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.

Serum and Soleus Metabolomics Signature of Klf10 Knockout Mice to Identify Potential Biomarkers.

The transcription factor Krüppel-like factor 10 (Klf10), also known as Tieg1 for TGFß (Inducible Early Gene-1) is known to control numerous genes in many cell types that are involved in various key biological processes (differentiation, proliferation, apoptosis, inflammation), including cell metabolism and human disease. In skeletal muscle, particularly in the soleus, deletion of the Klf10 gene (Klf10 KO) resulted in ultrastructure fiber disorganization and mitochondrial metabolism deficiencies, characterized by muscular hypertrophy. To determine the metabolic profile related to loss of Klf10 expression, we analyzed blood and soleus tissue using UHPLC-Mass Spectrometry. Metabolomics analyses on both serum and soleus revealed profound differences between wild-type (WT) and KO animals. Klf10 deficient mice exhibited alterations in metabolites associated with energetic metabolism. Additionally, chemical classes of aromatic and amino-acid compounds were disrupted, together with Krebs cycle intermediates, lipids and phospholipids. From variable importance in projection (VIP) analyses, the Warburg effect, citric acid cycle, gluconeogenesis and transfer of acetyl groups into mitochondria appeared to be possible pathways involved in the metabolic alterations observed in Klf10 KO mice. These studies have revealed essential roles for Klf10 in regulating multiple metabolic pathways whose alterations may underlie the observed skeletal muscle defects as well as other diseases.

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.

Improved Ultrasound Attenuation Measurement Method for the Non-invasive Evaluation of Hepatic Steatosis Using FibroScan.

Controlled attenuation parameter (CAP) is a measurement of ultrasound attenuation used to assess liver steatosis non-invasively. However, the standard method has some limitations. This study assessed the performance of a new CAP method by ex vivo and in vivo assessments. The major difference with the new method is that it uses ultrasound data continuously acquired during the imaging phase of the FibroScan examination. Seven reference tissue-mimicking phantoms were used to test the performance. In vivo performance was assessed in two cohorts (in total 195 patients) of patients using magnetic resonance imaging proton density fat fraction (MRI-PDFF) as a reference. The precision of CAP was improved by more than 50% on tissue-mimicking phantoms and 22%-41% in the in vivo cohort studies. The agreement between both methods was excellent, and the correlation between CAP and MRI-PDFF improved in both studies (0.71 to 0.74; 0.70 to 0.76). Using MRI-PDFF as a reference, the diagnostic performance of the new method was at least equal or superior (area under the receiver operating curve 0.889-0.900, 0.835-0.873). This study suggests that the new continuous CAP method can significantly improve the precision of CAP measurements ex vivo and in vivo.

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.

MRI-based finite element modeling of facial mimics: a case study on the paired zygomaticus major muscles.

Finite element simulation of facial mimics provides objective indicators about soft tissue functions for improving diagnosis, treatment and follow-up of facial disorders. There is a lack of in vivo experimental data for model development and validation. In this study, the contribution of the paired Zygomaticus Major (ZM) muscle contraction on the facial mimics was investigated using in vivo experimental data derived from MRI. Maximal relative differences of 7.7% and 37% were noted between MRI-based measurements and numerical outcomes for ZM and skin deformation behaviors respectively. This study opens a new direction to simulate facial mimics with in vivo data.

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.

Viscoelastic shear properties of in vivo thigh muscles measured by MR elastography.

PURPOSE: To measure the viscoelastic properties of passive thigh muscles using multifrequency magnetic resonance elastography (MMRE) and rheological models. MATERIALS AND METHODS: Four muscles in five volunteers underwent MMRE tests set up inside a 1.5T magnetic resonance imaging (MRI) scanner. Compression excitation was generated with a driver attached around the thigh, and waves were generated at 70, 90, and 110 Hz. In vivo experimental viscoelastic parameters (G(ω) = G' + i G″) were extracted from the wavelength and attenuation measurements along a local profile in the direction of the wave's displacement. The data-processing method was validated on a phantom using MMRE and RheoSpectris tests. The complex modulus (G(ω)) related to elasticity (µ) and viscosity (η) was then determined using four rheological models. RESULTS: Zener was the best-fit model (χ ∼0.35 kPa) for the rheological parameters of all muscles. Similar behaviors for the elastic components for each muscle were found for the Zener and springpot models. The gracilis muscle showed higher elastic values (about 2 kPa) in both models compared to other muscles. The α-values for each muscle was equivalent to the ratio G″/G' at 90 Hz. CONCLUSION: MMRE tests associated with data processing demonstrated that the complex shear modulus G(ω) of passive muscles could be analyzed using two rheological models. The viscoelastic data can be used as a reference for future assessment of muscular dysfunction. J. Magn. Reson. Imaging 2015. J. Magn. Reson. Imaging 2016;43:1423-1433.

In vivo assessment of nervous fiber distribution in the intervertebral disc.

This present study aims to assess in vivo the nervous fibers distribution in the intervertebral disc using diffusion tensor imaging technique. Five healthy subjects participated into the data acquisition. Fiber extraction and tracking algorithms were used. The number of fibers in L4/5 disc ranges from 314 to 679 and the mean fiber length L4/5 in disc ranges from 8.22 ± 2.36 mm to 11.24 ± 5.17 mm. This study showed the feasibility of using diffusion tensor imaging technique to detect and assess the nervous fibers in the intervertebral discs. This could be of great clinical interest for the study of the correlations between these useful characteristics with pain levels on the low back pain patients.

Analysis of shear wave propagation derived from MR elastography in 3D thigh skeletal muscle using subject specific finite element model.

The purpose of this study was to develop a subject specific finite element model derived from MRI images to numerically analyze the MRE (magnetic resonance elastography) shear wave propagation within skeletal thigh muscles. A sagittal T2 CUBE MRI sequence was performed on the 20-cm thigh segment of a healthy male subject. Skin, adipose tissue, femoral bone and 11 muscles were manually segmented in order to have 3D smoothed solid and meshed models. These tissues were modeled with different constitutive laws. A transient modal dynamics analysis was applied to simulate the shear wave propagation within the thigh tissues. The effects of MRE experimental parameters (frequency, force) and the muscle material properties (shear modulus: C10) were analyzed through the simulated shear wave displacement within the vastus medialis muscle. The results showed a plausible range of frequencies (from 90Hz to 120 Hz), which could be used for MRE muscle protocol. The wave amplitude increased with the level of the force, revealing the importance of the boundary condition. Moreover, different shear displacement patterns were obtained as a function of the muscle mechanical properties. The present study is the first to analyze the shear wave propagation in skeletal muscles using a 3D subject specific finite element model. This study could be of great value to assist the experimenters in the set-up of MRE protocols.

Facial mimics simulation using MRI and finite element analysis.

Recent research studies aimed to simulate facial expressions or motions due to muscle contraction using biomechanical models ranging from basic to advanced muscle constitutive models. However, these models are based on generic geometries and there is a lack of experimental data for the numerical simulation as well as for the model validation in a clinical context. The objective of our present study was to perform facial mimics simulation using subject specific data derived from MRI technique. Zygomaticus major muscle is modelled as a transversely isotropic hyperelastic material. Then the resulting effect of its shortening and lengthening process on the facial mimics simulation was performed using Finite Element Analysis. Simulation results were presented and discussed. Such study will be of interest for defining objective criteria to evaluate the facial disfigurement patients and to perform the functional rehabilitation.