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Potential Roles of Numb in Myogenesis, Mitochondrial Metabolism, and Calcium Signaling
Author(s) -
Mo Chenglin,
Wang Zhiying,
Harlow Lauren,
De Gasperi Rita,
Cardozo Christopher,
Brotto Marco
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.03025
Subject(s) - numb , biology , microbiology and biotechnology , mitochondrial fusion , myogenesis , skeletal muscle , myocyte , mitochondrion , calcium signaling , signal transduction , endocrinology , biochemistry , mitochondrial dna , gene
Numb, first discovered in Drosophila as a critical genetic determinant of central nervous system development, is a highly conserved protein expressed in other tissues and in all higher organisms. Reduced Numb mRNA levels was found in muscles of older individuals, suggesting the role of Numb in the pathogenesis of aging‐related decline of muscle strength. However, its molecular mechanisms remain incompletely understood. Since mitochondrial dysfunction has been linked to muscle aging, and mitochondria are organelles responsible for ATP production, lipid and overall metabolism, and calcium signaling, all closely associated with muscle function, our hypothesis in this study is that downregulation of Numb will perturbe mitochondrial functional, which will eventually contribute to muscle aging. To test this hypothesis, we employed both in vitro and in vivo models in our studies. Mouse primary myoblasts were transfected with control or vivo morpholino of Numb for 48 hours during myoblast differentiation, followed by morphological and functional evaluations. We quantified myogenic differentiation by fusion index, cytosolic calcium homeostasis by the Fura‐2 ratio method, and mitochondrial functional tests using the Seahorse analyzer using both the Phenotype test kit and the Mito stress test kit. In addition, lipidomic profiles of gastrocnemius muscles from mice with muscle‐specific conditional knockout (cKO) of Numb/NumbL and their control type littermates were measured using a liquid chromatography with tandem mass spectrometry (LC‐MS/MS) method recently developed. Our results demonstrated that transfection with Numb morpholino led to approximately 30% reduction in Numb protein levels, ~15% reduction in fusion index, a more quiescent energy phenotype, and a statistically significant decreased basal respiration, maximal respiration, and spare capacity in mitochondria. Moreover, myotubes treated with the Numb morpholino had a 50% reduction in the amplitude of calcium transient induced by caffeine as well as a prolongation of the calcium transients, along with the intriguing appearance of spontaneous small calcium transients similar to those observed in beating hearts. Lipidomic profiling results indicated a significant increase in the levels of key inflammation‐related lipid mediators, derived from polyunsaturated fatty acids through lipoxygenase metabolic pathways, in gastrocnemius muscles from cKO mice, such as 12‐hydroxyeicosapentaenoic acid (12‐HEPE, p =0.035) and 12‐hydroxyeicosatetraenoic acid (12‐HETE, p =0.024). These findings imply that Numb could be a critical factor in maintaining myogenic differentiation, mitochondrial function and calcium signaling in skeletal muscle. A decline in Numb level during aging could be one of the key drivers responsible for reduced muscle mass and strength that develops during aging.