Hyperglycemic and hyperlipidemic conditions alter cardiac cell biomechanical properties.

Currently, many diabetic cardiomyopathy (DC) studies focus on either in vitro molecular pathways or in vivo whole-heart properties such as ejection fraction. However, as DC is primarily a disease caused by changes in structural and functional properties, such studies may not precisely identify the influence of hyperglycemia or hyperlipidemia in producing specific cellular changes, such as increased myocardial stiffness or diastolic dysfunction. To address this need, we developed an in vitro approach to examine how structural and functional properties may change as a result of a diabetic environment. Particle-tracking microrheology was used to characterize the biomechanical properties of cardiac myocytes and fibroblasts under hyperglycemia or hyperlipidemic conditions. We showed that myocytes, but not fibroblasts, exhibited increased stiffness under diabetic conditions. Hyperlipidemia, but not hyperglycemia, led to increased cFos expression. Although direct application of reactive oxygen species had only limited effects that altered myocyte properties, the antioxidant N-acetylcysteine had broader effects in limiting glucose or fatty-acid alterations. Changes consistent with clinical DC alterations occur in cells cultured in elevated glucose or fatty acids. However, the individual roles of glucose, reactive oxygen species, and fatty acids are varied, suggesting multiple pathway involvement.

Rheological responses of cardiac fibroblasts to mechanical stretch.

Rheological characterization of cells using passive particle tracking techniques can yield substantial information regarding local cellular material properties. However, limited work has been done to establish the changes in material properties of mechanically-responsive cells that experience external stimuli. In this study, cardiac fibroblasts plated on either fibronectin or collagen were treated with cytochalasin, mechanically stretched, or both, and their trajectories and complex moduli were extracted. Results demonstrate that both solid and fluid components were altered by such treatments in a receptor-dependent manner, and that, interestingly, cells treated with cytochalasin were still capable of stiffening in response to mechanical stimuli despite gross stress fiber disruption. These results suggest that the material properties of cells are dependent on a variety of environmental cues and can provide insight into physiological and disease processes.

Keratinocyte cytoskeletal roles in cell sheet engineering.

BACKGROUND: There is an increasing need to understand cell-cell interactions for cell and tissue engineering purposes, such as optimizing cell sheet constructs, as well as for examining adhesion defect diseases. For cell-sheet engineering, one major obstacle to sheet function is that cell sheets in suspension are fragile and, over time, will contract. While the role of the cytoskeleton in maintaining the structure and adhesion of cells cultured on a rigid substrate is well-characterized, a systematic examination of the role played by different components of the cytoskeleton in regulating cell sheet contraction and cohesion in the absence of a substrate has been lacking. RESULTS: In this study, keratinocytes were cultured until confluent and cell sheets were generated using dispase to remove the influence of the substrate. The effects of disrupting actin, microtubules or intermediate filaments on cell-cell interactions were assessed by measuring cell sheet cohesion and contraction. Keratin intermediate filament disruption caused comparable effects on cell sheet cohesion and contraction, when compared to actin or microtubule disruption. Interfering with actomyosin contraction demonstrated that interfering with cell contraction can also diminish cell cohesion. CONCLUSIONS: All components of the cytoskeleton are involved in maintaining cell sheet cohesion and contraction, although not to the same extent. These findings demonstrate that substrate-free cell sheet biomechanical properties are dependent on the integrity of the cytoskeleton network.

Tetraspanin CD151 maintains vascular stability by balancing the forces of cell adhesion and cytoskeletal tension.

Tetraspanin CD151 is highly expressed in endothelial cells and regulates pathologic angiogenesis. However, the mechanism by which CD151 promotes vascular morphogenesis and whether CD151 engages other vascular functions are unclear. Here we report that CD151 is required for maintaining endothelial capillary-like structures formed in vitro and the integrity of endothelial cell-cell and cell-matrix contacts in vivo. In addition, vascular permeability is markedly enhanced in the absence of CD151. As a global regulator of endothelial cell-cell and cell-matrix adhesions, CD151 is needed for the optimal functions of various cell adhesion proteins. The loss of CD151 elevates actin cytoskeletal traction by up-regulating RhoA signaling and diminishes actin cortical meshwork by down-regulating Rac1 activity. The inhibition of RhoA or activation of cAMP signaling stabilizes CD151-silenced or -null endothelial structure in vascular morphogenesis. Together, our data demonstrate that CD151 maintains vascular stability by promoting endothelial cell adhesions, especially cell-cell adhesion, and confining cytoskeletal tension.

A new diagnostic test for arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND: The diagnosis of arrhythmogenic right ventricular cardiomyopathy (ARVC) can be challenging because the clinical presentation is highly variable and genetic penetrance is often low. METHODS: To determine whether a change in the distribution of desmosomal proteins can be used as a sensitive and specific diagnostic test for ARVC, we performed immunohistochemical analysis of human myocardial samples. RESULTS: We first tested myocardium from 11 subjects with ARVC; of these samples, 8 had desmosomal gene mutations. We also tested myocardium obtained at autopsy from 10 subjects with no clinical or pathological evidence of heart disease as control samples. All ARVC samples but no control samples showed a marked reduction in immunoreactive signal levels for plakoglobin (also known as gamma-catenin), a protein that links adhesion molecules at the intercalated disk to the cytoskeleton. Other desmosomal proteins showed variable changes, but signal levels for the nondesmosomal adhesion molecule N-cadherin were normal in all subjects with ARVC. To determine whether a diminished plakoglobin signal level was specific for ARVC, we analyzed myocardium from 15 subjects with hypertrophic, dilated, or ischemic cardiomyopathies. In every sample, levels of N-cadherin and plakoglobin signals at junctions were indistinguishable from those in control samples. Finally, we performed blinded immunohistochemical analysis of heart-biopsy samples from the Johns Hopkins ARVC registry. We made the correct diagnosis in 10 of 11 subjects with definite ARVC on the basis of clinical criteria and correctly ruled out ARVC in 10 of 11 subjects without ARVC, for a sensitivity of 91%, a specificity of 82%, a positive predictive value of 83%, and a negative predictive value of 90%. The plakoglobin signal level was reduced diffusely in ARVC samples, including those obtained in the left ventricle and the interventricular septum. CONCLUSIONS: Routine immunohistochemical analysis of a conventional endomyocardial-biopsy sample appears to be a highly sensitive and specific diagnostic test for ARVC.

A dynamic zone defines interneuron remodeling in the adult neocortex.

The contribution of structural remodeling to long-term adult brain plasticity is unclear. Here, we investigate features of GABAergic interneuron dendrite dynamics and extract clues regarding its potential role in cortical function and circuit plasticity. We show that remodeling interneurons are contained within a "dynamic zone" corresponding to a superficial strip of layers 2/3, and remodeling dendrites respect the lower border of this zone. Remodeling occurs primarily at the periphery of dendritic fields with addition and retraction of new branch tips. We further show that dendrite remodeling is not intrinsic to a specific interneuron class. These data suggest that interneuron remodeling is not a feature predetermined by genetic lineage, but rather, it is imposed by cortical laminar circuitry. Our findings are consistent with dynamic GABAergic modulation of feedforward and recurrent connections in response to top-down feedback and suggest a structural component to functional plasticity of supragranular neocortical laminae.

Genetically engineered resistance for MMP collagenases promotes abdominal aortic aneurysm formation in mice infused with angiotensin II.

Clinical evidence links increased aortic collagen content and stiffness to abdominal aortic aneurysm (AAA) formation. However, the possibility that excess collagen contributes to AAA formation remains untested. We investigated the hypothesis that augmented collagen promotes AAA formation, and employed apoE-null mice expressing collagenase-resistant mutant collagen (Col(R/R)/apoE(-/-)), heterozygote (Col(R/+)/apoE(-/-)), or wild-type collagen (Col(+/+)/apoE(-/-)) infused with angiotensin II to induce AAA. As expected, the aortas of Col(R/R)/apoE(-/-) mice contained more interstitial collagen than those from the other groups. Angiotensin II treatment elicited more AAA formation in Col(R/R)/apoE(-/-) mice than Col(R/+)/apoE(-/-) or Col(+/+)/apoE(-/-) mice. Aortic circumferences correlated positively with collagen content, determined by picrosirius red and Masson trichrome staining. Mechanical testing of aortas of Col(R/R)/apoE(-/-) mice showed increased stiffness and susceptibility to mechanical failure compared to those of Col(+/+)/apoE(-/-) mice. Optical analysis further indicated altered collagen fiber orientation in the adventitia of Col(R/R)/apoE(-/-) mice. These results demonstrate that collagen content regulates aortic biomechanical properties and influences AAA formation.

Dynamic remodeling of dendritic arbors in GABAergic interneurons of adult visual cortex.

Despite decades of evidence for functional plasticity in the adult brain, the role of structural plasticity in its manifestation remains unclear. To examine the extent of neuronal remodeling that occurs in the brain on a day-to-day basis, we used a multiphoton-based microscopy system for chronic in vivo imaging and reconstruction of entire neurons in the superficial layers of the rodent cerebral cortex. Here we show the first unambiguous evidence (to our knowledge) of dendrite growth and remodeling in adult neurons. Over a period of months, neurons could be seen extending and retracting existing branches, and in rare cases adding new branch tips. Neurons exhibiting dynamic arbor rearrangements were GABA-positive non-pyramidal interneurons, while pyramidal cells remained stable. These results are consistent with the idea that dendritic structural remodeling is a substrate for adult plasticity and they suggest that circuit rearrangement in the adult cortex is restricted by cell type-specific rules.

Disparate effects of different mutations in plakoglobin on cell mechanical behavior.

Mutations in genes encoding desmosomal proteins have been implicated in the pathogenesis of heart and skin diseases. This has led to the hypothesis that defective cell-cell adhesion is the underlying cause of injury in tissues that repeatedly bear high mechanical loads. In this study, we examined the effects of two different mutations in plakoglobin on cell migration, stiffness, and adhesion. One is a C-terminal mutation causing Naxos disease, a recessive syndrome of arrhythmogenic right ventricular cardiomyopathy (ARVC) and abnormal skin and hair. The other is an N-terminal mutation causing dominant inheritance of ARVC without cutaneous abnormalities. To assess the effects of plakoglobin mutations on a broad range of cell mechanical behavior, we characterized a model system consisting of stably transfected HEK cells which are particularly well suited for analyses of cell migration and adhesion. Both mutations increased the speed of wound healing which appeared to be related to increased cell motility rather than increased cell proliferation. However, the C-terminal mutation led to dramatically decreased cell-cell adhesion, whereas the N-terminal mutation caused a decrease in cell stiffness. These results indicate that different mutations in plakoglobin have markedly disparate effects on cell mechanical behavior, suggesting complex biomechanical roles for this protein.

Fast fluorescence laser tracking microrheometry, II: quantitative studies of cytoskeletal mechanotransduction.

Fluorescence laser tracking microrheometry (FLTM) is what we believe to be a novel method able to assess the local, frequency-dependent mechanical properties of living cells with nanometer spatial sensitivity at speeds up to 50 kHz. In an earlier article, we described the design, development, and optimization phases of the FLTM before reporting its performances in a variety of viscoelastic materials. In the work presented here, we demonstrate the suitability of FLTM to study local cellular rheology and obtain values for the storage and loss moduli G'(omega) and G''(omega) of fibroblasts consistent with past literature. We further establish that chemically induced cytoskeletal disruption is accompanied by reduced cellular stiffness and viscosity. Next, we provide a systematic study of some experimental variables that may critically influence microrheology measurements. First, we interrogate and justify the relevance of bead endocytosis as a method of cellular internalization of 1-microm probes in FLTM. Second, we show that as sample temperature increases, FLTM findings are elevated toward higher frequencies. Third, we confirm that relevant bead sizes (1 and 2 microm) have no effect on FLTM measurements. Fourth, we report the lack of influence of bead coatings (antiintegrin, antitransferrin, antidystroglycan, or uncoated tracers were surveyed) on their rheological readouts. Finally, we demonstrate the potential of FLTM in studying how substratum rigidity regulates cellular rheological properties. Interestingly, multiple, coupled strain relaxation mechanisms can be observed separated by two plateau moduli. Although these observations can be partly explained by rheological theories describing entangled actin filaments, there is a clear need to extend existing microrheology models to the cytoskeleton, including potentially important factors such as network geometry and remodeling.

Fast fluorescence laser tracking microrheometry. I: instrument development.

To gain insight into cellular mechanotransduction pathways, we have developed a fluorescence laser tracking microrheometer (FLTM) to measure material rheological features on micrometer length scales using fluorescent microspheres as tracer particles. The statistical analysis of the Brownian motion of a particle quantifies the viscoelastic properties of the probe's environment, parameterized by the frequency-dependent complex shear modulus G*(omega). This FLTM has nanometer spatial resolution over a frequency range extending from 1 Hz to 50 kHz. In this work, we first describe the consecutive stages of instrument design, development, and optimization. We subsequently demonstrate the accuracy of the FLTM by reproducing satisfactorily the known rheological characteristics of purely viscous glycerol solutions and cross-linked polyacrylamide polymer networks. An upcoming companion article will illustrate the use of FLTM in studying the solid-like versus liquid-like rheological properties of fibroblast cytoskeletons in living biological samples.

Time-Saving Benefits of Intravital Staining.

One of the challenges in labeling tissues for fluorescence microscopy is minimizing sample processing while maintaining or improving the information generated by the fluorescent label. Generally, tissues are extracted, fixed, and embedded in mounting media (such as paraffin), sectioned, and then postprocessed by removing the paraffin, blocking, labeling, and washing. Despite all of these steps, the consistency of labeling quality can vary as a result of several factors, including heterogeneity in labeling efficiency from slide to slide, the necessity of postprocessing to obtain information on sequential sections of tissue, interference from the mounting media, and loss of native three-dimensional structural information, especially in thicker sections. A method for embedding and processing tissues that have been labeled by intravital staining is described in this study. Intravital staining is the process in which live-cell dyes and other labels are injected into the bloodstream before fixation of the tissues. Tissues processed this way can be imaged upon sectioning without further staining and retain their native, three-dimensional information, thereby improving the information retained by the labels and speeding up sample processing.

High-resolution whole organ imaging using two-photon tissue cytometry.

Three-dimensional (3-D) tissue imaging offers substantial benefits to a wide range of biomedical investigations from cardiovascular biology, diabetes, Alzheimer's disease to cancer. Two-photon tissue cytometry is a novel technique based on high-speed multiphoton microscopy coupled with automated histological sectioning, which can quantify tissue morphology and physiology throughout entire organs with subcellular resolution. Furthermore, two-photon tissue cytometry offers all the benefits of fluorescence-based approaches including high specificity and sensitivity and appropriateness for molecular imaging of gene and protein expression. We use two-photon tissue cytometry to image an entire mouse heart at subcellular resolution to quantify the 3-D morphology of cardiac microvasculature and myocyte morphology spanning almost five orders of magnitude in length scales.

Thioredoxin-interacting protein controls cardiac hypertrophy through regulation of thioredoxin activity.

BACKGROUND: Although cellular redox balance plays an important role in mechanically induced cardiac hypertrophy, the mechanisms of regulation are incompletely defined. Because thioredoxin is a major intracellular antioxidant and can also regulate redox-dependent transcription, we explored the role of thioredoxin activity in mechanically overloaded cardiomyocytes in vitro and in vivo. METHODS AND RESULTS: Overexpression of thioredoxin induced protein synthesis in cardiomyocytes (127+/-5% of controls, P<0.01). Overexpression of thioredoxin-interacting protein (Txnip), an endogenous thioredoxin inhibitor, reduced protein synthesis in response to mechanical strain (89+/-5% reduction, P<0.01), phenylephrine (80+/-3% reduction, P<0.01), or angiotensin II (80+/-4% reduction, P<0.01). In vivo, myocardial thioredoxin activity increased 3.5-fold compared with sham controls after transverse aortic constriction (P<0.01). Aortic constriction did not change thioredoxin expression but reduced Txnip expression by 40% (P<0.05). Gene transfer studies showed that cells that overexpress Txnip develop less hypertrophy after aortic constriction than control cells in the same animals (28.1+/-5.2% reduction versus noninfected cells, P<0.01). CONCLUSIONS: Thus, even though thioredoxin is an antioxidant, activation of thioredoxin participates in the development of pressure-overload cardiac hypertrophy, demonstrating the dual function of thioredoxin as both an antioxidant and a signaling protein. These results also support the emerging concept that the thioredoxin inhibitor Txnip is a critical regulator of biomechanical signaling.

Characterization of partially lifted cell sheets.

There is a need to characterize biomechanical cell-cell interactions, but due to a lack of suitable experimental methods, relevant in vitro experimental data are often masked by cell-substrate interactions. This study describes a novel method to generate partially lifted substrate-free cell sheets that engage primarily in cell-cell interactions, yet are amenable to biological and chemical perturbations and, importantly, mechanical conditioning and characterization. A polydimethylsiloxane (PDMS) mold is used to isolate a patch of cells, and the patch is then enzymatically lifted. The cells outside the mold remain attached, creating a partially lifted cell sheet. This simple yet powerful tool enables the simultaneous examination of lifted and adherent cells. This tool was then deployed to test the hypothesis that the lifted cells would exhibit substantial reinforcement of key cytoskeletal and junctional components at cell-cell contacts, and that such reinforcement would be enhanced by mechanical conditioning. Results demonstrate that the mechanical strength and cohesion of the substrate-free cell sheets strongly depend on the integrity of the actomyosin cytoskeleton and the cell-cell junctional protein plakoglobin. Both actin and plakoglobin are significantly reinforced at junctions with mechanical conditioning. However, total cellular actin is significantly diminished on dissociation from a substrate and does not recover with mechanical conditioning. These results represent a first systematic examination of mechanical conditioning on cells with primarily intercellular interactions.

Dietary levels of acrylamide affect rat cardiomyocyte properties.

The toxic effects of acrylamide on cytoskeletal integrity and ion channel balance is well-established in many cell types, but there has been little examination regarding the effects of acrylamide on primary cardiomyocytes, despite the importance of such components in their function. Furthermore, acrylamide toxicity is generally examined using concentrations higher than those found in vivo under starch-rich diets. Accordingly, we sought to characterize the dose-dependent effects of acrylamide on various properties, including cell morphology, contraction patterns, and junctional connexin 43 staining, in primary cardiomyocytes. We show that several days exposure to 1-100 µM acrylamide resulted in altered morphology, irregular contraction patterns, and an increase in the amount of immunoreactive signal for connexin 43 at cell junctions. We conclude that dietary levels of acrylamide may alter cellular function with prolonged exposure, in primary cardiomyocytes.

Arrhythmogenic right ventricular cardiomyopathy mutations alter shear response without changes in cell-cell adhesion.

AIMS: The majority of patients diagnosed with arrhythmogenic right ventricular cardiomyopathy (ARVC) have mutations in genes encoding desmosomal proteins, raising the possibility that abnormal intercellular adhesion plays an important role in disease pathogenesis. We characterize cell mechanical properties and molecular responses to oscillatory shear stress in cardiac myocytes expressing mutant forms of the desmosomal proteins, plakoglobin and plakophilin, which are linked to ARVC in patients. METHODS AND RESULTS: Cells expressing mutant plakoglobin or plakophilin showed no differences in cell-cell adhesion relative to controls, while knocking down these proteins weakened cell-cell adhesion. However, cells expressing mutant plakoglobin failed to increase the amount of immunoreactive signal for plakoglobin or N-cadherin at cell-cell junctions in response to shear stress, as seen in control cells. Cells expressing mutant plakophilin exhibited a similar attenuation in the shear-induced increase in junctional plakoglobin immunoreactive signal in response to shear stress, suggesting that the phenotype is independent of the type of mutant protein being expressed. Cells expressing mutant plakoglobin also showed greater myocyte apoptosis compared with controls. Apoptosis rates increased greatly in response to shear stress in cells expressing mutant plakoglobin, but not in controls. Abnormal responses to shear stress in cells expressing either mutant plakoglobin or plakophilin could be reversed by SB216763, a GSK3ß inhibitor. CONCLUSIONS: Desmosomal mutations linked to ARVC do not significantly affect cell mechanical properties, but cause myocytes to respond abnormally to mechanical stress through a mechanism involving GSK3ß. These results may help explain why patients with ARVC experience disease exacerbations following strenuous exercise.

Folliculin regulates cell-cell adhesion, AMPK, and mTORC1 in a cell-type-specific manner in lung-derived cells.

Germline loss-of-function BHD mutations cause cystic lung disease and hereditary pneumothorax, yet little is known about the impact of BHD mutations in the lung. Folliculin (FLCN), the product of the Birt-Hogg-Dube (BHD) gene, has been linked to altered cell-cell adhesion and to the AMPK and mTORC1 signaling pathways. We found that downregulation of FLCN in human bronchial epithelial (HBE) cells decreased the phosphorylation of ACC, a marker of AMPK activation, while downregulation of FLCN in small airway epithelial (SAEC) cells increased the activity of phospho-S6, a marker of mTORC1 activation, highlighting the cell type-dependent functions of FLCN. Cell-cell adhesion forces were significantly increased in FLCN-deficient HBE cells, consistent with prior findings in FLCN-deficient human kidney-derived cells. To determine how these altered cell-cell adhesion forces impact the lung, we exposed mice with heterozygous inactivation of Bhd (similarly to humans with germline inactivation of one BHD allele) to mechanical ventilation at high tidal volumes. Bhd(+/-) mice exhibited a trend (P = 0.08) toward increased elastance after 6 h of ventilation at 24 cc/kg. Our results indicate that FLCN regulates the AMPK and mTORC1 pathways and cell-cell adhesion in a cell type-dependent manner. FLCN deficiency may impact the physiologic response to inflation-induced mechanical stress, but further investigation is required. We hypothesize that FLCN-dependent effects on signaling and cellular adhesion contribute to the pathogenesis of cystic lung disease in BHD patients.

Insights into the role of cell-cell junctions in physiology and disease.

Contacting cells establish different classes of intricate structures at the cell-cell junctions. These structures are of increasing research interest as they regulate a broad variety of processes in development and disease. Further, in vitro studies are revealing that various cell-cell interaction proteins are involved not only in cell-cell processes but also in many additional aspects of physiology, such as migration and apoptosis. This chapter reviews the basic classification of cell-cell junctional structures and some of their representative proteins. Their roles in development and disease are briefly outlined, followed by a section on contemporary methods for probing cell-cell interactions and some recent developments. This chapter concludes with a few suggestions for potential research directions to further develop this promising area of study.

Cell-cell junctional proteins in cardiovascular mechanotransduction.

Cell-cell junctional proteins play important structural and functional roles in several physiological systems. Recent studies have illuminated key aspects in the relationship of junctional proteins with normal cell and tissue function as well as various pathologies. In this review article, the roles of cell-cell junctional proteins will be presented in four classes: adherens junctions, desmosomes, gap junctions, and tight junctions, and discussed primarily in the context of cardiovascular cell and tissue physiology and pathophysiology. The functions of the proteins are described from the perspective of mechanotransductive regulation of physiological and disease processes, with focus being laid on more biomechanical aspects, such as cell adhesion, migration, and mechanosignaling.