Skeletal muscle phenotyping of Hippo gene-mutated mice reveals that Lats1 deletion increases the percentage of type I muscle fibers

The Hippo signal transduction network regulates transcription through Yap/Taz-Tead1-4 in many tissues including skeletal muscle. Whilst transgenic mice have been generated for many Hippo genes, the resultant skeletal muscle phenotypes were not always characterized. Here, we aimed to phenotype the hindlimb muscles of Hippo gene-mutated Lats1−/−, Mst2−/−, Vgll3−/−, and Vgll4+/− mice. This analysis revealed that Lats1−/− mice have 11% more slow type I fibers than age and sex-matched wild-type controls. Moreover, the mRNA expression of slow Myh7 increased by 50%, and the concentration of type I myosin heavy chain is 80% higher in Lats1−/− mice than in age and sex-matched wild-type controls. Second, to find out whether exercise-related stimuli affect Lats1, we stimulated C2C12 myotubes with the hypertrophy agent clenbuterol or the energy stress agent AICAR. We found that both stimulated Lats1 expression by 1.2 and 1.3 fold respectively. Third, we re-analyzed published datasets and found that Lats1 mRNA in muscle is 63% higher in muscular dystrophy, increases by 17–77% after cardiotoxin-induced muscle injury, by 41–71% in muscles during overload-induced hypertrophy, and by 19–21% after endurance exercise when compared to respective controls. To conclude, Lats1 contributes to the regulation of muscle fiber type proportions, and its expression is regulated by physiological and pathological situations in skeletal muscle.

Asymmetric and transient properties of reciprocal activity of antagonists during the paw-shake response in the cat

Mutually inhibitory populations of neurons, half-center oscillators (HCOs), are commonly involved in the dynamics of the central pattern generators (CPGs) driving various rhythmic movements. Previously, we developed a multifunctional, multistable symmetric HCO model which produced slow locomotor-like and fast paw-shake-like activity patterns. Here, we describe asymmetric features of paw-shake responses in a symmetric HCO model and test these predictions experimentally. We considered bursting properties of the two model half-centers during transient paw-shake-like responses to short perturbations during locomotor-like activity. We found that when a current pulse was applied during the spiking phase of one half-center, let’s call it #1, the consecutive burst durations (BDs) of that half-center increased throughout the paw-shake response, while BDs of the other half-center, let’s call it #2, only changed slightly. In contrast, the consecutive interburst intervals (IBIs) of half-center #1 changed little, while IBIs of half-center #2 increased. We demonstrated that this asymmetry between the half-centers depends on the phase of the locomotor-like rhythm at which the perturbation was applied. We suggest that the fast transient response reflects functional asymmetries of slow processes that underly the locomotor-like pattern; e.g., asymmetric levels of inactivation across the two half-centers for a slowly inactivating inward current. We compared model results with those of in-vivo paw-shake responses evoked in locomoting cats and found similar asymmetries. Electromyographic (EMG) BDs of anterior hindlimb muscles with flexor-related activity increased in consecutive paw-shake cycles, while BD of posterior muscles with extensor-related activity did not change, and vice versa for IBIs of anterior flexors and posterior extensors. We conclude that EMG activity patterns during paw-shaking are consistent with the proposed mechanism producing transient paw-shake-like bursting patterns found in our multistable HCO model. We suggest that the described asymmetry of paw-shaking responses could implicate a multifunctional CPG controlling both locomotion and paw-shaking.

Case Studies in Neuroscience: an epidural stimulating interface unveils the intrinsic modulation of electrically motor evoked potentials in behaving rats.

In intact and spinal injured anesthetized animals, stimulation levels that did not induce any visible muscle twitches, were used to elicit motor evoked potentials (MEPs) of varying amplitude, reflecting the temporal and amplitude dynamics of the background excitability of spinal networks. To characterize the physiological excitability states of neuronal networks driving movement, we designed five experiments in awake rats chronically implanted with an epidural stimulating interface, with and without a spinal cord injury (SCI). Firstly, an uninjured rat at rest underwent a series of single electrical pulses at sub motor-threshold intensity, which generated responses that were continuously recorded from flexor and extensor hindlimb muscles, showing an intrinsic patterned modulation of MEPs. Responses were recruited by increasing strengths of stimulation and the amplitudes were moderately correlated between flexors and extensors. Next, after SCI, four awake rats at rest showed electrically induced MEPs, varying largely in amplitude of both flexors and extensors that were mainly synchronously modulated. After full anesthesia, MEP amplitudes were largely reduced, although stimulation still generated random baseline changes, unveiling an intrinsic stochastic modulation. The current five cases demonstrate a methodology that can be feasibly replicated in a broader group of awake and behaving rats to further define experimental treatments involving neuroplasticity. Beside validating a new technology for a neural stimulating interface, the present data support the broader message that there were intrinsic patterned and stochastic modulation of baseline excitability reflecting the dynamics of physiological states of spinal networks.

Butyrate Feeding Reverses CypD-Related Mitoflash Phenotypes in Mouse Myofibers

Mitoflashes are spontaneous transients of the biosensor mt-cpYFP. In cardiomyocytes, mitoflashes are associated with the cyclophilin D (CypD) mediated opening of mitochondrial permeability transition pore (mPTP), while in skeletal muscle they are considered hallmarks of mitochondrial respiration burst under physiological conditions. Here, we evaluated the potential association between mitoflashes and the mPTP opening at different CypD levels and phosphorylation status by generating three CypD derived fusion constructs with a red shifted, pH stable Ca2+ sensor jRCaMP1b. We observed perinuclear mitochondrial Ca2+ efflux accompanying mitoflashes in CypD and CypDS42A (a phosphor-resistant mutation at Serine 42) overexpressed myofibers but not the control myofibers expressing the mitochondria-targeting sequence of CypD (CypDN30). Assisted by a newly developed analysis program, we identified shorter, more frequent mitoflash activities occurring over larger areas in CypD and CypDS42A overexpressed myofibers than the control CypDN30 myofibers. These observations provide an association between the elevated CypD expression and increased mitoflash activities in hindlimb muscles in an amyotrophic lateral sclerosis (ALS) mouse model previously observed. More importantly, feeding the mice with sodium butyrate reversed the CypD-associated mitoflash phenotypes and protected against ectopic upregulation of CypD, unveiling a novel molecular mechanism underlying butyrate mediated alleviation of ALS progression in the mouse model.

The KrasG12D;Trp53fl/fl murine model of undifferentiated pleomorphic sarcoma is macrophage dense, lymphocyte poor, and resistant to immune checkpoint blockade

Sarcomas are rare, difficult to treat, mesenchymal lineage tumours that affect children and adults. Immunologically-based therapies have improved outcomes for numerous adult cancers, however, these therapeutic strategies have been minimally effective in sarcoma so far. Clinically relevant, immunologically-competent, and transplantable pre-clinical sarcoma models are essential to advance sarcoma immunology research. Herein we show that Cre-mediated activation of KrasG12D, and deletion of Trp53, in the hindlimb muscles of C57Bl/6 mice results in the highly penetrant, rapid onset undifferentiated pleomorphic sarcomas (UPS), one of the most common human sarcoma subtypes. Cell lines derived from spontaneous UPS tumours can be reproducibly transplanted into the hindlimbs or lungs of naïve, immune competent syngeneic mice. Immunological characterization of both spontaneous and transplanted UPS tumours demonstrates an immunologically-‘quiescent’ microenvironment, characterized by a paucity of lymphocytes, limited spontaneous adaptive immune pathways, and dense macrophage infiltrates. Macrophages are the dominant immune population in both spontaneous and transplanted UPS tumours, although compared to spontaneous tumours, transplanted tumours demonstrate increased spontaneous lymphocytic infiltrates. The growth of transplanted UPS tumours is unaffected by host lymphocyte deficiency, and despite strong expression of PD-1 on tumour infiltrating lymphocytes, tumours are resistant to immunological checkpoint blockade. This spontaneous and transplantable immune competent UPS model will be an important experimental tool in the pre-clinical development and evaluation of novel immunotherapeutic approaches for immunologically cold soft tissue sarcomas.

COMMON AND DISTINCT MUSCLE SYNERGIES DURING LEVEL AND SLOPE LOCOMOTION IN THE CAT

Although it is well established that the motor control system is modular, the organization of muscle synergies during locomotion and their change with ground slope are not completely understood. For example, typical reciprocal flexor-extensor muscle synergies of level walking in cats break down in downslope: one-joint hip extensors are silent throughout the stride cycle, whereas hindlimb flexors demonstrate an additional stance phase-related EMG burst (Smith et al. 1998a). Here we investigated muscle synergies during Level, Upslope (27o) and Downslope (-27o) walking in adult cats to examine common and distinct features of modular organization of locomotor EMG activity. Cluster analysis of EMG burst onset-offset times of 12 hindlimb muscles revealed 5 flexor and extensor burst groups that were generally shared across slopes. Stance-related bursts of flexor muscles in downslope were placed in a burst group from Level and Upslope walking formed by the rectus femoris. Walking Upslope changed swing/stance phase durations of Level walking but not the cycle duration. Five muscle synergies computed using non-negative matrix factorization accounted for at least 95% of variance in EMG patterns in each slope. Five synergies were shared between Level and Upslope walking, whereas only 3 of those were shared with Downslope synergies; these synergies were active during the swing phase and phase transitions. Two stance-related synergies of downslope walking were distinct; they comprised a mixture of flexors and extensors. We suggest that the modular organization of muscle activity during Level and Slope walking results from interactions between motion-related sensory feedback, CPG, and supraspinal inputs.

Dynamic transcriptome and histomorphology analysis of developmental traits of hindlimb thigh muscle from Odorrana tormota and its adaptability to different life history stages

Background Systematic studies on the development and adaptation of hindlimb muscles in anura amphibians are rare. Here, we integrated analysis of transcriptome and histomorphological data for the hindlimb thigh muscle of Odorrana tormota (concave-eared torrent frog) at different developmental stages, to uncover the developmental traits of hindlimb thigh muscle from O. tormota and its adaptability to different life history stages. Results The development of hindlimb thigh muscle from O. tormota has the following characteristics. Before metamorphosis, myogenous cells proliferate and differentiate into myotubes, and form 11 muscle groups at G41; Primary myofibers and secondary myofibers appeared during metamorphosis; 11 muscle groups differentiated continuously to form myofibers, accompanied by myofibers hypertrophy after metamorphosis; During the growth process of O. tormota from G42 to G46, there were differences between the sexes in the muscle groups that differentiate into muscle fibers, indicating that there was sexual dimorphism in the hindlimb thigh muscles of O. tormota at the metamorphosis stages. Some genes and pathways related to growth, development, and movement ability of O. tormota at different developmental stages were obtained. In addition, some pathways associated with adaptation to metamorphosis and hibernation also were enriched. Furthermore, integrated analysis of the number of myofibers and transcriptome data suggested that myofibers of specific muscle groups in the hindlimbs may be degraded through lysosome and ubiquitin pathways to transform into energy metabolism and other energy-related substances to meet the physiological needs of hibernation. Conclusions These results provide further understanding the hindlimb thigh muscle development pattern of frogs and their adaption to life history stages.

Il-10 signaling reduces survival in mouse models of synucleinopathy

Parkinson’s disease (PD) and related synucleinopathies are characterized by chronic neuroinflammation leading to the premise that anti-inflammatory therapies could ameliorate synucleinopathy and associated sequelae. To test this idea, we used recombinant adeno-associated viruses (AAV) to express the anti-inflammatory cytokine, Interleukin (Il)-10, in Line M83 transgenic mice that expresses the PD-associated A53T mutant human α-synuclein (αSyn). Contrary to our expectations, we observed that intraspinal Il-10 expression initiated at birth upregulated microgliosis and led to early death in homozygous M83+/+ mice. We further observed that Il-10 preconditioning led to reduced lifespan in the hemizygous M83+/− mice injected with preformed αSyn aggregates in hindlimb muscles. To determine the mechanistic basis for these adverse effects, we took advantage of the I87A variant Il-10 (vIl-10) that has predominantly immunosuppressive properties. Sustained intraspinal expression of vIl-10 in preformed αSyn-aggregate seeded M83+/− mice resulted in earlier death, accelerated αSyn pathology, pronounced microgliosis, and increased apoptosis compared to control mice. AAV-vIl-10 expression robustly induced p62 and neuronal LC3B accumulation in these mice, indicating that Il-10 signaling mediated preconditioning of the neuraxis can potentially exacerbate αSyn accumulation through autophagy dysfunction in the neurons. Together, our data demonstrate unexpected adverse effects of both Il-10 and its immunosuppressive variant, vIl-10, in a mouse model of PD, highlighting the pleiotropic functions of immune mediators and their complex role in non-cell autonomous signaling in neurodegenerative proteinopathies.