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.

Appreciating force and shape­the rise of mechanotransduction in cell biology.

Although the shapes of organisms are encoded in their genome, the developmental processes that lead to the final form of vertebrates involve a constant feedback between dynamic mechanical forces, and cell growth and motility. Mechanobiology has emerged as a discipline dedicated to the study of the effects of mechanical forces and geometry on cell growth and motility­for example, during cell-matrix adhesion development­through the signalling process of mechanotransduction.

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.

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.

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.

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.

N-WASP-directed actin polymerization activates Cas phosphorylation and lamellipodium spreading.

Tyrosine phosphorylation of the substrate domain of Cas (CasSD) correlates with increased cell migration in healthy and diseased cells. Here, we address the mechanism leading to the phosphorylation of CasSD in the context of fibronectin-induced early spreading of fibroblasts. We have previously demonstrated that mechanical stretching of CasSD exposes phosphorylation sites for Src family kinases (SFKs). Surprisingly, phosphorylation of CasSD was independent of myosin contractile activity but dependent on actin polymerization. Furthermore, we found that CasSD phosphorylation in the early stages of cell spreading required: (1) integrin anchorage and integrin-mediated activation of SFKs, (2) association of Cas with focal adhesion kinase (FAK), and (3) N-WASP-driven actin-assembly activity. These findings, and analyses of the interactions of the Cas domains, indicate that the N-terminus of Cas associates with the FAK-N-WASP complex at the protrusive edge of the cell and that the C-terminus of Cas associates with the immobilized integrin-SFK cluster. Thus, extension of the leading edge mediated by actin polymerization could stretch Cas during early cell spreading, priming it for phosphorylation.

Actin polymerization-dependent activation of Cas-L promotes immunological synapse stability.

The immunological synapse formed between a T-cell and an antigen-presenting cell is important for cell-cell communication during T-cell-mediated immune responses. Immunological synapse formation begins with stimulation of the T-cell receptor (TCR). TCR microclusters are assembled and transported to the center of the immunological synapse in an actin polymerization-dependent process. However, the physical link between TCR and actin remains elusive. Here we show that lymphocyte-specific Crk-associated substrate (Cas-L), a member of a force sensing protein family, is required for transport of TCR microclusters and for establishing synapse stability. We found that Cas-L is phosphorylated at TCR microclusters in an actin polymerization-dependent fashion. Furthermore, Cas-L participates in a positive feedback loop leading to amplification of Ca2+ signaling, inside-out integrin activation, and actomyosin contraction. We propose a new role for Cas-L in T-cell activation as a mechanical transducer linking TCR microclusters to the underlying actin network and coordinating multiple actin-dependent structures in the immunological synapse. Our studies highlight the importance of mechanotransduction processes in T-cell-mediated immune responses.

Early events in cell spreading as a model for quantitative analysis of biomechanical events.

In this review, we focus on the early events in the process of fibroblast spreading on fibronectin matrices of different rigidities. We present a focused position piece that illustrates the many different tests that a cell makes of its environment before it establishes mature matrix adhesions. When a fibroblast is placed on fibronectin-coated glass surfaces at 37°C, it typically spreads and polarizes within 20-40 min primarily through αvß3 integrin binding to fibronectin. In that short period, the cell goes through three major phases that involve binding, integrin activation, spreading, and mechanical testing of the surface. The advantage of using the model system of cell spreading from the unattached state is that it is highly reproducible and the stages that the cell undergoes can thus be studied in a highly quantitative manner, in both space and time. The mechanical and biochemical parameters that matter in this example are often surprising because of both the large number of tests that occur and the precision of the tests. We discuss our current understanding of those tests, the decision tree that is involved in this process, and an extension to the behavior of the cells at longer time periods when mature adhesions develop. Because many other matrices and integrins are involved in cell-matrix adhesion, this model system gives us a limited view of a subset of cellular behaviors that can occur. However, by defining one cellular process at a molecular level, we know more of what to expect when defining other processes. Because each cellular process will involve some different proteins, a molecular understanding of multiple functions operating within a given cell can lead to strategies to selectively block a function.

Finding the weakest link: exploring integrin-mediated mechanical molecular pathways.

From the extracellular matrix to the cytoskeleton, a network of molecular links connects cells to their environment. Molecules in this network transmit and detect mechanical forces, which subsequently determine cell behavior and fate. Here, we reconstruct the mechanical pathway followed by these forces. From matrix proteins to actin through integrins and adaptor proteins, we review how forces affect the lifetime of bonds and stretch or alter the conformation of proteins, and how these mechanical changes are converted into biochemical signals in mechanotransduction events. We evaluate which of the proteins in the network can participate in mechanotransduction and which are simply responsible for transmitting forces in a dynamic network. Besides their individual properties, we also analyze how the mechanical responses of a protein are determined by their serial connections from the matrix to actin, their parallel connections in integrin clusters and by the rate at which force is applied to them. All these define mechanical molecular pathways in cells, which are emerging as key regulators of cell function alongside better studied biochemical pathways.

Two distinct phosphorylation events govern the function of muscle FHOD3.

Posttranslational modifications such as phosphorylation are universally acknowledged regulators of protein function. Recently we characterised a striated muscle-specific isoform of the formin FHOD3 that displays distinct subcellular targeting and protein half-life compared to its non-muscle counterpart and which is dependent on phosphorylation by CK2 (formerly casein kinase 2). We now show that the two isoforms of FHOD3 are already expressed in the vertebrate embryonic heart. Analysis of CK2 alpha knockout mice showed that phosphorylation by CK2 is also required for proper targeting of muscle FHOD3 to the myofibrils in embryonic cardiomyocytes in situ. The localisation of muscle FHOD3 in the sarcomere varies depending on the maturation state, being either broader or restricted to the Z-disc proper in the adult heart. Following myofibril disassembly, such as that in dedifferentiating adult rat cardiomyocytes in culture, the expression of non-muscle FHOD3 is up-regulated, which is reversed once the myofibrils are reassembled. The shift in expression levels of different isoforms is accompanied by an increased co-localisation with p62, which is involved in autophagy, and affects the half-life of FHOD3. Phosphorylation of three amino acids in the C-terminus of FHOD3 by ROCK1 is sufficient for activation, which results in increased actin filament synthesis in cardiomyocytes and also a broader localisation pattern of FHOD3 in the myofibrils. ROCK1 can directly phosphorylate FHOD3, and FHOD3 seems to be the downstream mediator of the exaggerated actin filament formation phenotype that is induced in cardiomyocytes upon the overexpression of constitutively active ROCK1. We conclude that the expression of the muscle FHOD3 isoform is characteristic of the healthy mature heart and that two distinct phosphorylation events are crucial to regulate the activity of this isoform in thin filament assembly and maintenance.

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.

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.

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.

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.

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.

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.

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.

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.

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'.