The desmin mutation R349P increases contractility and fragility of stem cell-generated muscle micro-tissues.

AIMS: Desminopathies comprise hereditary myopathies and cardiomyopathies caused by mutations in the intermediate filament protein desmin that lead to severe and often lethal degeneration of striated muscle tissue. Animal and single cell studies hinted that this degeneration process is associated with massive ultrastructural defects correlating with increased susceptibility of the muscle to acute mechanical stress. The underlying mechanism of mechanical susceptibility, and how muscle degeneration develops over time, however, has remained elusive. METHODS: Here, we investigated the effect of a desmin mutation on the formation, differentiation, and contractile function of in vitro-engineered three-dimensional micro-tissues grown from muscle stem cells (satellite cells) isolated from heterozygous R349P desmin knock-in mice. RESULTS: Micro-tissues grown from desmin-mutated cells exhibited spontaneous unsynchronised contractions, higher contractile forces in response to electrical stimulation, and faster force recovery compared with tissues grown from wild-type cells. Within 1 week of culture, the majority of R349P desmin-mutated tissues disintegrated, whereas wild-type tissues remained intact over at least three weeks. Moreover, under tetanic stimulation lasting less than 5 s, desmin-mutated tissues partially or completely ruptured, whereas wild-type tissues did not display signs of damage. CONCLUSIONS: Our results demonstrate that the progressive degeneration of desmin-mutated micro-tissues is closely linked to extracellular matrix fibre breakage associated with increased contractile forces and unevenly distributed tensile stress. This suggests that the age-related degeneration of skeletal and cardiac muscle in patients suffering from desminopathies may be similarly exacerbated by mechanical damage from high-intensity muscle contractions. We conclude that micro-tissues may provide a valuable tool for studying the organization of myocytes and the pathogenic mechanisms of myopathies.

Plectin ensures intestinal epithelial integrity and protects colon against colitis.

Plectin, a highly versatile cytolinker protein, provides tissues with mechanical stability through the integration of intermediate filaments (IFs) with cell junctions. Here, we hypothesize that plectin-controlled cytoarchitecture is a critical determinant of the intestinal barrier function and homeostasis. Mice lacking plectin in an intestinal epithelial cell (IEC; PleΔIEC) spontaneously developed colitis characterized by extensive detachment of IECs from the basement membrane (BM), increased intestinal permeability, and inflammatory lesions. Moreover, plectin expression was reduced in the colons of ulcerative colitis (UC) patients and negatively correlated with the severity of colitis. Mechanistically, plectin deficiency in IECs led to aberrant keratin filament (KF) network organization and the formation of dysfunctional hemidesmosomes (HDs) and intercellular junctions. In addition, the hemidesmosomal α6ß4 integrin (Itg) receptor showed attenuated association with KFs, and protein profiling revealed prominent downregulation of junctional constituents. Consistent with the effects of plectin loss in the intestinal epithelium, plectin-deficient IECs exhibited remarkably reduced mechanical stability and limited adhesion capacity in vitro. Feeding mice with a low-residue liquid diet that reduced mechanical stress and antibiotic treatment successfully mitigated epithelial damage in the PleΔIEC colon.

Treatment of keratinocytes with 4-phenylbutyrate in epidermolysis bullosa: Lessons for therapies in keratin disorders.

BACKGROUND: Missense mutations in keratin 5 and 14 genes cause the severe skin fragility disorder epidermolysis bullosa simplex (EBS) by collapsing of the keratin cytoskeleton into cytoplasmic protein aggregates. Despite intense efforts, no molecular therapies are available, mostly due to the complex phenotype of EBS, comprising cell fragility, diminished adhesion, skin inflammation and itch. METHODS: We extensively characterized KRT5 and KRT14 mutant keratinocytes from patients with severe generalized EBS following exposure to the chemical chaperone 4-phenylbutyrate (4-PBA). FINDINGS: 4-PBA diminished keratin aggregates within EBS cells and ameliorated their inflammatory phenotype. Chemoproteomics of 4-PBA-treated and untreated EBS cells revealed reduced IL1ß expression- but also showed activation of Wnt/ß-catenin and NF-kB pathways. The abundance of extracellular matrix and cytoskeletal proteins was significantly altered, coinciding with diminished keratinocyte adhesion and migration in a 4-PBA dose-dependent manner. INTERPRETATION: Together, our study reveals a complex interplay of benefits and disadvantages that challenge the use of 4-PBA in skin fragility disorders.

Early signs of architectural and biomechanical failure in isolated myofibers and immortalized myoblasts from desmin-mutant knock-in mice.

In striated muscle, desmin intermediate filaments interlink the contractile myofibrillar apparatus with mitochondria, nuclei, and the sarcolemma. The desmin network's pivotal role in myocytes is evident since mutations in the human desmin gene cause severe myopathies and cardiomyopathies. Here, we investigated skeletal muscle pathology in myofibers and myofibrils isolated from young hetero- and homozygous R349P desmin knock-in mice, which carry the orthologue of the most frequent human desmin missense mutation R350P. We demonstrate that mutant desmin alters myofibrillar cytoarchitecture, markedly disrupts the lateral sarcomere lattice and distorts myofibrillar angular axial orientation. Biomechanical assessment revealed a high predisposition to stretch-induced damage in fiber bundles of R349P mice. Notably, Ca2+-sensitivity and passive myofibrillar tension were decreased in heterozygous fiber bundles, but increased in homozygous fiber bundles compared to wildtype mice. In a parallel approach, we generated and subsequently subjected immortalized heterozygous R349P desmin knock-in myoblasts to magnetic tweezer experiments that revealed a significantly increased sarcolemmal lateral stiffness. Our data suggest that mutated desmin already markedly impedes myocyte structure and function at pre-symptomatic stages of myofibrillar myopathies.

The role of focal adhesion anchoring domains of CAS in mechanotransduction.

CAS is a docking protein, which was shown to act as a mechanosensor in focal adhesions. The unique assembly of structural domains in CAS is important for its function as a mechanosensor. The tension within focal adhesions is transmitted to a stretchable substrate domain of CAS by focal adhesion-targeting of SH3 and CCH domain of CAS, which anchor the CAS protein in focal adhesions. Mechanistic models of the stretching biosensor propose equal roles for both anchoring domains. Using deletion mutants and domain replacements, we have analyzed the relative importance of the focal adhesion anchoring domains on CAS localization and dynamics in focal adhesions as well as on CAS-mediated mechanotransduction. We confirmed the predicted prerequisite of the focal adhesion targeting for CAS-dependent mechanosensing and unraveled the critical importance of CAS SH3 domain in mechanosensing. We further show that CAS localizes to the force transduction layer of focal adhesions and that mechanical stress stabilizes CAS in focal adhesions.

Mechanical plasticity of cells.

Under mechanical loading, most living cells show a viscoelastic deformation that follows a power law in time. After removal of the mechanical load, the cell shape recovers only incompletely to its original undeformed configuration. Here, we show that incomplete shape recovery is due to an additive plastic deformation that displays the same power-law dynamics as the fully reversible viscoelastic deformation response. Moreover, the plastic deformation is a constant fraction of the total cell deformation and originates from bond ruptures within the cytoskeleton. A simple extension of the prevailing viscoelastic power-law response theory with a plastic element correctly predicts the cell behaviour under cyclic loading. Our findings show that plastic energy dissipation during cell deformation is tightly linked to elastic cytoskeletal stresses, which suggests the existence of an adaptive mechanism that protects the cell against mechanical damage.

Determining the mechanical properties of plectin in mouse myoblasts and keratinocytes.

Plectin is the prototype of an intermediate filament (IF)-based cytolinker protein. It affects cells mechanically by interlinking and anchoring cytoskeletal filaments and acts as scaffolding and docking platform for signaling proteins to control cytoskeleton dynamics. The most common disease caused by mutations in the human plectin gene, epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is characterized by severe skin blistering and progressive muscular dystrophy. Therefore, we compared the biomechanical properties and the response to mechanical stress of murine plectin-deficient myoblasts and keratinocytes with wild-type cells. Using a cell stretching device, plectin-deficient myoblasts exhibited lower mechanical vulnerability upon external stress compared to wild-type cells, which we attributed to lower cellular pre-stress. Contrary to myoblasts, wild-type and plectin-deficient keratinocytes showed no significant differences. In magnetic tweezer measurements using fibronectin-coated paramagnetic beads, the stiffness of keratinocytes was higher than of myoblasts. Interestingly, cell stiffness, adhesion strength, and cytoskeletal dynamics were strikingly altered in plectin-deficient compared to wild-type myoblasts, whereas smaller differences were observed between plectin-deficient and wild-type keratinocytes, indicating that plectin might be more important for stabilizing cytoskeletal structures in myoblasts than in keratinocytes. Traction forces strongly correlated with the stiffness of plectin-deficient and wild-type myoblasts and keratinocytes. Contrary to that cell motility was comparable in plectin-deficient and wild-type myoblasts, but was significantly increased in plectin-deficient compared to wild-type keratinocytes. Thus, we postulate that the lack of plectin has divergent implications on biomechanical properties depending on the respective cell type.

Measuring mechanical properties in cells: three easy methods for biologists.

The mechanism by which cells sense stresses and transmit them throughout the cytoplasm and the cytoskeleton (CSK) and by which these mechanical signals are converted into biochemical signaling responses is not clear. Specifically, there is little direct experimental evidence on how intracellular CSK structural elements in living cells deform and transmit stresses in response to external mechanical forces. Existing theories have invoked various biophysical and biochemical mechanisms to explain how cells spread, deform, divide, move, and change shape in response to mechanical inputs, but rigorous tests in cells are lacking. The lack of data and understanding is preventing the identification of mechanisms and sites of mechano-regulation in cells. Here, we introduce and describe three unique and easy methods for biologists to determine mechanical properties and signaling events in cells.