hiPSC-CMs and human stromal cells. The platform makes use of racks of silicone posts that fit a standard 24-well dish. Stromal cells and hiPSC-CMs are cast in a fibrin hydrogel suspended between two silicone posts, forming an engineered tissue that generates synchronous contractions. The platform described herein is amenable to various measures of cardiac function including measurement of contractile force and calcium handling, as well as molecular biology assays and immunostaining.

Full-length dystrophin deficiency leads to contractile and calcium transient defects in human engineered heart tissues

Cardiomyopathy is currently the leading cause of death for patients with Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder affecting young boys. Animal models have provided insight into the mechanisms by which dystrophin protein deficiency causes cardiomyopathy, but there remains a need to develop human models of DMD to validate pathogenic mechanisms and identify therapeutic targets. Here, we have developed human engineered heart tissues (EHTs) from CRISPR-edited, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing a truncated dystrophin protein lacking part of the actin-binding domain. The 3D EHT platform enables direct measurement of contractile force, simultaneous monitoring of Ca2+ transients, and assessment of myofibril structure. Dystrophin-mutant EHTs produced less contractile force as well as delayed kinetics of force generation and relaxation, as compared to isogenic controls. Contractile dysfunction was accompanied by reduced sarcomere length, increased resting cytosolic Ca2+ levels, delayed Ca2+ release and reuptake, and increased beat rate irregularity. Transcriptomic analysis revealed clear differences between dystrophin-deficient and control EHTs, including downregulation of genes related to Ca2+ homeostasis and extracellular matrix organization, and upregulation of genes related to regulation of membrane potential, cardiac muscle development, and heart contraction. These findings indicate that the EHT platform provides the cues necessary to expose the clinically-relevant, functional phenotype of force production as well as mechanistic insights into the role of Ca2+ handling and transcriptomic dysregulation in dystrophic cardiac function, ultimately providing a powerful platform for further studies in disease modeling and drug discovery.

Abstract 12305: Cardiomyocyte Alignment Control Promotes the Electrical Integration and Improve the Contractile Function of Human Cardiac Tissue

Introduction: Alignment, as seen in the native myocardium, is crucial for maintaining the functional cardiac tissue. However, it remains unclear how cardiomyocyte alignment control influences cardiac function and the underlying mechanisms. The aims of our study were to fabricate aligned human cardiac tissue using a micro-processed fibrin gel and elucidated the effect of alignment control on contractile properties. Methods: We fabricated aligned human cardiac tissue through cultivating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on a micro-processed fibrin gel with inverted V-shaped ridges (MFG). The orientation index was calculated based on the Fourier analysis using Phalloidin staining images. The cardiac tissue function was evaluated with a contractile force measurement system. Results: The orientation index of hiPSC-CMs on MFG was significantly higher than that on the control fibrin gel (1.5±0.1 vs. 1.2±0.1, p&lt;0.001, n=4) and that was close to the orientation index of an adult rat heart tissue (1.6±0.1), suggesting hiPSC-CMs on MFG were aligned more uniformly than the control. The contractile force, maximum contractile velocity, and relaxation velocity were significantly increased in aligned cardiac tissue compared with non-aligned cardiac tissue [contractile force (75bpm): 0.9±0.5 mN vs. 0.5±0.3 mN, p=0.02, n=11]. Motion capture analysis revealed cardiomyocytes in the aligned cardiac tissues showed more unidirectional contraction than the non-aligned cardiac tissues. Furthermore, the timing of contraction at five designated points in the tissues was significantly closed in the aligned tissue, suggesting cardiomyocyte alignment control might improve the systolic and relaxation function of cardiac tissue by promoting synchronous cardiomyocyte contraction. Conclusion: Cardiomyocyte alignment control promoted the electrical integration of cardiomyocytes and contributed to generate the strong contractile force. Understanding the molecular mechanisms of alignment control-mediated synchronous contraction of cardiomyocytes might provide us the new insights on electrophysiological properties of heart tissue.

Dynamic loading of human engineered heart tissue enhances contractile function and drives a desmosome-linked disease phenotype.

The role that mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes. However, most EHT systems cannot model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained contractile shortening of >10%. To do this, three-dimensional (3D) EHTs were integrated with an elastic polydimethylsiloxane strip providing mechanical preload and afterload in addition to enabling contractile force measurements based on strip bending. Our results demonstrated that dynamic loading improves the function of wild-type EHTs on the basis of the magnitude of the applied force, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we used hiPSC-derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy due to mutations in the desmoplakin gene. We demonstrated that manifestation of this desmosome-linked disease state required dyn-EHT conditioning and that it could not be induced using 2D or standard 3D EHT approaches. Thus, a dynamic loading strategy is necessary to provoke the disease phenotype of diastolic lengthening, reduction of desmosome counts, and reduced contractility, which are related to primary end points of clinical disease, such as chamber thinning and reduced cardiac output.

Microelectromechanical System Measurement of Platelet Contraction: Direct Interrogation of Myosin Light Chain Phosphorylation.

Myosin Light Chain (MLC) regulates platelet contraction through its phosphorylation by Myosin Light Chain Kinase (MLCK) or dephosphorylation by Myosin Light Chain Phosphatase (MLCP). The correlation between platelet contraction force and levels of MLC phosphorylation is unknown. We investigate the relationship between platelet contraction force and MLC phosphorylation using a novel microelectromechanical (MEMS) based clot contraction sensor (CCS). The MLCK and MLCP pair were interrogated by inhibitors and activators of platelet function. The CCS was fabricated from silicon using photolithography techniques and force was validated over a range of deflection for different chip spring constants. The force of platelet contraction measured by the clot contraction sensor (CCS) was compared to the degree of MLC phosphorylation by Western Blotting (WB) and ELISA. Stimulators of MLC phosphorylation produced higher contraction force, higher phosphorylated MLC signal in ELISA and higher intensity bands in WB. Inhibitors of MLC phosphorylation produced the opposite. Contraction force is linearly related to levels of phosphorylated MLC. Direct measurements of clot contractile force are possible using a MEMS sensor platform and correlate linearly with the degree of MLC phosphorylation during coagulation. Measured force represents the mechanical output of the actin/myosin motor in platelets regulated by myosin light chain phosphorylation.

Compliant 3D frameworks instrumented with strain sensors for characterization of millimeter-scale engineered muscle tissues

Tissue-on-chip systems represent promising platforms for monitoring and controlling tissue functions in vitro for various purposes in biomedical research. The two-dimensional (2D) layouts of these constructs constrain the types of interactions that can be studied and limit their relevance to three-dimensional (3D) tissues. The development of 3D electronic scaffolds and microphysiological devices with geometries and functions tailored to realistic 3D tissues has the potential to create important possibilities in advanced sensing and control. This study presents classes of compliant 3D frameworks that incorporate microscale strain sensors for high-sensitivity measurements of contractile forces of engineered optogenetic muscle tissue rings, supported by quantitative simulations. Compared with traditional approaches based on optical microscopy, these 3D mechanical frameworks and sensing systems can measure not only motions but also contractile forces with high accuracy and high temporal resolution. Results of active tension force measurements of engineered muscle rings under different stimulation conditions in long-term monitoring settings for over 5 wk and in response to various chemical and drug doses demonstrate the utility of such platforms in sensing and modulation of muscle and other tissues. Possibilities for applications range from drug screening and disease modeling to biohybrid robotic engineering.

Assessment of human bioengineered cardiac tissue function in hypoxic and re-oxygenized environments to understand functional recovery in heart failure.

IntroductionMyocardial recovery is one of the targets for heart failure treatment. A non-negligible number of heart failure with reduced ejection fraction (EF) patients experience myocardial recovery through treatment. Although myocardial hypoxia has been reported to contribute to the progression of heart failure even in non-ischemic cardiomyopathy, the relationship between contractile recovery and re-oxygenation and its underlying mechanisms remain unclear. The present study investigated the effects of hypoxia/re-oxygenation on bioengineered cardiac cell sheets-tissue function and the underlying mechanisms.MethodsBioengineered cardiac cell sheets-tissue was fabricated with human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) using temperature-responsive culture dishes. Cardiac tissue functions in the following conditions were evaluated with a contractile force measurement system: continuous normoxia (20% O2) for 12 days; hypoxia (1% O2) for 4 days followed by normoxia (20% O2) for 8 days; or continuous hypoxia (1% O2) for 8 days. Cell number, sarcomere structure, ATP levels, mRNA expressions and Ca2+ transients of hiPSC-CM in those conditions were also assessed.ResultsHypoxia (4 days) elicited progressive decreases in contractile force, maximum contraction velocity, maximum relaxation velocity, Ca2+ transient amplitude and ATP level, but sarcomere structure and cell number were not affected. Re-oxygenation (8 days) after hypoxia (4 days) was associated with progressive increases in contractile force, maximum contraction velocity and relaxation time to the similar extent levels of continuous normoxia group, while maximum relaxation velocity was still significantly low even after re-oxygenation. Ca2+ transient magnitude, cell number, sarcomere structure and ATP level after re-oxygenation were similar to those in the continuous normoxia group. Hypoxia/re-oxygenation up-regulated mRNA expression of PLN.ConclusionsHypoxia and re-oxygenation condition directly affected human bioengineered cardiac tissue function. Further understanding the molecular mechanisms of functional recovery of cardiac tissue after re-oxygenation might provide us the new insight on heart failure with recovered ejection fraction and preserved ejection fraction.

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues.

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitromodels closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as 'MyoTACTIC', and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.

Simultaneous measurement of contractile force and field potential of dynamically beating human iPS cell-derived cardiac cell sheet-tissue with flexible electronics.

Human induced pluripotent stem (iPS) cell-derived cardiomyocytes are used for in vitro pharmacological and pathological studies worldwide. In particular, the functional assessment of cardiac tissues created from iPS cell-derived cardiomyocytes is expected to provide precise prediction of drug effects and thus streamline the process of drug development. However, the current format of electrophysiological and contractile assessment of cardiomyocytes on a rigid substrate is not appropriate for cardiac tissues that beat dynamically. Here, we show a novel simultaneous measurement system for contractile force and extracellular field potential of iPS cell-derived cardiac cell sheet-tissues using 500 nm-thick flexible electronic sheets. It was confirmed that the developed system is applicable for pharmacological studies and assessments of excitation-contraction coupling-related parameters, such as the electro-mechanical window. Our results indicate that flexible electronics with cardiac tissue engineering provide an advanced platform for drug development. This system will contribute to gaining new insight in pharmacological study of human cardiac function.

Measurement of Contractile Force of Artificial Muscle using Soft Sensor for Feedback Control of bio-actuator

Measurement of Contractile Force of Artificial Muscle using Soft Sensor for Feedback Control of bio-actuator

Contractile Force Measurement of Engineered Cardiac Tissues Derived from Human iPS Cells.

Recent advances in stem cell technologies and tissue engineering are enabling the fabrication of dynamically beating cardiac tissues from human induced pluripotent stem cells. These engineered human cardiac tissues are expected to be used for cardiac regenerative therapies, in vitro drug testing, and pathological investigations. Here we describe the method to fabricate engineered cardiac tissues from human induced pluripotent stem cell-derived cardiomyocytes and to measure the contractile force.

Approaches to Quantification of Contractile Force Measurement Devices for Artificial Skeletal Muscle

Approaches to Quantification of Contractile Force Measurement Devices for Artificial Skeletal Muscle

Mechanical, topographicaland chemical cues combined with physical therapy for peripheral nerve injuries.

Aim: The aim of this paper is to evaluate biomaterial cues combined with physical therapy (PT) on functional recovery in a rat sciatic nerve injury model. Materials & methods: Nerve growth conduits were filled with longitudinally aligned hyaluronic acid fibers and microspheres containing neurotrophic factor (growth factor[GF]). All animals received behavior and functional testing throughout the study, which concluded with measurement of compound muscle action potentials and contractile force of the gastrocnemius muscle. Results & conclusion: Including GF improved recovery of gross motor function and increased sensory pain sensation. During the 4weeks that animals participated in PT, these groups showed higher static sciatic index scores. Including GF and PT has the potential to improve clinical outcomes following peripheral nerve injury.

SUPPRESSION OF THE SMALL INTESTINE SHRINKABLE ACTIVITY AFTER USAGE OF THE ANESTHETIC KETAMINE

The work is devoted to the problem of general anesthetics side effects during surgical interventions. In particular, some of the common complications of anesthetics usage are intestinal motility disorders (eg, ileus), nausea and vomiting. We analyzed a clinical data of 60 patients who underwent analgosedation (AS) during coronary artery stenting. They were divided into 2 groups (1 group - AS with Diazepam and Fentanyl, 2 group - AS with Ketamine, Fentanyl and Propofol). Significantly more frequent manifestations of nausea in the perioperative period were identified in the group where ketamine was used. This formed the basis of the study of the molecular and cellular mechanisms of the effects of ketamine on the contractile activity of the smooth muscles of the small intestine.Anesthetics are known to interact with receptors, G-proteins and ion channels, including transient receptor potential channels (TRP channels). The member of this superfamily, TRPC4 channel, which is coupled to muscarinic receptors (M2/M3 type) through G-protein activation and causes cholinergic excitation and contraction of small intestinal smooth muscles, may be a potential target for ketamine.Thus, we aimed to investigate the effects of ketamine (100 μM) on the muscarinic cation current (mICAT), which underlies cholinergic excitation-contraction coupling of visceral smooth muscles. All experiments were performed on single ileal myocytes freshly isolated from the longitudinal layer of the mouse ileum using patch-clamp techniques in the whole-cell configuration. mICAT was recorded using symmetrical Cs+ solutions (containing Cs+125 mM). [Ca2+]i was ‘clamped’ at 100 nM using 10 mM BAPTA/4.6 mM CaCl2 mixture. Measurements of isometric contractile force of the small intestinal smooth muscles were recorded using tensiometry techniques. It was showed that 100 μM ketamine inhibits mICAT . mICAT initiated by the application of CCh (50 μM) was suppressed on 64% (n=5) and mICAT, induced by intracellular GTP γ s (200 μM) that interacts directly with the G-proteins (when muscarinic receptors are bypassed) was inhibited on 42% (n=5). Ketamine inhibited intestinal smooth muscle contractions evoked by carbachol (50 μM) by about 40 % (n=5). Thus, we can conclude that both muscarinic receptors and G-proteins (or their coupling) are affected by ketamine, but the main sites of ketamine action appear to be the G-proteins. These data will provide basis for the molecular mechanisms of postoperative motility disorders and thus may be important for the development of novel approaches to the correction of such states.