Figure 1.
Nedd4-1 protein is absent from MyoCre;Nedd4-1flox/flox (Nedd4-1 SMS-KO) skeletal muscle.
(A) SDS-PAGE and Nedd4-1 Western blotting (WB) of 50 µg of gastrocnemius muscle lysates from representative Nedd4-1 SMS-KO mice (M4, M5, M6) and littermate MyoCre;Nedd4-1+/+ control mice (M1, M2, M3) reveal the absence of Nedd4-1 in the Nedd4-1 SMS-KO muscle. Nedd4-2 protein remains equally expressed in the muscle of Nedd4-1 SMS-KO and MyoCre;Nedd4-1+/+ control mice. GAPDH served as a loading control. (B) WB of heart, liver and kidney protein lysates reveal equal Nedd4-1 protein in these tissues in the Nedd4-1 SMS-KO and littermate MyoCre;Nedd4-1+/+ control mice. GAPDH served as a loading control for all blots; liver lysate is shown as a representative blot.
Figure 2.
Denervation induces increased Nedd4-1 protein expression in gastrocnemius muscle.
Nedd4-1 SMS-KO and littermate MyoCre;Nedd4-1+/+ control mice underwent right tibial nerve transection with the left hind limb serving as control. Western blotting (WB) of 50 µg gastrocnemius protein lysates denervated for 1 week (A) and 2 weeks (B) show increased Nedd4-1 protein in the denervated (Den) compared to the contralateral control (Con) muscle of MyoCre;Nedd4-1+/+ mice. Nedd4-1 is absent from the Nedd4-1 SMS-KO control gastrocnemius, but trace Nedd4-1 becomes evident with denervation. Representative blots are shown (n = 8 to 9 mice/cohort).
Figure 3.
Satellite cells/myoblasts isolated from Nedd4-1 SMS-KO mice express Nedd4-1.
Muscle satellite cells/myoblasts were isolated from Nedd4-1 SMS-KO mice and littermate MyoCre;Nedd4-1+/+ control mice and maintained in culture. Cultures were immunostained for Nedd4-1 (red) and MyoD (green) and nuclei were visualized with Hoechst (blue). MyoD, a specific marker of myoblasts, is expressed in nuclei; Nedd4-1 is predominantly cytoplasmic. Myoblasts derived from both Nedd4-1 SMS-KO and littermate control mice demonstrate co-expression of MyoD and Nedd4-1. Immunostaining with 2° antibodies alone served as a negative control and did not demonstrate non-specific staining (data not shown). Scale bar shown = 50 µm.
Figure 4.
Satellite cell/myoblast population is increased in denervated gastrocnemius muscle.
(A) Cross sections of gastrocnemius muscle from Nedd4-1 SMS-KO and littermate MyoCre;Nedd4-1+/+ mice were immunostained for the satellite cell/myoblast marker Pax 7 (brown nuclei, indicated by white arrows). Myonuclei are counterstained purple with hematoxylin. There is an apparent increase in satellite cell number in the denervated gastrocnemius of both Nedd4-1 SMS-KO and littermate MyoCre;Nedd4-1+/+ control mice. (B) Similarly, representative Western blots (WB) of gastrocnemius protein lysates denervated for 1 week (left panel) and 2 weeks (right panel) show increased expression of Pax 7 in the denervated (Den) compared to the contralateral control (Con) muscle of Nedd4-1 SMS-KO and littermate MyoCre;Nedd4-1+/+ mice. HPRT served as loading control. (C) The chemiluminescent signal was quantified and Pax-7 protein levels were normalized to corresponding HPRT levels (lane matched). Normalized Pax-7 levels in denervated (Exp) muscle were expressed as a fraction of the normalized Pax-7 level in control (Con) muscle and these values are depicted in the graph. Pax-7 is significantly increased in the denervated gastrocnemius muscle of both Nedd4-1 SMS-KO and MyoCre;Nedd4-1+/+ mice at both 1 and 2 weeks post-tibial nerve transection (n = 6 mice/group, p<0.05), but there is no difference in the magnitude of the increase between Nedd4-1 SMS-KO and MyoCre;Nedd4-1+/+ mice. Data are presented as the mean ± SEM.
Figure 5.
Nedd4-1 SMS-KO gastrocnemius muscle weights are partially spared from denervation induced atrophy.
(A) Total body weights (left panel) and gastrocnemius muscle weights (right panel) of Nedd4-1 SMS-KO and littermate MyoCre;Nedd4-1+/+ control mice. Nedd4-1 SMS-KO mice are slightly smaller, and their gastrocnemius muscle weigh less, than littermate controls (n = 16 pairs, * p<0.05). (B) There is no difference in the basal gastrocnemius muscle mass between the Nedd4-1 SMS-KO and control mice when muscle weight is normalized to body weight p>0.05). (C) Nedd4-1 SMS-KO and littermate control mice underwent right tibial nerve transection, denervating the gastrocnemius muscle, with the intact left hind limb serving as internal control. Denervated gastrocnemius muscle weights are normalized to the weight of the contralateral control muscle. Denervated muscle of Nedd4-1 SMS-KO (solid line, closed squares) mice demonstrates an attenuated atrophic response weighing significantly more than in MyoCre;Nedd4-1+/+ (dashed line, open circles) mice at 1 and 2 weeks post denervation, (*p<0.05, n = 8 or 9 mice/cohort. Data are presented as the mean ± SEM).
Figure 6.
Nedd4-1 deletion attenuates denervation induced decreases in gastrocnemius Type II fibre cross-sectional area.
(A) Cross sections of denervated and control gastrocnemius muscle of Nedd4-1 SMS-KO and MyoCre;Nedd4-1+/+ (littermate control) mice were immunostained with an anti-fast twitch myosin antibody and counterstained with hematoxylin. Fast twitch type II fibres stain brown, and slow twitch type I fibres stain light purple. Representative cross sections from control and 2 week denervated gastrocnemius muscle from both Nedd4-1 SMS-KO and littermate control mice are shown. (B) Histograms of type II fibre cross sectional areas (CSA) for control innervated (left panel), 1 week denervated (middle panel) and 2 week denervated (right panel) gastrocnemius muscles in Nedd4-1 SMS-KO (solid line, closed squares) and littermate control (dashed line, open circles) mice are shown. (C) At baseline, Nedd4-1 SMS-KO gastrocnemius type II fibre CSA is smaller than that of MyoCre;Nedd4-1+/+ mice (*). Following denervation, there is a significant decrease in type II fibre CSA for both cohorts of mice. However the CSA of Nedd4-1 SMS-KO muscle fibres denervated for 2 weeks is significantly larger than fibres of MyoCre;Nedd4-1+/+ mice (**) denervated for 2 weeks. (D) Similarly, when Type II fibre CSA in denervated gastrocnemius muscle is normalized to CSA in the contralateral control muscle, type II fibres are significantly larger in Nedd4-1 SMS-KO (solid line, closed squares) mice compared to MyoCre;Nedd4-1+/+ (dashed line, open circles) mice at both 1 week and 2 weeks post denervation. A minimum of 300 myofibres/muscle was measured, (** and *p<0.05, n = 8 or 9 mice/cohort. Data are presented as the mean ± SEM).
Figure 7.
Levels of MTMR4 and FGFR-1 are not maintained in the denervated gastrocnemius of Nedd4-1 SMS-KO mice, while cleaved Notch-1 expression is increased.
(A) Representative Western blots of MTMR4, FGFR1, cleaved Notch-1 (Notch-1 ICD) and HPRT (as a loading control) in protein lysates from denervated gastrocnemius muscle (Den) or the contralateral control gastrocnemius (Con) muscle in Nedd4-1 SMS-KO and littermate MyoCre;Nedd4-1+/+ control mice at 1 and 2 weeks post tibial nerve transection. (B) The chemiluminescent signal was quantified, MTMR4, FGFR1 and Notch-1 protein levels were normalized to the corresponding HPRT levels (lane matched) and numerical values are depicted in bar graphs. For each of MTMR4, FGFR1 and Notch-1, protein levels in denervated (Exp) muscle are expressed as a fraction of the protein level in control (Con) muscle. MTMR4 levels were significantly decreased in denervated compared to control gastrocnemius muscle in both Nedd4-1 SMS-KO (n = 6) and MyoCre;Nedd4-1+/+ mice (n = 6) mice at 2 weeks (p<0.05), but there was no difference in the magnitude of MTMR4 decrease between the Nedd4-1 SMS-KO compared to control mice. FGFR1 levels were similarly significantly decreased in denervated compared to control gastrocnemius muscle in both Nedd4-1 SMS-KO (n = 8) and littermate MyoCre;Nedd4-1+/+ control mice (n = 8) mice at 1 week and 2 weeks (p<0.05) post tibial nerve transection, and again there was no difference in the magnitude of the decrease between the 2 cohorts of mice at either timepoint. Cleaved Notch-1 levels (Notch ICD) were significantly increased in denervated compared to control gastrocnemius in both Nedd4-1 SMS-KO (n = 6) and MyoCre;Nedd4-1+/+ control mice (n = 6) mice at 2 weeks (p<0.05), but not at 1 week. There was no difference in the magnitude of the increase between the 2 cohorts of mice. Data are mean ± SEM.