Numb is required for optimal contraction of skeletal muscle

De Gasperi, R.; Mo, C.; Azulai, D.; Wang, Z.; Harlow, L. M.; Du, Y.; Graham, Z. A.; Liu, X.-h.; Guo, L.; Zhang, B.; Ko, F.; Raczkowski, A. M.; Bauman, W. A.; Zhao, W.; Brotto, M.; Cardozo, C. P.

doi:10.1101/2021.09.03.453960

bioRxiv · 10.1101/2021.09.03.453960

Numb is required for optimal contraction of skeletal muscle

De Gasperi, R.·Mo, C.·Azulai, D.·Wang, Z.·Harlow, L. M.·Du, Y.·Graham, Z. A.·Liu, X.-h.·Guo, L.·Zhang, B.·Ko, F.·Raczkowski, A. M.·Bauman, W. A.·Zhao, W.·Brotto, M.·Cardozo, C. P.

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Abstract

BackgroundThe role of Numb, a protein that is important for cell fate and development was investigated in adult skeletal muscle in mice using a conditional, inducible knockout (cKO) model. MethodsNumb expression was evaluated by Western blot. Numb localization was determined by confocal microscopy. The effects of cKO of Numb and the closely-related gene Numb-like in skeletal muscle fibers was evaluated by in-situ physiology; transmission and focused ion beam scanning electron microscopy; 3-dimensional reconstruction of mitochondrial; lipidomics; and bulk RNA-sequencing. Additional studies using primary mouse myotubes investigated the effects the effects of Numb knockdown on cell fusion, mitochondrial function and calcium transients. ResultsNumb protein expression was reduced by [~]70% (p < 0.01) at 24 as compared to 3 months of age. Numb was localized within muscle fibers as bands traversing fibers at regularly spaced intervals in close proximity to dihydropyridine receptors. The cKO of Numb and Numb-like reduced specific tetanic force by 36%, p < 0.01), altered mitochondrial spatial relationships to sarcomeric structures, increased Z-line spacing by 30% (p < 0.0001), perturbed sarcoplasmic reticulum organization and reduced mitochondrial volume by over 80% (p < 0.01). Only six genes were differentially expressed in cKO mice: Itga4, Sema7a, Irgm2, Vezf1, Mib1 and Tmem132a. Several lipid mediators derived from polyunsaturated fatty acid (PUFAs) through lipoxygenases were upregulated in Numb cKO skeletal muscle; 12-HEPE was increased by [~]250% (p < 0.05) and 17,18-EpETE by [~]240% (p < 0.05). In mouse primary myotubes, Numb knock-down reduced cell fusion ([~]20%, p < 0.01) and mitochondrial function and delayed the caffeine-induced rise in cytosolic calcium concentrations by more than 100% (p < 0.01). ConclusionsThese findings implicate Numb as a critical factor in skeletal muscle structure and function which appear to be critical for calcium release; we therefore speculate Numb plays critical roles in excitation-contraction coupling, one of the putative targets of aged skeletal muscles. These findings provide new insights into the molecular underpinnings of the loss of muscle function observed with sarcopenia.

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De Gasperi, R., Mo, C., Azulai, D., Wang, Z., Harlow, L. M., Du, Y., Graham, Z. A., Liu, X.-h., Guo, L., Zhang, B., Ko, F., Raczkowski, A. M., Bauman, W. A., Zhao, W., Brotto, M., Cardozo, C. P.. 2021-09-04. Numb is required for optimal contraction of skeletal muscle. https://doi.org/10.1101/2021.09.03.453960

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