LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model

Boccanegra, Brigida; Mantuano, Paola; Conte, Elena; Cerchiara, Alessandro Giovanni; Tulimiero, Lisamaura; Quarta, Raffaella; Caputo, E; Sanarica, Francesca; Forino, M.; Spadotto, Valeria; Cappellari, Ornella; Fossati, Gianluca; Steinkühler, Christian; A, De Luca

doi:10.1242/dmm.049930

Cited by 4 publications

(4 citation statements)

References 44 publications

(61 reference statements)

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“…This again suggests that DYS cells tend to get smaller, probably as a result of changes in membrane protein localization, possible lipid composition, epigenetic mechanisms, and/or delayed differentiation. 29 This would be consistent with a membrane that is less stable and more prone to damage due to the absence of dystrophin. 75,79 Few researchers have measured Cm in WT and DYS cells/fibers and there is no consensus on the relative Cm changes.…”

Section: Rmp and CMsupporting

confidence: 56%

“…The Cm of D11 DYS cells was significantly lower than D6 DYS and D11 WT cells. This again suggests that DYS cells tend to get smaller, probably as a result of changes in membrane protein localization, possible lipid composition, epigenetic mechanisms, and/or delayed differentiation 29 . This would be consistent with a membrane that is less stable and more prone to damage due to the absence of dystrophin 75,79 .…”

Section: Discussionmentioning

confidence: 63%

“…We used an immortalized myoblast-derived cell line (H2K-2B4) and its immortalized dystrophic counterpart (H2K-SF1) developed from healthy and mdx mice, respectively, by Muses et al as previously described. [27][28][29] 2B4 and SF1 cells were cultured in both proliferative (myoblasts) and differentiation (myocytes/myotubes) medium. protein that maintains cells in the proliferative state.…”

Section: Cell Culturesmentioning

confidence: 99%

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Ion channels as biomarkers of altered myogenesis in myofiber precursors of Duchenne muscular dystrophy

Cerchiara,

Imbrici,

Quarta

et al. 2024

Annals of the New York Academy of Sciences

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Myogenesis is essential for skeletal muscle formation, growth, and regeneration and can be altered in Duchenne muscular dystrophy (DMD), an X‐linked disorder due to the absence of the cytoskeletal protein dystrophin. Ion channels play a pivotal role in muscle differentiation and interact with the dystrophin complex. To investigate ion channel involvement in myogenesis in dystrophic settings, we performed electrophysiological characterization of two immortalized mouse cell lines, wild‐type (WT) H2K‐2B4 and the dystrophic (DYS) H2K‐SF1, and measured gene expression of differentiation markers and ion channels. Inward and outward currents/density increased as differentiation progressed in both WT and DYS cells. However, day‐11 DYS cells showed higher (27%) inward current density with an increased expression ratio of Scn5a/Scn4a and decreased (48%) barium‐sensitive outward current compared to WT. Furthermore, day‐11 DYS cells showed more positive resting membrane potential (+10 mV) and lower membrane capacitance (50%) compared to WT. DYS cells also had reduced Myog and Myf5 expression at days 6 and 11. Overall, ion channel profile and myogenesis appeared altered in DYS cells. These results are a first step in validating ion channels as potential drug targets to ameliorate muscle degeneration in DMD settings and as differentiation biomarkers in innovative platforms.

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Section: Rmp and CMsupporting

confidence: 56%

Section: Discussionmentioning

confidence: 63%

Section: Cell Culturesmentioning

confidence: 99%

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Ion channels as biomarkers of altered myogenesis in myofiber precursors of Duchenne muscular dystrophy

Cerchiara,

Imbrici,

Quarta

et al. 2024

Annals of the New York Academy of Sciences

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“…Total proteins were extracted from gastrocnemius (GC) muscles of different experimental groups, as described by Boccanegra et al (2023b) with some modifications. Briefly, all muscles were homogenized in ice-cold buffer containing 10 mM Tris·HCl (pH 7.4 at 4°C), 1% Triton X100, 10% glycerol, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, and protease inhibitor cocktail (Roche, Cat# 5056489001).…”

Section: Methodsmentioning

confidence: 99%

Branched-chain amino acids and L-alanine supplementation ameliorate calcium dyshomeostasis in sarcopenia: New insights for nutritional interventions

Conte,

Mantuano,

Boccanegra

et al. 2024

Front. Pharmacol.

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Introduction: During aging, sarcopenia and decline in physiological processes lead to partial loss of muscle strength, atrophy, and increased fatigability. Muscle changes may be related to a reduced intake of essential amino acids playing a role in proteostasis. We have recently shown that branched-chain amino acid (BCAA) supplements improve atrophy and weakness in models of muscle disuse and aging. Considering the key roles that the alteration of Ca2+-related homeostasis and store-operated calcium entry (SOCE) play in several muscle dysfunctions, this study has been aimed at gaining insight into the potential ability of BCAA-based dietary formulations in aged mice on various players of Ca2+ dyshomeostasis.Methods: Seventeen-month-old male C57BL/6J mice received a 12-week supplementation with BCAAs alone or boosted with two equivalents of L-alanine (2-Ala) or with dipeptide L-alanyl-L-alanine (Di-Ala) in drinking water. Outcomes were evaluated on ex vivo skeletal muscles indices vs. adult 3-month-old male C57BL/6J mice.Results: Ca2+ imaging confirmed a decrease in SOCE and an increase of resting Ca2+ concentration in aged vs. adult mice without alteration in the canonical components of SOCE. Aged muscles vs. adult muscles were characterized by a decrease in the expression of ryanodine receptor 1 (RyR1), the Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) pump, and sarcalumenin together with an alteration of the expression of mitsugumin 29 and mitsugumin 53, two recently recognized players in the SOCE mechanism. BCAAs, particularly the formulation BCAAs+2-Ala, were able to ameliorate all these alterations.Discussion: These results provide evidence that Ca2+ homeostasis dysfunction plays a role in the functional deficit observed in aged muscle and supports the interest of dietary BCAA supplementation in counteracting sarcopenia-related SOCE dysregulation.

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Determination of qPCR reference genes suitable for normalizing gene expression in a novel model of Duchenne muscular dystrophy, the D2-mdx mouse

Boccanegra,

Lenti,

Mantuano

et al. 2024

PLoS ONE

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Duchenne muscular dystrophy (DMD) is a X-linked neuromuscular disorder arising from mutations in the dystrophin gene, leading to a progressive muscle wasting and disability. Currently there is no universal therapy, and there is thus a strong interest in preclinical studies for finding novel treatments. The most widely used and characterized mouse model for DMD is the C57BL/10ScSn-Dmdmdx/J (BL10-mdx), but this model exhibits mild pathology and does not replicate key features of human disease. The D2.B10-Dmdmdx/J (D2-mdx) mouse is a more recent model which seems to better mimics the complex human DMD phenotype. However, the D2-mdx mouse remains less extensively characterised than its BL10-mdx counterpart. Quantitative PCR analysis of gene expression is an important tool to monitor disease progression and evaluate therapeutic efficacy, but measurements must be normalised to stably expressed reference genes, which should ideally be determined and validated empirically. We examined gene expression in the gastrocnemius (GC), diaphragm (DIA) and heart in the D2-mdx mouse, the BL10-mdx mouse, and appropriate strain-matched wild-type controls (D2-wt and BL10-wt), from 4 to 52 weeks of age, using a large panel of candidate references (ACTB, AP3D1, CSNK2A2, GAPDH, HPRT1, PAK1IP1, RPL13A, SDHA, and in the heart, also HTATSF1 and HMBS). Data was analyzed using GeNorm, Bestkeeper, deltaCt and Normfinder algorithms to identify stable references under multiple possible scenarios. We show that CSNK2A2, AP3D1 and ACTB represent strong universal reference genes in both GC and DIA, regardless of age, muscle type, strain and genotype, while HTATSF1 and SDHA are optimal for the heart. GAPDH, HPRT1 and RPL13A were conversely revealed to be poor references, showing tissue-, age- or disease-specific changes in expression. Our results illustrate the importance of determining appropriate reference genes for specific comparative scenarios, but also reconfirm that universal panels can nevertheless be identified for normalising gene expression studies in even complex pathological states.

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LKB1 signaling is altered in skeletal muscle of a Duchenne muscular dystrophy mouse model

Cited by 4 publications

References 44 publications

Ion channels as biomarkers of altered myogenesis in myofiber precursors of Duchenne muscular dystrophy

Ion channels as biomarkers of altered myogenesis in myofiber precursors of Duchenne muscular dystrophy

Branched-chain amino acids and L-alanine supplementation ameliorate calcium dyshomeostasis in sarcopenia: New insights for nutritional interventions

Determination of qPCR reference genes suitable for normalizing gene expression in a novel model of Duchenne muscular dystrophy, the D2-mdx mouse

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