SEPTIN 7 INTERACTS WITH NUMB TO PRESERVE SARCOMERE STRUCTURAL ORGANIZATION AND MUSCLE CONTRACTILE FUNCTION

Abstract

Here, we investigated mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development including asymmetric cell division, cell-type specification and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (NumbL) in mouse myofibers caused weakness, disorganization of sarcomeres and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, NumbL knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb, that Septin 7 is a potential Numb binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.

Figure 1.

Effect of Numb or Numb/NumbL cKO on contractile function of EDL muscle during ex-vivo physiological testing. (A) Specific tension generated during tetanic contraction at the indicated frequencies is shown for weight normalized EDL muscle harvested from HSA-MCM/Numb(^f/f)) or HSA-MCM/Numb^(f/f/NumbL^(f/f) mice at 14-days after starting injections of tamoxifen or vehicle. Statistical analysis was performed with repeated measure ANOVA followed by Sidak’s multiple comparison test. F = 28.29, DFn= 6, DFd= 22; ***p< 0.001, force X frequency interaction ***p< 0.001; (B) Maximum force generated during a single twitch is shown. Statistical analysis was performed with one-way ANOVA with Tukey’s post-hoc test (F=10.07, DFn=3, DFd=13). *p<0.05; **p<0.01. N=3–6

Figure 2.

Time to peak tension (A), half-relaxation time (B), and fatigue index (C) are shown for HSA-MCM/Numb(^f/f)) and double (HSA-MCM/Numb^(f/f/NumbL^(f/f) mice) at 14 days after starting injections of tamoxifen or vehicle. Data were analyzed by one-way ANOVA with Tukey post-hoc test. (A) F=4.561, DFn=3, DFd=13; *p<0.05; (B) F=3.679, DFn=3,DFd=13 ; (C) F=0.9772, DFn=3, DFd=13. N=3–6.

Figure 3.

A Western blot showing a representative co-immunoprecipitation of Septin7 by anti-Numb antibody. IgG: immunoprecipitation with control Ig; N: immunoprecipitation with anti-Numb; input: original C2C12 lysate. The upper panel was probed with anti-Septin 7, the lower panel with anti-Numb. B-D. Immunolocalization of Numb and Septin 7 in muscle. Longitudinal cryosections of TA muscle from C57Bl6 mice were immunostained for Numb and Septin 7: (B) merged image; (C) anti-Numb antibody (green); (D) anti-Septin 7 antibody (red); (E) DAPI staining of nuclei (blue). Scale bar, 10 μm. The inset in A shows higher magnification (x2.3) of the area in the black square.

Figure 4.

Septin 7 is enriched at neuromuscular junctions. A-B. Violin plots generated by Myoatlas showing abundance of Septin 7 (A) and Numb (B) mRNA in nuclei of TA muscle from 5-month old mice [30] C. Representative confocal microscopy image of the neuromuscular junction of an isolated myofiber immunostained with anti-Septin 7 (red) and with a fluorescently tagged α-bungarotoxin (green); nuclei were stained with DAPI (blue). Scale bar, 10 μm.

Figure 5.

Representative confocal images of isolated mouse hindlimb fibers immunostained with anti-Numb (green) (B and E) and anti-Septin 7 antibodies (red) (C-F); merge: merged images (A and D). Nuclei were labeled with DAPI (blue). A-C: myofiber from a control mouse (vehicle treated); D-F, myofiber from a Numb cKO mouse (tamoxifen treated). Scale bar, 10 μm.