Increase In Force Of Contraction Research Articles

Studies on the mechanisms of action of MR33317

MR33317 was synthesized as an acetylcholinesterase-inhibitor and an agonist at brain 5-HT4-receptors. MR33317 might be used to treat Morbus Alzheimer. This therapeutic action of MR33317 might be based on MR33317´s dual synergistic activity. We tested the hypothesis that MR33317 also stimulates 5-HT4-receptors in the heart. MR33317 (starting at 10 nM) increased force of contraction and beating rate in isolated atrial preparations from mice with cardiac confined overexpression of the human 5-HT4-serotonin receptor (5-HT4-TG) but was inactive in wild type mouse hearts (WT). Only in the presence of the phosphodiesterase III-inhibitor cilostamide, MR33317 raised force of contraction under isometric conditions in isolated paced (1 Hz) human right atrial preparations (HAP). This increase in force of contraction in human atrium by MR33317 was attenuated by 10 µM tropisetron or GR125487. These data suggest that MR33317 is an agonist at human 5-HT4-serotonin receptors in the human atrium. Clinically, one would predict that MR33317 may lead to atrial fibrillation.

Right atrial volumes and strains in healthy adults: is the Frank-Starling mechanism working?-detailed analysis from the three-dimensional speckle-tracking echocardiographic MAGYAR-Healthy Study.

With larger blood volume flowing into a cardiac chamber, by stretching muscle fibers, increased contraction force could be detected. This phenomenon is called Frank-Starling mechanism, allowing the output of a cardiac chamber to be synchronized without external regulation. The purpose of the present study was to investigate the Frank-Starling mechanism in the right atrium (RA) represented by its volumes, volume-based functional properties and strains respecting the cardiac cycle in healthy adults by three-dimensional (3D) speckle-tracking echocardiography (3DSTE). The present single center retrospective cohort study comprised 179 healthy adult volunteers (mean age: 33.2±12.0 years, 92 males), in whom complete two-dimensional Doppler echocardiography with 3DSTE was performed. Subjects were divided into 3 groups according to the mean value of maximum RA volume (Vmax) ± standard deviation: Vmax <30 mL, 30 mL ≤ Vmax <60 mL and Vmax ≥60 mL. All RA volumes respecting the cardiac cycle of all subjects and calculated separately for females and males and their indexed equivalents increased with Vmax. RA stroke volumes increased with Vmax regardless of the phase it was measured in. While total atrial emptying fraction representing the reservoir phase remained unchanged with the increase of Vmax, a significant increase in passive atrial emptying fraction representing the conduit phase could be detected, in case of Vmax >60 mL (28.9%±15.1% vs. 32.5%±12.6%, P<0.05). Active atrial emptying fraction representing the booster pump function did not change with the increase of Vmax. Most global and mean segmental peak RA strains did not show significant changes with increasing RA volumes except for the RA area strain, it was the largest when Vmax was larger than 60 mL (64.7%±44.9% vs. 83.3%±49.4%, P<0.05). RA circumferential, longitudinal and area strains at atrial contraction decreased with increasing Vmax, RA radial and 3D strains did not change significantly with increasing Vmax. Increasing RA volumes do not cause significant increase in RA contractility represented by strains, but reduction in strains in longitudinal and circumferential directions could be detected in end-diastolic booster pump function. In contrast to the left atrium, obvious signs of Frank-Starling mechanism could not be detected in case of the RA.

Proteome-wide systems genetics identifies UFMylation as a regulator of skeletal muscle function.

Improving muscle function has great potential to improve the quality of life. To identify novel regulators of skeletal muscle metabolism and function, we performed a proteomic analysis of gastrocnemius muscle from 73 genetically distinct inbred mouse strains, and integrated the data with previously acquired genomics and >300 molecular/phenotypic traits via quantitative trait loci mapping and correlation network analysis. These data identified thousands of associations between protein abundance and phenotypes and can be accessed online (https://muscle.coffeeprot.com/) to identify regulators of muscle function. We used this resource to prioritize targets for a functional genomic screen in human bioengineered skeletal muscle. This identified several negative regulators of muscle function including UFC1, an E2 ligase for protein UFMylation. We show UFMylation is up-regulated in a mouse model of amyotrophic lateral sclerosis, a disease that involves muscle atrophy. Furthermore, in vivo knockdown of UFMylation increased contraction force, implicating its role as a negative regulator of skeletal muscle function.

Effects of omecamtiv mecarbil and mavacamten in isolated human atrium

Heart failure is a syndrome that can result from impaired heart muscle contractions like in dilative cardiomyopathy but also from hypertrophic obstructive cardiomyopathy (HOCOM). A pharmacological therapy might lie in Ca2+-sensitizing or Ca2+-desensitizing drugs, respectively. Such drugs are thought to be omecamtiv mecarbil (OME) and mavacamten (MYK-461), respectively. Their function in contracting human muscle is not fully understood and was the focus of the present study. OME from 1 nM to 10 µM cumulatively applied failed to raise force of contraction in human right atrial preparations strips (HAP) or mouse left atrial preparations (LA). However, OME prolonged time to peak tension and time of relaxation in HAP and LA but did not alter the beating rate in right atrial preparations from mice (RA). In contrast, MYK-461 (10 nM to 10 µM) reduced concentration- and time-dependently force of contraction in HAP and LA. MYK-461 (10 µM) did not affect the beating rate in RA. In summary, the present data failed to detect an increase in force of contraction for OME, in human and mouse atrium. In contrast, a Ca2+ desensitizer studied for comparison was able to reduce force of contraction in HAP and LA. We conclude that putative beneficial effects of OME in dilated cardiomyopathy cannot be explained by positive inotropic effects in the HAP, whereas beneficial functional effects of MYK-461 in HOCOM can be explained by negative inotropic effects in HAP.

Long-Term Cultivation of Human Atrial Myocardium.

Organotypic culture of human ventricular myocardium is emerging in basic and translational cardiac research. However, few institutions have access to human ventricular tissue, whereas atrial tissue is more commonly available and important for studying atrial physiology. This study presents a method for long-term cultivation of beating human atrial myocardium. After written informed consent, tissues from the right-atrial appendage were obtained from patients with sinus rhythm undergoing open heart surgery with cardiopulmonary bypass. Trabeculae (pectinate muscles) prepared from the samples were installed into cultivation chambers at 37°C with a diastolic preload of 500 μN. After 2 days with 0.5 Hz pacing, stimulation frequency was set to 1 Hz. Contractile force was monitored continuously. Beta-adrenergic response, refractory period (RP) and maximum captured frequency (fmax) were assessed periodically. After cultivation, viability and electromechanical function were investigated, as well as the expression of several genes important for intracellular Ca2+ cycling and electrophysiology. Tissue microstructure was analyzed by confocal microscopy. We cultivated 19 constantly beating trabeculae from 8 patient samples for 12 days and 4 trabeculae from 3 specimen for 21 days. Functional parameters were compared directly after installation (0 d) with those after 12 d in culture. Contraction force was 384 ± 69 μN at 0 d and 255 ± 90 μN at 12 d (p = 0.8, n = 22), RP 480 ± 97 ms and 408 ± 78 ms (p = 0.3, n = 9), fmax 3.0 ± 0.5 Hz and 3.8 ± 0.5 Hz (p = 0.18, n = 9), respectively. Application of 100 nM isoprenaline to 11 trabeculae at 7 d increased contraction force from 168 ± 35 μN to 361 ± 60 μN (p < 0.01), fmax from 6.4 ± 0.6 Hz to 8.5 ± 0.4 Hz (p < 0.01) and lowered RP from 319 ± 22 ms to 223 ± 15 ms. CACNA1c (L-type Ca2+ channel subunit) and GJA1 (connexin-43) mRNA expressions were not significantly altered at 12 d vs 0 d, while ATP2A (SERCA) and KCNJ4 (Kir2.3) were downregulated, and KCNJ2 (Kir2.1) was upregulated. Simultaneous Ca2+ imaging and force recording showed preserved excitation-contraction coupling in cultivated trabeculae. Confocal microscopy indicated preserved cardiomyocyte structure, unaltered amounts of extracellular matrix and gap junctions. MTT assays confirmed viability at 12 d. We established a workflow that allows for stable cultivation and functional analysis of beating human atrial myocardium for up to 3 weeks. This method may lead to novel insights into the physiology and pathophysiology of human atrial myocardium.

Activation of synovial fibroblasts from patients at revision of their metal-on-metal total hip arthroplasty

BackgroundThe toxicity of released metallic particles generated in metal-on-metal (MoM) total hip arthroplasty (THA) using cobalt chromium (CoCr) has raised concerns regarding their safety amongst both surgeons and the public. Soft tissue changes such as pseudotumours and metallosis have been widely observed following the use of these implants, which release metallic by-products due to both wear and corrosion. Although activated fibroblasts, the dominant cell type in soft tissues, have been linked to many diseases, the role of synovial fibroblasts in the adverse reactions caused by CoCr implants remains unknown. To investigate the influence of implants manufactured from CoCr, the periprosthetic synovial tissues and synovial fibroblasts from patients with failed MoM THA, undergoing a revision operation, were analysed and compared with samples from patients undergoing a primary hip replacement, in order to elucidate histological and cellular changes.ResultsPeriprosthetic tissue from patients with MoM implants was characterized by marked fibrotic changes, notably an increase in collagen content from less than 20% to 45–55%, an increase in α-smooth muscle actin positive cells from 4 to 9% as well as immune cells infiltration. Primary cell culture results demonstrated that MoM synovial fibroblasts have a decreased apoptosis rate from 14 to 6% compared to control synovial fibroblasts. In addition, synovial fibroblasts from MoM patients retained higher contractility and increased responsiveness to chemotaxis in matrix contraction. Their mechanical properties at a single cell level increased as observed by a 60% increase in contraction force and higher cell stiffness (3.3 kPa in MoM vs 2.18 kPa in control), as measured by traction force microscopy and atomic force microscopy. Further, fibroblasts from MoM patients promoted immune cell invasion by secreting monocyte chemoattractant protein 1 (MCP-1, CCL2) and induced monocyte differentiation, which could also be associated with excess accumulation of synovial macrophages.ConclusionSynovial fibroblasts exposed in vivo to MoM THA implants that release CoCr wear debris displayed dramatic phenotypic alteration and functional changes. These findings unravelled an unexpected effect of the CoCr alloy and demonstrated an important role of synovial fibroblasts in the undesired tissue reactions caused by MoM THAs.

Hyponatraemia reversibly affects human myometrial contractility. An in vitro pilot study.

In a previous study we found a significant correlation between dystocia and hyponatraemia that developed during labour. The present study examined a possible causal relationship. In vitro studies often use area under the curve (AUC) determined by frequency and force of contractions as a measure of myometrial contractility. However, a phase portrait plot of isometric contraction, obtained by plotting the first derivate of contraction against force of contraction, could indicate that bi-or multiphasic contractions might be less effective compared to the smooth contractions. Myometrial biopsies were obtained from 17 women undergoing elective caesarean section at term. Each biopsy was divided into 8 strips and mounted isometrically in a force transducer. Seven biopsies were used in the first part of the study when half of the strips were immersed in the hyponatraemic study solution S containing Na+ 120 mmol/L and observed for 1 hour, followed by 1 hour in normonatraemic control solution C containing Na+ 136 mmol/L, then again in S for 1 hour, and finally 1 hour in C. The other half of the strips were studied in reverse order, C-S-C-S. The remaining ten biopsies were included in the second part of the study. Response to increasing doses of oxytocin (OT) in solutions S and C was studied. In the first part of the study we calculated AUC, and created phase portrait plots of two different contractions from the same strip, one smooth and one biphasic. In both parts of the study we registered frequency and force of contractions, and described appearance of the contractions. First part of the study: Mean (median) contractions per hour in C: 8.7 (7.6), in S 14,3 (13). Mean (SD) difference between groups 5.6 (4.2), p = 0.018. Force of contractions in C: 11.8 (10.2) mN, in S: 10.8 (9.2) mN, p = 0.09, AUC increased in S; p = 0.018. Bi-/multiphasic contractions increased from 8% in C to 18% in S, p = 0.001. All changes were reversible in C. Second part of the study: Frequency after OT 1.65 x 10-9 M in C:3.4 (2.9), in S: 3.8 (3.2), difference between groups: p = 0.48. After OT 1.65 x 10-7 M in C: 7.8 (8.9), increase from previous OT administration: p = 0.09, in S: 8.7 (9.0), p = 0.04, difference between groups, p = 0.32. Only at the highest dose of OT dose was there an increase in force of contraction in S, p = 0.05, difference between groups, p = 0.33. Initial response to OT was more frequently bi/multiphasic in S, reaching significance at the highest dose of OT(1.65 x 10-7 M), p = 0.015. when almost all contractions were bi/multiphasic. Hyponatraemia reversibly increased frequency of contractions and appearance of bi-or multiphasic contractions, that could reduce myometrial contractility. This could explain the correlation of hyponatraemia and instrumental delivery previously observed. Contractions in the hyponatraemic solution more frequently showed initial multiphasic contractions when OT was added in increasing doses. Longer lasting labours carry the risk both of hyponatraemia and OT administration, and their negative interaction could be significant. Further studies should address this possibility.

ІОННІ КАНАЛИ В КОНТЕКСТІ ПОШУКУ МОЛЕКУЛЯРНИХ МІШЕНЕЙ РЕГУЛЯЦІЇ СКОРОТЛИВОСТІ МІОМЕТРІЮ

Many women now have complications in childbirth due to poor labor, which often threatens both mother and fetus. Also,the problem of prevention and treatment of premature uterine contractions is unresolved. Therefore, in this work we investigated the influence of ion channels as the end stage effectors of the regulatory cascades in the contractility of myometrium. To better understand the participation of TRPC4, TRPV4 and BKCa ion channels in myometrial contractility, we conducted experiments, keeping in mind the fact that changes in ionic conductivity of the plasma membrane regulate spontaneous and agonist-induced contractions. On the myometrial preparations of pregnant rats usingisolated tissue tensiometry, the amplitude of contractile force was recorded under the activation of these ion channels by their selective agonists. Obtained results allow us to consider (-) – englerin A as a way to stimulate uterine contractions in case of insufficient response to oxytocin, because at a concentration of 1 nM a significant increase in contraction force was developed and did not differ statistically from the response to oxytocin or carbacholin. The use of an agonist at concentrations of 30-100 nM causes some suppression of contractility. Based on the results describing the role of TRPV4 channels, namely the reduction of uterine smooth muscle contractions in response to their selective agonist GSK1016790A administration, we suggest that the main effect of activation of these channels depends on the expression and activity of adjacent calcium-dependent potassium channels. Our experiments found that the use of the liposomal form of quercetin to activate BKCa channels inhibits the excitability of myometrial cells more effectively than that dissolved in DMSO, which is promising for the correction of premature or excessive uterine activity.

Contractile deficits in engineered cardiac microtissues as a result of MYBPC3 deficiency and mechanical overload

The integration of in vitro cardiac tissue models, human induced pluripotent stem cells (hiPSCs) and genome-editing tools allows for the enhanced interrogation of physiological phenotypes and the recapitulation of disease pathologies. Here, in a cardiac tissue model consisting of filamentous 3D matrices populated with cardiomyocytes (CMs) derived from healthy wild-type hiPSCs (WT hiPSC-CMs) or from isogenic hiPSCs deficient in the sarcomere protein cardiac myosin binding protein C (MYBPC3−/− hiPSC-CMs), we show that the WT microtissues adapted to the mechanical environment with increased contraction force commensurate to matrix stiffness, whereas the MYBPC3−/− microtissues exhibited impaired force-development kinetics regardless of matrix stiffness and deficient contraction force only when grown on matrices with high fiber stiffness. Under mechanical overload, the MYBPC3−/− microtissues had a higher degree of calcium transient abnormalities, and exhibited an accelerated decay of calcium dynamics as well as calcium desensitization, which accelerated when contracting against stiffer fibers. Our findings suggest that MYBPC3 deficiency and the presence of environmental stresses synergistically lead to contractile deficits in the cardiac tissues.

Steps toward Maturation of Embryonic Stem Cell-Derived Cardiomyocytes by Defined Physical Signals.

SummaryCardiovascular disease remains a leading cause of mortality and morbidity worldwide. Embryonic stem cell-derived cardiomyocytes (ESC-CMs) may offer significant advances in creating in vitro cardiac tissues for disease modeling, drug testing, and elucidating developmental processes; however, the induction of ESCs to a more adult-like CM phenotype remains challenging. In this study, we developed a bioreactor system to employ pulsatile flow (1.48 mL/min), cyclic strain (5%), and extended culture time to improve the maturation of murine and human ESC-CMs. Dynamically-cultured ESC-CMs showed an increased expression of cardiac-associated proteins and genes, cardiac ion channel genes, as well as increased SERCA activity and a Raman fingerprint with the presence of maturation-associated peaks similar to primary CMs. We present a bioreactor platform that can serve as a foundation for the development of human-based cardiac in vitro models to verify drug candidates, and facilitates the study of cardiovascular development and disease.

Ingestion of transient receptor potential channel agonists attenuates exercise-induced muscle cramps.

Exercise-associated muscle cramping (EAMC) is a poorly understood problem that is neuromuscular in origin. Ingestion of transient receptor potential (TRP) channel agonists has been efficacious in attenuating electrically induced muscle cramps. This study examines the effect of TRP agonist ingestion on voluntarily induced EAMC and motor function. Study 1: Thirty-nine participants completed 2 trials after ingesting TRP agonist-containing active treatment (A), or vehicle (V) control. Cramping in the triceps surae muscle was induced via voluntary isometric contraction. Study 2: After ingesting A or V, 31 participants performed kinematic and psychomotor tests of manual dexterity. A increased precramp contraction duration (A, 36.9 ± 4.1 s; V, 27.8 ± 3.1 s), decreased cramp EMG area under the curve (A, 37.3 ± 7.7 %EMGmax ·s; V, 77.2 ± 17.7 %EMGmax ·s), increased contraction force to produce the cramp (A, 13.8 ± 1.8 kg; V, 9.9 ± 1.6 kg), and decreased postcramp soreness (A, 4.1 ± 0.3 arbitrary units (a.u.); V, 4.7 ± 0.3 a.u.). Kinematic and psychomotor tests were not affected. TRP agonist ingestion attenuated EAMC characteristics without affecting motor function. Muscle Nerve 56: 379-385, 2017.

Mathematical model for β1-adrenergic regulation of the mouse ventricular myocyte contraction.

The β1-adrenergic regulation of cardiac myocyte contraction plays an important role in regulating heart function. Activation of this system leads to an increased heart rate and stronger myocyte contraction. However, chronic stimulation of the β1-adrenergic signaling system can lead to cardiac hypertrophy and heart failure. To understand the mechanisms of action of β1-adrenoceptors, a mathematical model of cardiac myocyte contraction that includes the β1-adrenergic system was developed and studied. The model was able to simulate major experimental protocols for measurements of steady-state force-calcium relationships, cross-bridge release rate and force development rate, force-velocity relationship, and force redevelopment rate. It also reproduced quite well frequency and isoproterenol dependencies for intracellular Ca2+ concentration ([Ca2+]i) transients, total contraction force, and sarcomere shortening. The mathematical model suggested the mechanisms of increased contraction force and myocyte shortening on stimulation of β1-adrenergic receptors is due to phosphorylation of troponin I and myosin-binding protein C and increased [Ca2+]i transient resulting from activation of the β1-adrenergic signaling system. The model was used to simulate work-loop contractions and estimate the power during the cardiac cycle as well as the effects of 4-aminopyridine and tedisamil on the myocyte contraction. The developed mathematical model can be used further for simulations of contraction of ventricular myocytes from genetically modified mice and myocytes from mice with chronic cardiac diseases.NEW & NOTEWORTHY A new mathematical model of mouse ventricular myocyte contraction that includes the β1-adrenergic system was developed. The model simulated major experimental protocols for myocyte contraction and predicted the effects of 4-aminopyridine and tedisamil on the myocyte contraction. The model also allowed for simulations of work-loop contractions and estimation of the power during the cardiac cycle.

Skeletal muscle: from birth to old age, routes to mechanical and metabolic failure.

Skeletal muscle (SM) is the most abundant tissue and the largest reservoir of protein in the body. It transports glucose in an insulin dependent manner by the glucose transporter type 4 (GLUT4) and contributes in the maintenance of serum amino acids concentration. By its mass and energetic requirements, it is fundamental for the systemic metabolic balance. In the present work, we present the effect of gestational undernourishment (GU) on the mechanical and metabolic properties of SM at birth and in old age in an animal model. Mechanical studies were performed on isolated muscles, while the GLUT4, amino acid transporters LAT2, SNAT2 and insulin receptors (IR) determination were performed on isolated transverse-tubule membranes (TT). The GU in offspring at birth, results in low muscle mass with increased contraction force and resistance to fatigue. However, in two-years old rats, there was muscle hypotrophy and sarcopenia, the force decreased between 50 and 70% in control rats and rats with GU respectively, accompanied by a lower expression of LAT2, SNAT2 and IR in TT. In conclusion, GU irreversibly affects the SM, an effect that could be similar in humans, which help us to understand the events that associate the GU with the metabolic debacle of SM and the metabolic diseases of human adulthood.

Dynamic culture yields engineered myocardium with near-adult functional output.

Engineered cardiac tissues hold promise for cell therapy and drug development, but exhibit inadequate function and maturity. In this study, we sought to significantly improve the function and maturation of rat and human engineered cardiac tissues. We developed dynamic, free-floating culture conditions for engineering "cardiobundles", 3-dimensional cylindrical tissues made from neonatal rat cardiomyocytes or human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) embedded in fibrin-based hydrogel. Compared to static culture, 2-week dynamic culture of neonatal rat cardiobundles significantly increased expression of sarcomeric proteins, cardiomyocyte size (∼2.1-fold), contractile force (∼3.5-fold), and conduction velocity of action potentials (∼1.4-fold). The average contractile force per cross-sectional area (59.7mN/mm2) and conduction velocity (52.5cm/s) matched or approached those of adult rat myocardium, respectively. The inferior function of statically cultured cardiobundles was rescued by transfer to dynamic conditions, which was accompanied by an increase in mTORC1 activity and decline in AMPK phosphorylation and was blocked by rapamycin. Furthermore, dynamic culture effects did not stimulate ERK1/2 pathway and were insensitive to blockers of mechanosensitive channels, suggesting increased nutrient availability rather than mechanical stimulation as the upstream activator of mTORC1. Direct comparison with phenylephrine treatment confirmed that dynamic culture promoted physiological cardiomyocyte growth rather than pathological hypertrophy. Optimized dynamic culture conditions also augmented function of human cardiobundles made reproducibly from cardiomyocytes derived from multiple hPSC lines, resulting in significantly increased contraction force (∼2.5-fold) and conduction velocity (∼1.4-fold). The average specific force of 23.2mN/mm2 and conduction velocity of 25.8cm/s approached the functional metrics of adult human myocardium. In conclusion, we have developed a versatile methodology for engineering cardiac tissues with a near-adult functional output without the need for exogenous electrical or mechanical stimulation, and have identified mTOR signaling as an important mechanism for advancing tissue maturation and function invitro.

Surface-patterned SU-8 cantilever arrays for preliminary screening of cardiac toxicity

Arrays of a μgrooved SU-8 cantilever were utilized to analyze changes in the contraction force and beating frequency of cardiomyocytes in vitro. The longitudinally patterned μgrooves facilitates alignment of cardiomyocytes on top of the SU-8 cantilever, which increases the contraction force of cardiomyocytes by a factor of about 2.5. The bending displacement of the SU-8 cantilever was precisely measured in nanoscale using a laser-based measurement system combined with a motorized xyz stage. The cantilever displacement due to contraction of the cardiomyocytes showed the maximum on day 8 after their cultivation. Following preliminary experiments, Isoproterenol, Verapamil, and Astemizole were used to investigate the effect of drug toxicity on the physiology of cardiomyocytes. The experimental results indicated that 1µM of Isoproterenol treatment increased contraction force and beating frequencies of cardiomyocytes by 30% and 200%, respectively, whereas 500nM of Verapamil treatment decreased contraction force and beating frequencies of cardiomyocytes by 56% and 42%, respectively. A concentration of less than 5nM of the hERG channel suppression drug Astemizole did not change the contraction forces in the displacement but slightly decreased the beating frequencies. However, irregular or abnormal heartbeats were observed at Astemizole concentrations of 5nM and higher. We experimentally conformed that the proposed SU-8 cantilever arrays combined with the laser-based measurement systems has the great potential for a high-throughput drug toxicity screening system in future.

Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure

One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation.

A novel collagen gel-based measurement technique for quantitation of cell contraction force.

Cell contraction force plays an important role in wound healing, inflammation,angiogenesis and metastasis. This study describes a novel method to quantify single cell contraction force in vitro using human aortic adventitial fibroblasts embedded in a collagen gel. The technique is based on a depth sensing nano-indentation tester to measure the thickness and elasticity of collagen gels containing stimulated fibroblasts and a microscopy imaging system to estimate the gel area. In parallel, a simple theoretical model has been developed to calculate cell contraction force based on the measured parameters. Histamine (100 mM) was used to stimulate fibroblast contraction while the myosin light chain kinase inhibitor ML-7 (25 mM) was used to inhibit cell contraction. The collagen matrix used in the model provides a physiological environment for fibroblast contraction studies. Measurement of changes in collagen gel elasticity and thickness arising from histamine treatments provides a novel convenient technique to measure cell contraction force within a collagen matrix. This study demonstrates that histamine can elicit a significant increase in contraction force of fibroblasts embedded in collagen,while the Young's modulus of the gel decreases due to the gel degradation.

METABOLISM OF SEROTONIN IN MOUSE AND HUMAN HEART

Serotonin (5‐HT) can exert cardiac effects like increase in force of contraction and increase in beating rate. It is generally assumed that 5‐HT is generated in non‐cardiac tissue and reaches the heart via the blood. However, we hypothesized that 5‐HT might be produced within the heart. Hence, we studied the effect of the direct precursor of 5‐HT, that is 5‐hydroxytryptophane. Indeed, 5‐hydroxytryptophane (1‐100 µM) exerted a concentration dependent increase in force of contraction and in beating rate in isolated left and right atrial preparations of transgenic mice overexpressing the human 5‐HT4‐receptor. But 5‐hydroxytryptophane was 30 to 100 fold less potent than 5‐HT. The effect could be blocked by pre‐incubation with an inhibitor (100 µM NSD 1015) of the pacemaker enzyme for formation of 5‐HT, that is aromatic L‐amino acid decarboxylase. Moreover, in isolated electrically driven (1 Hz) trabeculae (n=10) from five patients (undergoing bypass surgery), a positive inotropic effect to 100 µM 5‐hydroxytryptophane was noted which could be completely abolished by a 5‐HT4‐receptor antagonist (GR113808). These data combined argue for the possibility that intracardiac formation of serotonin can occur and might be relevant in health and disease.

Functional maturation of human pluripotent stem cell derived cardiomyocytes in vitro – Correlation between contraction force and electrophysiology

Cardiomyocytes from human pluripotent stem cells (hPSC-CM) have many potential applications in disease modelling and drug target discovery but their phenotypic similarity to early fetal stages of cardiac development limits their applicability. In this study we compared contraction stresses of hPSC-CM to 2nd trimester human fetal derived cardiomyocytes (hFetal-CM) by imaging displacement of fluorescent beads by single contracting hPSC-CM, aligned by microcontact-printing on polyacrylamide gels. hPSC-CM showed distinctly lower contraction stress than cardiomyocytes isolated from hFetal-CM. To improve maturation of hPSC-CM in vitro we made use of commercial media optimized for cardiomyocyte maturation, which promoted significantly higher contraction stress in hPSC-compared with hFetal-CM. Accordingly, other features of cardiomyocyte maturation were observed, most strikingly increased upstroke velocities and action potential amplitudes, lower resting membrane potentials, improved sarcomeric organization and alterations in cardiac-specific gene expression. Performing contraction force and electrophysiology measurements on individual cardiomyocytes revealed strong correlations between an increase in contraction force and a rise of the upstroke velocity and action potential amplitude and with a decrease in the resting membrane potential.We showed that under standard differentiation conditions hPSC-CM display lower contractile force than primary hFetal-CM and identified conditions under which a commercially available culture medium could induce molecular, morphological and functional maturation of hPSC-CM in vitro. These results are an important contribution for full implementation of hPSC-CM in cardiac disease modelling and drug discovery.

Cystathionine gamma-lyase of perivascular adipose tissue with reversed regulatory effect in diabetic rat artery

The aim of this study is to reveal the regulatory role of cystathionine gamma-lyase (CSE), the main source of hydrogen sulphide (H2S) in perivascular adipose tissue (PVAT), of diabetic rats. Diabetes was induced in male rats by a single intraperitoneal injection of streptozotocin. Animals with glucose levels above 20 mmol/L were determined as diabetic. The rat gracilis arteries (a. gracilis) were dissected with or without PVAT. In all in vitro experiments endothelium-denuded preparations were used for isometric contraction measurements. Increasing concentrations of 5-hydroxytryptamine (5-HT) from 10−10 to 10−5 mol/L were applied to induce gradual increase in force of contractions of circular artery segments. The relaxing effect of CSE was inhibited by DL-propargyl glycine (PGG). The presence of PVAT decreases the contractile response to 5-HT of a. gracilis from control rats. This response is reversed in contraction studies in the same rat artery from diabetic rats. DL-PPG (1 mmol/L) induced significant increase of the force of contraction in artery preparations with PVAT from control rats in the whole range of 5-HT. In contrast, PGG had a relaxing effect in high concentrations of 5-HT (10−6 and 10−5 mol/L) in diabetic rat arteries with PVAT. It is concluded that in skeletal muscle artery from diabetic rats, a mediator related to H2S is released from PVAT. This paracrine mediator increases the maximal force of contraction of endothelium-denuded preparations at higher concentrations of 5-HT.