Biophysical regulation of histone acetylation in mesenchymal stem cells

Histone deacetylation and acetylation are catalyzed by histone deacetylase (HDAC) and histone acetyltransferase, respectively, which play important roles in the regulation of chromatin remodeling, gene expression, and cell functions. However, whether and how biophysical cues modulate HDAC activity and histone acetylation is not well understood. Here, we tested the hypothesis that microtopographic patterning and mechanical strain on the substrate regulate nuclear shape, HDAC activity, and histone acetylation. Bone marrow mesenchymal stem cells (MSCs) were cultured on elastic membranes patterned with parallel microgrooves 10 μm wide that kept MSCs aligned along the axis of the grooves. Compared with MSCs on an unpatterned substrate, MSCs on microgrooves had elongated nuclear shape, a decrease in HDAC activity, and an increase of histone acetylation. To investigate anisotropic mechanical sensing by MSCs, cells on the elastic micropatterned membranes were subjected to static uniaxial mechanical compression or stretch in the direction parallel or perpendicular to the microgrooves. Among the four types of loads, compression or stretch perpendicular to the microgrooves caused a decrease in HDAC activity, accompanied by the increase in histone acetylation and slight changes of nuclear shape. Knocking down nuclear matrix protein lamin A/C abolished mechanical strain-induced changes in HDAC activity. These results demonstrate that micropattern and mechanical strain on the substrate can modulate nuclear shape, HDAC activity, and histone acetylation in an anisotropic manner and that nuclear matrix mediates mechanotransduction. These findings reveal a new mechanism, to our knowledge, by which extracellular biophysical signals are translated into biochemical signaling events in the nucleus, and they will have significant impact in the area of mechanobiology and mechanotransduction.     

N-cadherin-mediated cell adhesion determines the plasticity for cell alignment in response to mechanical stretch in cultured cardiomyocytes

Mechanical stretch has been implicated as the growth stimuli in the heart. Physiologically, mechanical stretch is reported to contribute to the orientation of cardiomyocytes, though the molecular mechanism remains to be elucidated. This study was designed to make clear functional significances of N-cadherin in plasticity of cell alignment in response to mechanical stretch. Neonatal rat cardiomyocytes, cultured on silicone dishes, were subjected to artificial uniaxial cyclic stretch. Mechanical stretch was started at certain times (3-75h) after seeding and continued for 24h. Stretch stimulation in 3h after cultivation promoted cell orientation running parallel to tension direction. In contrast, cardiac myocytes fail to align when exposed to stretch 24-75h after cultivation. To address the importance of N-cadherin in the responsiveness to stretch, the expression and distribution of N-cadherin were analyzed. Immediately after seeding, N-cadherin showed dispersed distributions. During cultivation, N-cadherin localized to cell-cell contacts accompanied by the upregulation of its protein. Next, to investigate influence of cell-cell adhesion, cardiomyocytes cultured for 72h were replated by trypsin treatment and exposed to stretch 3h after replating. The cardiomyocytes replated by trypsinization were oriented in parallel to tension direction by mechanical stretch. Finally, adenoviral transfection of dominant-negative N-cadherin recovered the ability to exhibit cell orientation in response to stretch. Our results suggested that N-cadherin was involved in the oriented responses of cardiomyocytes induced by mechanical stretch.     

N-cadherin-mediated cell adhesion determines the plasticity for cell alignment in response to mechanical stretch in cultured cardiomyocytes

Mechanical stretch has been implicated as the growth stimuli in the heart. Physiologically, mechanical stretch is reported to contribute to the orientation of cardiomyocytes, though the molecular mechanism remains to be elucidated. This study was designed to make clear functional significances of N-cadherin in plasticity of cell alignment in response to mechanical stretch. Neonatal rat cardiomyocytes, cultured on silicone dishes, were subjected to artificial uniaxial cyclic stretch. Mechanical stretch was started at certain times (3-75 h) after seeding and continued for 24 h. Stretch stimulation in 3 h after cultivation promoted cell orientation running parallel to tension direction. In contrast, cardiac myocytes fail to align when exposed to stretch 24-75 h after cultivation. To address the importance of N-cadherin in the responsiveness to stretch, the expression and distribution of N-cadherin were analyzed. Immediately after seeding, N-cadherin showed dispersed distributions. During cultivation, N-cadherin localized to cell-cell contacts accompanied by the upregulation of its protein. Next, to investigate influence of cell-cell adhesion, cardiomyocytes cultured for 72 h were replated by trypsin treatment and exposed to stretch 3 h after replating. The cardiomyocytes replated by trypsinization were oriented in parallel to tension direction by mechanical stretch. Finally, adenoviral transfection of dominant-negative N-cadherin recovered the ability to exhibit cell orientation in response to stretch. Our results suggested that N-cadherin was involved in the oriented responses of cardiomyocytes induced by mechanical stretch.     

Calcium not strain regulates localization of alpha-myosin heavy chain mRNA in oriented cardiac myocytes

Our aim was to test a hypothesis that localization of the alpha-myosin heavy chain (alpha-MyHC) mRNA in oriented neonatal rat cardiomyocytes is regulated either by calcium, or by mechanical strain, or by both. Myocytes, grown on collagen aligned on stretchable silicone membranes, were elongated and had an increased length to width ratio (L/W) compared with randomly oriented myocytes grown on conventional substrata. Oriented cells were stretched by 10% in the longitudinal direction, in the transverse direction or passively unloaded for 6 h. As expected, shape changes followed these mechanical deformations. In situ hybridization was used to determine the localization of alpha-MyHC mRNA by quantitative analysis of optical density under various mechanical perturbations in myocytes that were either spontaneously beating or treated with verapamil (10 mM) to block influx of calcium. Unstretched, longitudinally stretched, and cells stretched transversely all had mRNA dispersed to their extremities. Verapamil treatment resulted in a perinuclear pattern of mRNA under all three mechanical perturbations. Additionally, mRNA distribution was examined in myocytes that were passively unloaded in the presence and absence of verapamil. Unloading myocytes with intact calcium cycling does not result in a perinuclear accumulation of mRNA. These data suggest that calcium is essential for alpha-MyHC mRNA distribution throughout the cell whereas stretch and alignment affect myocyte shape but have little effect on mRNA localization.     

Microtubule dynamics regulate cyclic stretch-induced cell alignment in human airway smooth muscle cells

Microtubules are structural components of the cytoskeleton that determine cell shape, polarity, and motility in cooperation with the actin filaments. In order to determine the role of microtubules in cell alignment, human airway smooth muscle cells were exposed to cyclic uniaxial stretch. Human airway smooth muscle cells, cultured on type I collagen-coated elastic silicone membranes, were stretched uniaxially (20% in strain, 30 cycles/min) for 2 h. The population of airway smooth muscle cells which were originally oriented randomly aligned near perpendicular to the stretch axis in a time-dependent manner. However, when the cells treated with microtubule disruptors, nocodazole and colchicine, were subjected to the same cyclic uniaxial stretch, the cells failed to align. Lack of alignment was also observed for airway smooth muscle cells treated with a microtubule stabilizer, paclitaxel. To understand the intracellular mechanisms involved, we developed a computational model in which microtubule polymerization and attachment to focal adhesions were regulated by the preexisting tensile stress, pre-stress, on actin stress fibers. We demonstrate that microtubules play a central role in cell re-orientation when cells experience cyclic uniaxial stretching. Our findings further suggest that cell alignment and cytoskeletal reorganization in response to cyclic stretch results from the ability of the microtubule-stress fiber assembly to maintain a homeostatic strain on the stress fiber at focal adhesions. The mechanism of stretch-induced alignment we uncovered is likely involved in various airway functions as well as in the pathophysiology of airway remodeling in asthma.     

Altered PKC expression and phosphorylation in response to the nature, direction, and magnitude of mechanical stretch

Protein kinase C (PKC) isozymes have been shown to play a role in mechanotransduction in a variety of cell types. We sought to identify the PKC isozymes involved in transducing mechanical (cyclic vs. static), direction and intensity of stretch by examining changes in protein expression and phosphorylation. We used a 3-dimensional culture system with aligned neonatal rat cardiac myocytes on silastic membranes. Myocytes were subjected to either cyclic stretch at 5 cycles/min or static stretch for a period of 24 h at intensities of 0%, 2.5%, 5%, or 10% of full membrane length. Stretch was applied in perpendicular or parallel directions to myocyte alignment. PKC delta was most sensitive to stretch applied perpendicular to myocyte alignment regardless of the nature of stretch, while phospho PKC delta T505 increased in response to static-perpendicular stretch. PKC epsilon expression was altered by cyclic stretch but not static stretch, while phospho PKC epsilon S719 remained unchanged. PKC alpha expression was not altered by stretch; however, phospho PKC alpha S657 increased in a dose-dependent manner following cyclic-perpendicular stretch. Our results indicate that changes in PKC expression and phosphorylation state may be a mechanism for cardiac myocytes to discriminate between the nature, direction, and intensity of mechanical stretch.     

Aortic valve leaflet mechanical properties facilitate diastolic valve function

This work was concerned with the numerical simulation of the behaviour of aortic valves whose material can be modelled as non-linear elastic anisotropic. Linear elastic models for the valve leaflets with parameters used in previous studies were compared with hyperelastic models, incorporating leaflet anisotropy with pronounced stiffness in the circumferential direction through a transverse isotropic model. The parameters for the hyperelastic models were obtained from fits to results of orthogonal uniaxial tensile tests on porcine aortic valve leaflets. The computational results indicated the significant impact of transverse isotropy and hyperelastic effects on leaflet mechanics; in particular, increased coaptation with peak values of stress and strain in the elastic limit. The alignment of maximum principal stresses in all models follows approximately the coarse collagen fibre distribution found in aortic valve leaflets. The non-linear elastic leaflets also demonstrated more evenly distributed stress and strain which appears relevant to long-term scaffold stability and mechanotransduction.     

Orientation of neurites influences severity of mechanically induced tau pathology

Chronic traumatic encephalopathy is a neurodegenerative disease associated with repeated traumatic brain injury (TBI). Chronic traumatic encephalopathy is a tauopathy, in which cognitive decline is accompanied by the accumulation of neurofibrillary tangles of the protein tau in patients’ brains. We recently found that mechanical force alone can induce tau mislocalization to dendritic spines and loss of synaptic function in in vitro neuronal cultures with random cell organization. However, in the brain, neurons are highly aligned, so here we aimed to determine how neuronal organization influences early-stage tauopathy caused by mechanical injury. Using microfabricated cell culture constructs to control the growth of neurites and an in vitro simulated TBI device to apply controlled mechanical deformation, we found that neuronal orientation with respect to the direction of a uniaxial high-strain-rate stretch injury influences the degree of tau pathology in injured neurons. We found that a mechanical stretch applied parallel to the neurite alignment induces greater mislocalization of tau proteins to dendritic spines than does a stretch with the same strain applied perpendicular to the neurites. Synaptic function, characterized by the amplitude of miniature excitatory postsynaptic currents, was similarly decreased in neurons with neurites aligned parallel to stretch, whereas in neurons aligned perpendicular to stretch, it had little to no functional loss. Experimental injury parameters (strain, strain rate, direction of stretch) were combined with a standard viscoelastic solid model to show that in our in vitro model, neurite work density during stretch correlates with tau mislocalization. These findings suggest that in a TBI, the magnitude of brain deformation is not wholly predictive of neurodegenerative consequences of TBI but that deformation relative to local neuronal architecture and the neurite mechanical energy during injury are better metrics for predicting trauma-induced tauopathy.     

Analysis and interpretation of stress fiber organization in cells subject to cyclic stretch

Numerical simulations that incorporate a biochemomechanical model for the contractility of the cytoskeleton have been used to rationalize the following observations. Uniaxial cyclic stretching of cells causes stress fibers to align perpendicular to the stretch direction, with degree of alignment dependent on the stretch strain magnitude, as well as the frequency and the transverse contraction of the substrate. Conversely, equibiaxial cyclic stretching induces a uniform distribution of stress fiber orientations. Demonstrations that the model successfully predicts the alignments experimentally found are followed by a parameter study to investigate the influence of a range of key variables including the stretch magnitude, the intrinsic rate sensitivity of the stress fibers, the straining frequency, and the transverse contraction of the substrate. The primary predictions are as follows. The rate sensitivity has a strong influence on alignment, equivalent to that attained by a few percent of additional stretch. The fiber alignment increases with increasing cycling frequency. Transverse contraction of the substrate causes the stress fibers to organize into two symmetrical orientations with respect to the primary stretch direction.     

Rac1 activity is required for cardiac myocyte alignment in response to mechanical stress

Mechanical stretch is essential for the cardiac growth. The exposure of cardiac myocytes to the mechanical stretch leads to the cell alignment in parallel to the stretch direction, determining the cell polarity, though it remains to be addressed how mechanical stretch regulates cell orientation. In the present study, we investigated the signal transduction pathways responsible for the cell orientation response to mechanical stretch, focusing on Rho family proteins. Neonatal rat cardiomyocytes were cultured on silicon chambers and exposed to artificial uniaxial cyclic stretch. The pull-down assays revealed that Rac1 was rapidly activated by stretch, but not RhoA. To analyze the roles of Rho family proteins in cardiomyocyte orientation, adenoviral vectors expressing dominant-negative (dn) RhoA and Rac1 were generated. The transfection with adenovirus vector expressing dnRac1, but not dnRhoA, inhibited stretch-induced cell alignment. In conclusion, Rac1 activity is necessary for cardiomyocyte alignment in response to directional stretch.     

Micropatterned cell cultures on elastic membranes as an in vitro model of myocardium

We describe here a new in vitro protocol for structuring cardiac cell cultures to mimic important aspects of the in vivo ventricular myocardial phenotype by controlling the location and mechanical environment of cultured cells. Microlithography is used to engineer microstructured silicon metal wafers. Those are used to fabricate either microgrooved silicone membranes or silicone molds for microfluidic application of extracellular matrix proteins onto elastic membranes (involving flow control at micrometer resolution). The physically or microfluidically structured membranes serve as a cell culture growth substrate that supports cell alignment and allows the application of stretch. The latter is achieved with a stretching device that can deliver isotropic or anisotropic stretch. Neonatal ventricular cardiomyocytes, grown on these micropatterned membranes, develop an in vivo-like morphology with regular sarcomeric patterns. The entire process from fabrication of the micropatterned silicon metal wafers to casting of silicone molds, microfluidic patterning and cell isolation and seeding takes approximately 7 days.     

Fluidization, resolidification, and reorientation of the endothelial cell in response to slow tidal stretches

Mechanical stretch plays an important role in regulating shape and orientation of the vascular endothelial cell. This morphological response to stretch is basic to angiogenesis, neovascularization, and vascular homeostasis, but mechanism remains unclear. To elucidate mechanisms, we used cell mapping rheometry to measure traction forces in primary human umbilical vein endothelial cells subjected to periodic uniaxial stretches. Onset of periodic stretch of 10% strain amplitude caused a fluidization response typified by attenuation of traction forces almost to zero. As periodic stretch continued, the prompt fluidization response was followed by a slow resolidification response typified by recovery of the traction forces, but now aligned along the axis perpendicular to the imposed stretch. Reorientation of the cell body lagged reorientation of the traction forces, however. Together, these observations demonstrate that cellular reorientation in response to periodic stretch is preceded by traction attenuation by means of cytoskeletal fluidization and subsequent traction recovery transverse to the stretch direction by means of cytoskeletal resolidification.     

Fibronectin fibril alignment is established upon initiation of extracellular matrix assembly

The physical structure of the extracellular matrix (ECM) is tissue-specific and fundamental to normal tissue function. Proper alignment of ECM fibers is essential for the functioning of a variety of tissues. While matrix assembly in general has been intensively investigated, little is known about the mechanisms required for formation of aligned ECM fibrils. We investigated the initiation of fibronectin (FN) matrix assembly using fibroblasts that assemble parallel ECM fibrils and found that matrix assembly sites, where FN fibrillogenesis is initiated, were oriented in parallel at the cell poles. We show that these polarized matrix assembly sites progress into fibrillar adhesions and ultimately into aligned FN fibrils. Cells that assemble an unaligned meshwork matrix form matrix assembly sites around the cell periphery, but the distribution of matrix assembly sites in these cells could be modulated through micropatterning or mechanical stretch. While an elongated cell shape corresponds with a polarized matrix assembly site distribution, these two features are not absolutely linked, since we discovered that transforming growth factor beta (TGF-β1) enhances matrix assembly site polarity and assembly of aligned fibrils independent of cell elongation. We conclude that the ultimate orientation of FN fibrils is determined by the alignment and distribution of matrix assembly sites that form during the initial stages of cell–FN interactions.     

Generation of hierarchical nano- and microwrinkle structure for smooth muscle cell alignment

On the contrary to the complicated and high-cost photolithography based topographical patterning, the non-lithographical strain responsive wrinkling method can generate a variety of wrinkle structures with ease, high uniformity, and cost-effectiveness. The strain responsive wrinkling approach relied on the modulus difference between a thin and stiff film and a soft substrate, resulting in a periodic out-of-plane buckling deformation upon the release of the compressive stress. While the previous reports were dedicated to the micropattern generation, we, in this study, investigated the effect of the UV/O exposure time (10, 20, 30, 40, 50, and 60 min) and the stretching rate (10, 20, 30, and 40%) of the PDMS substrate on the wavelength and the amplitude of the wrinkle patterns in details. Sole nanowrinkle as well as hierarchical nano/microwrinkle patterns could be fabricated through the fine control of those factors. Furthermore, we examined the human aortic smooth muscle cell (HASMCs) alignment on the topographical patterned surface, and found that more than 80% of the HASMCs were cultured and aligned well along with the hierarchical nano/microwrinkle rather than the nanowrinkle pattern.     

Chronic stretch of engineered heart tissue induces hypertrophy and functional improvement.

To examine the influence of chronic mechanical stretch on functional behavior of cardiac myocytes, we reconstituted embryonic chick or neonatal rat cardiac myocytes to a 3-dimensional engineered heart tissue (EHT) by mixing freshly isolated cells with neutralized collagen I and culturing them between two Velcro-coated silicone tubes, held at a fixed distance with a metal spacer. After 4 days, EHTs were subjected to a phasic unidirectional stretch for 6 days in serum-containing medium. Compared to unstretched controls, RNA/DNA and protein/cell ratios increased by 100% and 50%, respectively. ANF mRNA and alpha-sarcomeric actin increased by 98% and 40%, respectively. Morphologically, stretched EHTs exhibited improved organization of cardiac myocytes into parallel arrays of rod-shaped cells, increased cell length and width, longer myofilaments, and increased mitochondrial density. Thus, stretch induced phenotypic changes, generally referred to as hypertrophy. Concomitantly, force of contraction was two- to fourfold higher both under basal conditions and after stimulation with calcium or the beta-adrenergic agonist isoprenaline. Contraction kinetics were accelerated with a 14-44% decrease in twitch duration under all those conditions. In summary, we have developed a new in vitro model that allows morphological, molecular, and functional consequences of stretch to be studied under defined conditions. The main finding was that stretch of EHTs induced cardiac myocyte hypertrophy, which was accompanied by marked improvement of contractile function.     

Computer simulations of mitosis and interdependencies between mitosis orientation, cell shape and epithelia reshaping

Finite element-based computer simulations are used to investigate mitosis and how mitosis, cell shape, and epithelium reshaping depend on each other. Frame- and cell-oriented patterns of mitosis with growing and non-growing daughter cells are considered. Previous simulations have shown that applied stresses or strains can reshape cells so that their long axes are aligned in the principal stretch direction. The simulations reported here show that this can produce global alignment of the mitosis cleavage planes. Other simulations reported here show that mitoses with suitably aligned cleavage planes can drive epithelium reshaping. Formulas that quantify these and other dependencies are derived. These formulas provide quantitative relationships against which current hypotheses regarding epithelia reshaping in real biological systems can be evaluated.     

Three-dimensional cell and tissue patterning in a strained fibrin gel system

Techniques developed for the in vitro reproduction of three-dimensional (3D) biomimetic tissue will be valuable for investigating changes in cell function in tissues and for fabricating cell/matrix composites for applications in tissue engineering techniques. In this study, we show that the simple application of a continuous strain to a fibrin gel facilitates the development of fibril alignment and bundle-like structures in the fibrin gel in the direction of the applied strain. Myoblasts cultured in this gel also exhibited well-aligned cell patterning in a direction parallel to the direction of the strain. Interestingly, the direction of cell proliferation was identical to that of cell alignment. Finally, the oriented cells formed linear groups that were aligned parallel to the direction of the strain and replicated the native skeletal muscle cell patterning. In addition, vein endothelial cells formed a linear, aligned vessel-like structure in this system. Thus, the system enables the in vitro reproduction of 3D aligned cell sets replicating biological tissue patterns.     

Topographic modulation of the orientation and shape of cell nuclei and their influence on the measured elastic modulus of epithelial cells

The influence of nucleus shape and orientation on the elastic modulus of epithelial cells was investigated with atomic force microscopy. The shape and orientation were controlled by presenting the epithelial cells with anisotropic parallel ridges and grooves of varying pitch at the cell substratum. As the cells oriented to the underlying topography, the volume of the nucleus increased as the pitch of the topography increased from 400 nm to 2000 nm. The increase in nucleus volume was reflected by an increase in the measured elastic modulus of the topographically aligned cells. Significant alterations in the shape of the nucleus, by intimate contact with the topographic ridge and grooves of the underlying cell, were also observed via confocal microscopy, indicating that the nucleus may also act as a direct mechanosensor of substratum topography.     

Optical acquisition and polar decomposition of the full-field deformation gradient tensor within a fracture callus

Tracking tissue deformation is often hampered by material inhomogeneity, so local measurements tend to be insufficient thus lending to the necessity of full-field optical measurements. This study presents a novel approach to factoring heterogeneous deformation of soft and hard tissues in a fracture callus by introducing an anisotropic metric derived from the deformation gradient tensor (F). The deformation gradient tensor contains all the information available in a Green–Lagrange strain tensor, plus the rigid-body rotational components. A recent study [Bottlang et al., Journal of Biomechanics 41(3), 2008] produced full-field strains within ovine fracture calluses acquired through the application of electronic speckle pattern interferometery (ESPI). The technique is based on infinitesimal strain approximation (Engineering Strain) whose scheme is not independent of rigid-body rotation. In this work, for rotation extraction, the stretch and rotation tensors were separately determined from F by the polar decomposition theorem. Interfragmentary motions in a fracture gap were characterized by the two distinct mechanical factors (stretch and rotation) at each material point through full-field mapping. In the composite nature of bone and soft tissue, collagen arrangements are hypothesized such that fibers locally aligned with principal directions will stretch and fibers not aligned with the principal direction will rotate and stretch. This approach has revealed the deformation gradient tensor as an appropriate quantification of strain within callus bony and fibrous tissue via optical measurements.     

Cellular signals regulating the release of ANF.

Atrial natriuretic factor (ANF), a peptide hormone that regulates salt and water balance and blood pressure, is synthesized, stored, and secreted from mammalian myocytes. Stretching of atrial myocytes stimulates ANF secretion, but the cellular processes involved in linking mechanical distension to ANF release are unknown. We reported that phorbol esters, which mimic the action of diacylglycerol by acting directly on protein kinase C and the Ca2+ ionophore A23187, which introduces free Ca2+ into the cell, both increase basal ANF secretion in the isolated perfused rat heart. Phorbol ester also increased responsiveness to Ca2+ channel agonists, such as Bay k8644, and to agents that increase cAMP, such as forskolin and membrane-permeable cAMP analogs. In neonatal cultured rat atrial myocytes, protein kinase C activation by 12-O-tetradecanoylphorbol 13-acetate stimulated ANF secretion, whereas the release was unresponsive to changes in intracellular Ca2+. Endothelin, which stimulates phospholipase C mediated hydrolysis of phosphoinositides and activates protein kinase C, increased both basal and atrial stretch-induced ANF secretion from isolated perfused rat hearts. Similarly, phorbol ester enhanced atrial stretch-stimulated ANF secretion, while the increase in intracellular Ca2+ appeared to be negatively coupled to the stretch-induced ANF release. Finally, phorbol ester stimulated ANF release from the severely hypertrophied ventricles of hypertensive animals but not from normal rat myocardium. These results suggest that the protein kinase C activity may play an important role in the regulation of basal ANF secretion both from atria and ventricular cells, and that stretch of atrial myocytes appears to be positively modulated by phorbol esters.