Mechanical stretch inhibits oxidized low density lipoprotein-induced apoptosis in vascular smooth muscle cells by up-regulating integrin alphavbeta3 and stablization of PINCH-1

To determine the mechanisms involved in regulating the balance between apoptosis and survival in vascular smooth muscle cells (VSMC), we studied anti-apoptotic stimuli that can counteract pro-apoptotic events in the process of early atherosclerotic lesions formation. Such a process involves VSMC accumulation even in the presence of oxidized low density lipoprotein (Ox-LDL). In the arch of the aorta, we find that integrin beta3 is higher than in descending arteries. In the advanced atherosclerosis lesion, we found an inverse correlation between the level of integrin beta3 and apoptosis (deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive). We also found an increase in integrin alphaVbeta3 (but not integrin beta1) expression in VSMC that are subjected to cyclic stretch. VSMC subjected to stretch as well as VSMC with forced expression of alphaVbeta3 were demonstrated to be resistant to Ox-LDL-induced cytoskeleton disruption and apoptosis. The anti-apoptotic effect of stretch was abolished by treatment of VSMC with small interfering RNA against integrin beta3 as well as VSMC isolated from integrin beta3 knock-out mice. Disruption of the cytoskeleton abolished the protective effect of stretch or alphaVbeta3 overexpression on Ox-LDL-induced activation of Bax and apoptosis. We also demonstrated that stretch-mediated protection of Ox-LDL-induced apoptosis involved stabilization of PINCH-1; Ox-LDL decreased the level of PINCH-1, but the application of mechanical stretch or overexpression of either integrin beta1 or integrin beta3 prevented its down-regulation. In the arteries of integrin beta3 null mice, there were lower levels of PINCH-1 and ILK-1. Moreover, deletion of integrin beta3 in VSMC abolished the stretch protective effect on PINCH-1. Small interfering RNA-mediated knockdown of PINCH-1 disrupted the cytoskeleton and caused apoptosis of VSMC. These findings provided experimental evidence that mechanical stretch acted as a survival factor in the arches of aortas. Furthermore, mechanical stretch prevented VSMC from apoptosis via a mechanism that involves alphaVbeta3 integrin expression, stabilization of PINCH-1, and remodeling of the cytoskeleton.     

Genetic Models in Applied Physiology. Merosin deficiency leads to alterations in passive and active skeletal muscle mechanics.

The role of extracellular elements on the mechanical properties of skeletal muscles is unknown. Merosin is an essential extracellular matrix protein that forms a mechanical junction between the sarcolemma and collagen. Therefore, it is possible that merosin plays a role in force transmission between muscle fibers and collagen. We hypothesized that deficiency in merosin may alter passive muscle stiffness, viscoelastic properties, and contractile muscle force in skeletal muscles. We used the dy/dy mouse, a merosin-deficient mouse model, to examine changes in passive and active muscle mechanics. After mice were anesthetized and the diaphragm or the biceps femoris hindlimb muscle was excised, passive length-tension relationships, stress-relaxation curves, or isometric contractile properties were determined with an in vitro biaxial mechanical testing apparatus. Compared with controls, extensibility was smaller in the muscle fiber direction and the transverse fiber direction of the mutant mice. The relaxed elastic modulus was smaller in merosin-deficient diaphragms compared with controls. Interestingly, maximal muscle tetanic stress was depressed in muscles from the mutant mice during uniaxial loading but not during biaxial loading. However, presence of transverse passive stretch increases maximal contractile stress in both the mutant and normal mice. Our data suggest that merosin contributes to muscle passive stiffness, viscoelasticity, and contractility and that the mechanism by which force is transmitted between adjacent myofibers via merosin possibly in shear.     

Loss of desmin triggers mechanosensitivity and up-regulation of Ankrd1 expression through Akt-NF-κB signaling pathway in smooth muscle cells

Muscle cells, including human airway smooth muscle cells (HASMCs) express ankyrin repeat protein 1 (Ankrd1), a member of ankyrin repeat protein family. Ankrd1 efficiently interacts with the type III intermediate filament desmin. Our earlier study showed that desmin is an intracellular load-bearing protein that influences airway compliance, lung recoil, and airway contractile responsiveness. These results suggest that Ankrd1 and desmin may play important roles on ASMC homeostasis. Here we show that small interfering (si)RNA-mediated knockdown of the desmin gene in HASMCs, recombinant HASMCs (reHASMCs), up-regulates Ankrd1 expression. Moreover, loss of desmin in HASMCs increases the phosphorylation of Akt, inhibitor of κB kinase (IKK)-α, and inhibitor of κB (IκB)-α proteins, leading to NF-κB activation. Treatment of reHASMCs with Akt, IKKα, IκBα, or NF-κB inhibitor inhibits the loss of desmin-induced Ankrd1 up-regulation, suggesting Akt/NF-κB-mediated Ankrd1 regulation. Transfection of reHASMCs with siRNA specific for p50 or p65 corroborates the NF-κB-mediated Ankrd1 regulation. Luciferase reporter assays show that NF-κB directly binds on Ankrd1 promoter and up-regulates Ankrd1 levels. Overall, our data provide a new link between desmin and Ankrd1 regulation, which may be important for ASMC homeostasis.     

Effects of transverse fiber stiffness and central tendon on displacement and shape of a simple diaphragm model

Boriek, Aladin M., and Joseph R. Rodarte. Effects oftransverse fiber stiffness and central tendon on displacement and shapeof a simple diaphragm model. J. Appl. Physiol. 82(5): 1626-1636, 1997.Our previous experimental results (A. M. Boriek, S. Lui, and J. R. Rodarte. J. Appl. Physiol. 75:527-533, 1993 and A. M. Boriek, T. A. Wilson, and J. R. Rodarte.J. Appl. Physiol. 76: 223-229, 1994) showed that1) costal diaphragm shape is similar at functional residualcapacity and end inspiration regardless of whether the diaphragm muscleshortens actively (increased tension) or passively (decreased tension);2) diaphragmatic muscle length changes minimally in thedirection transverse to the muscle fibers, suggesting the diaphragm maybe inextensible in that direction; and 3) the central tendon isnot stretched by physiological stresses. A two-dimensional orthotropicmaterial has two different stiffnesses in orthogonal directions. In theplane tangent to the muscle surface, these directions are along thefibers and transverse to the fibers. We wondered whether orthotropicmaterial properties in the muscular region of the diaphragm andinextensibility of the central tendon might contribute to the constancyof diaphragm shape. Therefore, in the present study, we examined theeffects of stiffness transverse to muscle fibers and inextensibility ofthe central tendon on diaphragmatic displacement and shape. Finiteelement hemispherical models of the diaphragm were developed by usingpressurized isotropic and orthotropic membranes with a wide range ofstiffness ratios. We also tested heterogeneous models, in which themuscle sheet was an orthotropic material, having transverse fiberstiffness greater than that along the fibers, with the central tendonbeing an inextensible isotropic cap. These models revealed thatincreased transverse stiffness limits the shape change of thediaphragm. Furthermore, an inextensible cap simulating the centraltendon dramatically limits the change in shape as well as the membrane displacement in response to pressure. These findings provide a plausible mechanism by which the diaphragm maintains similar shapes despite different physiological loads. This study suggests that changesof diaphragm shape are restricted because the central tendon isessentially inextensible and stiffness in the direction transverse tothe muscle fibers is greater than stiffness along the fibers.

     

Mechanical advantage of the canine diaphragm

The mechanical advantage (µ) of a respiratorymuscle is defined as the respiratory pressure generated per unit musclemass and per unit active stress. The value of µ can be obtained by measuring the change in the length of the muscle during inflation ofthe passive lung and chest wall. We report values of µ for themuscles of the canine diaphragm that were obtained by measuring thelengths of the muscles during a passive quasistatic vital capacitymaneuver. Radiopaque markers were attached along six muscle bundles ofthe costal and two muscle bundles of the crural left hemidiaphragms offour bred-for-research beagle dogs. The three-dimensional locations ofthe markers were obtained from biplane video-fluoroscopic images takenat four volumes during a passive relaxation maneuver from total lungcapacity to functional residual capacity in the prone and supinepostures. Muscle lengths were determined as a function of lung volume,and from these data, values of µ were obtained. Values of µ arefairly uniform around the ventral midcostal and crural diaphragm butsignificantly lower at the dorsal end of the costal diaphragm. Theaverage values of µ are 0.35 ± 0.18 and 0.27 ± 0.16 cmH₂O · g¹ · kg¹ · cm²in the prone and supine dog, respectively. These values are 1.5-2 times larger than the largest values of µ of the intercostal muscles in the supine dog. From these data we estimate that during spontaneous breathing the diaphragm contributes ~40% of inspiratory pressure inthe prone posture and ~30% in the supine posture. Passiveshortening, and hence µ, in the upper one-third of inspiratorycapacity is less than one-half of that at lower lung volume. The lower µ is attributed primarily to a lower abdominal compliance at highlung volume.

     

Shape and tension distribution of the active canine diaphragm

Both diaphragm shape and tension contribute to transdiaphragmatic pressure, but of the three variables, tension is most difficult to measure. We measured transdiaphragmatic pressure and the global shape of the in vivo canine diaphragm and used principles of mechanics to compute the tension distribution. Our hypotheses were that 1) tension in the active diaphragm is nonuniform with greater tension in the central tendon than in the muscular regions; 2) maximum tension is essentially oriented in the muscle fiber direction, whereas minimum tension is orthogonal to the fiber direction; and 3) during submaximal activation change in the in vivo global shape is small. Metallic markers, each 2 mm in length, were implanted surgically on the peritoneal surface of the diaphragm at 1.5- to 2.0-cm intervals along the muscle bundles at the midline, ventral, middle, and dorsal regions of the left costal diaphragm and along a muscle bundle of the crural diaphragm. Postsurgery, a biplane videofluoroscopic system was used to determine the in vivo three-dimensional coordinates of the markers at end expiration and end inspiration during quiet breathing as well as at end-inspiratory efforts against an occluded airway at lung volumes of functional residual capacity and at one-third maximum inspiratory capacity increments in volume to total lung capacity. A surface was fit to the marker locations using a two-dimensional spline algorithm. Diaphragm surface was modeled as a pressurized membrane, and tension distribution in the active diaphragm was computed using the ANSYS finite element program. We showed that the peak of the diaphragm dome was closer to the ventral surface than to the dorsal surface and that there was a depression or valley in the crural region. In the supine position, during inspiratory efforts, the caudal displacement of the dorsal region of the diaphragm was greater than that of the dome, and the valley along the crural diaphragm was accentuated. In contrast, at lower lung volumes in the prone posture, the caudal displacement of the dome was greater than that of the crural region. At end of inspiration, transdiaphragmatic pressure was approximately 6.5 cmH2O, and tensions were nonuniform in the diaphragm. Maximum principal stress sigma(1) of central tendon was found to be greater than sigma(1) of the costal region, and that was greater than sigma(1) of the crural region, with values of 14-34, 14-29, and 4-14 g/cm, respectively. The corresponding data of the minimum principal stress sigma(2) were 9-18, 3-9, and 0-1.5 g/cm, respectively. Maximum principal tension was approximately parallel to the muscle fibers, whereas minimum tension was essentially orthogonal to the longitudinal direction of the muscle fibers. In the muscular region, sigma(1) was approximately 3-fold sigma(2), whereas in the central tendon, sigma(1) was only approximately 1.5-fold sigma(2.).     

Desmin integrates the three-dimensional mechanical properties of muscles

Striated muscle is a linear motor whose properties have been defined in terms of uniaxial structures. The question addressed here is what contribution is made to the properties of this motor by extramyofilament cytoskeletal structures that are not aligned in parallel with the myofilaments. This question arose from observations that transverse loads increase muscle force production in diaphragm but not in the hindlimb muscle, thereby indicating the presence of structures that couple longitudinal and transverse properties of diaphragmatic muscle. Furthermore, we find that the diaphragms of null mutants for the cytoskeletal protein desmin show 1) significant reductions in coupling between the longitudinal and transverse properties, indicating for the first time a role for a specific protein in integrating the three-dimensional mechanical properties of muscle, 2) significant reductions in the stiffness and viscoelasticity of muscle, and 3) significant increases in tetanic force production. Thus desmin serves a complex mechanical function in diaphragm muscle by contributing both to passive stiffness and viscoelasticity and to modulation of active force production in a three-dimensional structural network. Our finding changes the paradigm of force transmission among cells by placing our understanding of the function of the cytoskeleton in the context of the structural and mechanical complexity of muscles.