Non-destructive Characterization of Skeletal Muscle Extracellular Matrix Morphology by Combining Optical Coherence Tomography (OCT) Imaging with Tissue Clearing.

Histology is an essential step to visualize and analyze the microstructure of any biological tissue; however, histological processing is often irreversible, and histological samples are unable to be imaged or tested further. In this work, a novel non-destructive protocol is proposed for morphological analysis of skeletal muscles, combining Optical Coherence Tomography (OCT) imaging with Tissue Clearing. Imaging combining OCT and Propylene Glycol (PG) as a tissue-clearing agent, was performed on rat tail and extensor digitorum longus (EDL) muscle. The results show that the extracellular matrix morphology of skeletal muscles, including muscular fibers and the whole microstructure architecture were clearly identified. PG improved OCT imaging as measured by image quality metric Contrast Per Pixel CPP (increases by 3.9%), Naturalness Image Quality Evaluator NIQE (decreases by 23%), and Volume of Interest VOI size (higher for CPP and lower for NIQE values). The tendon microstructure was observed with less precision, as collagen fibers could not be clearly detected. The reversibility of the optical effects of the PG on the immersed tissue (in a Phosphate-Buffered Saline solution) was studied comparing native and rehydrated OCT image acquisition from a single EDL sample. Optical properties and microstructure visibility (CPP and NIQE) have been recovered to 99% of the native sample values. Moreover, clearing process caused shrinkage of the tissue recovered to 86% of the original width. Future work will aim to employ the proposed experimental protocol to identify the local mechanical properties of biological tissues.

Deciphering the Role of Klf10 in the Cerebellum.

Recent studies have demonstrated a new role for Klf10, a Krüppel-like transcription factor, in skeletal muscle, specifically relating to mitochondrial function. Thus, it was of interest to analyze additional tissues that are highly reliant on optimal mitochondrial function such as the cerebellum and to decipher the role of Klf10 in the functional and structural properties of this brain region. In vivo (magnetic resonance imaging and localized spectroscopy, behavior analysis) and in vitro (histology, spectroscopy analysis, enzymatic activity) techniques were applied to comprehensively assess the cerebellum of wild type (WT) and Klf10 knockout (KO) mice. Histology analysis and assessment of locomotion revealed no significant difference in Klf10 KO mice. Diffusion and texture results obtained using MRI revealed structural changes in KO mice characterized as defects in the organization of axons. These modifications may be explained by differences in the levels of specific metabolites (myo-inositol, lactate) within the KO cerebellum. Loss of Klf10 expression also led to changes in mitochondrial activity as reflected by a significant increase in the activity of citrate synthase, complexes I and IV. In summary, this study has provided evidence that Klf10 plays an important role in energy production and mitochondrial function in the cerebellum.

Krüppel-like factor 10 regulates the contractile properties of skeletal muscle fibers in mice.

INTRODUCTION/AIMS: Klf10 is a member of the Krüppel-like family of transcription factors, which is implicated in mediating muscle structure (fiber size, organization of the sarcomere), muscle metabolic activity (respiratory chain), and passive force. The aim of this study was to further characterize the roles of Klf10 in the contractile properties of skeletal muscle fibers. METHODS: Fifty-two single fibers were extracted from female wild-type (WT) and Klf10 knockout (KO) oxidative (soleus) and glycolytic (extensor digitorum longus [EDL]) skinned muscles. Each fiber was immersed successively in relaxing (R), washing (W), and activating (A) solutions. Calcium was included in the activating solution to induce a maximum contraction of the fiber. The maximum force (Fmax ) was measured and normalized to the cross-sectional area to obtain the maximum stress (Stressmax ). After a steady state in contraction was reached, a quick stretch-release was performed; the force at the maximum stretch (Fstretch ) was measured and the stiffness was assessed. RESULTS: Deletion of the Klf10 gene induced changes in the contractile parameters (Fmax , Stressmax , Stiffness), which were lower and higher for soleus and EDL fibers compared with littermates, respectively. These measurements also revealed changes in the proportion and resistance of attached cross-bridges. DISCUSSION: Klf10 plays a major role in the homeostasis of the contractile behavior of skeletal muscle fibers in a muscle fiber type-specific manner. These findings further implicate important roles for Klf10 in skeletal muscle function and shed new light on understanding the molecular processes regulating the contractility of skeletal muscle fibers.

Ultrasound image processing to estimate the structural and functional properties of mouse skeletal muscle.

Noninvasive imaging techniques are increasingly used for monitoring muscle behavior in mice. However, muscle is a complex tissue that exhibits different properties under passive and active conditions. In addition to structural properties, it is also important to analyze functional characteristics. At present, such information can be obtained with ultrasound elastography. However, this technique is poorly used for small rodent models (mice and gerbils). Thus, this study aims at establish referent hindlimb muscle data, and experimental guidelines, for wild-type (WT) control mice as well as the TIEG1 knockout (KO) mouse model that is known to exhibit skeletal muscle defects. Ultrasound was performed with the Aixplorer machine using a SLH 20-6 linear transducer probe (2.8 cm footprint). A region of interest (ROI) was placed around a superficial group of muscles. Subsequently, from the B-mode image, a classification of all the muscles and ultrasound biomarkers such as echo intensity and texture anisotropy have been determined. The influence of the gain setting (from 40% to 70%) was analyzed on these parameters. Moreover, the elasticity (E) was also measured within the ROI. This study provides a suitable methodology for collecting experimental data: 1) the correct range of gain (between 50% and 70%) to apply for the ultrasound measurement of muscle structure, 2) the structural and functional referent data for a group of healthy muscles, 3) the gray scale index, the texture anisotropy and the elasticity (ETIEG1 KO = 36.1 ± 10.3 kPa, EWT = 44.4 ± 13.4 kPa) parameters, which were obtained for a group of muscles as a function of genotype.

Novel role of Tieg1 in muscle metabolism and mitochondrial oxidative capacities.

AIM: Tieg1 is involved in multiple signalling pathways, human diseases, and is highly expressed in muscle where its functions are poorly understood. METHODS: We have utilized Tieg1 knockout (KO) mice to identify novel and important roles for this transcription factor in regulating muscle ultrastructure, metabolism and mitochondrial functions in the soleus and extensor digitorum longus (EDL) muscles. RNA sequencing, immunoblotting, transmission electron microscopy, MRI, NMR, histochemical and mitochondrial function assays were performed. RESULTS: Loss of Tieg1 expression resulted in altered sarcomere organization and a significant decrease in mitochondrial number. Histochemical analyses demonstrated an absence of succinate dehydrogenase staining and a decrease in cytochrome c oxidase (COX) enzyme activity in KO soleus with similar, but diminished, effects in the EDL. Decreased complex I, COX and citrate synthase (CS) activities were detected in the soleus muscle of KO mice indicating altered mitochondrial function. Complex I activity was also diminished in KO EDL. Significant decreases in CS and respiratory chain complex activities were identified in KO soleus. 1 H-NMR spectra revealed no significant metabolic difference between wild-type and KO muscles. However, 31 P spectra revealed a significant decrease in phosphocreatine and ATPγ. Altered expression of 279 genes, many of which play roles in mitochondrial and muscle function, were identified in KO soleus muscle. Ultimately, all of these changes resulted in an exercise intolerance phenotype in Tieg1 KO mice. CONCLUSION: Our findings have implicated novel roles for Tieg1 in muscle including regulation of gene expression, metabolic activity and organization of tissue ultrastructure. This muscle phenotype resembles diseases associated with exercise intolerance and myopathies of unknown consequence.

Development of a novel multiphysical approach for the characterization of mechanical properties of musculotendinous tissues.

At present, there is a lack of well-validated protocols that allow for the analysis of the mechanical properties of muscle and tendon tissues. Further, there are no reports regarding characterization of mouse skeletal muscle and tendon mechanical properties in vivo using elastography thereby limiting the ability to monitor changes in these tissues during disease progression or response to therapy. Therefore, we sought to develop novel protocols for the characterization of mechanical properties in musculotendinous tissues using atomic force microscopy (AFM) and ultrasound elastography. Given that TIEG1 knockout (KO) mice exhibit well characterized defects in the mechanical properties of skeletal muscle and tendon tissue, we have chosen to use this model system in the present study. Using TIEG1 knockout and wild-type mice, we have devised an AFM protocol that does not rely on the use of glue or chemical agents for muscle and tendon fiber immobilization during acquisition of transversal cartographies of elasticity and topography. Additionally, since AFM cannot be employed on live animals, we have also developed an ultrasound elastography protocol using a new linear transducer, SLH20-6 (resolution: 38 µm, footprint: 2.38 cm), to characterize the musculotendinous system in vivo. This protocol allows for the identification of changes in muscle and tendon elasticities. Such innovative technological approaches have no equivalent to date, promise to accelerate our understanding of musculotendinous mechanical properties and have numerous research and clinical applications.

Impact of TIEG1 on the structural properties of fast- and slow-twitch skeletal muscle.

INTRODUCTION: Transforming growth factor-beta (TGF-ß)-inducible early gene-1 (TIEG1) is a transcription factor that is highly expressed in skeletal muscle. The purpose of this study was to characterize the structural properties of both fast-twitch (EDL) and slow-twitch (soleus) muscles in the hindlimb of TIEG1-deficient (TIEG1-/- ) mice. METHODS: Ten slow and 10 fast muscles were analyzed from TIEG1-/- and wild-type (WT) mice using MRI texture (MRI-TA) and histological analyses. RESULTS: MRI-TA could discriminate between WT slow and fast muscles. Deletion of the TIEG1 gene led to changes in the texture profile within both muscle types. Specifically, muscle isolated from TIEG1-/- mice displayed hypertrophy, hyperplasia, and a modification of fiber area distribution. CONCLUSIONS: We demonstrated that TIEG1 plays an important role in the structural properties of skeletal muscle. This study further implicates important roles for TIEG1 in the development of skeletal muscle and suggests that defects in TIEG1 expression and/or function may be associated with muscle disease. Muscle Nerve 55: 410-416, 2017.

Impact of TIEG1 Deletion on the Passive Mechanical Properties of Fast and Slow Twitch Skeletal Muscles in Female Mice.

As transforming growth factor (TGF)-ß inducible early gene-1 is highly expressed in skeletal muscle, the effect of TIEG1 gene deletion on the passive mechanical properties of slow and fast twitch muscle fibers was analyzed. Twenty five muscle fibers were harvested from soleus (Sol) and extensor digitorum longus (EDL) muscles from TIEG1-/- (N = 5) and control (N = 5) mice. Mechanical tests were performed on fibers and the dynamic and static stresses were measured. A viscoelastic Hill model of 3rd order was used to fit the experimental relaxation test data. In parallel, immunohistochemical analyses were performed on three serial transverse sections to detect the myosin isoforms within the slow and fast muscles. The percentage and the mean cross sectional area of each fiber type were calculated. These tests revealed a significant increase in the mechanical stress properties for the TIEG1-/- Sol fibers while a significant decrease appeared for the TIEG1-/- EDL fibers. Hill model tracked the shape of the experimental relaxation curve for both genotypes and both fiber types. Immunohistochemical results showed hypertrophy of all fiber types for TIEG1-/- muscles with an increase in the percentage of glycolytic fibers (IIX, and IIB) and a decrease of oxidative fibers (I, and IIA). This study has provided new insights into the role of TIEG1, known as KLF10, in the functional (SoltypeI: more resistant, EDLtypeIIB: less resistant) and morphological (glycolytic hypertrophy) properties of fast and slow twitch skeletal muscles. Further investigation at the cellular level will better reveal the role of the TIEG1 gene in skeletal muscle tissue.

The Invalidation of HspB1 Gene in Mouse Alters the Ultrastructural Phenotype of Muscles.

Even though abundance of Hsp27 is the highest in skeletal muscle, the relationships between the expression of HspB1 (encoding Hsp27) and muscle characteristics are not fully understood. In this study, we have analysed the effect of Hsp27 inactivation on mouse development and phenotype. We generated a mouse strain devoid of Hsp27 protein by homologous recombination of the HspB1 gene. The HspB1-/- mouse was viable and fertile, showing neither apparent morphological nor anatomical alterations. We detected a gender dimorphism with marked effects in males, a lower body weight (P < 0.05) with no obvious changes in the growth rate, and a lower plasma lipids profile (cholesterol, HDL and triglycerides, 0.001 < P< 0.05). The muscle structure of the animals was examined by optical microscopy and transmission electron microscopy. Not any differences in the characteristics of muscle fibres (contractile and metabolic type, shape, perimeter, cross-sectional area) were detected except a trend for a higher proportion of small fibres. Different myosin heavy chains electrophoretic profiles were observed in the HspB1-/- mouse especially the presence of an additional isoform. Electron microscopy revealed ultrastructural abnormalities in the myofibrillar structure of the HspB1-/- mouse mutant mice (e.g. destructured myofibrils and higher gaps between myofibrils) especially in the m. Soleus. Combined with our previous data, these findings suggest that Hsp27 could directly impact the organization of muscle cytoskeleton at the molecular and ultrastructural levels.

Calcium Homeostasis and Muscle Energy Metabolism Are Modified in HspB1-Null Mice.

Hsp27-encoded by HspB1-is a member of the small heat shock proteins (sHsp, 12-43 kDa (kilodalton)) family. This protein is constitutively present in a wide variety of tissues and in many cell lines. The abundance of Hsp27 is highest in skeletal muscle, indicating a crucial role for muscle physiology. The protein identified as a beef tenderness biomarker was found at a crucial hub in a functional network involved in beef tenderness. The aim of this study was to analyze the proteins impacted by the targeted invalidation of HspB1 in the Tibialis anterior muscle of the mouse. Comparative proteomics using two-dimensional gel electrophoresis revealed 22 spots that were differentially abundant between HspB1-null mice and their controls that could be identified by mass spectrometry. Eighteen spots were more abundant in the muscle of the mutant mice, and four were less abundant. The proteins impacted by the absence of Hsp27 belonged mainly to calcium homeostasis (Srl and Calsq1), contraction (TnnT3), energy metabolism (Tpi1, Mdh1, PdhB, Ckm, Pygm, ApoA1) and the Hsp proteins family (HspA9). These data suggest a crucial role for these proteins in meat tenderization. The information gained by this study could also be helpful to predict the side effects of Hsp27 depletion in muscle development and pathologies linked to small Hsps.

A network-based approach for predicting Hsp27 knock-out targets in mouse skeletal muscles.

Thanks to genomics, we have previously identified markers of beef tenderness, and computed a bioinformatic analysis that enabled us to build an interactome in which we found Hsp27 at a crucial node. Here, we have used a network-based approach for understanding the contribution of Hsp27 to tenderness through the prediction of its interactors related to tenderness. We have revealed the direct interactors of Hsp27. The predicted partners of Hsp27 included proteins involved in different functions, e.g. members of Hsp families (Hsp20, Cryab, Hsp70a1a, and Hsp90aa1), regulators of apoptosis (Fas, Chuk, and caspase-3), translation factors (Eif4E, and Eif4G1), cytoskeletal proteins (Desmin) and antioxidants (Sod1). The abundances of 15 proteins were quantified by Western blotting in two muscles of HspB1-null mice and their controls. We observed changes in the amount of most of the Hsp27 predicted targets in mice devoid of Hsp27 mainly in the most oxidative muscle. Our study demonstrates the functional links between Hsp27 and its predicted targets. It suggests that Hsp status, apoptotic processes and protection against oxidative stress are crucial for post-mortem muscle metabolism, subsequent proteolysis, and therefore for beef tenderness.