Biology subjects

Zammit, P. S.

Publications and source records attributed to Zammit, P. S..

12 recordsLinked to original sources

bioRxiv2026

Metformin enhances differentiation and function of skeletal muscle in models of Facioscapulohumeral Muscular Dystrophy (FSHD)

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent inherited muscular dystrophies, for which there are no disease-modifying therapies. Metabolic perturbation, mitochondrial dysfunction, and oxidative stress are key contributors to FSHD pathology. Here, the effects of the metabolic regulator and anti-diabetic drug Metformin on myogenesis and muscle function in human and murine models of FSHD were investigated. Metformin did not affect the proliferation rate of human control or patient-derived FSHD myoblasts but promoted their myogenic differentiation, increasing myotube formation and maturation. Metformin also enhanced the metabolic health and viability of myotubes. Mechanistic interrogation revealed reduced levels of mitochondrial reactive oxygen species and modified mitochondrial turnover. These cellular investigations were complemented with in vivo functional assessment in a murine model of FSHD, in which Metformin treated mice exhibited significantly improved muscle strength. Collectively, these findings identify metabolic regulation as a therapeutically tractable feature of FSHD and demonstrate that Metformin improves muscle function in multiple models of FSHD via reduction of oxidative stress and augmentation of cellular metabolic fitness. These results provide insight into the therapeutic actions of Metformin and pre-clinical data to support its testing for repurposing in FSHD.

cell biology↗

bioRxiv2025

Mitochondrial Respiratory Chain Function is crucial for Muscle Toxicity in Facioscapulohumeral Muscular Dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is driven by DUX4-induced toxicity, yet the pathomechanisms remain unclear. Here, we identify persistent transcriptional suppression of the mitochondrial respiratory chain and metabolic rewiring in FSHD muscle biopsies and myotubes. Using DUX4-inducible human myogenic cells, we show that DUX4 target gene activation is accompanied by mitochondrial function impairment and Caspase 9-mediated apoptosis. Reverse electron transfer (RET) at complex I is the dominant oxidative stress generating mechanism in FSHD muscle cells. RET-driven mitochondrial reactive oxygen species (mitoROS) are the trigger for oxidative stress and apoptosis, which is a unique feature of FSHD mitochondria. Pharmacological inhibition of RET suppresses mitoROS, reduces Caspase 9 activation, and rescues abnormal myogenesis in FSHD cell lines. Importantly, DUX4 is non-toxic in oxidative phosphorylation-deficient human myogenic cells. Our findings identify the mitochondrial respiratory chain as a key mediator of DUX4 toxicity and highlight RET inhibition as a potential therapeutic approach for FSHD. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/690559v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@254927org.highwire.dtl.DTLVardef@1e5c67forg.highwire.dtl.DTLVardef@4ace42org.highwire.dtl.DTLVardef@12b710e_HPS_FORMAT_FIGEXP M_FIG (Created with BioRender.com) C_FIG

biochemistry↗

bioRxiv2025

CEBPβ regulates myoblast proliferation and myogenic differentiation during human myogenesis and rescues defective differentiation in FSHD

CCAAT enhancer binding protein {beta} (CEBPB) is a member of the bZIP transcription factor family and is expressed in many cell types. Whilst CEBPB has been shown to regulate the function of murine satellite cells, the resident skeletal muscle stem cell, however little is known of its role in human myogenesis and muscle disease. We report that CEBPB is expressed in proliferating human myoblasts but swiftly down-regulated upon myogenic differentiation. Interestingly, either over-expression or knock-down of CEBPB inhibit the proliferation rate of human myoblasts. Down-regulation of CEBPB upon myogenic differentiation is then necessary for subsequent myogenic differentiation and fusion into multinucleated myotubes. In the muscle disease context, CEBPB protein levels are increased in myoblasts derived from facioscapulohumeral muscular dystrophy (FSHD) patients. Knock-down of CEBPB in FSHD myoblasts improves myogenic differentiation. In summary, we show that CEBPB is a regulator of human myogenesis and altered CEBPB expression may contribute to defective myogenesis in FSHD.

cell biology↗

bioRxiv2025

Advanced human iPSC-based modelling of LMNA-related congenital muscular dystrophy enables development of targeted genetic therapies for muscle laminopathies

LMNA-related congenital muscular dystrophy (L-CMD) is amongst the most severe forms of laminopathies, which are diseases caused by pathogenic variants in the LMNA gene. LMNA encodes the proteins Lamin A and C, which assemble with Lamin B1 and B2 to form the nuclear lamina: a meshwork providing structural stability to the nucleus that also regulates chromatin organisation and gene expression. Research into L-CMD mechanisms and therapies is hindered by lack of humanised, tissue-specific models that accurately recapitulate disease phenotypes. We previously reported that LMNA-mutant induced pluripotent stem cell (iPSC)-derived skeletal muscle cells have nuclear shape abnormalities and Lamin A/C protein mislocalisation. Here, we expand the selection of L-CMD patient- derived iPSCs and validate disease-associated readouts using a transgene-free based protocol which more accurately mimics skeletal myogenesis. Results showed no overt defects in developmental myogenesis, but recapitulation of pathological nuclear shape abnormalities in 2D and 3D cultures, nuclear envelope protein mislocalisation and transcriptomic alterations across multiple pathogenic LMNA variants. We then utilised this platform to assess LMNA gene editing strategies. CRISPR-based exon removal generated stable RNA and protein Lamin A/C species, without significant normalisation of nuclear morphological phenotypes or transcriptomic profile. Conversely, precise editing of the same mutation showed complete reversal of disease-associated nuclear morphometrics, alongside normalisation of the pro-inflammatory transcriptomic signature. Our data provide the foundation for a humanised in vitro disease and therapy modelling platform for this complex and severe muscle disorder. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/660928v2_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@134276forg.highwire.dtl.DTLVardef@108f44forg.highwire.dtl.DTLVardef@dfa04eorg.highwire.dtl.DTLVardef@1956a90_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LILMNA-mutant iPSCs undergo efficient skeletal myogenesis upon transgene-free, small molecule-based lineage-directed differentiation C_LIO_LIL-CMD iPSCs recapitulate hallmark disease-associated nuclear phenotypes and show a pro-inflammatory transcriptional profile C_LIO_LIDisease modelling platforms based on iPSC-derived skeletal muscle cells enable comparative testing of gene editing strategies C_LIO_LICRISPR-edited L-CMD iPSC-derived myogenic cells show amelioration of disease-associated readouts C_LI

cell biology↗

bioRxiv2024

Quantifying anti-DUX4 therapy for facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited skeletal myopathy with no cure. Expression of the myotoxic transcription factor double homeobox 4 (DUX4) is believed to underlie FSHD pathogenesis and many proposed therapies target DUX4 generation or function. Which of these therapies will be the most effective is unclear. Here, by constructing a Markov-chain-based mathematical model of DUX4-mediated myotoxity in FSHD, we interrogate various anti-DUX4 FSHD therapeutic strategies. We derive an analytical function for myonuclear life expectancy in terms of the parameters of DUX4 expression and function. In a biologically relevant parameter regime, therapeutically decreasing the DUX4 protein diffusion rate is, surprisingly, predicted to be more effective at increasing myonuclear life expectancy than reducing the rate of myonuclear apoptosis caused by the expression of DUX4-target genes. We find that targeting elements of DUX4 transcription/translation, such as mRNA stability via siRNA therapy, has a limited predicted impact on DUX4-meditated toxicity when performed in isolation. However, our model predicts a super-additive effect from combining transcription/translation targeting strategies with approaches that minimise DUX4 diffusion-mediated import into neighbouring myonuclei. Importantly, we provide a computational tool to test and inform therapeutic designs, enabling pre-clinical screening of FSHD treatment approaches.

cell biology↗

bioRxiv2022

An in silico FSHD muscle fibre for modelling DUX4 dynamics and predicting the impact of therapy

Facioscapulohumeral muscular dystrophy (FSHD) is an incurable myopathy linked to over-expression of the myotoxic transcription factor DUX4. Targeting DUX4 is the leading therapeutic approach, however it is only detectable in 0.1-3.8% of FSHD myonuclei. How rare DUX4 drives FSHD and the optimal anti-DUX4 strategy is unclear. We combine stochastic gene expression with compartment models of cell states, building a simulation of DUX4 expression and consequences in FSHD muscle fibres. Investigating iDUX4 myoblasts, scRNAseq and snRNAseq of FSHD muscle we estimate parameters including DUX4 mRNA degradation, transcription and translation rates and DUX4 target gene activation rates. Our model accurately recreates the distribution of DUX4 and target gene positive cells seen in scRNAseq of FSHD myocytes. Importantly we show DUX4 drives significant cell death despite expression in only 0.8% of live cells. Comparing scRNAseq of unfused FSHD myocytes to snRNAseq of fused FSHD myonuclei, we find evidence of DUX4 protein syncytial diffusion and estimate its rate via genetic algorithms. We package our model into freely available tools, to rapidly investigate consequences of anti-DUX4 therapy.

cell biology↗

bioRxiv2022

A circulating biomarker of facioscapulohumeral muscular dystrophy clinical severity, valid in skeletal muscle and blood

Facioscapulohumeral muscular dystrophy (FSHD) is incurable. DUX4 mis-expression is believed to underlie FSHD pathogenesis, alongside PAX7 target gene repression, yet clinical trials lack robust biomarkers of severity. FSHD entails fatty replacement of muscle, accelerated by inflammation, we thus performed RNA-sequencing on both an MRI guided inflamed (TIRM+) and non-inflamed (TIRM-) muscle biopsies from clinically-characterised FSHD patients, alongside peripheral blood mononucleated cells (PBMCs). PAX7 target gene repression in TIRM-muscle associates with severity. DUX4 target gene biomarkers associate with lower limb fat fraction and D4Z4 repeat length, but not severity. PAX7 target gene repression in muscle correlates with levels in matched PBMCs. A refined biomarker computed in PBMCs associates with severity in FSHD patients, and also validates as a classifier of severity in an independent set of 54 FSHD patient blood samples. In summary, we present a minimally-invasive, circulating, transcriptomic biomarker of FSHD clinical severity valid in muscle and blood.

bioinformatics↗

bioRxiv2021

Interplay between mitochondria and reactive oxygen and nitrogen species in metabolic adaptation to hypoxia in facioscapulohumeral muscular dystrophy: potential therapeutic targets.

Facioscapulohumeral muscular dystrophy (FSHD) is characterised by descending skeletal muscle weakness and wasting. FSHD is caused by mis-expression of the transcription factor DUX4, which is linked to oxidative stress, a condition especially detrimental to skeletal muscle with its high metabolic activity and energy demands. Oxidative damage characterises FSHD and recent work suggests metabolic dysfunction and perturbed hypoxia signalling as novel pathomechanisms. However, redox biology of FSHD remains poorly understood, and integrating the complex dynamics of DUX4-induced metabolic changes is lacking. Here we pinpoint the kinetic involvement of altered mitochondrial RONS metabolism and impaired mitochondrial function in aetiology of oxidative stress in FSHD. Transcriptomic analysis in FSHD muscle biopsies reveals strong enrichment for pathways involved in mitochondrial complex I assembly, nitrogen metabolism, oxidative stress response and hypoxia signalling. We found elevated ROS levels correlate with increases in steady-state mitochondrial membrane potential in FSHD myogenic cells. DUX4 triggers mitochondrial membrane polarisation prior to oxidative stress generation and apoptosis through mitochondrial ROS, and affects NO{middle dot} bioavailability via mitochondrial peroxidation. We identify complex I as the primary target for DUX4-induced mitochondrial dysfunction, with strong correlation between complex I-linked respiration and cellular oxygenation/hypoxia signalling activity in environmental hypoxia. Thus, FSHD myogenesis is uniquely susceptible to hypoxia-induced oxidative stress as a consequence of metabolic mis-adaptation. Importantly, mitochondria-targeted antioxidants rescue FSHD pathology more effectively than conventional antioxidants, highlighting the central involvement of disturbed mitochondrial RONS metabolism. This work provides a pathomechanistic model by which DUX4-induced changes in oxidative metabolism impair muscle function in FSHD, amplified when metabolic adaptation to varying O2 tension is required. HighlightsO_LITranscriptomics data from FSHD muscle indicates enrichment for disturbed mitochondrial pathways C_LIO_LIDisturbed RONS metabolism correlates with mitochondrial membrane polarisation and myotube hypotrophy C_LIO_LIDUX4-induced changes in mitochondrial function precede oxidative stress through mitoROS and affect hypoxia signalling via complex I C_LIO_LIFSHD is sensitive to environmental hypoxia, which increases ROS levels in FSHD myotubes C_LIO_LIHypotrophy in hypoxic FSHD myotubes is efficiently rescued with mitochondria-targeted antioxidants C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/459509v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@16ae1a8org.highwire.dtl.DTLVardef@517caorg.highwire.dtl.DTLVardef@5d0734org.highwire.dtl.DTLVardef@183ef50_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2020

Mechanosensitive Ion Channel Piezo1 Regulates Myocyte Fusion during Skeletal Myogenesis

Mechanical stimuli such as stretch and resistance training are essential to regulate growth and function of skeletal muscle. However, the molecular mechanisms involved in sensing mechanical stress during muscle formation remain unclear. Here, we investigate the role of the mechano-sensitive ion channel Piezo1 during myogenic progression. Direct manipulation of Piezo1 in muscle stem cells alters their myogenic progression. Indeed, Piezo1 knockdown suppresses myoblast fusion leading to smaller myotubes. Such event is accompanied by significant downregulation of the fusogenic protein Myomaker. In parallel, while Piezo1 knockdown also lowers Ca2+ influx in response to stretch, Piezo1 activation increases Ca2+ influx in response to stretch and enhances myoblasts fusion. We believe these findings may help understand molecular defects present in some muscle diseases. Altogether our study shows that Piezo1 is essential for terminal muscle differentiation acting on myoblast fusion, suggesting that Piezo1 deregulation may have implications in muscle aging and degenerative diseases including muscular dystrophies.

molecular biology↗

bioRxiv2020

Myogenin is an Essential Regulator of Adult Myofibre Growth and Muscle Stem Cell Homeostasis

Growth and maintenance of skeletal muscle fibres depend on coordinated activation and return to quiescence of resident muscle stem-cells (MuSCs). The transcription factor Myogenin (Myog) regulates myocyte fusion during development, but its role in adult myogenesis remains unclear. In contrast to mice, myog-/- zebrafish are viable, but have hypotrophic muscles. By isolating adult myofibres with associated MuSCs we found that myog-/- myofibres have severely reduced nuclear number, but increased myonuclear domain size. Expression of fusogenic genes is decreased, pax7 upregulated, MuSCs are fivefold more numerous and mis-positioned throughout the length of myog-/- myofibers instead of localising at myofibre ends as in wild-type. Loss of Myog dysregulates mTORC1 signalling, resulting in an alerted state of MuSCs, which display precocious activation and faster cell cycle entry ex vivo, concomitant with myod upregulation. Thus, beyond controlling myocyte fusion, Myog influences the MuSC:niche relationship, demonstrating a multi-level contribution to muscle homeostasis throughout life.

developmental biology↗

bioRxiv2019

Lymphocytes contribute to DUX4 target genes in FSHD muscle biopsies

Facioscapulohumeral muscular dystrophy (FSHD) is an incurable myopathy linked to overexpression of DUX4. However, DUX4 is difficult to detect in FSHD myoblasts and target gene expression is not a consistent FSHD muscle biopsy biomarker, displaying efficacy only on pathologically inflamed samples. Immune gene misregulation occurs in FSHD muscle biopsies with DUX4 targets enriched for inflammatory processes. However, assessment of the FSHD immune cell transcriptome, and the evaluation of DUX4 and target gene expression has not yet been performed. We show that FSHD lymphoblastoid cell lines (LCLs) display robust DUX4 expression, and express early and late DUX4 targets. Moreover, genes elevated on FSHD LCLs are elevated in FSHD muscle biopsies, correlating with DUX4 target activation and histological inflammation. These genes are importantly unaltered in FSHD myoblasts/myotubes, implying a non-muscle source in biopsies. Our results indicate an immune cell source of DUX4 and target gene expression in FSHD muscle biopsies.

bioinformatics↗

bioRxiv2018

Accumulation of prelamin A drives inflammation in the heart with implications for treatment of inherited and acquired cardiomyopathies

Cardiomyopathies are complex heart muscle diseases that can be inherited e.g. dilated cardiomyopathy resulting from LMNA gene mutations, or acquired, e.g. cardiomyopathy associated with HIV. In both cases the lamin A precursor, prelamin A, may play a central role: mutations in LMNA and certain HIV protease inhibitors acting via the enzyme ZMPSTE24 both inhibit prelamin A processing. Firstly, we show that myocardial prelamin A accumulation occurs in both these cardiomyopathies in patients. Secondly, we developed a novel mouse model of cardiac specific prelamin A accumulation which mimicked tissue and molecular features of HIV associated cardiomyopathy, including inflammation. These findings: (1) confirm a central pathological role of prelamin A common to genetic and acquired cardiomyopathies; (2) have implications for the management of HIV patients with cardiac disease in whom protease inhibitors with low/no binding to ZMPSTE24 may be preferred; and (3) suggest that targeting inflammation may be a useful treatment strategy for some forms of inherited cardiomyopathy.

pathology↗