CaMKIIß deregulation contributes to neuromuscular junction destabilization in Myotonic Dystrophy type I.

BACKGROUND: Myotonic Dystrophy type I (DM1) is the most common muscular dystrophy in adults. Previous reports have highlighted that neuromuscular junctions (NMJs) deteriorate in skeletal muscle from DM1 patients and mouse models thereof. However, the underlying pathomechanisms and their contribution to muscle dysfunction remain unknown. METHODS: We compared changes in NMJs and activity-dependent signalling pathways in HSALR and Mbnl1ΔE3/ΔE3 mice, two established mouse models of DM1. RESULTS: Muscle from DM1 mouse models showed major deregulation of calcium/calmodulin-dependent protein kinases II (CaMKIIs), which are key activity sensors regulating synaptic gene expression and acetylcholine receptor (AChR) recycling at the NMJ. Both mouse models exhibited increased fragmentation of the endplate, which preceded muscle degeneration. Endplate fragmentation was not accompanied by changes in AChR turnover at the NMJ. However, the expression of synaptic genes was up-regulated in mutant innervated muscle, together with an abnormal accumulation of histone deacetylase 4 (HDAC4), a known target of CaMKII. Interestingly, denervation-induced increase in synaptic gene expression and AChR turnover was hampered in DM1 muscle. Importantly, CaMKIIß/ßM overexpression normalized endplate fragmentation and synaptic gene expression in innervated Mbnl1ΔE3/ΔE3 muscle, but it did not restore denervation-induced synaptic gene up-regulation. CONCLUSIONS: Our results indicate that CaMKIIß-dependent and -independent mechanisms perturb synaptic gene regulation and muscle response to denervation in DM1 mouse models. Changes in these signalling pathways may contribute to NMJ destabilization and muscle dysfunction in DM1 patients.

Cervical and thoracic spinal cord gray matter atrophy is associated with disability in patients with amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: In amyotrophic lateral sclerosis (ALS), there is an unmet need for more precise patient characterization through quantitative, ideally operator-independent, assessments of disease extent and severity. Radially sampled averaged magnetization inversion recovery acquisitions (rAMIRA) magnetic resonance imaging enables gray matter (GM) and white matter (WM) area quantitation in the cervical and thoracic spinal cord (SC) with optimized contrast. We aimed to investigate rAMIRA-derived SC GM and SC WM areas and their association with clinical phenotype and disability in ALS. METHODS: A total of 36 patients with ALS (mean [SD] age 61.7 [12.6] years, 14 women) and 36 healthy, age- and sex-matched controls (HCs; mean [SD] age 63.1 [12.1] years, 14 women) underwent two-dimensional axial rAMIRA imaging at the inter-vertebral disc levels C2/3-C5/C6 and the lumbar enlargement level Tmax. ALS Functional Rating Scale-revised (ALSFRS-R) score, muscle strength, and sniff nasal inspiratory pressure (SNIP) were assessed. RESULTS: Compared to HCs, GM and WM areas were reduced in patients at all cervical levels (p < 0.0001). GM area (p = 0.0001), but not WM area, was reduced at Tmax. Patients with King's Stage 3 showed significant GM atrophy at all levels, while patients with King's Stage 1 showed significant GM atrophy selectively at Tmax. SC GM area was significantly associated with muscle force at corresponding myotomes. GM area at C3/C4 was associated with ALSFRS-R (p < 0.001) and SNIP (p = 0.0016). CONCLUSION: Patients with ALS assessed by rAMIRA imaging show significant cervical and thoracic SC GM and SC WM atrophy. SC GM area correlates with muscle strength and clinical disability. GM area reduction at Tmax may be an early disease sign. Longitudinal studies are warranted.

Targeted transcript analysis in muscles from patients with genetically diverse congenital myopathies.

Congenital myopathies are a group of early onset muscle diseases of variable severity often with characteristic muscle biopsy findings and involvement of specific muscle types. The clinical diagnosis of patients typically relies on histopathological findings and is confirmed by genetic analysis. The most commonly mutated genes encode proteins involved in skeletal muscle excitation-contraction coupling, calcium regulation, sarcomeric proteins and thin-thick filament interaction. However, mutations in genes encoding proteins involved in other physiological functions (for example mutations in SELENON and MTM1, which encode for ubiquitously expressed proteins of low tissue specificity) have also been identified. This intriguing observation indicates that the presence of a genetic mutation impacts the expression of other genes whose product is important for skeletal muscle function. The aim of the present investigation was to verify if there are common changes in transcript and microRNA expression in muscles from patients with genetically heterogeneous congenital myopathies, focusing on genes encoding proteins involved in excitation-contraction coupling and calcium homeostasis, sarcomeric proteins, transcription factors and epigenetic enzymes. Our results identify RYR1, ATPB2B and miRNA-22 as common transcripts whose expression is decreased in muscles from congenital myopathy patients. The resulting protein deficiency may contribute to the muscle weakness observed in these patients. This study also provides information regarding potential biomarkers for monitoring disease progression and response to pharmacological treatments in patients with congenital myopathies.

Distinct and additive effects of calorie restriction and rapamycin in aging skeletal muscle.

Preserving skeletal muscle function is essential to maintain life quality at high age. Calorie restriction (CR) potently extends health and lifespan, but is largely unachievable in humans, making "CR mimetics" of great interest. CR targets nutrient-sensing pathways centering on mTORC1. The mTORC1 inhibitor, rapamycin, is considered a potential CR mimetic and is proven to counteract age-related muscle loss. Therefore, we tested whether rapamycin acts via similar mechanisms as CR to slow muscle aging. Here we show that long-term CR and rapamycin unexpectedly display distinct gene expression profiles in geriatric mouse skeletal muscle, despite both benefiting aging muscles. Furthermore, CR improves muscle integrity in mice with nutrient-insensitive, sustained muscle mTORC1 activity and rapamycin provides additive benefits to CR in naturally aging mouse muscles. We conclude that rapamycin and CR exert distinct, compounding effects in aging skeletal muscle, thus opening the possibility of parallel interventions to counteract muscle aging.

Skeletal Muscle Disorders: A Noncardiac Source of Cardiac Troponin T.

BACKGROUND: Cardiac troponin (cTn) T and cTnI are considered cardiac specific and equivalent in the diagnosis of acute myocardial infarction. Previous studies suggested rare skeletal myopathies as a noncardiac source of cTnT. We aimed to confirm the reliability/cardiac specificity of cTnT in patients with various skeletal muscle disorders (SMDs). METHODS: We prospectively enrolled patients presenting with muscular complaints (≥2 weeks) for elective evaluation in 4 hospitals in 2 countries. After a cardiac workup, patients were adjudicated into 3 predefined cardiac disease categories. Concentrations of cTnT/I and resulting cTnT/I mismatches were assessed with high-sensitivity (hs-) cTnT (hs-cTnT-Elecsys) and 3 hs-cTnI assays (hs-cTnI-Architect, hs-cTnI-Access, hs-cTnI-Vista) and compared with those of control subjects without SMD presenting with adjudicated noncardiac chest pain to the emergency department (n=3508; mean age, 55 years; 37% female). In patients with available skeletal muscle biopsies, TNNT/I1-3 mRNA differential gene expression was compared with biopsies obtained in control subjects without SMD. RESULTS: Among 211 patients (mean age, 57 years; 42% female), 108 (51%) were adjudicated to having no cardiac disease, 44 (21%) to having mild disease, and 59 (28%) to having severe cardiac disease. hs-cTnT/I concentrations significantly increased from patients with no to those with mild and severe cardiac disease for all assays (all P<0.001). hs-cTnT-Elecsys concentrations were significantly higher in patients with SMD versus control subjects (median, 16 ng/L [interquartile range (IQR), 7-32.5 ng/L] versus 5 ng/L [IQR, 3-9 ng/L]; P<0.001), whereas hs-cTnI concentrations were mostly similar (hs-cTnI-Architect, 2.5 ng/L [IQR, 1.2-6.2 ng/L] versus 2.9 ng/L [IQR, 1.8-5.0 ng/L]; hs-cTnI-Access, 3.3 ng/L [IQR, 2.4-6.1 ng/L] versus 2.7 ng/L [IQR, 1.6-5.0 ng/L]; and hs-cTnI-Vista, 7.4 ng/L [IQR, 5.2-13.4 ng/L] versus 7.5 ng/L [IQR, 6-10 ng/L]). hs-cTnT-Elecsys concentrations were above the upper limit of normal in 55% of patients with SMD versus 13% of control subjects (P<0.01). mRNA analyses in skeletal muscle biopsies (n=33), mostly (n=24) from individuals with noninflammatory myopathy and myositis, showed 8-fold upregulation of TNNT2, encoding cTnT (but none for TNNI3, encoding cTnI) versus control subjects (n=16, PWald<0.001); the expression correlated with pathological disease activity (R=0.59, Pt-statistic<0.001) and circulating hs-cTnT concentrations (R=0.26, Pt-statistic=0.031). CONCLUSIONS: In patients with active chronic SMD, elevations in cTnT concentrations are common and not attributable to cardiac disease in the majority. This was not observed for cTnI and may be explained in part by re-expression of cTnT in skeletal muscle. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03660969.

The neuromuscular junction is a focal point of mTORC1 signaling in sarcopenia.

With human median lifespan extending into the 80s in many developed countries, the societal burden of age-related muscle loss (sarcopenia) is increasing. mTORC1 promotes skeletal muscle hypertrophy, but also drives organismal aging. Here, we address the question of whether mTORC1 activation or suppression is beneficial for skeletal muscle aging. We demonstrate that chronic mTORC1 inhibition with rapamycin is overwhelmingly, but not entirely, positive for aging mouse skeletal muscle, while genetic, muscle fiber-specific activation of mTORC1 is sufficient to induce molecular signatures of sarcopenia. Through integration of comprehensive physiological and extensive gene expression profiling in young and old mice, and following genetic activation or pharmacological inhibition of mTORC1, we establish the phenotypically-backed, mTORC1-focused, multi-muscle gene expression atlas, SarcoAtlas (https://sarcoatlas.scicore.unibas.ch/), as a user-friendly gene discovery tool. We uncover inter-muscle divergence in the primary drivers of sarcopenia and identify the neuromuscular junction as a focal point of mTORC1-driven muscle aging.

Protein delivery in intermittent and continuous enteral nutrition with a protein-rich formula in critically ill patients-a protocol for the prospective randomized controlled proof-of-concept Protein Bolus Nutrition (Pro BoNo) study.

BACKGROUND: Critically ill patients rapidly develop muscle wasting resulting in sarcopenia, long-term disability and higher mortality. Bolus nutrition (30-60 min period), whilst having a similar incidence of aspiration as continuous feeding, seems to provide metabolic benefits through increased muscle protein synthesis due to higher leucine peaks. To date, clinical evidence on achievement of nutritional goals and influence of bolus nutrition on skeletal muscle metabolism in ICU patients is lacking. The aim of the Pro BoNo study (Protein Bolus Nutrition) is to compare intermittent and continuous enteral feeding with a specific high-protein formula. We hypothesise that target quantity of protein is reached earlier (within 36 h) by an intermittent feeding protocol with a favourable influence on muscle protein synthesis. METHODS: Pro BoNo is a prospective randomised controlled study aiming to compare the impact of intermittent and continuous enteral feeding on preventing muscle wasting in 60 critically ill patients recruited during the first 48 h after ICU admission. The primary outcome measure is the time until the daily protein target (≥ 1.5 g protein/kg bodyweight/24 h) is achieved. Secondary outcome measures include tolerance of enteral feeding and evolution of glucose, urea and IGF-1. Ultrasound and muscle biopsy of the quadriceps will be performed. DISCUSSION: The Basel Pro BoNo study aims to collect innovative data on the effect of intermittent enteral feeding of critically ill patients on muscle wasting. TRIAL REGISTRATION: ClinicalTrials.gov NCT03587870 . Registered on July 16, 2018. Swiss National Clinical Trials Portal SNCTP000003234. Last updated on July 24, 2019.

AIMTOR, a BRET biosensor for live imaging, reveals subcellular mTOR signaling and dysfunctions.

BACKGROUND: mTOR signaling is an essential nutrient and energetic sensing pathway. Here we describe AIMTOR, a sensitive genetically encoded BRET (Bioluminescent Resonance Energy Transfer) biosensor to study mTOR activity in living cells. RESULTS: As a proof of principle, we show in both cell lines and primary cell cultures that AIMTOR BRET intensities are modified by mTOR activity changes induced by specific inhibitors and activators of mTORC1 including amino acids and insulin. We further engineered several versions of AIMTOR enabling subcellular-specific assessment of mTOR activities. We then used AIMTOR to decipher mTOR signaling in physio-pathological conditions. First, we show that mTORC1 activity increases during muscle cell differentiation and in response to leucine stimulation in different subcellular compartments such as the cytosol and at the surface of the lysosome, the nucleus, and near the mitochondria. Second, in hippocampal neurons, we found that the enhancement of neuronal activity increases mTOR signaling. AIMTOR further reveals mTOR-signaling dysfunctions in neurons from mouse models of autism spectrum disorder. CONCLUSIONS: Altogether, our results demonstrate that AIMTOR is a sensitive and specific tool to investigate mTOR-signaling dynamics in living cells and phenotype mTORopathies.

DNA aptamers against the DUX4 protein reveal novel therapeutic implications for FSHD.

Aberrant expression of the transcription factor double homeobox protein 4 (DUX4) can lead to a number of diseases including facio-scapulo-humeral muscular dystrophy (FSHD), acute lymphoblastic leukemia, and sarcomas. Inhibition of DUX4 may represent a therapeutic strategy for these diseases. By applying Systematic Evolution of Ligands by EXponential Enrichment (SELEX), we identified aptamers against DUX4 with specific secondary structural elements conveying high affinity to DUX4 as assessed by fluorescence resonance energy transfer and fluorescence polarization techniques. Sequences analysis of these aptamers revealed the presence of two consensus DUX4 motifs in a reverse complementary fashion forming hairpins interspersed with bulge loops at distinct positions that enlarged the binding surface with the DUX4 protein, as determined by crystal structure analysis. We demonstrate that insertion of specific structural elements into transcription factor binding oligonucleotides can enhance specificity and affinity.

mTORC1 signalling is not essential for the maintenance of muscle mass and function in adult sedentary mice.

BACKGROUND: The balance between protein synthesis and degradation (proteostasis) is a determining factor for muscle size and function. Signalling via the mammalian target of rapamycin complex 1 (mTORC1) regulates proteostasis in skeletal muscle by affecting protein synthesis and autophagosomal protein degradation. Indeed, genetic inactivation of mTORC1 in developing and growing muscle causes atrophy resulting in a lethal myopathy. However, systemic dampening of mTORC1 signalling by its allosteric inhibitor rapamycin is beneficial at the organismal level and increases lifespan. Whether the beneficial effect of rapamycin comes at the expense of muscle mass and function is yet to be established. METHODS: We conditionally ablated the gene coding for the mTORC1-essential component raptor in muscle fibres of adult mice [inducible raptor muscle-specific knockout (iRAmKO)]. We performed detailed phenotypic and biochemical analyses of iRAmKO mice and compared them with muscle-specific raptor knockout (RAmKO) mice, which lack raptor in developing muscle fibres. We also used polysome profiling and proteomics to assess protein translation and associated signalling in skeletal muscle of iRAmKO mice. RESULTS: Analysis at different time points reveal that, as in RAmKO mice, the proportion of oxidative fibres decreases, but slow-type fibres increase in iRAmKO mice. Nevertheless, no significant decrease in body and muscle mass or muscle fibre area was detected up to 5 months post-raptor depletion. Similarly, ex vivo muscle force was not significantly reduced in iRAmKO mice. Despite stable muscle size and function, inducible raptor depletion significantly reduced the expression of key components of the translation machinery and overall translation rates. CONCLUSIONS: Raptor depletion and hence complete inhibition of mTORC1 signalling in fully grown muscle leads to metabolic and morphological changes without inducing muscle atrophy even after 5 months. Together, our data indicate that maintenance of muscle size does not require mTORC1 signalling, suggesting that rapamycin treatment is unlikely to negatively affect muscle mass and function.

mTORC1 and PKB/Akt control the muscle response to denervation by regulating autophagy and HDAC4.

Loss of innervation of skeletal muscle is a determinant event in several muscle diseases. Although several effectors have been identified, the pathways controlling the integrated muscle response to denervation remain largely unknown. Here, we demonstrate that PKB/Akt and mTORC1 play important roles in regulating muscle homeostasis and maintaining neuromuscular endplates after nerve injury. To allow dynamic changes in autophagy, mTORC1 activation must be tightly balanced following denervation. Acutely activating or inhibiting mTORC1 impairs autophagy regulation and alters homeostasis in denervated muscle. Importantly, PKB/Akt inhibition, conferred by sustained mTORC1 activation, abrogates denervation-induced synaptic remodeling and causes neuromuscular endplate degeneration. We establish that PKB/Akt activation promotes the nuclear import of HDAC4 and is thereby required for epigenetic changes and synaptic gene up-regulation upon denervation. Hence, our study unveils yet-unknown functions of PKB/Akt-mTORC1 signaling in the muscle response to nerve injury, with important implications for neuromuscular integrity in various pathological conditions.

Diagnosis of adult-onset MELAS syndrome in a 63-year-old patient with suspected recurrent strokes - a case report.

BACKGROUND: Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) is a mitochondrial cytopathy caused by mutations in mitochondrial DNA. Clinical manifestation is typically before the age of 40. CASE PRESENTATION: We present the case of a 63-year-old female in whom the symptoms of MELAS were initially misdiagnosed as episodes of recurrent ischemic strokes. Brain imaging including MRI, clinical and laboratory findings that lent cues to the diagnosis of MELAS are discussed. In addition, MRI findings in MELAS in comparison to imaging mimics of MELAS are presented. CONCLUSIONS: This case underscores the importance of considering MELAS as a potential cause of recurrent stroke-like events if imaging findings are untypical for cerebral infarction, even among middle-aged patients with vascular risk factors.

Targeting deregulated AMPK/mTORC1 pathways improves muscle function in myotonic dystrophy type I.

Myotonic dystrophy type I (DM1) is a disabling multisystemic disease that predominantly affects skeletal muscle. It is caused by expanded CTG repeats in the 3'-UTR of the dystrophia myotonica protein kinase (DMPK) gene. RNA hairpins formed by elongated DMPK transcripts sequester RNA-binding proteins, leading to mis-splicing of numerous pre-mRNAs. Here, we have investigated whether DM1-associated muscle pathology is related to deregulation of central metabolic pathways, which may identify potential therapeutic targets for the disease. In a well-characterized mouse model for DM1 (HSALR mice), activation of AMPK signaling in muscle was impaired under starved conditions, while mTORC1 signaling remained active. In parallel, autophagic flux was perturbed in HSALR muscle and in cultured human DM1 myotubes. Pharmacological approaches targeting AMPK/mTORC1 signaling greatly ameliorated muscle function in HSALR mice. AICAR, an AMPK activator, led to a strong reduction of myotonia, which was accompanied by partial correction of misregulated alternative splicing. Rapamycin, an mTORC1 inhibitor, improved muscle relaxation and increased muscle force in HSALR mice without affecting splicing. These findings highlight the involvement of AMPK/mTORC1 deregulation in DM1 muscle pathophysiology and may open potential avenues for the treatment of this disease.

"Get the Balance Right": Pathological Significance of Autophagy Perturbation in Neuromuscular Disorders.

Recent research has revealed that autophagy, a major catabolic process in cells, is dysregulated in several neuromuscular diseases and contributes to the muscle wasting caused by non-muscle disorders (e.g. cancer cachexia) or during aging (i.e. sarcopenia). From there, the idea arose to interfere with autophagy or manipulate its regulatory signalling to help restore muscle homeostasis and attenuate disease progression. The major difficulty for the development of therapeutic strategies is to restore a balanced autophagic flux, due to the dynamic nature of autophagy. Thus, it is essential to better understand the mechanisms and identify the signalling pathways at play in the control of autophagy in skeletal muscle. A comprehensive analysis of the autophagic flux and of the causes of its dysregulation is required to assess the pathogenic role of autophagy in diseased muscle. Furthermore, it is essential that experiments distinguish between primary dysregulation of autophagy (prior to disease onset) and impairments as a consequence of the pathology. Of note, in most muscle disorders, autophagy perturbation is not caused by genetic modification of an autophagy-related protein, but rather through indirect alteration of regulatory signalling or lysosomal function. In this review, we will present the mechanisms involved in autophagy, and those ensuring its tight regulation in skeletal muscle. We will then discuss as to how autophagy dysregulation contributes to the pathogenesis of neuromuscular disorders and possible ways to interfere with this process to limit disease progression.

Mitochondrial cytopathy with common MELAS mutation presenting as multiple system atrophy mimic.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome1 is one of the most frequently inherited mitochondrial disorders. MELAS syndrome is a systemic disease with multiple organ involvement.2 The most common mutation in MELAS is the m.3243A>G mutation in the MT-TL1 gene.2.

Identification of Plant-derived Alkaloids with Therapeutic Potential for Myotonic Dystrophy Type I.

Myotonic dystrophy type I (DM1) is a disabling neuromuscular disease with no causal treatment available. This disease is caused by expanded CTG trinucleotide repeats in the 3' UTR of the dystrophia myotonica protein kinase gene. On the RNA level, expanded (CUG)n repeats form hairpin structures that sequester splicing factors such as muscleblind-like 1 (MBNL1). Lack of available MBNL1 leads to misregulated alternative splicing of many target pre-mRNAs, leading to the multisystemic symptoms in DM1. Many studies aiming to identify small molecules that target the (CUG)n-MBNL1 complex focused on synthetic molecules. In an effort to identify new small molecules that liberate sequestered MBNL1 from (CUG)n RNA, we focused specifically on small molecules of natural origin. Natural products remain an important source for drugs and play a significant role in providing novel leads and pharmacophores for medicinal chemistry. In a new DM1 mechanism-based biochemical assay, we screened a collection of isolated natural compounds and a library of over 2100 extracts from plants and fungal strains. HPLC-based activity profiling in combination with spectroscopic methods were used to identify the active principles in the extracts. The bioactivity of the identified compounds was investigated in a human cell model and in a mouse model of DM1. We identified several alkaloids, including the ß-carboline harmine and the isoquinoline berberine, that ameliorated certain aspects of the DM1 pathology in these models. Alkaloids as a compound class may have potential for drug discovery in other RNA-mediated diseases.