Isolated Muscle Preparations Research Articles

A novel and unique refraction-based optical recording system for pharmacological investigations on isolated muscle preparations

In the field of neuroscience and ecotoxicology, there is a great need for investigating the effect(s) of a variety of different chemicals (e.g., pharmacologically active compounds, pesticides, neurotransmitters, modulators) at different biological levels. Different contractile tissue preparations have provided excellent model systems for in vitro pharmacological experiments for a long time. However, such investigations usually apply mechanical force transducer-based approaches. Thus, a rapid, easy, cheap, digital, and reproducible in vitro pharmacological method based on an effective, ‘non-invasive’ (compared to the force-transducer approaches), refraction-based optical recording approach and isolated heart preparations was developed.•A versatile and unique refraction-based optical recording system with a Java application was developed.•The recording system was tested and validated on isolated heart preparations obtained from the widely used invertebrate model organism, the great pond snail (Lymnaea stagnalis).•The recording system illustrates the progression of technology from the mechanical force transducer system and can represent a suitable tool in ecotoxicology or neuroscience.

Effect of hypothyroidism on contractile performance of isolated end-stage failing human myocardium.

The relationship between hypothyroidism and the occurrence and progression of heart failure (HF) has had increased interest over the past years. The low T3 syndrome, a reduced T3 in the presence of normal thyroid stimulating hormone (TSH), and free T4 concentration, is a strong predictor of all-cause mortality in HF patients. Still, the impact of hypothyroidism on the contractile properties of failing human myocardium is unknown. Our study aimed to investigate that impact using ex-vivo assessment of force and kinetics of contraction/relaxation in left ventricular intact human myocardial muscle preparations. Trabeculae were dissected from non-failing (NF; n = 9), failing with no hypothyroidism (FNH; n = 9), and failing with hypothyroidism (FH; n = 9) hearts. Isolated muscle preparations were transferred into a custom-made setup where baseline conditions as well as the three main physiological modulators that regulate the contractile strength, length-dependent and frequency-dependent activation, as well as β-adrenergic stimulation, were assessed under near-physiological conditions. Hypothyroidism did not show any additional significant impact on the contractile properties different from the recognized alterations usually detected in such parameters in any end-stage failing heart without thyroid dysfunction. Clinical information for FH patients in our study revealed they were all receiving levothyroxine. Absence of any difference between failing hearts with or without hypothyroidism, may possibly be due to the profound effects of the advanced stage of heart failure that concealed any changes between the groups. Still, we cannot exclude the possibility of differences that may have been present at earlier stages. The effects of THs supplementation such as levothyroxine on contractile force and kinetic parameters of failing human myocardium require further investigation to explore its full potential in improving cardiovascular performance and cardiovascular outcomes of HF associated with hypothyroidism.

Understanding the Mechanism Underlie the Antidiabetic Activity of Oleuropein Using Ex-Vivo Approach.

Oleuropein, the main constituent of olive fruit and leaves, has been reported to protect against insulin resistance and diabetes. While many experimental investigations have examined the mechanisms by which oleuropein improves insulin resistance and diabetes, much of these investigations have been carried out in either muscle cell lines or in vivo models two scenarios with many drawbacks. Accordingly, to simplify identification of mechanisms by which oleuropein regulates specific cellular processes, we resort, in the present study, to isolated muscle preparation which enables better metabolic milieu control and permit more detailed analyses. For this purpose, soleus muscles were incubated for 12 h without or with palmitate (1.5 mM) in the presence or absence of oleuropein (1.5 mM), and compound C. Insulin-stimulated glucose transport, glucose transporter type 4 (GLUT4) translocation, Akt substrate of 160 kDa (AS160) phosphorylation and adenosine monophosphate-activated protein kinase (AMPK) phosphorylation were examined. Palmitate treatment reduced insulin-stimulated glucose transport, GLUT4 translocation and AS160 phosphorylation, but AMPK phosphorylation was not changed. Oleuropein administration (12 h) fully rescued insulin-stimulated glucose transport, but partially restored GLUT4 translocation. However, it fully restored AS160 phosphorylation, raising the possibility that oleuropein may also have contributed to the restoration of glucose transport by increased GLUT4 intrinsic activity. Inhibition of AMPK phosphorylation with compound C (50 µM) prevented oleuropein -induced improvements in insulin-stimulated glucose transport, GLUT4 translocation, and AS160 phosphorylation. Our results clearly indicate that oleuropein alleviates palmitate-induced insulin resistance appears to occur via an AMPK-dependent mechanism involving improvements in the functionality of the AS160-GLUT4 signaling system.

Increased force following muscle stretching and simultaneous fibre shortening: Residual force enhancement or force depression – That is the question?

Residual force enhancement (rFE) describes the increase in isometric force following muscle stretching compared to the corresponding isometric force. Even though rFE is consistently observed in isolated muscle preparations, it is not always observed in human skeletal muscle. This inconsistency might be associated with disociations between length changes in muscle tendon units (MTUs) and fibres. This prompted the question if there is rFE for conditions where the MTU is stretched while fibres shorten. Rabbit tibialis anterior (TA) MTUs (n = 4) were stretched and the isometric forces following stretching were compared to corresponding forces from isometric reference contractions. Unique combinations of stretch speed and activation were used to create conditions of continuous fibre shortening during MTU stretch.Mean force was increased (18 ± 2%) following MTU stretching compared to the isometric reference forces. Without fibre length measurements, this result would be referred to as rFE. However, fibre shortening in the reference contractions was always greater than for the eccentric stretch contractions, suggesting that the observed increase in force might be caused by less residual force depression (rFD) in the stretch tests compared to the reference contractions. However, the work performed by fibre shortening was similar between the reference and the MTU stretch contractions, suggesting that rFD was similar for both experimental conditions. Therefore, we conclude that we observed rFE in the absence of contractile element stretching. However, a lack of knowledge of the molecular mechanisms that distinguish rFE from rFD prevents an unequivocal pronouncement of what caused the enhanced forces after active muscle stretching.

Effect of acute nitrite infusion on contractile economy and metabolism in isolated skeletal muscle in situ during hypoxia.

Increased plasma nitrite concentrations may have beneficial effects on skeletal muscle function. The physiological basis explaining these observations has not been clearly defined and it may involve positive effects on muscle contraction force, microvascular O2 delivery and skeletal muscle oxidative metabolism. In the isolated canine gastrocnemius model, we evaluated the effects of acute nitrite infusion on muscle force and skeletal muscle oxidative metabolism. Under hypoxic conditions, but in the presence of normal convective O2 delivery, an elevated plasma nitrite concentration affects neither muscle force, nor muscle contractile economy. In accordance with previous results suggesting limited or no effects of nitrate/nitrite administrations in highly oxidative and highly perfused muscle, our data suggest that neither mitochondrial respiration, nor muscle force generation are affected by acute increased concentrations of NO precursors in hypoxia. Contrasting findings have been reported concerning the effects of augmented nitric oxide (NO) on skeletal muscle force production and oxygen consumption ( ). The present study examined skeletal muscle mitochondrial respiration and contractile economy in an isolated muscle preparation during hypoxia (but normal convective O2 delivery) with nitrite infusion. Isolated canine gastrocnemius muscles in situ (n=8) were studied during 3min of electrically stimulated isometric tetanic contractions corresponding to ∼35% of . During contractions, sodium nitrite (NITRITE) or sodium chloride (SALINE) was infused into the popliteal artery. was calculated from the Fick principle. Experiments were carried out in hypoxia ( =0.12), whereas convective O2 delivery was maintained at normal levels under both conditions by pump-driven blood flow ( ). Muscle biopsies were taken and mitochondrial respiration was evaluated by respirometry. Nitrite infusion significantly increased both nitrite and nitrate concentrations in plasma. No differences in force were observed between conditions. was not significantly different between NITRITE (6.1±1.8mL 100g-1 min-1 ) and SALINE (6.2±1.8mL100g-1 min-1 ), even after being 'normalized' per unit of developed force (muscle contractile economy). No differences between conditions were found for maximal ADP-stimulated mitochondrial respiration (both for complex I and complex II), leak respiration and oxidative phosphorylation coupling. In conclusion, in the absence of changes in convective O2 delivery, muscle force, muscle contractile economy and mitochondrial respiration were not affected by acute infusion of nitrite. The previously reported positive effects of elevated plasma nitrite concentrations are presumably mediated by the increased microvascular O2 availability.

Metabolic Cost of Activation and Mechanical Efficiency of Mouse Soleus Muscle Fiber Bundles During Repetitive Concentric and Eccentric Contractions.

Currently available data on the energetics of isolated muscle preparations are based on bouts of less than 10 muscle contractions, whereas metabolic energy consumption is mostly relevant during steady state tasks such as locomotion. In this study we quantified the energetics of small fiber bundles of mouse soleus muscle during prolonged (2 min) series of contractions. Bundles (N = 9) were subjected to sinusoidal length changes, while measuring force and oxygen consumption. Stimulation (five pulses at 100 Hz) occurred either during shortening or during lengthening. Movement frequency (2–3 Hz) and amplitude (0.25–0.50 mm; corresponding to ± 4–8% muscle fiber strain) were close to that reported for mouse soleus muscle during locomotion. The experiments were performed at 32°C. The contributions of cross-bridge cycling and muscle activation to total metabolic energy expenditure were separated using blebbistatin. The mechanical work per contraction cycle decreased sharply during the first 10 cycles, emphasizing the importance of prolonged series of contractions. The mean ± SD fraction of metabolic energy required for activation was 0.37 ± 0.07 and 0.56 ± 0.17 for concentric and eccentric contractions, respectively (both 0.25 mm, 2 Hz). The mechanical efficiency during concentric contractions increased with contraction velocity from 0.12 ± 0.03 (0.25 mm 2 Hz) to 0.15 ± 0.03 (0.25 mm, 3 Hz) and 0.16 ± 0.02 (0.50 mm, 2 Hz) and was -0.22 ± 0.08 during eccentric contractions (0.25 mm, 2 Hz). The percentage of type I fibers correlated positively with mechanical efficiency during concentric contractions, but did not correlate with the fraction of metabolic energy required for activation.

The effects of obesity on skeletal muscle contractile function.

Obesity can cause a decline in contractile function of skeletal muscle, thereby reducing mobility and promoting obesity-associated health risks. We reviewed the literature to establish the current state-of-knowledge of how obesity affects skeletal muscle contraction and relaxation. At a cellular level, the dominant effects of obesity are disrupted calcium signalling and 5'-adenosine monophosphate-activated protein kinase (AMPK) activity. As a result, there is a shift from slow to fast muscle fibre types. Decreased AMPK activity promotes the class II histone deacetylase (HDAC)-mediated inhibition of the myocyte enhancer factor 2 (MEF2). MEF2 promotes slow fibre type expression, and its activity is stimulated by the calcium-dependent phosphatase calcineurin. Obesity-induced attenuation of calcium signalling via its effects on calcineurin, as well as on adiponectin and actinin affects excitation-contraction coupling and excitation-transcription coupling in the myocyte. These molecular changes affect muscle contractile function and phenotype, and thereby in vivo and in vitro muscle performance. In vivo, obesity can increase the absolute force and power produced by increasing the demand on weight-supporting muscle. However, when normalised to body mass, muscle performance of obese individuals is reduced. Isolated muscle preparations show that obesity often leads to a decrease in force produced per muscle cross-sectional area, and power produced per muscle mass. Obesity and ageing have similar physiological consequences. The synergistic effects of obesity and ageing on muscle function may exacerbate morbidity and mortality. Important future research directions include determining: the relationship between time course of weight gain and changes in muscle function; the relative effects of weight gain and high-fat diet feeding per se; the effects of obesity on muscle function during ageing; and if the effects of obesity on muscle function are reversible.

Stretching the limits of maximal voluntary eccentric force production in vivo

During eccentric contractions, muscular force production capacity is enhanced compared to isometric contractions. Although this is well accepted in terms of muscle mechanics, maximal voluntary eccentric contractions are associated with neural inhibition that prevents increased force production of in vivo human muscles. However, because it was shown that maximal voluntary eccentric forces can exceed maximum isometric forces by a factor of 1.2–1.4, this review focuses on the question of whether the absent eccentric force enhancement, as observed in many studies, can unambiguously be attributed to an inherent neural inhibition. First, we demonstrate that participant familiarization, preload, and fascicle behavior are crucial factors influencing maximal voluntary eccentric force production. Second, we show that muscle mechanics such as muscle length, lengthening velocity, and stretch amplitude interact when it comes to maximal voluntary eccentric force production. Finally, we discuss the diverging findings on neural inhibition during maximal voluntary eccentric contractions. Because there was no inhibition of the major motor pathways in the presence of enhanced maximal voluntary eccentric forces, further research is needed to test the concept of neural inhibition and to understand why maximal voluntary force production is reduced compared to the force capacity of isolated muscle preparations.

Design and Validation of New Data Acquisition System for Isolated Muscle Experiments: Application to Pharmacology Teaching and Research

In this paper we describe a new data acquisition (DAQ) system for pharmacology experiments involving isolated muscle contraction. The system is composed of hardware acquisition unit and computer software for analysis of signals representing muscle contractions. As compared to mechanoelectrical DAQ systems of other constructions, the advantages of the new system include a high sensitivity (in the order of 1 µN), a wide range of forces recorded (1 µN to 100 mN), linearity throughout the whole range, easy assembly, low costs, long-term calibration stability, good dynamic characteristics and full control graphical interface. The device can be readily adjusted to actual experimental conditions and a variety of muscle preparations. Experimental validation of our DAQ inside the Clinical Pharmacology Department at Mansoura University Faculty of Medicine showed that it can record contractions of isolated muscle preparations with good precision and accuracy. Accordingly, the DAQ system design presented herein with its dedicated software is largely suitable for isolated muscle experiments in both teaching and research in biomedical fields.

О физиологических механизмах действия секретов слюнных желез клещей Argas persicus Oken, 1818 (Argasidae, Argas)

The anticoagulant effect of salivary secretion of ticks Argas persicus was studied. The experiments were conducted on aorta preparations isolated from the chest of white outbread rats. Isometric contraction in an isolated muscle preparation was registered by mechanotron. To determine the anticoagulant mechanism of blood plasma at deficiency of factors X, XI, IX and VIII we used the activated partial thromboplastin time. It was determined that the process of blood coagulation in rats slows down. The coagulation values depend on the concentration of tested secretions. It was found that at concentration 150 mkg /ml, the blood coagulation rate fell to 78,4 %. The role of calcium ions in regulation of vascular smooth muscle cells (VSMCs) contraction was determined. The components of salivary glands A. Persicus have a relaxation effect on the contraction of rat aortic ring preparation.

Calcium Sensitivity after Active Shortening in Rabbit Psoas Fibres

Introduction It has been shown that isometric force after active shortening of muscles is lower than the purely isometric force performed at the corresponding lengths. This property is termed force depression. Force depression has been observed in isolated muscle preparations and muscles in situ and in vivo (Rassier et al., J Applied Physiol 2004). The origin of force depression is still unknown. It has been suggested that active shortening may affect calcium sensitivity by changing calcium-troponin binding equilibrium leading to decreased force after active shortening (Ekelund and Edman, Acta Physiol Scand 1982). Our aim in the present study was to test whether calcium sensitivity was decreased after active shortening. Methods Experiments were performed using skinned fibres (n=7) isolated from rabbit psoas muscle. Calcium sensitivity was characterized by establishing the force-pCa curves for isometric reference contractions performed at an average sarcomere length of 2.4 µm and active shortening contractions from an average sarcomere length of 3.0 µm to an average sarcomere length of 2.4 µm. pCa50 and the coefficient of cooperativity (nH) were compared between reference and active shortening contractions. Results and Discussion pCa50 for the reference contractions was 5.99±0.02. No change in pCa50 was observed in the force depressed state (6.00±0.01). Furthermore, the coefficient of cooperativity did not change after active shortening (3.9±0.3 versus 4.3±0.3 for the reference and force depressed states respectively). These results suggest that calcium sensitivity does not decrease after active shortening and that the binding interactions between troponin and calcium are not altered in the force depressed state.

Alternative Approach for Studying Contractility of the Isolated Muscle Preparations in Real-Time Mode

The real-time control over the observed parameters is known to be necessary during the experiments with biological objects. The use of approaches, such as real-time operating systems, often requires significant material or human resources (operating time, the special administration and programming skills and etc.). In this paper we propose an alternative approach for the implementation of real-time mode in the experiments on muscle preparations (papillary muscles, trabeculae, walls of vessels) based on an external input-output cards with built-in analog-to-digital/digital-to-analog converters and signal processor. As a result, we developed a hardware-software complex for studying the mechanical properties of biological materials (muscles) in real-time mode. This solution has a convenient and easy-to-use user interface, and requires a minimum of computing resources and minimal cost of electricity.

Let's get real about the regulation of TBC1D1 and TBC1D4 phosphorylation in skeletal muscle

Let's get real about the regulation of TBC1D1 and TBC1D4 phosphorylation in skeletal muscle

An active learning mammalian skeletal muscle lab demonstrating contractile and kinetic properties of fast- and slow-twitch muscle

The fact that humans possess fast- and slow-twitch muscle in the ratio of ∼50% has profound implications for designing exercise training strategies for power and endurance activities. With the growth of exercise and sport science courses, we have seen the need to develop an undergraduate student laboratory that demonstrates the basic properties of fast- and slow-twitch mammalian skeletal muscle. This laboratory illustrates the major differences in contractile properties and fatigue profiles exhibited by the two muscle types. Students compare and contrast twitch kinetics, fused tetanus characteristics, force-frequency relationships, and fatigue properties of fast- and slow-twitch muscles. Examples of results collected by students during class are used to illustrate the type of data collected and analysis performed. During the laboratory, students are encouraged to connect factual information from their skeletal muscle lectures to their laboratory findings. This enables student learning in an active fashion; in particular, the isolated muscle preparation demonstrates that much of what makes muscle fast or slow is myogenic and not the product of the nervous or circulatory systems. This has far-reaching implications for motor control and exercise behavior and therefore is a crucial element in exercise science, with its focus on power and endurance sport activities. To measure student satisfaction with this active learning technique, a questionnaire was administered after the laboratory; 96% of the comments were positive in their support of active versus passive learning strategies.

Muscle fascicle strains in human gastrocnemius during backward downhill walking

Extensive muscle damage can be induced in isolated muscle preparations by performing a small number of stretches during muscle activation. While typically these fiber strains are large and occur over long lengths, the extent of exercise-induced muscle damage (EIMD) observed in humans is normally less even when multiple high-force lengthening actions are performed. This apparent discrepancy may be due to differences in muscle fiber and tendon dynamics in vivo; however, muscle and tendon strains have not been quantified during muscle-damaging exercise in humans. Ultrasound and an infrared motion analysis system were used to measure medial gastrocnemius fascicle length and lower limb kinematics while humans walked backward, downhill for 1 h (inducing muscle damage), and while they walked briefly forward on the flat (inducing no damage). Supramaximal tibial nerve stimulation, ultrasound, and an isokinetic dynamometer were used to quantify the fascicle length-torque relationship pre- and 2 h postexercise. Torque decreased ~23%, and optimal fascicle length shifted rightward ~10%, indicating that EIMD occurred during the damage protocol even though medial gastrocnemius fascicle stretch amplitude was relatively small (~18% of optimal fascicle length) and occurred predominantly within the ascending limb and plateau region of the length-torque curve. Furthermore, tendon contribution to overall muscle-tendon unit stretch was ~91%. The data suggest the compliant tendon plays a role in attenuating muscle fascicle strain during backward walking in humans, thus minimizing the extent of EIMD. As such, in situ or in vitro mechanisms of muscle damage may not be applicable to EIMD of the human gastrocnemius muscle.

Sex differences in myocardial metabolism and cardiac function: an emerging concept

There is substantial evidence that there are dramatic sex-related differences in the incidence of cardiovascular disease, apparently related to the presence of steroid hormones. This is supported by the discovery of steroid hormone receptors in the heart and vasculature. More controversial is the area of sex-related differences in cardiac metabolism and function. A number of human and animal studies have demonstrated that estrogen and testosterone have cardiac metabolic effects. Additionally, research shows females have higher heart rates and various indices of function, including cardiac output and stroke volume, compared with males. However, some controversy exists, as other studies report that function in isolated muscle preparations is lower in females versus males. The reasons for these differences may reflect effects of sex hormones that are dependent on the conditions being studied. Cardiac function is reduced in postmenopausal females, suggesting that female sex hormones, specifically estrogen and progesterone, influence cardiac function. Apart from its well-documented vasodilatory effects, estrogen has also been shown to have negative inotropic effects and to reduce Ca(2+) transients in cardiomyocytes. Similar results have been found for progesterone. Several studies show that testosterone administration appears to increase cardiac performance, while others show that it increases the stiffness of the ventricle due to increased collagen synthesis, thereby reducing diastolic performance. This review will discuss current evidence suggesting sex-related differences in cardiac metabolism and its energetics and function and will present the potential role of the principal sex steroid hormones.

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Contractile parameters and occurrence of alternans in isolated rat myocardium at supra-physiological stimulation frequency

The cardiac refractory period prevents the heart from tetanic activation that is typically used in noncardiac striated muscle tissue. To what extent the refractory period prevents successive action potentials to activate the excitation-contraction coupling process and contractile machinery at supra-physiological rates, such as those present during ventricular fibrillation, is unknown. Using multicellular trabeculae isolated from rat hearts, we studied amplitude and kinetics of contraction at rates well above the normal in vivo rat heart range. We show that even at twice the maximal heart rate of the rat, little or no mechanical instability is observed; twitch contractions are at steady state, albeit with an elevated active diastolic force. Although the amplitude of contraction increased within in vivo heart rates (positive force-frequency response), at frequencies beyond the maximal heart rate (10-30 Hz) a steady decline of contractile amplitude is observed. Not until 30 Hz do the majority of the isolated muscle preparations show mechanical alternans, where strong and weak beats alternate. Interestingly, unlike striated limb skeletal muscle, fusing of twitch contractions did not cause a continuous increase in peak force: at frequencies of 10 Hz and above, systolic force declines with relatively little elevation in diastolic force. Contractile kinetics continued to accelerate, from 1 Hz up to 30 Hz, whereas the relative speed of contraction and relaxation remained closely coupled, reflected by a singular linear relationship between the maximal and minimal derivative of force (dF/dt). We conclude that cardiac muscle can produce mechanically stable steady-state contractions at supra-physiological pacing rates, while these contractions continue to decline in amplitude and increase in diastolic force past maximal heart rate.

Developmental toxicity of the environmental pollutant 4-nonylphenol in zebrafish

4-Nonylphenol (4-NP), an estrogen mimicking compound is produced by biodegradation of alkylethoxylates. It is well established that 4-NP can affect the development of aquatic animals by disrupting the endocrine signals. Here we show for the first time in zebrafish that 4-NP does not only target the neuroendocrine system but also the notochord and the muscle. The notochord malformation was first evident as distortions at 24 hours post fertilization (hpf) which within 24 h appeared as kinks and herniations. The notochord phenotype was accompanied by reduced motility and impaired swimming behavior. Whole-mount in situ hybridization using chordamesoderm markers and electron microscopic analysis showed failure in the notochord differentiation and disruption of the perinotochordal basement membrane. Late larval stages of 4-NP treated embryos displayed abnormal mineralization, vertebral curvature, fusion of vertebral bodies and abnormal extension of haemal arches. The muscle structure and the maximal active force in isolated muscle preparations were similar between 4-NP exposed and of control embryos, suggesting that 4-NP did not induce major changes in striated muscle function. However, repeated electrical stimulation (> 40 Hz) of the 4-NP exposed larvae revealed an impaired relaxation between stimuli, possibly reflecting an alteration in the relaxant mechanisms (e.g. in cellular Ca 2+ removal) which could explain the abnormal swimming pattern exhibited by 4-NP exposed larvae. Additionally, we demonstrate that the expression levels of the stress hormone, corticotropin releasing hormonewere elevated in the brain following 4-NP treatment. We also observed a significant decrease in the transcript levels of luteinizing hormone b at early larval stages. Collectively, our results show that 4-NP is able to disrupt the notochord morphogenesis, muscle function and the neuroendocrine system. These data suggest that 4-NP enduringly affects the embryonic development in zebrafish and that this compound might exert these deleterious effects through diverse signaling pathways.