Lung and alveolar wall elastic and hysteretic behavior in rats: effects of in vivo elastase treatment.

We investigated the relationship between the microscopic elastic and hysteretic behavior of the alveolar walls and the macroscopic mechanical properties of the whole lung in an in vivo elastase-treated rat model of emphysema. We measured the input impedance of isolated lungs at three levels of transpulmonary pressure (Ptp) and used a linear model to estimate the dynamic elastance and hysteresivity of the lungs. The elastance of the normal lungs increased steeply with Ptp, whereas this dependence diminished in the treated lungs. Hysteresivity decreased significantly with Ptp in the normal lungs, but this dependence disappeared in the treated lungs. To investigate the microscopic origins of these changes, the alveolar walls were immunofluorescently labeled in small tissue strips. By using a fluorescent microscope, the lengths and angular orientations of individual alveolar walls were followed during cyclic uniaxial stretching of the tissue strips. The microstrains (relative change in segment length) and changes in angle of the alveolar walls showed considerable heterogeneity, which was interpreted in terms of a network model. In the normal strips, the alveolar walls showed larger angular changes compared with the treated tissue, whereas the alveolar walls of the treated tissue tended to be more extensible. Hysteresis in the average angle change was also larger in the treated tissue than in the normal tissue. We conclude that the decreased Ptp dependence of elastance and the constant hysteresivity in the treated lungs are related to microstructural remodeling and network phenomena at the level of the alveolar walls.     

Changes in proteoglycans and lung tissue mechanics during excessive mechanical ventilation in rats

Excessive mechanical ventilation results in changes in lung tissue mechanics. We hypothesized that changes in tissue properties might be related to changes in the extracellular matrix component proteoglycans (PGs). The effect of different ventilation regimens on lung tissue mechanics and PGs was examined in an in vivo rat model. Animals were anesthetized, tracheostomized, and ventilated at a tidal volume of 8 (VT(8)), 20, or 30 (VT(30)) ml/kg, positive end-expiratory pressure of 0 (PEEP(0)) or 1.5 (PEEP(1.5)) cmH(2)O, and frequency of 1.5 Hz for 2 h. The constant-phase model was used to derive airway resistance, tissue elastance, and tissue damping. After physiological measurements, one lung was frozen for immunohistochemistry and the other was reserved for PG extraction and Western blotting. After 2 h of mechanical ventilation, tissue elastance and damping were significantly increased in rats ventilated at VT(30)PEEP(0) compared with control rats (ventilated at VT(8)PEEP(1.5)). Versican, basement membrane heparan sulfate PG, and biglycan were all increased in rat lungs ventilated at VT(30)PEEP(0) compared with control rats. At VT(30)PEEP(0), heparan sulfate PG and versican staining became prominent in the alveolar wall and airspace; biglycan was mostly localized in the airway wall. These data demonstrate that alterations in lung tissue mechanics with excessive mechanical ventilation are accompanied by changes in all classes of extracellular matrix PG.     

Effect of glycosaminoglycan degradation on lung tissue viscoelasticity

We tested the hypothesis that matrix glycosaminoglycans contribute to lung tissue viscoelasticity. We exposed lung parenchymal strips to specific degradative enzymes (chondroitinase ABC, heparitinase I, and hyaluronidase) and determined whether the mechanical properties of the tissue were affected. Subpleural parenchymal strips were obtained from Sprague-Dawley rats and suspended in a Krebs-filled organ bath. One end of the strip was attached to a force transducer and the other to a servo-controlled lever arm that effected sinusoidal oscillations. Recordings of tension and length at different amplitudes and frequencies of oscillation were recorded before and after enzyme exposure. Resistance, dynamic elastance, and hysteresivity were estimated by fitting the equation of motion to changes in tension and length. Quasi-static stress-strain curves were also obtained. Exposure to chondroitinase and heparitinase I caused significant increases in hysteresivity, no decrement in resistance, and similar decreases in dynamic elastance relative to control strips exposed to Krebs solution only. Conversely, measures of static elastance were different in treated versus control strips. Hyaluronidase treatment did not alter any of the mechanical measures. These data demonstrate that digestion of chondroitin sulfate and heparan sulfate alters the mechanical behavior of lung parenchymal tissues.     

Volume dependence of airway and tissue impedances in mice.

We measured respiratory input impedance (1-25 Hz) in mice and obtained parameters for airway and tissue mechanics by model fitting. Lung volume was varied by inflating to airway opening pressure (Pao) between 0 and 20 cm H2O. The expected pattern of changes in respiratory mechanics with increasing lung volume was seen: a progressive fall in airway resistance and increases in the coefficients of tissue damping and elastance. A surprising pattern was seen in hysteresivity (eta), with a plateau at low lung volumes (Pao < 10 cm H2O), a sharp fall occurring between 10 and 15 cm H2O, and eta approaching a second (lower) plateau at higher lung volumes. Studies designed to elucidate the mechanism(s) behind this behavior revealed that this was not due to chest wall properties, differences in volume history at low lung volume, time dependence of volume recruitment, or surface-acting forces. Our data are consistent with the notion that at low lung volumes the mechanics of the tissue matrix determine eta, whereas at high lung volumes the properties of individual fibers (collagen) become more important.     

Effects of heterogeneities on the partitioning of airway and tissue properties in normal mice.

We measured the mechanical properties of the respiratory system of C57BL/6 mice using the optimal ventilation waveform method in closed- and open-chest conditions at different positive end-expiratory pressures. The tissue damping (G), tissue elastance (H), airway resistance (Raw), and hysteresivity were obtained by fitting the impedance data to three different models: a constant-phase model by Hantos et al. (Hantos Z, Daroczy B, Suki B, Nagy S, Fredberg JJ. J Appl Physiol 72: 168-178, 1992), a heterogeneous Raw model by Suki et al. (Suki B, Yuan H, Zhang Q, Lutchen KR. J Appl Physiol 82: 1349-1359, 1997), and a heterogeneous H model by Ito et al. (Ito S, Ingenito EP, Arold SP, Parameswaran H, Tgavalekos NT, Lutchen KR, Suki B. J Appl Physiol 97: 204-212, 2004). Both in the closed- and open-chest conditions, G and hysteresivity were the lowest and Raw the highest in the heterogeneous Raw model, and G and H were the largest in the heterogeneous H model. Values of G, Raw, and hysteresivity were significantly higher in the closed-chest than in the open-chest condition. However, H was not affected by the conditions. When the tidal volume of the optimal ventilation waveform was decreased from 8 to 4 ml/kg in the closed-chest condition, G and hysteresivity significantly increased, but there were smaller changes in H or Raw. In summary, values of the obtained mechanical properties varied among these models, primarily due to heterogeneity. Moreover, the mechanical parameters were significantly affected by the chest wall and tidal volume in mice. Contribution of the chest wall and heterogeneity to the mechanical properties should be carefully considered in physiological studies in which partitioning of airway and tissue properties are attempted.     

Hysteresivity of the lung and tissue strip in the normal rat: effects of heterogeneities.

We measured lung impedance in rats in closed chest (CC), open chest (OC), and isolated lungs (IL) at four transpulmonary pressures with a optimal ventilator waveform. Data were analyzed with an homogeneous linear or an inhomogeneous linear model. Both models include tissue damping and elastance and airway inertance. The homogeneous linear model includes airway resistance (Raw), whereas the inhomogeneous linear model has a continuous distribution of Raw characterized by the mean Raw and the standard deviation of Raw (SDR). Lung mechanics were compared with tissue strip mechanics at frequencies and operating stresses comparable to those during lung impedance measurements. The hysteresivity (eta) was calculated as tissue damping/elastance. We found that 1) airway and tissue parameters were different in the IL than in the CC and OC conditions; 2) SDR was lowest in the IL; and 3) eta in IL at low transpulmonary pressure was similar to eta in the tissue strip. We conclude that eta is primarily determined by lung connective tissue, and its elevated estimates from impedance data in the CC and OC conditions are a consequence of compartment-like heterogeneity being greater in CC and OC conditions than in the IL.     

Dynamic nonlinearity of lung tissue: frequency dependence and harmonic distortion

Harmonic distortion (HD) is a simple approach to analyze lung tissue nonlinear phenomena. This study aimed to characterize frequency-dependent behavior of HD at several amplitudes in lung tissue strips from healthy rats and its influence on the parameters of linear analysis. Lung strips (n = 17) were subjected to sinusoidal deformation at three different strain amplitudes (Δε) and fixed operational stress (12 hPa) among various frequencies, between 0.03 and 3 Hz. Input HD was <2% in all cases. The main findings in our study can be summarized as follows: 1) harmonic distortion of stress (HD) showed a positive frequency and amplitude dependence following a power law with frequency; 2) HD correlated significantly with the frequency response of dynamic elastance, seeming to converge to a limited range at an extrapolated point where HD=0; 3) the relationship between tissue damping (G) and HD(ω=1) (the harmonic distortion at ω=1 rad/s) was linear and accounted for a large part of the interindividual variability of G; 4) hysteresivity depended linearly on κ (the power law exponent of HD with ω); and 5) the error of the constant phase model could be corrected by taking into account the frequency dependence of harmonic distortion. We concluded that tissue elasticity and tissue damping are coupled at the level of the stress-bearing element and to the mechanisms underlying dynamic nonlinearity of lung tissue.     

Variability in preterm lamb lung mechanics after intra-amniotic endotoxin is associated with changes in surfactant pool size and morphometry

Antenatal exposure to intra-amniotic (i.a.) endotoxin initiates a complex series of events, including an inflammatory cascade, increased surfactant production, and alterations to lung structure. Using the low frequency forced oscillation technique as a sensitive tool for measurement of respiratory impedance, we aimed to determine which factors contributed most to measured changes in lung mechanics. Respiratory impedance data obtained from sedated preterm lambs exposed to either i.a. injection with saline or 20 mg of endotoxin 1, 2, 4, and 15 days before delivery at 125 days gestation were studied, and association with indexes of standard lung morphometry, inflammatory response, and alveolar surfactant-saturated phosphatidylcholine (Sat PC) pool size was demonstrated. Reduction in tissue impedance with increasing interval between exposure and delivery was evident as early as 4 days after i.a. endotoxin injection, coinciding with resolution of inflammatory reaction, increased alveolar surfactant pools, and contribution of alveolar ducts to the parenchymal fraction, and a later decrease in the tissue component of the parenchymal fraction. Decreases in tissue damping (resistance) were more marked than decreases in tissue elastance. Log alveolar Sat PC accounted for most variability in tissue damping (88.9%) and tissue elastance (73.4%), whereas tissue fraction contributed 2 and 6.4%, respectively. The alveolar Sat PC pool size was the sole factor contributing to change in tissue hysteresivity. No changes were observed in airway resistance. Despite the complex cascade of events initiated by antenatal endotoxin exposure, variability in lung tissue mechanics is associated primarily with changes in alveolar Sat PC pool and lung morphology.     

Mechanics, nonlinearity, and failure strength of lung tissue in a mouse model of emphysema: possible role of collagen remodeling.

Enlargement of the respiratory air spaces is associated with the breakdown and reorganization of the connective tissue fiber network during the development of pulmonary emphysema. In this study, a mouse (C57BL/6) model of emphysema was developed by direct instillation of 1.2 IU of porcine pancreatic elastase (PPE) and compared with control mice treated with saline. The PPE treatment caused 95% alveolar enlargement (P = 0.001) associated with a 29% lower elastance along the quasi-static pressure-volume curves (P < 0.001). Respiratory mechanics were measured at several positive end-expiratory pressures in the closed-chest condition. The dynamic tissue elastance was 19% lower (P < 0.001), hysteresivity was 9% higher (P < 0.05), and harmonic distortion, a measure of collagen-related dynamic nonlinearity, was 33% higher in the PPE-treated group (P < 0.001). Whole lung hydroxyproline content, which represents the total collagen content, was 48% higher (P < 0.01), and alpha-elastin content was 13% lower (P = 0.16) in the PPE-treated group. There was no significant difference in airway resistance (P = 0.7). The failure stress at which isolated parenchymal tissues break during stretching was 40% lower in the PPE-treated mice (P = 0.002). These findings suggest that, after elastolytic injury, abnormal collagen remodeling may play a significant role in all aspects of lung functional changes and mechanical forces, leading to progressive emphysema.     

Desmin modulates lung elastic recoil and airway responsiveness

Desmin is a structural protein that is expressed in smooth muscle cells of both airways and alveolar ducts. Therefore, desmin could be well situated to participate in passive and contractile force transmission in the lung. We hypothesized that desmin modulates lung compliance, lung recoil pressure, and airway contractile response. To test this hypothesis, respiratory system complex impedance (Zin,rs) at different positive end-expiratory pressure (PEEP) levels and quasi-static pressure-volume data were obtained in desmin-null and wild-type mice at baseline and during methacholine administration. Airways and lung tissue properties were partitioned by fitting Zin,rs to a constant-phase model. Relative to controls, desmin-null mice showed 1) lower values for lung stiffness and recoil pressure at baseline and induced airway constriction, 2) greater negative PEEP dependence of H and airway resistance under baseline conditions and cholinergic stimulation, and 3) airway hyporesponsiveness. These results demonstrate that desmin is a load-bearing protein that stiffens the airways and consequently the lung and modulates airway contractile response.     

Lung tissue behavior during methacholine challenge in rabbits in vivo.

Previous studies have shown that lung challenge with smooth muscle agonists increases tissue viscance (Vti), which is the pressure drop between the alveolus and the pleura divided by the flow. Passive inflation also increases Vti. The purpose of the present study was to measure the changes in Vti during positive end-expiratory pressure- (PEEP) induced changes in lung volume and with a concentration-response curve to methacholine (MCh) in rabbits and to compare the effects of induced constriction vs. passive lung inflation on tissue mechanics. Measurements were made in 10 anesthetized open-chest mechanically ventilated New Zealand male rabbits exposed first to increasing levels of PEEP (3-12 cmH2O) and then to increasing concentrations of MCh aerosol (0.5-128 mg/ml). Lung elastance (EL), lung resistance (RL), and Vti were determined by adjusting the equation of motion to tracheal and alveolar pressures during tidal ventilation. Our results show that under baseline conditions, Vti accounted for a major proportion of RL; during both passive lung inflation and MCh challenge this proportion increased progressively. For the same level of change in EL, however, the increase in Vti was larger during MCh challenge than during passive inflation; i.e., the relationship between energy storage and energy dissipation or hysteresivity was dramatically altered. These results are consistent with a MCh-induced change in the intrinsic rheological properties of lung tissues unrelated to lung volume change per se. Lung tissue constriction is one possible explanation.     

Renal NF-kappaB activation and TNF-alpha upregulation correlate with salt-sensitive hypertension in Dahl salt-sensitive rats

Molecular mechanisms of salt-sensitive (SS) hypertension related to renal inflammation have not been defined. We seek to determine whether a high-salt (HS) diet induces renal activation of NF-kappaB and upregulation of TNF-alpha related to the development of hypertension in Dahl SS rats. Six 8-wk-old male Dahl SS rats received a HS diet (4%), and six Dahl SS rats received a low-sodium diet (LS, 0.3%) for 5 wk. In the end, mean arterial pressure was determined in conscious rats by continuous monitoring through a catheter placed in the carotid artery. Mean arterial pressure was significantly higher in the HS than the LS group (177.9 +/- 3.7 vs. 109.4 +/- 2.9 mmHg, P < 0.001). There was a significant increase in urinary albumin secretion in the HS group compared with the LS group (22.3 +/- 2.6 vs. 6.1 +/- 0.7 mg/day; P < 0.001). Electrophoretic mobility shift assay demonstrated that the binding activity of NF-kappaB p65 proteins in the kidneys of Dahl SS rats was significantly increased by 53% in the HS group compared with the LS group (P = 0.007). ELISA indicated that renal protein levels of TNF-alpha, but not IL-6, interferon-gamma, and CCL28, were significantly higher in the HS than the LS group (2.3 +/- 0.8 vs. 0.7 +/- 0.2 pg/mg; P = 0.036). We demonstrated that plasma levels of TNF-alpha were significantly increased by fivefold in Dahl SS rats on a HS diet compared with a LS diet. Also, we found that increased physiologically relevant sodium concentration (10 mmol/l) directly stimulated NF-kappaB activation in cultured human renal proximal tubular epithelial cells. These findings support the hypothesis that activation of NF-kappaB and upregulation of TNF-alpha are the important renal mechanisms linking proinflammatory response to SS hypertension.     

Sequential use of human-derived medium supplements favours cardiovascular tissue engineering

For clinical application of tissue engineering strategies, the use of animal-derived serum in culture medium is not recommended, because it can evoke immune responses in patients. We previously observed that human platelet-lysate (PL) is favourable for cell expansion, but generates weaker tissue as compared to culture in foetal bovine serum (FBS). We investigated if human serum (HS) is a better human supplement to increase tissue strength. Cells were isolated from venous grafts of 10 patients and expanded in media supplemented with PL or HS, to determine proliferation rates and expression of genes related to collagen production and maturation. Zymography was used to assess protease expression. Collagen contraction assays were used as a two-dimensional (2D) model for matrix contraction. As a prove of principle, 3D tissue culture and tensile testing was performed for two patients, to determine tissue strength. Cell proliferation was lower in HS-supplemented medium than in PL medium. The HS cells produced less active matrix metallo-proteinase 2 (MMP2) and showed increased matrix contraction as indicated by gel contraction assays and 3D-tissue culture. Tensile testing showed increased strength for tissues cultured in HS when compared to PL. This effect was more pronounced if cells were sequentially cultured in PL, followed by tissue culture in HS. These data suggest that sequential use of PL and HS as substitutes for FBS in culture medium for cardiovascular tissue engineering results in improved cell proliferation and tissue mechanical properties, as compared to use of PL or HS apart.     

THE EFFECT OF HUMAN ENDOTHELIAL CELL-DERIVED PROTEOGLYCANS ON HUMAN SMOOTH MUSCLE CELL GROWTH

Extracellular proteoglycans (PGs) purified from cultured human arterial endothelial cells were tested for their effects on the proliferation of human vascular smooth muscle cells (VSMC). Fractions containing perlecan, the basement membrane heparan sulphate (HS) PG, the large chondrotin sulphate (CS) proteoglycan from connective tissue and other immunoreactive CS did not inhibit the proliferation of human VSMC. Native endothelial extracellular matrix, which was shown to contain the same PGs, demonstrated a pronounced stimulatory effect on the proliferation of human VSMCs. This stimulatory effect was not removed by pre-incubation of the matrix with 1M NaCl, heparin, platelet extract or plasmin. These experiments demonstrate that PGs produced by human arterial endothelial cells do not inhibit the proliferation of VSMC. These data do not support the hypothesis that human endothelial cells, in vivo control the activation or proliferation of VSMCs directly by the secretion of a non-proliferative molecule. Instead they support the hypothesis that the endothelial cells counteract intimal hyperplasia of VSMC indirectly by providing a barrier from activating factors in the plasma.     

Antiproliferative heparan sulfate inhibiting hyaluronan and transforming growth factor-β expression in human lung fibroblast cells

The objective of this study was to examine the effects of heparan sulfate (HS) on factors involved in the remodeling of connective tissue observed in patients with fibrotic respiratory disorders such as asthma. A suitable working model is to stimulate human fetal lung fibroblasts in vitro with structurally different forms of HS. Highly sulfated and iduronic acid (IdoUA)-rich HS specifically decreased cell proliferaton, production of jyaluronan (HA), transforming growth factor (TGF)-β₁, and TFF-β-induced α-smooth muscle actin but did not affect the overall proteoglycan production in the cells. These repressed factors are suggested to play a critical role in the early stages of remodeling and myofibroblast activation. Low sulfated and IdoUA-poor HS did not display any effects on these factors. Furthermore, analysis of the protein expression pattern by two-dimensional gel electrophoresis revealed a 70% increased expression of annexin II, which has previously been shown to have a high affinity for both heparin and HS. Heat-shock protein 27 and arsenite translocating factor, both involved in actin organization and polymerization, were also increased in the HS-stimulated cells. Thus, the reduced expression of HA and TGF-β₁, both important in the development of fibrosis, seems to be mediated by pecific changes in protein expression of the fibroblast. The observed inhibition of cell proliferation, HA, and TGF-β₁ allows speculation of highly sulfated HS as a antifibrotic candidate in the early stage of remodeling.     

Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging

Background

Heparan sulfate (HS) is present on the surface of virtually all mammalian cells and is a major component of the extracellular matrix (ECM), where it plays a pivotal role in cell-cell and cell-matrix cross-talk through its large interactome. Disruption of HS biosynthesis in mice results in neonatal death as a consequence of malformed lungs, indicating that HS is crucial for airway morphogenesis. Neonatal mortality (~50%) in newborns with congenital diaphragmatic hernia (CDH) is principally associated with lung hypoplasia and pulmonary hypertension. Given the importance of HS for lung morphogenesis, we investigated developmental changes in HS structure in normal and hypoplastic lungs using the nitrofen rat model of CDH and semi-synthetic bacteriophage ('phage) display antibodies, which identify distinct HS structures.

Results

The pulmonary pattern of elaborated HS structures is developmentally regulated. For example, the HS4E4V epitope is highly expressed in sub-epithelial mesenchyme of E15.5 - E17.5 lungs and at a lower level in more distal mesenchyme. However, by E19.5, this epitope is expressed similarly throughout the lung mesenchyme. We also reveal abnormalities in HS fine structure and spatiotemporal distribution of HS epitopes in hypoplastic CDH lungs. These changes involve structures recognised by key growth factors, FGF2 and FGF9. For example, the EV3C3V epitope, which was abnormally distributed in the mesenchyme of hypoplastic lungs, is recognised by FGF2.

Conclusions

The observed spatiotemporal changes in HS structure during normal lung development will likely reflect altered activities of many HS-binding proteins regulating lung morphogenesis. Abnormalities in HS structure and distribution in hypoplastic lungs can be expected to perturb HS:protein interactions, ECM microenvironments and crucial epithelial-mesenchyme communication, which may contribute to lung dysmorphogenesis. Indeed, a number of epitopes correlate with structures recognised by FGFs, suggesting a functional consequence of the observed changes in HS in these lungs. These results identify a novel, significant molecular defect in hypoplastic lungs and reveals HS as a potential contributor to hypoplastic lung development in CDH. Finally, these results afford the prospect that HS-mimetic therapeutics could repair defective signalling in hypoplastic lungs, improve lung growth, and reduce CDH mortality.     

Active remodeling in idiopathic interstitial pneumonias: evaluation of collagen types XII and XIV.

Fibril-associated collagens with interrupted triple helices (FACITs) XII and XIV act as fibril organizers and assist in the maintenance of uniform fibril size. We investigated the spatial expression patterns of collagens XII and XIV in cryptogenic organizing pneumonia (COP)/organizing pneumonia (OP) and in idiopathic pulmonary fibrosis (IPF)/usual interstitial pneumonia (UIP) and compared them to normal human lung. Study subjects included 10 patients with COP/OP, 10 patients with IPF/UIP, and 8 control subjects. Immunostaining for collagens XII and XIV was carried out in paraffin-embedded human lung tissue sections. Picrosirius red histochemical staining for collagen I expression and electron microcopy to evaluate fibril diameter were also performed. In normal lung, collagens XII and XIV were expressed in perivascular and subpleural connective tissue. In COP/OP, both collagens showed intense staining in perivascular connective tissue, thickened alveolar septae, and subpleural areas. In IPF/UIP, XII and XIV were expressed in perivascular connective tissue, in areas of established fibrosis, and in areas of subpleural thickening. Only collagen XII was expressed in granulation tissue plugs in COP/OP and in fibroblastic foci in IPF/UIP. Collagen type I was overexpressed in fibrotic areas. Electron micrographs revealed obvious fibril diameter alteration and fusion in the same areas. FACITs XII and XIV are expressed in normal and fibrotic lung. Unlike collagen XIV, collagen XII was expressed in granulation tissue plugs in COP/OP and in fibroblast foci in IPF/UIP. This may suggest a possible distinct role for both collagens in the modulation of the extracellular matrix during the onset of fibrotic process.     

Paxillin phosphorylation, actin polymerization, noise temperature, and the sustained phase of swine carotid artery contraction

Histamine stimulation of swine carotid artery induces both contraction and actin polymerization. The importance of stimulus-induced actin polymerization is not known. Tyrosine phosphorylation of the scaffolding protein paxillin is thought to be an important regulator of actin polymerization. Noise temperature, hysteresivity, and phase angle are rheological measures of the fluidity of a tissue, i.e., whether the muscle is more a "Hookean solid" or a "Newtonian liquid." Y118 paxillin phosphorylation, crossbridge phosphorylation, actin polymerization, noise temperature, hysteresivity, phase angle, real stiffness, and stress were measured in intact swine carotid arteries that were depolarized with high K⁺ or stimulated with histamine. The initial rapid force development phase of high-K⁺ or histamine-induced contraction was associated with increased crossbridge phosphorylation but no significant change in Y118 paxillin phosphorylation, actin polymerization, noise temperature, hysteresivity, or phase angle. This suggests that the initial contraction was caused by the increase in crossbridge phosphorylation and did not alter the tissue's rheology. Only after full force development was there a significant increase in Y118 paxillin phosphorylation and actin polymerization associated with a significant decrease in noise temperature and hysteresivity. These data suggest that some part of the sustained contraction may depend on stimulated actin polymerization and/or a transition to a more "solid" rheology. Supporting this contention was the finding that an inhibitor of actin polymerization, latrunculin-A, reduced force while increasing noise temperature/hysteresivity. Further research is needed to determine whether Y118 paxillin phosphorylation, actin polymerization, and changes in rheology could have a role in arterial smooth muscle contraction. cytoskeleton; hysteresivity; latch hypothesis; vascular smooth muscle     

Mesenteric lymph from rats with trauma-hemorrhagic shock causes abnormal cardiac myocyte function and induces myocardial contractile dysfunction

Myocardial contractile dysfunction develops following trauma-hemorrhagic shock (T/HS). We have previously shown that, in a rat fixed pressure model of T/HS (mean arterial pressure of 30-35 mmHg for 90 min), mesenteric lymph duct ligation before T/HS prevented T/HS-induced myocardial contractile depression. To determine whether T/HS lymph directly alters myocardial contractility, we examined the functional effects of physiologically relevant concentrations of mesenteric lymph collected from rats undergoing trauma-sham shock (T/SS) or T/HS on both isolated cardiac myocytes and Langendorff-perfused whole hearts. Acute application of T/HS lymph (0.1-2%), but not T/SS lymph, induced dual inotropic effects on myocytes with an immediate increase in the amplitude of cell shortening (1.4 ± 0.1-fold) followed by a complete block of contraction. Similarly, T/HS lymph caused dual, positive and negative effects on cellular Ca2? transients. These effects were associated with changes in the electrophysiological properties of cardiac myocytes; T/HS lymph initially prolonged the action potential duration (action potential duration at 90% repolarization, 3.3 ± 0.4-fold), and this was followed by a decrease in the plateau potential and membrane depolarization. Furthermore, intravenous infusion of T/HS lymph, but not T/SS lymph, caused myocardial contractile dysfunction at 24 h after injection, which mimicked actual T/HS-induced changes; left ventricular developed pressure (LVDP) and the maximal rate of LVDP rise and fall (±dP/dt(max)) were decreased and inotropic response to Ca2? was blunted. However, the contractile responsiveness to β-adrenergic receptor stimulation in the T/HS lymph-infused hearts remained unchanged. These results suggest that T/HS lymph directly causes negative inotropic effects on the myocardium and that T/HS lymph-induced changes in myocyte function are likely to contribute to the development of T/HS-induced myocardial dysfunction.