Contractile Behavior Research Articles

Characterization of the teneurin C-terminal associated peptide (TCAP) in the vase tunicate, Ciona intestinalis: A novel peptide system associated with energy metabolism and reproduction

The vase tunicate, Ciona intestinalis, is a protochordate and is considered a sister lineage to the chordates. The recent sequencing of its genome has made this species a particularly important model to understand the genetic basis of vertebrate evolution. However, C. intestinalis is also a highly invasive species along the Atlantic coast of North America and other regions of the world which have caused considerable economic stress due to its biofouling actions and, in particular, negative impacts on the mussel- and oyster-based aquaculture industry. Despite this background, little is known about C. intestinalis physiology. The teneurin C-terminal associated peptides (TCAP) are a family of highly conserved peptide hormones found in most metazoans. Moreover, these peptides have been implicated in the inhibition of stress and stimulation of feeding-based metabolism. We have, therefore, identified this peptide using an in silico approach and characterized its immunological expression in tissues using a mouse polyclonal antiserum. These data indicate that its primary structure is more similar to invertebrate TCAPs relative to vertebrate TCAPs. Immunological expression indicates that it is highly expressed in the digestive tract and gonads consistent with findings in vertebrates. Synthetic mouse TCAP-1 administered into the brachial basket significantly increases the incidence of non-stress contractile behaviors. These findings support the hypothesis that TCAP is a bioactive peptide in C. intestinalis. Thus, C. intestinalis and tunicates in general may offer a simple model to investigate peptide interaction while providing information on how to control this invasive species.

Muscle thickness measurements of the lower trapezius with rehabilitative ultrasound imaging are confounded by scapular dyskinesis

Alterations in scapular muscle activity have been theorized to contribute to abnormal scapular motion and shoulder pain, but pose challenges to quantify in the clinic. Rehabilitative Ultrasound Imaging (RUSI) has proved useful identifying dysfunction of lumbar regional stabilizing muscle activity, specifically contractile behavior. Although, recent examinations of scapular stabilizing trapezius muscle function using RUSI did not detect alterations individuals with shoulder pain or differences in muscle thickness between varying external loads in asymptomatic individuals, a potential confounder to prior results, scapular dyskinesis has not been controlled. It is unknown if dyskinesis alters scapular muscle thickness during activation measured with RUSI. Thus, the purpose of this study was to compare change in scapular muscle thickness between individuals with and without scapular dyskinesis. Thirty-nine asymptomatic adults with (n = 19) and without (n = 20) scapular dyskinesis, defined with a reliable and validated method, participated. Two separate ultrasound images of the serratus anterior (SA) and lower trapezius (LT) were captured under two randomized conditions, rest and isometric contraction against gravity, and saved for blinded measurement. Change in thickness with contraction was calculated and expressed as a percentage. The dyskinesis group demonstrated a greater increase (p = 0.005) in LT thickness with the isometric contraction than the group without (mean difference = 31.6%; 95%CI = 10.3, 53.0). No differences in SA or resting thickness of either muscle were found between groups. The presence of scapular dyskinesis alters thickness changes of the lower trapezius during activation. Furthermore, potential underlying reasons beyond muscle contractile behavior must be considered.

Bone Marrow-Derived Regenerated Smooth Muscle Cells Have Ion Channels and Properties Characteristic of Vascular Smooth Muscle Cells

Rationale: Numerous reports, including our own, have recently suggested the presence of putative smooth muscle progenitor cells in the bone marrow (BM) and those smooth muscle-like cells may be differentiated from BM stromal cells (BMSCs). However, few studies have addressed whether the differentiated cells also possess the functional properties of smooth muscle cells (SMCs). Contractility is the primary function of native vascular SMCs. Objective: The aim of this electrophysiological study was to characterize BM-derived SMCs using the patchclamp technique and Ca2+ imaging with fura-2. Methods and results: To investigate whether BM-derived SMCs exhibit functional vascular SMC properties, we measured Ca2+ and K+ currents in BM-derived SMCs using the whole-cell patch-clamp method. The cells showed L-type and T-type Ca2+ channel currents, Ca2+-activated K+ channel (KCa) currents, and delayed rectifier K+ channel (KV) currents. We also measured agonist-evoked [Ca2+]i transients in BM-derived SMCs using fura-2 imaging. Such [Ca2+] i transients were observed in response to the vascular SMC-specific agonists, bradykinin (10-6 M) and angiotensin II (10-7 M). Conclusions: BM-derived SMCs displayed contractile activity and expressed several ion channels critical for contractile behavior in a manner compatible with native vascular SMCs. BMSC-derived cells thus have the potential to differentiate into functional vascular SMCs, suggesting bone marrow stromal tissue as a useful source of cells for the treatment of injured arteries and to construct tissue-engineered grafts for adult arterial revascularization.

Prospects for In Vitro Myofilament Maturation in Stem Cell-Derived Cardiac Myocytes.

Cardiomyocytes derived from human stem cells are quickly becoming mainstays of cardiac regenerative medicine, in vitro disease modeling, and drug screening. Their suitability for such roles may seem obvious, but assessments of their contractile behavior suggest that they have not achieved a completely mature cardiac muscle phenotype. This could be explained in part by an incomplete transition from fetal to adult myofilament protein isoform expression. In this commentary, we review evidence that supports this hypothesis and discuss prospects for ultimately generating engineered heart tissue specimens that behave similarly to adult human myocardium. We suggest approaches to better characterize myofilament maturation level in these in vitro systems, and illustrate how new computational models could be used to better understand complex relationships between muscle contraction, myofilament protein isoform expression, and maturation.

Metabolic regulation of collagen gel contraction by porcine aortic valvular interstitial cells.

Despite a high incidence of calcific aortic valve disease in metabolic syndrome, there is little information about the fundamental metabolism of heart valves. Cell metabolism is a first responder to chemical and mechanical stimuli, but it is unknown how such signals employed in valve tissue engineering impact valvular interstitial cell (VIC) biology and valvular disease pathogenesis. In this study porcine aortic VICs were seeded into three-dimensional collagen gels and analysed for gel contraction, lactate production and glucose consumption in response to manipulation of metabolic substrates, including glucose, galactose, pyruvate and glutamine. Cell viability was also assessed in two-dimensional culture. We found that gel contraction was sensitive to metabolic manipulation, particularly in nutrient-depleted medium. Contraction was optimal at an intermediate glucose concentration (2 g l(-1)) with less contraction with excess (4.5 g l(-1)) or reduced glucose (1 g l(-1)). Substitution with galactose delayed contraction and decreased lactate production. In low sugar concentrations, pyruvate depletion reduced contraction. Glutamine depletion reduced cell metabolism and viability. Our results suggest that nutrient depletion and manipulation of metabolic substrates impacts the viability, metabolism and contractile behaviour of VICs. Particularly, hyperglycaemic conditions can reduce VIC interaction with and remodelling of the extracellular matrix. These results begin to link VIC metabolism and macroscopic behaviour such as cell-matrix interaction.

Abstract 13200: Acute Inhibition of Phosphodiesterase Type 5A (PDE5) with Sildenafil Augments Contractility of LV and Myocyte in Heart Failure

Background: Sildenafil (SIL), a selective inhibitor of cGMP-specific PDE5 has been shown to exert profound cardioprotection in various cardiac pathologies. This beneficial action has been attributed to its selective vasodilatation effect. However, recent studies indicate that SIL may augment cGMP signaling and produce direct cardiac inotropic modulation, thus affords its cardioprotection in heart failure (HF). Whether SIL plays a role in cardiac contractile behavior in HF remains undetermined. SIL has been reported variably to have a positive, negative or no inotropic effect on cardiac contractility in HF. These disparate observations may be due to the confounding effects of loading conditions and dosage of SIL. We tested the hypothesis that SIL evokes positive modulation on cardiac contractility which importantly contributes to its beneficial action in HF. Methods: We measured Left ventricular (LV) contractility changes after SIL (0.4 mg/kg, iv), and compared myocyte contractile and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses to SIL (10 -6 M) in 8 rats with isoproterenol (ISO)-induced HF (3 months after 170 mg/kg sq for 2 days). LV contractility was measured by pressure-volume analysis. Results: Compared with normal controls, ISO-treated rats had established HF after ISO at 3 months with significantly elevated plasma norepinephrine, LV dilatation, and decreased ejection fraction (EF, 29% vs 61%). Compared with baseline, acute administration of SIL caused no change in LV systolic blood pressure and heart rate, but increased EF (43% vs 29 %). LV time constant of relaxation (t) (12.2 vs 15.1 ms) decreased. The slopes of LV pressure-volume relations of E ES (49%, 0.92 vs 0.61 mmHg/μl) and M SW (42%, 97.4 vs 68.6 mmHg) were increased, indicating significantly enhanced LV contractility. These responses were accompanied with about 24% increases in cell contraction (dL/dt max , 97.1 vs 78.3 μm/s) and relaxation (dR/dt max , 63.7 vs 51.5 μm/s) and relatively unchanged [Ca 2+ ] iT (0.149 vs 0.137), while myocyte Ca 2+ sensitivity was enhanced. Conclusions: In HF, positive inotropic effect is induced by acute inhibiting PDE5 with clinically-relevant concentrations of SIL. The cardiostimulatory and vasodilatory actions of SIL make it suitable for the treatment of HF.

Human melanocytes mitigate keratinocyte-dependent contraction in an in vitro collagen contraction assay

Scarring is an extensive problem in burn care, and treatment can be especially complicated in cases of hypertrophic scarring. Contraction is an important factor in scarring but the contribution of different cell types remains unclear. We have investigated the contractile behavior of keratinocytes, melanocytes and fibroblasts by using an in vitro collagen gel assay aimed at identifying a modulating role of melanocytes in keratinocyte-mediated contraction. Cells were seeded on a collagen type I gel substrate and the change in gel dimensions were measured over time. Hematoxylin & Eosin-staining and immunohistochemistry against pan-cytokeratin and microphthalmia-associated transcription factor showed that melanocytes integrated between keratinocytes and remained there throughout the experiments. Keratinocyte- and fibroblast-seeded gels contracted significantly over time, whereas melanocyte-seeded gels did not. Co-culture assays showed that melanocytes mitigate the keratinocyte-dependent contraction (significantly slower and 18–32% less). Fibroblasts augmented the contraction in most assays (approximately 6% more). Non-contact co-cultures showed some influence on the keratinocyte-dependent contraction. Results show that mechanisms attributable to melanocytes, but not fibroblasts, can mitigate keratinocyte contractile behavior. Contact-dependent mechanisms are stronger modulators than non-contact dependent mechanisms, but both modes carry significance to the contraction modulation of keratinocytes. Further investigations are required to determine the mechanisms involved and to determine the utility of melanocytes beyond hypopigmentation in improved clinical regimes of burn wounds and wound healing.

Control system design for the mock ventricle with aortic and mitral valve resistance uncertainty

Reproducing the native left ventricle’s contractile behavior is the key ingredient for the mock cardiovascular circulatory system. It can be achieved by controlling either mock ventricle’s pressure or volume so that it could follow the reference signal calculated by the Elastance waveform. However, due to inherent uncertainties of the parameter values of the mock ventricle, such as check valve resistance, it is difficult to achieve high quality control performance. In this paper, we present an adaptive control scheme to overcome this parameter value uncertainty and to achieve high quality control performance. To the best knowledge of the author, it is the first report that reproduces the mock ventricle pumping dynamics precisely considering the resistance of the aortic/mitral check valves and overcoming the uncertainty of them. In the paper, along with the detailed design of the controller, rigorous proof of the stability and the convergence is presented as well. Computer simulation was performed using an electrical-analog cardiovascular circulatory system model and a piston pump mock ventricle model. Results confirmed that adaptive control achieved almost perfect tracking performance in spite of large parameter uncertainty and high bandwidth dynamic characteristics of the mock ventricle. Performance comparison verified the superiority of the adaptive controller over PID controllers.

Interleukin-6 Contributes to the Paracrine Effects of Cardiospheres Cultured from Human, Murine and Rat Hearts

Cardiosphere-derived cells (CDCs) were cultured from human, murine, and rat hearts. Diluted supernatant (conditioned-medium) of the cultures improved the contractile behavior of isolated rat cardiomyocytes (CMCs). This effect is mediated by the paracrine release of cytokines. The present study tested the hypothesis, that the cardiovascular state of the donor's heart influences this effect on CMCs and tries to identify the responsible factors. CDCs were cultured from human tissue samples of cardiac surgery and from murine and rat hearts. The supernatants of cultured CDCs from hypertensive humans and rats showed a higher improvement of the contractile behavior of CMCs compared to CDCs of normotensive origin. Subsequently, the cytokine profile of the supernatants was analyzed. Among the cytokines elevated in supernatants originating from hypertensive humans or rats was Interleukin-6. CDCs were also generated from Interleukin-6(-/-) -mice and their wildtype littermates. The supernatant of the cultured Interleukin-6(-/-) -CDCs had no effect on the contractile behavior, whereas the supernatant of the Interleukin-6(+/+) -CDCs showed a positive effect. To confirm the hypothesis that Interleukin-6 contributes to the paracrine effects, CMCs were incubated with Interleukin-6. It improved the contractile function in a concentration dependent way. Finally, the effect of the supernatant of cultured CDCs derived from a hypertensive human sample could be abolished by simultaneous incubation with a specific Interleukin-6 antibody. CDCs release cytokines that improve the contractile behavior of CMCs. This effect is more intense in CDCs from hypertensive donors. Interleukin-6 is involved in this phenomenon.

Spatiotemporal control of epithelial remodeling by regulated myosin phosphorylation.

Spatiotemporally regulated actomyosin contractility generates the forces that drive epithelial cell rearrangements and tissue remodeling. Phosphorylation of the myosin II regulatory light chain (RLC) promotes the assembly of myosin monomers into active contractile filaments and is an essential mechanism regulating the level of myosin activity. However, the effects of phosphorylation on myosin localization, dynamics, and function during epithelial remodeling are not well understood. In Drosophila, planar polarized myosin contractility is required for oriented cell rearrangements during elongation of the body axis. We show that regulated myosin phosphorylation influences spatial and temporal properties of contractile behavior at molecular, cellular, and tissue length scales. Expression of myosin RLC variants that prevent or mimic phosphorylation both disrupt axis elongation, but have distinct effects at the molecular and cellular levels. Unphosphorylatable RLC produces fewer, slower cell rearrangements, whereas phosphomimetic RLC accelerates rearrangement and promotes higher-order cell interactions. Quantitative live imaging and biophysical approaches reveal that both phosphovariants reduce myosin planar polarity and mechanical anisotropy, altering the orientation of cell rearrangements during axis elongation. Moreover, the localized myosin activator Rho-kinase is required for spatially regulated myosin activity, even when the requirement for phosphorylation is bypassed by the expression of phosphomimetic myosin RLC. These results indicate that myosin phosphorylation influences both the level and the spatiotemporal regulation of myosin activity, linking molecular properties of myosin activity to tissue morphogenesis.

Abstract 299: Pulmonary Hypertension Activates Cardiac Fibroblasts To Release Paracrine Factors Regulating Cardiac Myocyte Dedifferentiation

In virtually all models of heart failure, prognosis is ultimately determined by right ventricular (RV) function. Thus understanding the unique cellular mechanisms that contribute to RV dysfunction is of critical importance. We have approached this question by studying a large animal model that has significant resonance with human disease: exposure of the neonatal calf to hypobaric hypoxia. This model demonstrates rapid onset of severe pulmonary hypertension (PH) and significant RV dysfunction. Strikingly, the RV of these animals shows a chamber-specific increase in markers of inflammation and local infiltration of activated pro-remodeling macrophages that are proportionate to the rise in PA pressure. In order to test the hypothesis that this inflammatory milieu directly contributes to myocyte dysfunction, we examined the effects of conditioned media (CM) from cardiac fibroblasts isolated from the PH calf on normal adult rat ventricular myocytes (ARVM) in culture. Even brief exposure (<2 days) to RV-CM results in rapid and marked dedifferentiation of ARVM to a neonatal-like phenotype that exhibits spontaneous contractile behavior. Dedifferentiated cells maintain viability for over 30 days with continued expression of cardiomyocyte proteins including TnI, sarcomeric actinin, and desmin. This response is not seen in ARVM exposed to CM from fibroblasts collected from the hypoxic LV or from the normoxic RV. We have characterized the factor(s) that are present in the RV-CM and have established that the active components are proteins with a mol wt >30 kDa and that inflammatory cytokines such as IL-18 are necessary but not sufficient to effect this phenotypic change. These data suggest that local and perhaps focal inflammation in the RV, induced by the combination of pressure overload and hypoxia, has the capacity to signal a phenotypic transformation in a population of RV cardiocytes, which likely contributes to chamber specific dysfunction. Therapies that target and interrupt this inflammatory process either by preventing in-migration of inflammatory cells or by blocking cell-cell signaling have the potential to prevent RV dysfunction.

Hepatic organoids for microfluidic drug screening.

We introduce the microfluidic organoids for drug screening (MODS) platform, a digital microfluidic system that is capable of generating arrays of individually addressable, free-floating, three-dimensional hydrogel-based microtissues (or 'organoids'). Here, we focused on liver organoids, driven by the need for early-stage screening methods for hepatotoxicity that enable a "fail early, fail cheaply" strategy in drug discovery. We demonstrate that arrays of hepatic organoids can be formed from co-cultures of HepG2 and NIH-3T3 cells embedded in hydrogel matrices. The organoids exhibit fibroblast-dependent contractile behaviour, and their albumin secretion profiles and cytochrome P450 3A4 activities are better mimics of in vivo liver tissue than comparable two-dimensional cell culture systems. As proof of principle for screening, MODS was used to generate and analyze the effects of a dilution series of acetaminophen on apoptosis and necrosis. With further development, we propose that the MODS platform may be a cost-effective tool in a "fail early, fail cheaply" paradigm of drug development.

Photophysical characterisation and studies of the effect of palladium(II) 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrin on isometric contraction of isolated human mesenteric artery: Good news for photodynamic therapy

Considering the important roles of porphyrins in biological systems and their promising use in photodynamic therapy (PDT), the present work investigated the photophysical properties of palladium(II) 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrin (PdTSPP) and the effects of non-activated by light form of this porphyrin on contractile behaviour of isolated healthy endothelium-denuded human mesenteric arteries. The photophysical characterisation of PdTSPP: the formation of the triplet states and the singlet oxygen were studied using laser flash photolysis. The effect of PdTSPP on the isometric contraction of artery segments from human mesentery was assessed utilising the precise method of artery isometric tension recording using Mulvany-Halpern wire myograph. We found that PdTSPP had a high lifetime of the triplet states τT=270μs. The calculated Stern Volmer rate constant kq=1.7×10(9)M(-1)s(-1) showed an efficient quenching by oxygen that indicated formation of singlet oxygen, O2((1)Δg). The photophysical parameters of PdTSPP, in particular its ability to generate O2((1)Δg) has defined it as an exceptionally interesting molecule for PDT. The results of the contraction study showed that PdTSPP applied in increasing concentrations (1-100μM) had no effect on the basal tone of human mesenteric artery under isometric condition. Furthermore, PdTSPP failed to potentiate or to attenuate the isometric contraction of the artery preparations precontracted with high extracellular potassium (42mM KCl) or with 1nM endothelin-1. The excellent photophysical properties of PdTSPP as well as the lack of an effect on the contractility of human vasculature in vitro characterise PdTSPP as a suitable compound for potential medical applications.

Multifidus muscle size and percentage thickness changes among patients with unilateral chronic low back pain (CLBP) and healthy controls in prone and standing

The aim of this study was to determine if differences exist in lumbar multifidus (LM) thickness at rest and during activation, between individuals with chronic low back pain (CLBP) and controls. Lumbar multifidus thickness was assessed via rehabilitative ultrasound imaging (RUSI), and was performed in prone and standing both at rest and during muscle activation i.e. with a contralateral arm lift (CAL). Twenty participants were assessed; ten CLBP participants and ten controls. Rehabilitative ultrasound imaging was used to measure LM thickness and percentage thickness change at L4/L5 and L5/S1 in four positions; prone at rest, prone during activation with a CAL, standing at rest, and standing during activation with a CAL. Independent and paired t-tests were used to calculate differences in LM thickness and percentage thickness change between groups, and also between sides in the CLBP group. There was a significant difference in LM percentage thickness change in standing during activation with a CAL; the CLBP demonstrated a greater percentage thickness increase at L5/S1 compared to the controls, (p = 0.05). There were no differences between groups at the L4/L5 level for this position. There were no differences between the groups for LM thickness or percentage thickness change in prone or standing at rest, or during activation with a CAL in prone. Within the CLBP group, no significant between side differences were found. These results give preliminary insight into possible differences in LM contractile behaviour during functional movements in CLBP, however, larger scale research is warranted.

Effect of Oxidative Stress on the Expression of Thin Filament-Associated Proteins in Gastric Smooth Muscle Cells

Thin filament-associated proteins such as calponin, caldesmon, and smoothelin are believed to regulate acto-myosin interaction and thus, muscle contraction. Oxidative stress has been found to affect the normal contractile behavior of smooth muscle and is involved in the pathogenesis of a number of human diseases such as diabetes mellitus, hypertension, and atherosclerosis. However, very little is known about the effect of oxidative stress on the expression of smooth muscle contractile proteins. The aim of the current study is to investigate the effect of oxidative stress on the expression of thin filament-associated proteins in rat gastric smooth muscle. Single smooth muscle cells of the stomach obtained from Sprague-Dawley rats were used. Muscle cells were treated with hydrogen peroxide (H2O2) (500 μM) for 30 min or the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) (1 mM) for 90 min to induce oxidative stress. Calponin, caldesmon, and smoothelin expressions were measured via specifically designed enzyme-linked immunosorbent assay. We found that exposure to exogenous H2O2 or incubation of dispersed gastric muscle cells with SIN-1 significantly increased the expression of calponin, caldesmon, and smoothelin proteins. In conclusion: oxidative stress increases the expression of thin filament-associated proteins in gastric smooth muscle, suggesting an important role in gastrointestinal motility disorders associated with oxidative stress.

Vasoactive agonists exert dynamic and coordinated effects on vascular smooth muscle cell elasticity, cytoskeletal remodelling and adhesion

In this study, we examined the ability of vasoactive agonists to induce dynamic changes in vascular smooth muscle cell (VSMC) elasticity and adhesion, and tested the hypothesis that these events are coordinated with rapid remodelling of the cortical cytoskeleton. Real-time measurement of cell elasticity was performed with atomic force microscopy (AFM) and adhesion was assessed with AFM probes coated with fibronectin (FN). Temporal data were analysed using an Eigen-decomposition method. Elasticity in VSMCs displayed temporal oscillations with three components at approximately 0.001, 0.004 and 0.07 Hz, respectively. Similarly, adhesion displayed a similar oscillatory pattern. Angiotensin II (ANG II, 10(-6) M) increased (+100%) the amplitude of the oscillations, whereas the vasodilator adenosine (ADO, 10(-4) M) reduced oscillation amplitude (-30%). To test whether the oscillatory changes were related to the architectural alterations in cortical cytoskeleton, the topography of the submembranous actin cytoskeleton (100-300 nm depth) was acquired with AFM. These data were analysed to compare cortical actin fibre distribution and orientation before and after treatment with vasoactive agonists. The results showed that ANG II increased the density of stress fibres by 23%, while ADO decreased the density of the stress fibres by 45%. AFM data were supported by Western blot and confocal microscopy. Collectively, these observations indicate that VSMC cytoskeletal structure and adhesion to the extracellular matrix are dynamically altered in response to agonist stimulation. Thus, vasoactive agonists probably invoke unique mechanisms that dynamically alter the behaviour and structure of both the VSMC cytoskeleton and focal adhesions to efficiently support the normal contractile behaviour of VSMCs.

Phenotypic Modulation of Smooth Muscle Cells by Chemical and Mechanical Cues of Electrospun Tecophilic/Gelatin Nanofibers

The ability of mature smooth muscle cells (SMCs) to modulate their phenotype in response to environmental cues is a critical issue related to vascular diseases. A tissue engineered vascular graft shall promote the contractile phenotype of vascular SMCs. To this aim, Tecophilic/gelatin (TP/gel) was electrospun at different weight ratios of TP/gelatin (100:0, 70:30, 50:50, 30:70), leading to differences in biochemical and mechanical properties of the nanofibers which in turn influenced the phenotype of SMCs. Results indicated that both the substrate with higher ligand density and lower stiffness could enhance SMC contractility and reduce cell proliferation. However, observing the highest SMCs contractility on electrospun TP(70)/gel(30) among the composite scaffolds demonstrated stiffness as the most critical parameter. Due to conflicting effects of softness versus minor fraction of gelatin (reduced ligand density) within TP(70)/gel(30) fibers, a relatively high proliferation of SMCs was still observed on TP(70)/gel(30) scaffold. The surface of TP(70)/gel(30) scaffold was further modified through physical adsorption of gelatin molecules so as to increase the ligand density on its surface, whereby a functional vascular construct that promotes the contractile behavior of SMCs with low cell proliferation was developed.

Correction: Corrigendum: Innexin gap junctions in nerve cells coordinate spontaneous contractile behavior in Hydra polyps

CORRIGENDUM: Innexin gap junctions in nerve cells coordinate spontaneous contractile behavior in Hydra polyps

Human skeletal muscle xenograft as a new preclinical model for muscle disorders

Development of novel therapeutics requires good animal models of disease. Disorders for which good animal models do not exist have very few drugs in development or clinical trial. Even where there are accepted, albeit imperfect models, the leap from promising preclinical drug results to positive clinical trials commonly fails, including in disorders of skeletal muscle. The main alternative model for early drug development, tissue culture, lacks both the architecture and, usually, the metabolic fidelity of the normal tissue in vivo. Herein, we demonstrate the feasibility and validity of human to mouse xenografts as a preclinical model of myopathy. Human skeletal muscle biopsies transplanted into the anterior tibial compartment of the hindlimbs of NOD-Rag1(null) IL2rγ(null) immunodeficient host mice regenerate new vascularized and innervated myofibers from human myogenic precursor cells. The grafts exhibit contractile and calcium release behavior, characteristic of functional muscle tissue. The validity of the human graft as a model of facioscapulohumeral muscular dystrophy is demonstrated in disease biomarker studies, showing that gene expression profiles of xenografts mirror those of the fresh donor biopsies. These findings illustrate the value of a new experimental model of muscle disease, the human muscle xenograft in mice, as a feasible and valid preclinical tool to better investigate the pathogenesis of human genetic myopathies and to more accurately predict their response to novel therapeutics.

PKC activation increases Ca2+sensitivity of permeabilized lymphatic muscle via myosin light chain 20 phosphorylation-dependent and -independent mechanisms

The contractile activity of muscle cells lining the walls of collecting lymphatics is responsible for generating and regulating flow within the lymphatic system. Activation of PKC signaling contributes to the regulation of smooth muscle contraction by enhancing sensitivity of the contractile apparatus to Ca(2+). It is currently unknown whether PKC signaling contributes to the regulation of lymphatic muscle contraction. We hypothesized that the activation of PKC signaling would increase the sensitivity of the lymphatic myofilament to Ca(2+). To test this hypothesis, we determined the effects of PKC activation with phorbol esters [PMA or phorbol dibutyrate (PDBu)] on the contractile behavior of α-toxin-permeabilized rat mesenteric and cervical lymphatics or the thoracic duct. The addition of PMA or PDBu induced a significant increase in the contractile force of submaximally activated α-toxin-permeabilized lymphatic muscle independent of a change in intracellular Ca(2+) concentration, and the Ca(2+)-force relationship of lymphatic muscle was significantly left shifted, indicating greater myofilament Ca(2+) sensitivity. Phorbol esters increased the maximal rate of force development, whereas the rate of relaxation was reduced. Western blot and immunohistochemistry data indicated that the initial rapid increase in tension development after stimulation by PDBu was associated with myosin light chain (MLC)20 phosphorylation; however, the later, steady-state Ca(2+) sensitization of permeabilized lymphatic muscle was not associated with increased phosphorylation of MLC20 at Ser(19), 17-kDa C-kinase-potentiated protein phosphatase-1 inhibitor at Thr(38), or caldesmon at Ser(789). Thus, these data indicate that PKC-dependent Ca(2+) sensitization of lymphatic muscle may involve MLC20 phosphorylation-dependent and -independent mechanism(s).