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Hyperglycemia Negatively Affects IPSC-Derived Myoblast Proliferation and Skeletal Muscle Regeneration and Function.

ABSTRACT: Diabetic myopathy is a co-morbidity diagnosed in most diabetes mellitus patients, yet its pathogenesis is still understudied, which hinders the development of effective therapies. This project aimed to investigate the effect of hyperglycemia on human myoblast physiology, devoid of other complicating factors, by utilizing human myoblasts derived from induced pluripotent stem cells (iPSCs), in a defined in vitro system. IPSC-derived myoblasts were expanded under three glucose conditions: low (5 mM), medium (17.5 mM) or high (25 mM). While hyperglycemic myoblasts demonstrated upregulation of Glut4 relative to the euglycemic control, myoblast proliferation demonstrated a glucose dose-dependent impedance. Further cellular analysis revealed a retarded cell cycle progression trapped at the S phase and G2/M phase and an impaired mitochondrial function in hyperglycemic myoblasts. Terminal differentiation of these hyperglycemic myoblasts resulted in significantly hypertrophic and highly branched myotubes with disturbed myosin heavy chain arrangement. Lastly, functional assessment of these myofibers derived from hyperglycemic myoblasts demonstrated comparatively increased fatigability. Collectively, the hyperglycemic myoblasts demonstrated deficient muscle regeneration capability and functionality, which falls in line with the sarcopenia symptoms observed in diabetic myopathy patients. This human-based iPSC-derived skeletal muscle hyperglycemic model provides a valuable platform for mechanistic investigation of diabetic myopathy and therapeutic development.

SUBMITTER: Badu-Mensah A

PROVIDER: S-EPMC9688533 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Hyperglycemia Negatively Affects IPSC-Derived Myoblast Proliferation and Skeletal Muscle Regeneration and Function.

Badu-Mensah Agnes A Valinski Paola P Parsaud Hemant H Hickman James J JJ Guo Xiufang X

Cells 20221118 22

Diabetic myopathy is a co-morbidity diagnosed in most diabetes mellitus patients, yet its pathogenesis is still understudied, which hinders the development of effective therapies. This project aimed to investigate the effect of hyperglycemia on human myoblast physiology, devoid of other complicating factors, by utilizing human myoblasts derived from induced pluripotent stem cells (iPSCs), in a defined in vitro system. IPSC-derived myoblasts were expanded under three glucose conditions: low (5 mM ...[more]

PMID: 36429100

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Project description:BackgroundCREG1 (cellular repressor of E1A-stimulated genes 1) is a protein involved in cellular differentiation and homeostasis regulation. However, its role in skeletal muscle satellite cells differentiation and muscle regeneration is poorly understood. This study aimed to investigate the role of CREG1 in myogenesis and muscle regeneration.MethodsRNA sequencing data (GSE8479) was analysed from the Gene Expression Omnibus database (GEO, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi). We generated Creg1 knockdown and skeletal muscle satellite cells specific Creg1 overexpression mice mediated by adeno-associated virus serotype 9 (AAV9), skeletal muscle mature myofibre Creg1 knockout mice (myoblast/Creg1MKO), and control mice Creg1flox/flox (Creg1fl/fl) as in vivo models. The mice were injected into tibialis anterior (TA) muscle with 100 μL of 10 μM cardiotoxin to establish a muscle regeneration model. Creg1fl/fl and Creg1MKO mice were treated with AAV-sh-C-Cbl (2 × 1010 genomic copies/mouse) to silence C-Cbl in the TA muscle. 293T and C2C12 cells were transfected with plasmids using lipofectamine RNAi MAX in vitro. Mass spectrometry analyses and RNA sequencing transcriptomic assay were performed.ResultsWe analysed the transcriptional profiles of the skeletal muscle biopsies from healthy older (N = 25) and younger (N = 26) adult men and women in GSE8479 database, and the results showed that Creg1 was associated with human sarcopenia. We found that Creg1 knockdown mice regenerated less newly formed fibres in response to cardiotoxin injection (~30% reduction, P < 0.01); however, muscle satellite cells specific Creg1 overexpression mice regenerated more newly formed fibres (~20% increase, P < 0.05). AMPKa1 is known as a key mediator in the muscle regeneration process. Our results revealed that CREG1 deficiency inhibited AMPKa1 signalling through C-CBL E3-ubiquitin ligase-mediated AMPKa1 degradation (P < 0.01). C-CBL-mediated AMPKa1 ubiquitination was attributed to the K48-linked polyubiquitination of AMPKa1 at K396 and that the modification played an important role in the regulation of AMPKa1 protein stability. We also found that Creg1MKO mice regenerated less newly formed fibres compared with Creg1fl/fl mice (~30% reduction, P < 0.01). RNA-seq analysis showed that CREG1 deletion in impaired muscles led to the upregulation of inflammation and DKK3 expression. The TA muscles of Creg1MKO mice were injected with AAV-vector or AAV-shC-Cbl, silencing C-CBL (P < 0.01) in the skeletal muscles of Creg1MKO mice significantly improved muscle regeneration induced by CTX injury (P < 0.01).ConclusionsOur findings suggest that CREG1 may be a potential therapeutic target for skeletal muscle regeneration.

Dataset Information

Hyperglycemia Negatively Affects IPSC-Derived Myoblast Proliferation and Skeletal Muscle Regeneration and Function.

Publications

Hyperglycemia Negatively Affects IPSC-Derived Myoblast Proliferation and Skeletal Muscle Regeneration and Function.

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