Tunable electrospun scaffolds of polyacrylonitrile loaded with carbon nanotubes: from synthesis to biological applications.

Growing cells in a biomimetic environment is critical for tissue engineering as well as for studying the cell biology underlying disease mechanisms. To this aim a range of 3D matrices have been developed, from hydrogels to decellularized matrices. They need to mimic the extracellular matrix to ensure the optimal growth and function of cells. Electrospinning has gained in popularity due to its capacity to individually tune chemistry and mechanical properties and as such influence cell attachment, differentiation or maturation. Polyacrylonitrile (PAN) derived electrospun fibres scaffolds have shown exciting potential due to reports of mechanical tunability and biocompatibility. Building on previous work we fabricate here a range of PAN fibre scaffolds with different concentrations of carbon nanotubes. We characterize them in-depth in respect to their structure, surface chemistry and mechanical properties, using scanning electron microscopy, image processing, ultramicrotomic transmission electron microscopy, x-ray nanotomography, infrared spectroscopy, atomic force microscopy and nanoindentation. Together the data demonstrate this approach to enable finetuning the mechanical properties, while keeping the structure and chemistry unaltered and hence offering ideal properties for comparative studies of the cellular mechanobiology. Finally, we confirm the biocompatibility of the scaffolds using primary rat cardiomyocytes, vascular smooth muscle (A7r5) and myoblast (C2C12) cell lines.

Sox9 Accelerates Vascular Aging by Regulating Extracellular Matrix Composition and Stiffness.

BACKGROUND: Vascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular aging. Sox9 (SRY-box transcription factor 9) has been implicated in vascular smooth muscle cell (VSMC) osteo/chondrogenic conversion; however, its relationship with aging and calcification has not been studied. METHODS: Immunohistochemistry was performed on human aortic samples from young and aged patients. Young and senescent primary human VSMCs were induced to produce ECM, and Sox9 expression was manipulated using adenoviral overexpression and depletion. ECM properties were characterized using atomic force microscopy and proteomics, and VSMC phenotype on hydrogels and the ECM were examined using confocal microscopy. RESULTS: In vivo, Sox9 was not spatially associated with vascular calcification but correlated with the senescence marker p16 (cyclin-dependent kinase inhibitor 2A). In vitro Sox9 showed mechanosensitive responses with increased expression and nuclear translocation in senescent cells and on stiff matrices. Sox9 was found to regulate ECM stiffness and organization by orchestrating changes in collagen (Col) expression and reducing VSMC contractility, leading to the formation of an ECM that mirrored that of senescent cells. These ECM changes promoted phenotypic modulation of VSMCs, whereby senescent cells plated on ECM synthesized from cells depleted of Sox9 returned to a proliferative state, while proliferating cells on a matrix produced by Sox9 expressing cells showed reduced proliferation and increased DNA damage, reiterating features of senescent cells. LH3 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3) was identified as an Sox9 target and key regulator of ECM stiffness. LH3 is packaged into extracellular vesicles and Sox9 promotes extracellular vesicle secretion, leading to increased LH3 deposition within the ECM. CONCLUSIONS: These findings highlight the crucial role of ECM structure and composition in regulating VSMC phenotype. We identify a positive feedback cycle, whereby cellular senescence and increased ECM stiffening promote Sox9 expression, which, in turn, drives further ECM modifications to further accelerate stiffening and senescence.

Hypertensive Pressure Mechanosensing Alone Triggers Lipid Droplet Accumulation and Transdifferentiation of Vascular Smooth Muscle Cells to Foam Cells.

Arterial Vascular smooth muscle cells (VSMCs) play a central role in the onset and progression of atherosclerosis. Upon exposure to pathological stimuli, they can take on alternative phenotypes that, among others, have been described as macrophage like, or foam cells. VSMC foam cells make up >50% of all arterial foam cells and have been suggested to retain an even higher proportion of the cell stored lipid droplets, further leading to apoptosis, secondary necrosis, and an inflammatory response. However, the mechanism of VSMC foam cell formation is still unclear. Here, it is identified that mechanical stimulation through hypertensive pressure alone is sufficient for the phenotypic switch. Hyperspectral stimulated Raman scattering imaging demonstrates rapid lipid droplet formation and changes to lipid metabolism and changes are confirmed in ABCA1, KLF4, LDLR, and CD68 expression, cell proliferation, and migration. Further, a mechanosignaling route is identified involving Piezo1, phospholipid, and arachidonic acid signaling, as well as epigenetic regulation, whereby CUT&Tag epigenomic analysis confirms changes in the cells (lipid) metabolism and atherosclerotic pathways. Overall, the results show for the first time that VSMC foam cell formation can be triggered by mechanical stimulation alone, suggesting modulation of mechanosignaling can be harnessed as potential therapeutic strategy.

Extracellular vesicles stimulate smooth muscle cell migration by presenting collagen VI.

The extracellular matrix (ECM) supports blood vessel architecture and functionality and undergoes active remodelling during vascular repair and atherogenesis. Vascular smooth muscle cells (VSMCs) are essential for vessel repair and, via their secretome, are able to invade from the vessel media into the intima to mediate ECM remodelling. Accumulation of fibronectin (FN) is a hallmark of early vascular repair and atherosclerosis and here we show that FN stimulates VSMCs to secrete small extracellular vesicles (sEVs) by activating the ß1 integrin/FAK/Src pathway as well as Arp2/3-dependent branching of the actin cytoskeleton. Spatially, sEV were secreted via filopodia-like cellular protrusions at the leading edge of migrating cells. We found that sEVs are trapped by the ECM in vitro and colocalise with FN in symptomatic atherosclerotic plaques in vivo. Functionally, ECM-trapped sEVs induced the formation of focal adhesions (FA) with enhanced pulling forces at the cellular periphery. Proteomic and GO pathway analysis revealed that VSMC-derived sEVs display a cell adhesion signature and are specifically enriched with collagen VI. In vitro assays identified collagen VI as playing the key role in cell adhesion and invasion. Taken together our data suggests that the accumulation of FN is a key early event in vessel repair acting to promote secretion of collage VI enriched sEVs by VSMCs. These sEVs stimulate migration and invasion by triggering peripheral focal adhesion formation and actomyosin contraction to exert sufficient traction forces to enable VSMC movement within the complex vascular ECM network.

Lem2 is essential for cardiac development by maintaining nuclear integrity.

AIMS: Nuclear envelope integrity is essential for the compartmentalization of the nucleus and cytoplasm. Importantly, mutations in genes encoding nuclear envelope (NE) and associated proteins are the second highest cause of familial dilated cardiomyopathy. One such NE protein that causes cardiomyopathy in humans and affects mouse heart development is Lem2. However, its role in the heart remains poorly understood. METHODS AND RESULTS: We generated mice in which Lem2 was specifically ablated either in embryonic cardiomyocytes (Lem2 cKO) or in adult cardiomyocytes (Lem2 iCKO) and carried out detailed physiological, tissue, and cellular analyses. High-resolution episcopic microscopy was used for three-dimensional reconstructions and detailed morphological analyses. RNA-sequencing and immunofluorescence identified altered pathways and cellular phenotypes, and cardiomyocytes were isolated to interrogate nuclear integrity in more detail. In addition, echocardiography provided a physiological assessment of Lem2 iCKO adult mice. We found that Lem2 was essential for cardiac development, and hearts from Lem2 cKO mice were morphologically and transcriptionally underdeveloped. Lem2 cKO hearts displayed high levels of DNA damage, nuclear rupture, and apoptosis. Crucially, we found that these defects were driven by muscle contraction as they were ameliorated by inhibiting myosin contraction and L-type calcium channels. Conversely, reducing Lem2 levels to ∼45% in adult cardiomyocytes did not lead to overt cardiac dysfunction up to 18 months of age. CONCLUSIONS: Our data suggest that Lem2 is critical for integrity at the nascent NE in foetal hearts, and protects the nucleus from the mechanical forces of muscle contraction. In contrast, the adult heart is not detectably affected by partial Lem2 depletion, perhaps owing to a more established NE and increased adaptation to mechanical stress. Taken together, these data provide insights into mechanisms underlying cardiomyopathy in patients with mutations in Lem2 and cardio-laminopathies in general.

Cardiovascular Mechano-Epigenetics: Force-Dependent Regulation of Histone Modifications and Gene Regulation.

The local mechanical microenvironment impacts on the cell behavior. In the cardiovascular system, cells in both the heart and the vessels are exposed to continuous blood flow, blood pressure, stretching forces, and changing extracellular matrix stiffness. The force-induced signals travel all the way to the nucleus regulating epigenetic changes such as chromatin dynamics and gene expression. Mechanical cues are needed at the very early stage for a faultless embryological development, while later in life, aberrant mechanical signaling can lead to a range of pathologies, including diverse cardiovascular diseases. Hence, an investigation of force-generated epigenetic alteration at different time scales is needed to understand fully the phenotypic changes in disease onset and progression. That being so, cardiovascular mechano-epigenetics emerges as an attractive field of study. Given the rapid advances in this emergent field of research, this short review aims to provide an analysis of the state of knowledge of force-induced epigenetic changes in the cardiovascular field.

Photoelectrochemical imaging of single cardiomyocytes and monitoring of their action potentials through contact force manipulation of organoids.

Accurate monitoring of cardiomyocyte action potentials (APs) is essential to understand disease propagation and for trials of novel therapeutics. Patch clamp techniques offer 'gold standard' measurements in this field, but are notoriously difficult to operate and only provide measurements of a single cell. Here we propose photoelectrochemical imaging (PEI) with light-addressable potentiometric sensors (LAPS) in conjunction with a setup for controlling the contact force between the cardiomyocyte organoids and the sensor surface for measuring APs with high sensitivity. The method was validated through measuring the responses to drugs, and the results successfully visualized the expected electrophysiological changes to the APs. PEI allows for several cells to be monitored simultaneously, opening further research to the electrophysiological interactions of adjoining cells. This method expands the applications of PEI to three-dimensional geometries and provides the fields of stem cell research, drug trials and heart disease modelling with an invaluable tool to further investigate the role of APs.

Editors' roundup: October 2022.

This commentary constitutes the October edition of the 'Editors' roundup'-a multi-author omnibus of personal recommendations to interesting biophysics-related articles contributed by members of the editorial boards of leading international biophysics journals. The present commentary contains contributions from Progress in Biochemistry and Biophysics (an official journal of the Biophysical Society of China), European Biophysics Journal (the official journal of the European Biophysical Societies Association), Biophysical Reviews (the official IUPAB journal), and Biophysics (an official journal of the Russian Academy of Sciences). This edition of the Editors' Roundup also contains a new section from an editor at large who has provided selections from a number of journals on a single thematic topic.

Regulation of cardiomyocyte adhesion and mechanosignalling through distinct nanoscale behaviour of integrin ligands mimicking healthy or fibrotic extracellular matrix.

The stiffness of the cardiovascular environment changes during ageing and in disease and contributes to disease incidence and progression. Changing collagen expression and cross-linking regulate the rigidity of the cardiac extracellular matrix (ECM). Additionally, basal lamina glycoproteins, especially laminin and fibronectin regulate cardiomyocyte adhesion formation, mechanics and mechanosignalling. Laminin is abundant in the healthy heart, but fibronectin is increasingly expressed in the fibrotic heart. ECM receptors are co-regulated with the changing ECM. Owing to differences in integrin dynamics, clustering and downstream adhesion formation this is expected to ultimately influence cardiomyocyte mechanosignalling; however, details remain elusive. Here, we sought to investigate how different cardiomyocyte integrin/ligand combinations affect adhesion formation, traction forces and mechanosignalling, using a combination of uniformly coated surfaces with defined stiffness, polydimethylsiloxane nanopillars, micropatterning and specifically designed bionanoarrays for precise ligand presentation. Thereby we found that the adhesion nanoscale organization, signalling and traction force generation of neonatal rat cardiomyocytes (which express both laminin and fibronectin binding integrins) are strongly dependent on the integrin/ligand combination. Together our data indicate that the presence of fibronectin in combination with the enhanced stiffness in fibrotic areas will strongly impact on the cardiomyocyte behaviour and influence disease progression. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

The role of the dystrophin glycoprotein complex in muscle cell mechanotransduction.

Dystrophin is the central protein of the dystrophin-glycoprotein complex (DGC) in skeletal and heart muscle cells. Dystrophin connects the actin cytoskeleton to the extracellular matrix (ECM). Severing the link between the ECM and the intracellular cytoskeleton has a devastating impact on the homeostasis of skeletal muscle cells, leading to a range of muscular dystrophies. In addition, the loss of a functional DGC leads to progressive dilated cardiomyopathy and premature death. Dystrophin functions as a molecular spring and the DGC plays a critical role in maintaining the integrity of the sarcolemma. Additionally, evidence is accumulating, linking the DGC to mechanosignalling, albeit this role is still less understood. This review article aims at providing an up-to-date perspective on the DGC and its role in mechanotransduction. We first discuss the intricate relationship between muscle cell mechanics and function, before examining the recent research for a role of the dystrophin glycoprotein complex in mechanotransduction and maintaining the biomechanical integrity of muscle cells. Finally, we review the current literature to map out how DGC signalling intersects with mechanical signalling pathways to highlight potential future points of intervention, especially with a focus on cardiomyopathies.

Pressure and stiffness sensing together regulate vascular smooth muscle cell phenotype switching.

Vascular smooth muscle cells (VSMCs) play a central role in the progression of atherosclerosis, where they switch from a contractile to a synthetic phenotype. Because of their role as risk factors for atherosclerosis, we sought here to systematically study the impact of matrix stiffness and (hemodynamic) pressure on VSMCs. Thereby, we find that pressure and stiffness individually affect the VSMC phenotype. However, only the combination of hypertensive pressure and matrix compliance, and as such mechanical stimuli that are prevalent during atherosclerosis, leads to a full phenotypic switch including the formation of matrix-degrading podosomes. We further analyze the molecular mechanism in stiffness and pressure sensing and identify a regulation through different but overlapping pathways culminating in the regulation of the actin cytoskeleton through cofilin. Together, our data show how different pathological mechanical signals combined but through distinct pathways accelerate a phenotypic switch that will ultimately contribute to atherosclerotic disease progression.

Factoring in the force: A novel role for eIF6.

eIF6 is known for its role as a stimulatory translation initiation factor. In this issue, Keen et al. (2022. J. Cell Biol. https://doi.org/10.1083/jcb.202005213) identify a novel, noncanonical role, whereby eIF6 regulates focal adhesion formation, mechanosensing, and cell mechanics, independent of its translational role.

Cardiovascular mechanobiology-a Special Issue to look at the state of the art and the newest insights into the role of mechanical forces in cardiovascular development, physiology and disease.

There has been much progress recently in the area of cardiovascular mechanobiology and this Special Issue aims at taking stock. This editorial gives context of the main motivation for this special issue as well as a brief summary of its content.

Tools for studying and modulating (cardiac muscle) cell mechanics and mechanosensing across the scales.

Cardiomyocytes generate force for the contraction of the heart to pump blood into the lungs and body. At the same time, they are exquisitely tuned to the mechanical environment and react to e.g. changes in cell and extracellular matrix stiffness or altered stretching due to reduced ejection fraction in heart disease, by adapting their cytoskeleton, force generation and cell mechanics. Both mechanical sensing and cell mechanical adaptations are multiscale processes. Receptor interactions with the extracellular matrix at the nanoscale will lead to clustering of receptors and modification of the cytoskeleton. This in turn alters mechanosensing, force generation, cell and nuclear stiffness and viscoelasticity at the microscale. Further, this affects cell shape, orientation, maturation and tissue integration at the microscale to macroscale. A variety of tools have been developed and adapted to measure cardiomyocyte receptor-ligand interactions and forces or mechanics at the different ranges, resulting in a wealth of new information about cardiomyocyte mechanobiology. Here, we take stock at the different tools for exploring cardiomyocyte mechanosensing and cell mechanics at the different scales from the nanoscale to microscale and macroscale.

Announcing the call for the Special Issue on "Cardiovascular mechanobiology-a special issue to look at the state of the art and the newest insights into the role of mechanical forces in cardiovascular development, physiology, and disease".

This Commentary describes a call for submissions for the upcoming special issue focused on the state of the art of cardiovascular mechanobiology research and the newest insights into the role of mechanical forces in cardiovascular development, physiology, and disease.

Glioma stem cells invasive phenotype at optimal stiffness is driven by MGAT5 dependent mechanosensing.

BACKGROUND: Glioblastomas stem-like cells (GSCs) by invading the brain parenchyma, remains after resection and radiotherapy and the tumoral microenvironment become stiffer. GSC invasion is reported as stiffness sensitive and associated with altered N-glycosylation pattern. Glycocalyx thickness modulates integrins mechanosensing, but details remain elusive and glycosylation enzymes involved are unknown. Here, we studied the association between matrix stiffness modulation, GSC migration and MGAT5 induced N-glycosylation in fibrillar 3D context. METHOD: To mimic the extracellular matrix fibrillar microenvironments, we designed 3D-ex-polyacrylonitrile nanofibers scaffolds (NFS) with adjustable stiffnesses by loading multiwall carbon nanotubes (MWCNT). GSCs neurosphere were plated on NFSs, allowing GSCs migration and MGAT5 was deleted using CRISPR-Cas9. RESULTS: We found that migration of GSCs was maximum at 166 kPa. Migration rate was correlated with cell shape, expression and maturation of focal adhesion (FA), Epithelial to Mesenchymal Transition (EMT) proteins and (ß1,6) branched N-glycan binding, galectin-3. Mutation of MGAT5 in GSC inhibited N-glycans (ß1-6) branching, suppressed the stiffness dependence of migration on 166 kPa NFS as well as the associated FA and EMT protein expression. CONCLUSION: MGAT5 catalysing multibranched N-glycans is a critical regulators of stiffness induced invasion and GSCs mechanotransduction, underpinning MGAT5 as a serious target to treat cancer.

Contractile myosin rings and cofilin-mediated actin disassembly orchestrate ECM nanotopography sensing.

The nanotopography and nanoscale geometry of the extra-cellular matrix (ECM) are important regulators of cell adhesion, motility and fate decision. However, unlike the sensing of matrix mechanics and ECM density, the molecular processes regulating the direct sensing of the ECM nanotopography and nanoscale geometry are not well understood. Here, we use nanotopographical patterns generated via electrospun nanofibre lithography (ENL) to investigate the mechanisms of nanotopography sensing by cells. We observe the dysregulation of actin dynamics, resulting in the surprising formation of actin foci. This alteration of actin organisation is regulated by myosin contractility but independent of adapter proteins such as vinculin. This process is highly dependent on differential integrin expression as ß3 integrin expressing cells, more sensitive to nanopattern dimensions than ß1 integrin expressing cells, also display increased perturbation of actin assembly and actin foci formation. We propose that, in ß3 integrin expressing cells, contractility results in the destabilisation of nanopatterned actin networks, collapsing into foci and sequestering regulators of actin dynamics such as cofilin that orchestrate disassembly. Therefore, in contrast to the sensing of substrate mechanics and ECM ligand density, which are directly orchestrated by focal adhesion assembly, we propose that nanotopography sensing is regulated by a long-range sensing mechanism, remote from focal adhesions and mediated by the actin architecture.

Mix and (mis-)match - The mechanosensing machinery in the changing environment of the developing, healthy adult and diseased heart.

The composition and the stiffness of cardiac microenvironment change during development and/or in heart disease. Cardiomyocytes (CMs) and their progenitors sense these changes, which decides over the cell fate and can trigger CM (progenitor) proliferation, differentiation, de-differentiation or death. The field of mechanobiology has seen a constant increase in output that also includes a wealth of new studies specific to cardiac or cardiomyocyte mechanosensing. As a result, mechanosensing and transduction in the heart is increasingly being recognised as a main driver of regulating the heart formation and function. Recent work has for instance focused on measuring the molecular, physical and mechanical changes of the cellular environment - as well as intracellular contributors to the passive stiffness of the heart. On the other hand, a variety of new studies shed light into the molecular machinery that allow the cardiomyocytes to sense these properties. Here we want to discuss the recent work on this topic, but also specifically focus on how the different components are regulated at various stages during development, in health or disease in order to highlight changes that might contribute to disease progression and heart failure.

Probing the nanoscale organisation and multivalency of cell surface receptors: DNA origami nanoarrays for cellular studies with single-molecule control.

Nanoscale organisation of receptor ligands has become an important approach to study the clustering behaviour of cell-surface receptors. Biomimetic substrates fabricated via different nanopatterning strategies have so far been applied to investigate specific integrins and cell types, but without multivalent control. Here we use DNA origami to surpass the limits of current approaches and fabricate nanoarrays to study different cell adhesion processes, with nanoscale spatial resolution and single-molecule control. Notably, DNA nanostructures enable the display of receptor ligands in a highly customisable manner, with modifiable parameters including ligand number, ligand spacing and most importantly, multivalency. To test the adaptability and robustness of the system we combined it with focused ion beam and electron-beam lithography nanopatterning to additionally control the distance between the origami structures (i.e. receptor clusters). Moreover, we demonstrate how the platform can be used to interrogate two different biological questions: (1) the cooperative effect of integrin and growth factor receptor in cancer cell spreading, and (2) the role of integrin clustering in cardiomyocyte adhesion and maturation. Thereby we find previously unknown clustering behaviour of different integrins, further outlining the importance for such customisable platforms for future investigations of specific receptor organisation at the nanoscale.

Costameres, dense plaques and podosomes: the cell matrix adhesions in cardiovascular mechanosensing.

The stiffness of the cardiovascular environment changes during ageing and in disease and contributes to disease incidence and progression. For instance, increased arterial stiffness can lead to atherosclerosis, while stiffening of the heart due to fibrosis can increase the chances of heart failure. Cells can sense the stiffness of the extracellular matrix through integrin adhesions and other mechanosensitive structures and in response to this initiate mechanosignalling pathways that ultimately change the cellular behaviour. Over the past decades, interest in mechanobiology has steadily increased and with this also our understanding of the molecular basis of mechanosensing and transduction. However, much of our knowledge about the mechanisms is derived from studies investigating focal adhesions in non-muscle cells, which are distinct in several regards from the cell-matrix adhesions in cardiomyocytes (costameres) or vascular smooth muscle cells (dense plaques or podosomes). Therefore, we will look here first at the evidence for mechanical sensing in the cardiovascular system, before comparing the different cytoskeletal arrangements and adhesion sites in cardiomyocytes and vascular smooth muscle cells and what is known about mechanical sensing through the various structures.