Enzymatically dissociated muscle fibers display rapid dedifferentiation and impaired mitochondrial calcium control.

Cells rapidly lose their physiological phenotype upon disruption of their extracellular matrix (ECM)-intracellular cytoskeleton interactions. By comparing adult mouse skeletal muscle fibers, isolated either by mechanical dissection or by collagenase-induced ECM digestion, we investigated acute effects of ECM disruption on cellular and mitochondrial morphology, transcriptomic signatures, and Ca2+ handling. RNA-sequencing showed striking differences in gene expression patterns between the two isolation methods with enzymatically dissociated fibers resembling myopathic phenotypes. Mitochondrial appearance was grossly similar in the two groups, but 3D electron microscopy revealed shorter and less branched mitochondria following enzymatic dissociation. Repeated contractions resulted in a prolonged mitochondrial Ca2+ accumulation in enzymatically dissociated fibers, which was partially prevented by cyclophilin inhibitors. Of importance, muscle fibers of mice with severe mitochondrial myopathy show pathognomonic mitochondrial Ca2+ accumulation during repeated contractions and this accumulation was concealed with enzymatic dissociation, making this an ambiguous method in studies of native intracellular Ca2+ fluxes.

Myofibrillar Lattice Remodeling Is a Structural Cytoskeletal Predictor of Diaphragm Muscle Weakness in a Fibrotic mdx (mdx Cmah-/-) Model.

Duchenne muscular dystrophy (DMD) is a degenerative genetic myopathy characterized by complete absence of dystrophin. Although the mdx mouse lacks dystrophin, its phenotype is milder compared to DMD patients. The incorporation of a null mutation in the Cmah gene led to a more DMD-like phenotype (i.e., more fibrosis). Although fibrosis is thought to be the major determinant of 'structural weakness', intracellular remodeling of myofibrillar geometry was shown to be a major cellular determinant thereof. To dissect the respective contribution to muscle weakness, we assessed biomechanics and extra- and intracellular architecture of whole muscle and single fibers from extensor digitorum longus (EDL) and diaphragm. Despite increased collagen contents in both muscles, passive stiffness in mdx Cmah-/- diaphragm was similar to wt mice (EDL muscles were twice as stiff). Isometric twitch and tetanic stresses were 50% reduced in mdx Cmah-/- diaphragm (15% in EDL). Myofibrillar architecture was severely compromised in mdx Cmah-/- single fibers of both muscle types, but more pronounced in diaphragm. Our results show that the mdx Cmah-/- genotype reproduces DMD-like fibrosis but is not associated with changes in passive visco-elastic muscle stiffness. Furthermore, detriments in active isometric force are compatible with the pronounced myofibrillar disarray of the dystrophic background.

In mammalian skeletal muscle, phosphorylation of TOMM22 by protein kinase CSNK2/CK2 controls mitophagy.

In yeast, Tom22, the central component of the TOMM (translocase of outer mitochondrial membrane) receptor complex, is responsible for the recognition and translocation of synthesized mitochondrial precursor proteins, and its protein kinase CK2-dependent phosphorylation is mandatory for TOMM complex biogenesis and proper mitochondrial protein import. In mammals, the biological function of protein kinase CSNK2/CK2 remains vastly elusive and it is unknown whether CSNK2-dependent phosphorylation of TOMM protein subunits has a similar role as that in yeast. To address this issue, we used a skeletal muscle-specific Csnk2b/Ck2ß-conditional knockout (cKO) mouse model. Phenotypically, these skeletal muscle Csnk2b cKO mice showed reduced muscle strength and abnormal metabolic activity of mainly oxidative muscle fibers, which point towards mitochondrial dysfunction. Enzymatically, active muscle lysates from skeletal muscle Csnk2b cKO mice phosphorylate murine TOMM22, the mammalian ortholog of yeast Tom22, to a lower extent than lysates prepared from controls. Mechanistically, CSNK2-mediated phosphorylation of TOMM22 changes its binding affinity for mitochondrial precursor proteins. However, in contrast to yeast, mitochondrial protein import seems not to be affected in vitro using mitochondria isolated from muscles of skeletal muscle Csnk2b cKO mice. PINK1, a mitochondrial health sensor that undergoes constitutive import under physiological conditions, accumulates within skeletal muscle Csnk2b cKO fibers and labels abnormal mitochondria for removal by mitophagy as demonstrated by the appearance of mitochondria-containing autophagosomes through electron microscopy. Mitophagy can be normalized by either introduction of a phosphomimetic TOMM22 mutant in cultured myotubes, or by in vivo electroporation of phosphomimetic Tomm22 into muscles of mice. Importantly, transfection of the phosphomimetic Tomm22 mutant in muscle cells with ablated Csnk2b restored their oxygen consumption rate comparable to wild-type levels. In sum, our data show that mammalian CSNK2-dependent phosphorylation of TOMM22 is a critical switch for mitophagy and reveal CSNK2-dependent physiological implications on metabolism, muscle integrity and behavior.

Preaged remodeling of myofibrillar cytoarchitecture in skeletal muscle expressing R349P mutant desmin.

The majority of hereditary and acquired myopathies are clinically characterized by progressive muscle weakness. We hypothesized that ongoing derangement of skeletal muscle cytoarchitecture at the single fiber level may precede and be responsible for the progressive muscle weakness. Here, we analyzed the effects of aging in wild-type (wt) and heterozygous (het) and homozygous (hom) R349P desmin knock-in mice. The latter harbor the ortholog of the most frequently encountered human R350P desmin missense mutation. We quantitatively analyzed the subcellular cytoarchitecture of fast- and slow-twitch muscles from young, intermediate, and aged wt as well as desminopathy mice. We recorded multiphoton second harmonic generation and nuclear fluorescence signals in single muscle fibers to compare aging-related effects in all genotypes. The analysis of wt mice revealed that the myofibrillar cytoarchitecture remained stable with aging in fast-twitch muscles, whereas slow-twitch muscle fibers displayed structural derangements during aging. In contrast, the myofibrillar cytoarchitecture and nuclear density were severely compromised in fast- and slow-twitch muscle fibers of hom R349P desmin mice at all ages. Het mice only showed a clear degradation in their fiber structure in fast-twitch muscles from the adult to the presenescent age bin. Our study documents distinct signs of normal and R349P mutant desmin-related remodeling of the 3D myofibrillar architecture during aging, which provides a structural basis for the progressive muscle weakness.

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.

Second Harmonic Generation Microscopy of Muscle Cell Morphology and Dynamics.

Microscopy in combination with contrast-increasing dyes allows the visualization and analysis of organs, tissues, and various cells. Because of their better resolution, the development of confocal and laser microscopes enables the investigations of cell components, which are labeled with fluorescent dyes. The imaging of living cells on subcellular level (also in vivo) needs a labeling by gene transfection of GFP or similar labeled proteins. We present a method for visualization of cell structure in skeletal and heart muscle by label-free Second Harmonic Generation (SHG) microscopy and describe analytic methods for quantitative measurements of morphology and dynamics in skeletal muscle fibers.

Store-Operated Ca2+ Entry (SOCE) and Purinergic Receptor-Mediated Ca2+ Homeostasis in Murine bv2 Microglia Cells: Early Cellular Responses to ATP-Mediated Microglia Activation.

Microglia activation is a neuroinflammatory response to parenchymal damage with release of intracellular metabolites, e.g., purines, and signaling molecules from damaged cells. Extracellular purines can elicit Ca2+-mediated microglia activation involving P2X/P2Y receptors with metabotropic (P2Y) and ionotropic (P2X) cell signaling in target cells. Such microglia activation results in increased phagocytic activity, activation of their inflammasome and release of cytokines to sustain neuroinflammatory (so-called M1/M2 polarization). ATP-induced activation of ionotropic P2X4 and P2X7 receptors differentially induces receptor-operated Ca2+ entry (ROCE). Although store-operated Ca2+ entry (SOCE) was identified to modulate ROCE in primary microglia, its existence and role in one of the most common murine microglia cell line, BV2, is unknown. To dissect SOCE from ROCE in BV2 cells, we applied high-resolution multiphoton Ca2+ imaging. After depleting internal Ca2+ stores, SOCE was clearly detectable. High ATP concentrations (1 mM) elicited sustained increases in intracellular [Ca2+]i whereas lower concentrations (≤100 µM) also induced Ca2+ oscillations. These differential responses were assigned to P2X7 and P2X4 activation, respectively. Pharmacologically inhibiting P2Y and P2X responses did not affect SOCE, and in fact, P2Y-responses were barely detectable in BV2 cells. STIM1S content was significantly upregulated by 1 mM ATP. As P2X-mediated Ca2+ oscillations were rare events in single cells, we implemented a high-content screening approach that allows to record Ca2+ signal patterns from a large number of individual cells at lower optical resolution. Using automated classifier analysis, several drugs (minocycline, U73122, U73343, wortmannin, LY294002, AZ10606120) were tested on their profile to act on Ca2+ oscillations (P2X4) and sustained [Ca2+]i increases. We demonstrate specific drug effects on purinergic Ca2+ pathways and provide new pharmacological insights into Ca2+ oscillations in BV2 cells. For example, minocycline inhibits both P2X7- and P2X4-mediated Ca2+-responses, and this may explain its anti-inflammatory action in neuroinflammatory disease. As a technical result, our novel automated bio-screening approach provides a biomedical engineering platform to allow high-content drug library screens to study neuro-inflammation in vitro.

A novel quantitative morphometry approach to assess regeneration in dystrophic skeletal muscle.

Duchenne muscular dystrophy is an inherited degenerative muscle disease with progressive weakness of skeletal and cardiac muscle. Disturbed calcium homeostasis and signalling pathways result in degeneration/regeneration cycles with fibrotic remodelling of muscle tissue, sustained by chronic inflammation. In addition to altered microarchitecture, regeneration in dystrophic muscle fibres is often only classified by centrally located nuclei but correlation of the regeneration process to nuclear volumes, myosin amounts, architecture and functional quality are missing, in particular in old muscles where the regenerative capacity is exhausted. Such information could yield novel regeneration-to-function biomarkers. Here we used second harmonic generation and multi photon fluorescence microscopy in intact single muscle fibres from wild-type, dystrophic mdx and transgenic mdx mice expressing an Δex 17-48 mini-dystrophin to determine the percentage of centronucleated fibres and nucleus-to-myosin volume ratio as a function of age. Based on this ratio we define a 'biomotoric efficiency' as an optical measure for fibre maturation, which is close to unity in adult wild-type and mini-dystrophin fibres, but smaller in very young and old mdx mice as a result of ongoing cell maturation (young) and regeneration (aged). With these parameters it is possible to provide a quantitative measure about muscle fibre regeneration.

IL-1α reversibly inhibits skeletal muscle ryanodine receptor. a novel mechanism for critical illness myopathy?

Critical illness myopathies in patients with sepsis or sustained mechanical ventilation prolong intensive care treatment and threaten both patients and health budgets; no specific therapy is available. Underlying pathophysiological mechanisms are still patchy. We characterized IL-1α action on muscle performance in "skinned" muscle fibers using force transducers and confocal Ca(2+) fluorescence microscopy for force/Ca(2+) transients and Ca(2+) sparks. Association of IL-1α with sarcoplasmic reticulum (SR) release channel, ryanodine receptor (RyR) 1, was investigated with coimmunoprecipitation and confocal immunofluorescence colocalization. Membrane integrity was studied in single, intact fibers challenged with IL-1α. IL-1α reversibly stabilized Mg(2+) inhibition of Ca(2+) release. Low Mg(2+)-induced force and Ca(2+) transients were reversibly abolished by IL-1α. At normal Mg(2+), IL-1α reversibly increased caffeine-induced force and Ca(2+) transients. IL-1α reduced SR Ca(2+) leak via RyR1, as judged by (1) increased SR Ca(2+) retention, (2) increased IL-1α force transients being reproduced by 25 µM tetracaine, and (3) reduced Ca(2+) spark frequencies by IL-1α or tetracaine. Coimmunoprecipitation confirmed RyR1/IL-1 association. RyR1/IL-1 immunofluorescence patterns perfectly colocalized. Long-term, 8-hour IL-1α challenge of intact muscle fibers compromised membrane integrity in approximately 50% of fibers, and confirmed intracellular IL-1α deposition. IL-1α exerts a novel, specific, and reversible interaction mechanism with the skeletal muscle RyR1 macromolecular release complex without the need to act via its membrane IL-1 receptor, as IL-1R membrane expression levels were not detectable in Western blots or immunostaining of single fibers. We present a potential explanation of how the inflammatory mediator, IL-1α, may contribute to muscle weakness in critical illness.

From chaos to split-ups--SHG microscopy reveals a specific remodelling mechanism in ageing dystrophic muscle.

Duchenne muscular dystrophy (DMD) is a common inherited muscle disease showing chronic inflammation and progressive muscle weakness. Absent dystrophin renders sarcolemma more Ca(2+) -permeable, disturbs signalling and triggers inflammation. Sustained degeneration/regeneration cycles render muscle cytoarchitecture susceptible to remodelling. Quantitative morphometry was introduced in living cells using second-harmonic generation (SHG) microscopy of myosin. As the time course of cellular remodelling is not known, we used SHG microscopy in mdx muscle fibres over a wide age range for three-dimensional (3D) rendering and detection of verniers and cosine angle sums (CASs). Wild-type (wt) and transgenic mini-dystrophin mice (MinD) were also studied. Vernier densities (VDs) declined in wt and MinD fibres until adulthood, while in mdx fibres, VDs remained significantly elevated during the life span. CAS values were close to unity in adult wt and MinD fibres, in agreement with tight regular myofibril orientation, while always smaller in mdx fibres. Using SHG 3D morphometry, we identified two types of altered ultrastructure: branched fibres and a novel, previously undetected 'chaotic' fibre type, both of which can be classified by distinct CAS and VD combinations. We present a novel model of tissue remodelling in dystrophic progression with age that involves the transition from normal to chaotic to branched fibres. Our model predicts a ~50% contribution of altered cytoarchitecture to progressive force loss with age. We also provide an improved automated image algorithm that is suitable for future ageing studies in human myopathies.

Automated multiscale morphometry of muscle disease from second harmonic generation microscopy using tensor-based image processing.

Practically, all chronic diseases are characterized by tissue remodeling that alters organ and cellular function through changes to normal organ architecture. Some morphometric alterations become irreversible and account for disease progression even on cellular levels. Early diagnostics to categorize tissue alterations, as well as monitoring progression or remission of disturbed cytoarchitecture upon treatment in the same individual, are a new emerging field. They strongly challenge spatial resolution and require advanced imaging techniques and strategies for detecting morphological changes. We use a combined second harmonic generation (SHG) microscopy and automated image processing approach to quantify morphology in an animal model of inherited Duchenne muscular dystrophy (mdx mouse) with age. Multiphoton XYZ image stacks from tissue slices reveal vast morphological deviation in muscles from old mdx mice at different scales of cytoskeleton architecture: cell calibers are irregular, myofibrils within cells are twisted, and sarcomere lattice disruptions (detected as "verniers") are larger in number compared to samples from healthy mice. In young mdx mice, such alterations are only minor. The boundary-tensor approach, adapted and optimized for SHG data, is a suitable approach to allow quick quantitative morphometry in whole tissue slices. The overall detection performance of the automated algorithm compares very well with manual "by eye" detection, the latter being time consuming and prone to subjective errors. Our algorithm outperfoms manual detection by time with similar reliability. This approach will be an important prerequisite for the implementation of a clinical image databases to diagnose and monitor specific morphological alterations in chronic (muscle) diseases.