Characterization of a novel zebrafish model of <i>SPEG</i>-related centronuclear myopathy

Espinosa, Karla G; Geissah, Salma; Groom, Linda; Volpatti, Jonathan; Scott, Ian C.; Dirksen, Robert T.; Zhao, Mo; Dowling, James J.

doi:10.1242/dmm.049437

Cited by 6 publications

(6 citation statements)

References 69 publications

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“…A previous study has reported the colocalization of SPEG and RyR1 in zebrafish myofibres, indicating a potential interaction between the two proteins. 33 Here, we confirm their interaction using co-IP (Figure 1E). RyR1 is part of the myospryn complex, and it was noted to be decreased in SPEG-deficient mice, but the reduction was not statistically significant (Figure 2E).…”

Section: Speg Phosphorylates S2902 Residue Of Ryr1supporting

confidence: 67%

Integrated multi‐omics approach reveals the role of striated muscle preferentially expressed protein kinase in skeletal muscle including its relationship with myospryn complex

Li,

Lin,

Luo

et al. 2024

J cachexia sarcopenia muscle

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BackgroundAutosomal‐recessive mutations in SPEG (striated muscle preferentially expressed protein kinase) have been linked to centronuclear myopathy with or without dilated cardiomyopathy (CNM5). Loss of SPEG is associated with defective triad formation, abnormal excitation–contraction coupling, calcium mishandling and disruption of the focal adhesion complex in skeletal muscles. To elucidate the underlying molecular pathways, we have utilized multi‐omics tools and analysis to obtain a comprehensive view of the complex biological processes and molecular functions.MethodsSkeletal muscles from 2‐month‐old SPEG‐deficient (Speg‐CKO) and wild‐type (WT) mice were used for RNA sequencing (n = 4 per genotype) to profile transcriptomics and mass spectrometry (n = 4 for WT; n = 3 for Speg‐CKO mice) to profile proteomics and phosphoproteomics. In addition, interactomics was performed using the SPEG antibody on pooled muscle lysates (quadriceps, gastrocnemius and triceps) from WT and Speg‐CKO mice. Based on the multi‐omics results, we performed quantitative real‐time PCR, co‐immunoprecipitation and immunoblot to verify the findings.ResultsWe identified that SPEG interacts with myospryn complex proteins CMYA5, FSD2 and RyR1, which are critical for triad formation, and that SPEG deficiency results in myospryn complex abnormalities (protein levels decreased to 22 ± 3% for CMYA5 [P < 0.05] and 18 ± 3% for FSD2 [P < 0.01]). Furthermore, SPEG phosphorylates RyR1 at S2902 (phosphorylation level decreased to 55 ± 15% at S2902 in Speg‐CKO mice; P < 0.05), and its loss affects JPH2 phosphorylation at multiple sites (increased phosphorylation at T161 [1.90 ± 0.24‐fold], S162 [1.61 ± 0.37‐fold] and S165 [1.66 ± 0.13‐fold]; decreased phosphorylation at S228 and S231 [39 ± 6%], S234 [50 ± 12%], S593 [48 ± 3%] and S613 [66 ± 10%]; P < 0.05 for S162 and P < 0.01 for other sites). On analysing the transcriptome, the most dysregulated pathways affected by SPEG deficiency included extracellular matrix–receptor interaction (P < 1e−15) and peroxisome proliferator‐activated receptor signalling (P < 9e−14).ConclusionsWe have elucidated the critical role of SPEG in the triad as it works closely with myospryn complex proteins (CMYA5, FSD2 and RyR1), it regulates phosphorylation levels of various residues in JPH2 and S2902 in RyR1, and its deficiency is associated with dysregulation of several pathways. The study identifies unique SPEG‐interacting proteins and their phosphorylation functions and emphasizes the importance of using a multi‐omics approach to comprehensively evaluate the molecular function of proteins involved in various genetic disorders.

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Section: Speg Phosphorylates S2902 Residue Of Ryr1supporting

confidence: 67%

Integrated multi‐omics approach reveals the role of striated muscle preferentially expressed protein kinase in skeletal muscle including its relationship with myospryn complex

Li,

Lin,

Luo

et al. 2024

J cachexia sarcopenia muscle

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“…Animal models are the gold standard for gaining pre-clinical insights into various diseases, due to their anatomical and morphological resemblance to humans 66 . Of late, modeling of human myopathies is achieved by knocking out disease-causing genes by CRISPR/Cas9 technology 67 .…”

Section: Discussionmentioning

confidence: 99%

Early life lipid overload in Native American myopathy is phenocopied bystac3knock out in zebrafish

Donaka¹,

Zheng

Karasik³

2023

Preprint

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Understanding the early stages of human congenital myopathies is critical for proposing strategies for improving skeletal muscle performance by the functional integrity of cytoskeleton. SH3 and cysteine-rich domain 3 (Stac3) is a protein involved in nutrient sensing, and is an essential component of the excitation-contraction (EC) coupling machinery for Ca2+ releasing. A mutation in STAC3 causes debilitating Native American myopathy (NAM) in humans, and loss of this gene in mice and zebrafish resulted in death in early life. Previously, NAM patients demonstrated increased lipids in skeletal muscle biopsy. However, elevated neutral lipids could alter muscle function in NAM disease via EC coupling apparatus is yet undiscovered in early development. Here, using a CRISPR/Cas9 induced stac3 knockout (KO) zebrafish model, we determined that loss of stac3 led to muscle weakness, as evidenced by delayed larval hatching. We observed decreased whole-body Ca2+ level at 5 days post-fertilization (dpf) and defects in the skeletal 35 muscle cytoskeleton, i.e., F-actin and slow muscle fibers at 5 and 7 dpf. Homozygous larvae 36 exhibited elevated neutral lipid levels at 5 dpf, which persisted beyond 7 dpf. Myogenesis regulators such as myoD and myf5, were significantly altered in stac3-/- larvae at 5 dpf, thus a progressive death of the KO larva by 11 dpf. In summary, the presented findings suggest that stac3-/- can serve as a non-mammalian model to identify lipid-lowering molecules for refining muscle function in NAM patients.

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“…Thus, downregulation of dynamin 2 by gene silencing tunes its relative amount for BIN1 protein resulting in normal survival, muscular force and triad structures [ 34 , 129 ]. In zebrafish, knockout of a CNM causal gene SPEG (striated preferentially expressed protein kinase) that encodes a myosin light chain kinase family protein show T-tubule abnormalities with the increased expression level of dynamin 2 protein [ 130 ]. Since SPEG has been shown to interact with MTM1 [ 5 ], SPEG may regulate dynamin 2 function together with MTM1 and BIN1 in skeletal muscle.…”

Section: Dynamin: a Membrane Fission Catalyser In Endocytosismentioning

confidence: 99%

Centronuclear Myopathy Caused by Defective Membrane Remodelling of Dynamin 2 and BIN1 Variants

Fujise

Noguchi

Takeda

2022

IJMS

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Centronuclear myopathy (CNM) is a congenital myopathy characterised by centralised nuclei in skeletal myofibers. T-tubules, sarcolemmal invaginations required for excitation-contraction coupling, are disorganised in the skeletal muscles of CNM patients. Previous studies showed that various endocytic proteins are involved in T-tubule biogenesis and their dysfunction is tightly associated with CNM pathogenesis. DNM2 and BIN1 are two causative genes for CNM that encode essential membrane remodelling proteins in endocytosis, dynamin 2 and BIN1, respectively. In this review, we overview the functions of dynamin 2 and BIN1 in T-tubule biogenesis and discuss how their dysfunction in membrane remodelling leads to CNM pathogenesis.

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Characterization of a novel zebrafish model of SPEG-related centronuclear myopathy

Cited by 6 publications

References 69 publications

Integrated multi‐omics approach reveals the role of striated muscle preferentially expressed protein kinase in skeletal muscle including its relationship with myospryn complex

Integrated multi‐omics approach reveals the role of striated muscle preferentially expressed protein kinase in skeletal muscle including its relationship with myospryn complex

Early life lipid overload in Native American myopathy is phenocopied bystac3knock out in zebrafish

Centronuclear Myopathy Caused by Defective Membrane Remodelling of Dynamin 2 and BIN1 Variants

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