Force and kinetics of fast and slow muscle myosin determined with a synthetic sarcomere-like nanomachine.

Myosin II is the muscle molecular motor that works in two bipolar arrays in each thick filament of the striated (skeletal and cardiac) muscle, converting the chemical energy into steady force and shortening by cyclic ATP-driven interactions with the nearby actin filaments. Different isoforms of the myosin motor in the skeletal muscles account for the different functional requirements of the slow muscles (primarily responsible for the posture) and fast muscles (responsible for voluntary movements). To clarify the molecular basis of the differences, here the isoform-dependent mechanokinetic parameters underpinning the force of slow and fast muscles are defined with a unidimensional synthetic nanomachine powered by pure myosin isoforms from either slow or fast rabbit skeletal muscle. Data fitting with a stochastic model provides a self-consistent estimate of all the mechanokinetic properties of the motor ensemble including the motor force, the fraction of actin-attached motors and the rate of transition through the attachment-detachment cycle. The achievements in this paper set the stage for any future study on the emergent mechanokinetic properties of an ensemble of myosin molecules either engineered or purified from mutant animal models or human biopsies.

Matching Mechanics and Energetics of Muscle Contraction Suggests Unconventional Chemomechanical Coupling during the Actin-Myosin Interaction.

The mechanical performances of the vertebrate skeletal muscle during isometric and isotonic contractions are interfaced with the corresponding energy consumptions to define the coupling between mechanical and biochemical steps in the myosin-actin energy transduction cycle. The analysis is extended to a simplified synthetic nanomachine in which eight HMM molecules purified from fast mammalian skeletal muscle are brought to interact with an actin filament in the presence of 2 mM ATP, to assess the emergent properties of a minimum number of motors working in ensemble without the effects of both the higher hierarchical levels of striated muscle organization and other sarcomeric, regulatory and cytoskeleton proteins. A three-state model of myosin-actin interaction is able to predict the known relationships between energetics and transient and steady-state mechanical properties of fast skeletal muscle either in vivo or in vitro only under the assumption that during shortening a myosin motor can interact with two actin sites during one ATP hydrolysis cycle. Implementation of the molecular details of the model should be achieved by exploiting kinetic and structural constraints present in the transients elicited by stepwise perturbations in length or force superimposed on the isometric contraction.

Titin activates myosin filaments in skeletal muscle by switching from an extensible spring to a mechanical rectifier.

Titin is a molecular spring in parallel with myosin motors in each muscle half-sarcomere, responsible for passive force development at sarcomere length (SL) above the physiological range (>2.7 µm). The role of titin at physiological SL is unclear and is investigated here in single intact muscle cells of the frog (Rana esculenta), by combining half-sarcomere mechanics and synchrotron X-ray diffraction in the presence of 20 µM para-nitro-blebbistatin, which abolishes the activity of myosin motors and maintains them in the resting state even during activation of the cell by electrical stimulation. We show that, during cell activation at physiological SL, titin in the I-band switches from an SL-dependent extensible spring (OFF-state) to an SL-independent rectifier (ON-state) that allows free shortening while resisting stretch with an effective stiffness of ~3 pN nm-1 per half-thick filament. In this way, I-band titin efficiently transmits any load increase to the myosin filament in the A-band. Small-angle X-ray diffraction signals reveal that, with I-band titin ON, the periodic interactions of A-band titin with myosin motors alter their resting disposition in a load-dependent manner, biasing the azimuthal orientation of the motors toward actin. This work sets the stage for future investigations on scaffold and mechanosensing-based signaling functions of titin in health and disease.

The force of the myosin motor sets cooperativity in thin filament activation of skeletal muscles.

Contraction of striated muscle is regulated by a dual mechanism involving both thin, actin-containing filament and thick, myosin-containing filament. Thin filament is activated by Ca2+ binding to troponin, leading to tropomyosin displacement that exposes actin sites for interaction with myosin motors, extending from the neighbouring stress-activated thick filaments. Motor attachment to actin contributes to spreading activation along the thin filament, through a cooperative mechanism, still unclear, that determines the slope of the sigmoidal relation between isometric force and pCa (-log[Ca2+]), estimated by Hill coefficient nH. We use sarcomere-level mechanics in demembranated fibres of rabbit skeletal muscle activated by Ca2+ at different temperatures (12-35 °C) to show that nH depends on the motor force at constant number of attached motors. The definition of the role of motor force provides fundamental constraints for modelling the dynamics of thin filament activation and defining the action of small molecules as possible therapeutic tools.

Allosteric modulation of cardiac myosin mechanics and kinetics by the conjugated omega-7,9 trans-fat rumenic acid.

KEY POINTS: Direct binding of rumenic acid to the cardiac myosin-2 motor domain increases the release rate for orthophosphate and increases the Ca2+ responsiveness of cardiac muscle at low load. Physiological cellular concentrations of rumenic acid affect the ATP turnover rates of the super-relaxed and disordered relaxed states of ß-cardiac myosin, leading to a net increase in myocardial metabolic load. In Ca2+ -activated trabeculae, rumenic acid exerts a direct inhibitory effect on the force-generating mechanism without affecting the number of force-generating motors. In the presence of saturating actin concentrations rumenic acid binds to the ß-cardiac myosin-2 motor domain with an EC50 of 200 nM. Molecular docking studies provide information about the binding site, the mode of binding, and associated allosteric communication pathways. Free rumenic acid may exceed thresholds in cardiomyocytes above which contractile efficiency is reduced and interference with small molecule therapeutics, targeting cardiac myosin, occurs. ABSTRACT: Based on experiments using purified myosin motor domains, reconstituted actomyosin complexes and rat heart ventricular trabeculae, we demonstrate direct binding of rumenic acid, the cis-delta-9-trans-delta-11 isomer of conjugated linoleic acid, to an allosteric site located in motor domain of mammalian cardiac myosin-2 isoforms. In the case of porcine ß-cardiac myosin, the EC50 for rumenic acid varies from 10.5 µM in the absence of actin to 200 nM in the presence of saturating concentrations of actin. Saturating concentrations of rumenic acid increase the maximum turnover of basal and actin-activated ATPase activity of ß-cardiac myosin approximately 2-fold but decrease the force output per motor by 23% during isometric contraction. The increase in ATP turnover is linked to an acceleration of the release of the hydrolysis product orthophosphate. In the presence of 5 µM rumenic acid, the difference in the rate of ATP turnover by the super-relaxed and disordered relaxed states of cardiac myosin increases from 4-fold to 20-fold. The equilibrium between the two functional myosin states is not affected by rumenic acid. Calcium responsiveness is increased under zero-load conditions but unchanged under load. Molecular docking studies provide information about the rumenic acid binding site, the mode of binding, and associated allosteric communication pathways. They show how the isoform-specific replacement of residues in the binding cleft induces a different mode of rumenic acid binding in the case of non-muscle myosin-2C and blocks binding to skeletal muscle and smooth muscle myosin-2 isoforms.

Muscle myosin performance measured with a synthetic nanomachine reveals a class-specific Ca2+ -sensitivity of the frog myosin II isoform.

KEY POINTS: A nanomachine made of an ensemble of seven heavy-meromyosin (HMM) fragments of muscle myosin interacting with an actin filament is able to mimic the half-sarcomere generating steady force and constant-velocity shortening. To preserve Ca2+ as a free parameter, the Ca2+ -insensitive gelsolin fragment TL40 is used to attach the correctly oriented actin filament to the laser-trapped bead acting as a force transducer. The new method reveals that the performance of the nanomachine powered by myosin from frog hind-limb muscles depends on [Ca2+ ], an effect mediated by a Ca2+ -binding site in the regulatory light chain of HMM. The Ca2+ -sensitivity is class-specific because the performance of the nanomachine powered by mammalian skeletal muscle myosin is Ca2+ independent. A model simulation is able to interface the nanomachine performance with that of the muscle of origin and provides a molecular explanation of the functional diversity of muscles with different orthologue isoforms of myosin. ABSTRACT: An ensemble of seven heavy-meromyosin (HMM) fragments of myosin-II purified from the hindlimb muscles of the frog (Rana esculenta) is used to drive a synthetic nanomachine that pulls an actin filament in the absence of confounding effects of other sarcomeric proteins. In the present version of the nanomachine the +end of the actin filament is attached to the laser trapped bead via the Ca2+ -insensitive gelsolin fragment TL40, making [Ca2+ ] a free parameter. Frog myosin performance in 2 mm ATP is affected by Ca2+ : in 0.1 mm Ca2+ , the isometric steady force (F0 , 15.25 pN) is increased by 50% (P = 0.004) with respect to that in Ca2+ -free solution, the maximum shortening velocity (V0 , 4.6 µm s-1 ) is reduced by 27% (P = 0.46) and the maximum power (Pmax , 7.6 aW) is increased by 21% (P = 0.17). V0 reduction is not significant for the paucity of data at low force, although it is solidified by a similar decrease (33%, P < 0.0001) in the velocity of actin sliding as indicated by an in vitro motility assay (Vf ). The rate of ATP-hydrolysis in solution (φ) exhibits a similar calcium dependence. Ca2+ titration curves for Vf and φ give Kd values of ∼30 µm. All the above mechanical and kinetic parameters are independent of Ca2+ when HMM from rabbit psoas myosin is used, indicating that the Ca2+ -sensitivity is a class-specific property of muscle myosin. A unique multiscale model allows interfacing of the nanomachine performance to that of the muscle of origin and identifies the kinetic steps responsible for the Ca2+ -sensitivity of frog myosin.

A Myosin II-Based Nanomachine Devised for the Study of Ca2+-Dependent Mechanisms of Muscle Regulation.

The emergent properties of the array arrangement of the molecular motor myosin II in the sarcomere of the striated muscle, the generation of steady force and shortening, can be studied in vitro with a synthetic nanomachine made of an ensemble of eight heavy-meromyosin (HMM) fragments of myosin from rabbit psoas muscle, carried on a piezoelectric nanopositioner and brought to interact with a properly oriented actin filament attached via gelsolin (a Ca2+-regulated actin binding protein) to a bead trapped by dual laser optical tweezers. However, the application of the original version of the nanomachine to investigate the Ca2+-dependent regulation mechanisms of the other sarcomeric (regulatory or cytoskeleton) proteins, adding them one at a time, was prevented by the impossibility to preserve [Ca2+] as a free parameter. Here, the nanomachine is implemented by assembling the bead-attached actin filament with the Ca2+-insensitive gelsolin fragment TL40. The performance of the nanomachine is determined both in the absence and in the presence of Ca2+ (0.1 mM, the concentration required for actin attachment to the bead with gelsolin). The nanomachine exhibits a maximum power output of 5.4 aW, independently of [Ca2+], opening the possibility for future studies of the Ca2+-dependent function/dysfunction of regulatory and cytoskeletal proteins.

Orthophosphate increases the efficiency of slow muscle-myosin isoform in the presence of omecamtiv mecarbil.

Omecamtiv mecarbil (OM) is a putative positive inotropic tool for treatment of systolic heart dysfunction, based on the finding that in vivo it increases the ejection fraction and in vitro it prolongs the actin-bond life time of the cardiac and slow-skeletal muscle isoforms of myosin. OM action in situ, however, is still poorly understood as the enhanced Ca2+-sensitivity of the myofilaments is at odds with the reduction of force and rate of force development observed at saturating Ca2+. Here we show, by combining fast sarcomere-level mechanics and ATPase measurements in single slow demembranated fibres from rabbit soleus, that the depressant effect of OM on the force per attached motor is reversed, without effect on the ATPase rate, by physiological concentrations of inorganic phosphate (Pi) (1-10 mM). This mechanism could underpin an energetically efficient reduction of systolic tension cost in OM-treated patients, whenever [Pi] increases with heart-beat frequency.

Straightening Out the Elasticity of Myosin Cross-Bridges.

In a contracting muscle, myosin cross-bridges extending from thick filaments pull the interdigitating thin (actin-containing) filaments during cyclical ATP-driven interactions toward the center of the sarcomere, the structural unit of striated muscle. Cross-bridge attachments in the sarcomere have been reported to exhibit a similar stiffness under both positive and negative forces. However, in vitro measurements on filaments with a sparse complement of heads detected a decrease of the cross-bridge stiffness at negative forces attributed to the buckling of the subfragment 2 tail portion. Here, we review some old and new data that confirm that cross-bridge stiffness is nearly linear in the muscle filament lattice. The implications of high myosin stiffness at positive and negative strains are considered in muscle fibers and in nonmuscle intracellular cargo transport.

Myopalladin promotes muscle growth through modulation of the serum response factor pathway.

BACKGROUND: Myopalladin (MYPN) is a striated muscle-specific, immunoglobulin-containing protein located in the Z-line and I-band of the sarcomere as well as the nucleus. Heterozygous MYPN gene mutations are associated with hypertrophic, dilated, and restrictive cardiomyopathy, and homozygous loss-of-function truncating mutations have recently been identified in patients with cap myopathy, nemaline myopathy, and congenital myopathy with hanging big toe. METHODS: Constitutive MYPN knockout (MKO) mice were generated, and the role of MYPN in skeletal muscle was studied through molecular, cellular, biochemical, structural, biomechanical, and physiological studies in vivo and in vitro. RESULTS: MKO mice were 13% smaller compared with wild-type controls and exhibited a 48% reduction in myofibre cross-sectional area (CSA) and significantly increased fibre number. Similarly, reduced myotube width was observed in MKO primary myoblast cultures. Biomechanical studies showed reduced isometric force and power output in MKO mice as a result of the reduced CSA, whereas the force developed by each myosin molecular motor was unaffected. While the performance by treadmill running was similar in MKO and wild-type mice, MKO mice showed progressively decreased exercise capability, Z-line damage, and signs of muscle regeneration following consecutive days of downhill running. Additionally, MKO muscle exhibited progressive Z-line widening starting from 8 months of age. RNA-sequencing analysis revealed down-regulation of serum response factor (SRF)-target genes in muscles from postnatal MKO mice, important for muscle growth and differentiation. The SRF pathway is regulated by actin dynamics as binding of globular actin to the SRF-cofactor myocardin-related transcription factor A (MRTF-A) prevents its translocation to the nucleus where it binds and activates SRF. MYPN was found to bind and bundle filamentous actin as well as interact with MRTF-A. In particular, while MYPN reduced actin polymerization, it strongly inhibited actin depolymerization and consequently increased MRTF-A-mediated activation of SRF signalling in myogenic cells. Reduced myotube width in MKO primary myoblast cultures was rescued by transduction with constitutive active SRF, demonstrating that MYPN promotes skeletal muscle growth through activation of the SRF pathway. CONCLUSIONS: Myopalladin plays a critical role in the control of skeletal muscle growth through its effect on actin dynamics and consequently the SRF pathway. In addition, MYPN is important for the maintenance of Z-line integrity during exercise and aging. These results suggest that muscle weakness in patients with biallelic MYPN mutations may be associated with reduced myofibre CSA and SRF signalling and that the disease phenotype may be aggravated by exercise.

Contracting striated muscle has a dynamic I-band spring with an undamped stiffness 100 times larger than the passive stiffness.

KEY POINTS: Fast sarcomere-level mechanics in contracting intact fibres from frog skeletal muscle reveal an I-band spring with an undamped stiffness 100 times larger than the known static stiffness. This undamped stiffness remains constant in the range of sarcomere length 2.7-3.1 µm, showing the ability of the I-band spring to adapt its length to the width of the I-band. The stiffness and tunability of the I-band spring implicate titin as a force contributor that, during contraction, allows weaker half-sarcomeres to equilibrate with in-series stronger half-sarcomeres, preventing the development of sarcomere length inhomogeneity. This work opens new possibilities for the detailed in situ description of the structural-functional basis of muscle dysfunctions related to mutations or site-directed mutagenesis in titin that alter the I-band stiffness. ABSTRACT: Force and shortening in the muscle sarcomere are due to myosin motors from thick filaments pulling nearby actin filaments toward the sarcomere centre. Thousands of serially linked sarcomeres in muscle make the shortening (and the shortening speed) macroscopic, while the intrinsic instability of in-series force generators is likely prevented by the cytoskeletal protein titin that connects the thick filament with the sarcomere end, working as an I-band spring that accounts for the rise of passive force with sarcomere length (SL). However, current estimates of titin stiffness, deduced from the passive force-SL relation and single molecule mechanics, are much smaller than what is required to avoid the development of large inhomogeneities among sarcomeres. In this work, using 4 kHz stiffness measurements on a population of sarcomeres selected along an intact fibre isolated from frog skeletal muscle contracting at different SLs (temperature 4°C), we measure the undamped stiffness of an I-band spring that at SL > 2.7 µm attains a maximum constant value of ∼6 pN nm-1 per half-thick filament, two orders of magnitude larger than expected from titin-related passive force. We conclude that a titin-like dynamic spring in the I-band, made by an undamped elastic element in-series with damped elastic elements, adapts its length to the SL with kinetics that provide force balancing among serially linked sarcomeres during contraction. In this way, the I-band spring plays a fundamental role in preventing the development of SL inhomogeneity.

A mechanical model of the half-sarcomere which includes the contribution of titin.

The evidence, in both resting and active muscle, for the presence of an I-band spring element like titin that anchors the Z line to the end of the thick filament did not yet produce a proper theoretical treatment in a complete model of the half-sarcomere. The textbook model developed by A. F. Huxley and his collaborators in 1981, which provides that the half-sarcomere (hs) compliance is due to the contribution of the compliances of the thin and thick filaments and actin-attached myosin motors, predicts that at any sarcomere length (SL) the absence of attached motors results in an infinite half-sarcomere compliance, in contrast with the observations. Growing evidence for the presence of a titin-like I-band spring urges the 1981 model to be implemented to include the contribution of this element in the mechanical model of the half-sarcomere. The model described here represents a tool for the interpretation of measurements of hs stiffness at increasing SL, which is important either in relation to the mechanism of stabilisation of SL against the consequence of sarcomere inhomogeneity in active force generation, or for investigations on the role of titin as mechano-sensor in thick filament regulation. Moreover the model opens the possibility for understanding the functional differences related to the titin isoform of various muscle types and the mechanism by which mutations in titin gene lead to myopathies.

A myosin II nanomachine mimicking the striated muscle.

The contraction of striated muscle (skeletal and cardiac muscle) is generated by ATP-dependent interactions between the molecular motor myosin II and the actin filament. The myosin motors are mechanically coupled along the thick filament in a geometry not achievable by single-molecule experiments. Here we show that a synthetic one-dimensional nanomachine, comprising fewer than ten myosin II dimers purified from rabbit psoas, performs isometric and isotonic contractions at 2 mM ATP, delivering a maximum power of 5 aW. The results are explained with a kinetic model fitted to the performance of mammalian skeletal muscle, showing that the condition for the motor coordination that maximises the efficiency in striated muscle is a minimum of 32 myosin heads sharing a common mechanical ground. The nanomachine offers a powerful tool for investigating muscle contractile-protein physiology, pathology and pharmacology without the potentially disturbing effects of the cytoskeletal-and regulatory-protein environment.

The force and stiffness of myosin motors in the isometric twitch of a cardiac trabecula and the effect of the extracellular calcium concentration.

KEY POINTS: Fast sarcomere-level mechanics in intact trabeculae, which allows the definition of the mechano-kinetic properties of cardiac myosin in situ, is a fundamental tool not only for understanding the molecular mechanisms of heart performance and regulation, but also for investigating the mechanisms of the cardiomyopathy-causing mutations in the myosin and testing small molecules for therapeutic interventions. The approach has been applied to measure the stiffness and force of the myosin motor and the fraction of motors attached during isometric twitches of electrically paced trabeculae under different extracellular Ca2+ concentrations. Although the average force of the cardiac myosin motor (∼6 pN) is similar to that of the fast myosin isoform of skeletal muscle, the stiffness (1.07 pN nm-1 ) is 2- to 3-fold smaller. The increase in the twitch force developed in the presence of larger extracellular Ca2+ concentrations is fully accounted for by a proportional increase in the number of attached motors. ABSTRACT: The mechano-kinetic properties of the cardiac myosin were studied in situ, in trabeculae dissected from the right ventricle of the rat heart, by measuring the stiffness of the half-sarcomere both at the twitch force peak (Tp ) of an electrically paced intact trabecula at different extracellular Ca2+ concentrations ([Ca2+ ]o ), and in the same trabecula after skinning and induction of rigor. Taking into account the contribution of filament compliance to half-sarcomere compliance and the lattice geometry, we found that the stiffness of the cardiac myosin motor is 1.07 ± 0.09 pN nm-1 , which is slightly larger than that of the slow myosin isoform of skeletal muscle (0.6-0.8 pN nm-1 ) and 2- to 3-fold smaller than that of the fast skeletal muscle isoform. The increase in Tp from 61 ± 4 kPa to 93 ± 9 kPa, induced by raising [Ca2+ ]o from 1 to 2.5 mm at sarcomere length ∼2.2 µm, is accompanied by an increase of the half-sarcomere stiffness that is explained by an increase of the fraction of actin-attached motors from 0.08 ± 0.01 to 0.12 ± 0.02, proportional to Tp . Consequently, each myosin motor bears an average force of 6.14 ± 0.52 pN independently of Tp and [Ca2+ ]o . The application of fast sarcomere-level mechanics to intact trabeculae to define the mechano-kinetic properties of the cardiac myosin in situ represents a powerful tool for investigating cardiomyopathy-causing mutations in the myosin motor and testing specific therapeutic interventions.

Mechanical parameters of the molecular motor myosin II determined in permeabilised fibres from slow and fast skeletal muscles of the rabbit.

KEY POINTS: The different performance of slow and fast muscles is mainly attributed to diversity of the myosin heavy chain (MHC) isoform expressed within them. In this study fast sarcomere-level mechanics has been applied to Ca2+ -activated single permeabilised fibres isolated from soleus (containing the slow myosin isoform) and psoas (containing the fast myosin isoform) muscles of rabbit for a comparative definition of the mechano-kinetics of force generation by slow and fast myosin isoforms in situ. The stiffness and the force of the slow myosin isoform are three times smaller than those of the fast isoform, suggesting that the stiffness of the myosin motor is a determinant of the isoform-dependent functional diversity between skeletal muscles. These results open the question of the mechanism that can reconcile the reduced performance of the slow MHC with the higher efficiency of the slow muscle. ABSTRACT: The skeletal muscle exhibits large functional differences depending on the myosin heavy chain (MHC) isoform expressed in its molecular motor, myosin II. The differences in the mechanical features of force generation by myosin isoforms were investigated in situ by using fast sarcomere-level mechanical methods in permeabilised fibres (sarcomere length 2.4 µm, temperature 12°C, 4% dextran T-500) from slow (soleus, containing the MHC-1 isoform) and fast (psoas, containing the MHC-2X isoform) skeletal muscle of the rabbit. The stiffness of the half-sarcomere was determined at the plateau of Ca2+ -activated isometric contractions and in rigor and analysed with a model that accounted for the filament compliance to estimate the stiffness of the myosin motor (ε). ε was 0.56 ± 0.04 and 1.70 ± 0.37 pN nm-1 for the slow and fast isoform, respectively, while the average strain per attached motor (s0 ) was similar (∼3.3 nm) in both isoforms. Consequently the force per motor (F0  = Îµs0 ) was three times smaller in the slow isoform than in the fast isoform (1.89 ± 0.43 versus 5.35 ± 1.51 pN). The fraction of actin-attached motors responsible for maximum isometric force at saturating Ca2+ (T0,4.5 ) was 0.47 ± 0.09 in soleus fibres, 70% larger than that in psoas fibres (0.29 ± 0.08), so that F0 in slow fibres was decreased by only 53%. The lower stiffness and force of the slow myosin isoform open the question of the molecular basis of the higher efficiency of slow muscle with respect to fast muscle.

CTGF/CCN2 exerts profibrotic action in myoblasts via the up-regulation of sphingosine kinase-1/S1P3 signaling axis: Implications in the action mechanism of TGFß.

The matricellular protein connective tissue growth factor (CTGF/CCN2) is recognized as key player in the onset of fibrosis in various tissues, including skeletal muscle. In many circumstances, CTGF has been shown to be induced by transforming growth factor beta (TGFß) and accounting, at least in part, for its biological action. In this study it was verified that in cultured myoblasts CTGF/CCN2 causes their transdifferentiation into myofibroblasts by up-regulating the expression of fibrosis marker proteins α-smooth muscle actin and transgelin. Interestingly, it was also found that the profibrotic effect exerted by CTGF/CCN2 was mediated by the sphingosine kinase (SK)-1/S1P3 signaling axis specifically induced by the treatment with the profibrotic cue. Following CTGF/CCN2-induced up-regulation, S1P3 became the S1P receptor subtype expressed at the highest degree, at least at mRNA level, and was thus capable of readdressing the sphingosine 1-phosphate signaling towards fibrosis rather than myogenic differentiation. Another interesting finding is that CTGF/CCN2 silencing prevented the TGFß-dependent up-regulation of SK1/S1P3 signaling axis and strongly reduced the profibrotic effect exerted by TGFß, pointing at a crucial role of endogenous CTGF/CCN2 generated following TGFß challenge in the transmission of at least part of its profibrotic effect. These results provide new insights into the molecular mechanism by which CTGF/CCN2 drives its biological action and strengthen the concept that SK1/S1P3 axis plays a critical role in the onset of fibrotic cell phenotype.