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. 2021 Apr 20;10:e61138. doi: 10.7554/eLife.61138

Figure 5. Gene delivery of Klotho enhances functional muscle regeneration following an acute injury.

(A) Experimental design using old (21–24 months) male mice. (B) Quantification of TA average myofiber cross-sectional area (N = 13). (C) Collagen IV expression in the TA muscle of GFP- versus KL-treated mice (one-tailed Mann-Whitney test, N = 13). (D) Top: Representative images of injured TA muscles stained for collagen IV (green) and DAPI (blue, scale bars = 50 µm). Bottom: Masson’s Trichrome staining of the TA (scale bars = 50 µm). (E) Collagen area percentage in the TA quantified from Masson’s Trichrome staining (one-tailed student’s t-test, N = 16). (F) FAPs to MuSCs ratio in injured TA muscles, as determined by flow cytometry (N = 6, one-tailed student’s t-test). (G) Representative TEM images showing mitochondria in the TA muscle fibers of AAV-GFP vs. KL-treated mice. Aberrant and empty mitochondria show abnormal shape and high proportion of white space respectively (scale bars = 1μm). (H) Quantification of the quality of mitochondria (two-way ANOVA, N = 13). (I) TA specific twitch force produced 14 days post-injury (dpi) (one-tailed Student’s t-test, N = 20). (J) TA maximum specific tetanic force 14 dpi (one-tailed Student’s t-test, N = 20). (K) Change in force production of the TA over time as mice underwent a fatigue protocol consisting of repeated TA stimulation for a total of 7 min, followed by recovery over two 5-min intervals (two-way ANOVA, N = 19). (L) Fold change in whole body endurance compared to one day post injury hang impulse score (Mixed-effects analysis, N = 16). (M) TA peak tetanic specific force for mice 7 days after an eccentric injury treadmill protocol (N = 19). All data presented as mean ± SD (*p<0.05, **p<0.01).

Figure 5—source data 1. Raw Data for Figure 5B-C, E-F, H-M, and Figure 5—figure supplement 1C, E.

Figure 5.

Figure 5—figure supplement 1. Validation of the eccentric exercise injury model.

Figure 5—figure supplement 1.

(A) Experimental design for the eccentric exercise injury model using old (21–24 months) male mice. (B) Summary of parameters for the acclimation period and injury protocol. (C) Assessment of adherence to the exercise protocol showing similar performance between mice receiving AAV-GFP and AAV-Kl (N = 24). Red symbols represent underperforming animals that were removed from further analysis (N = 2 per group). (D) Representative images in the TA showing the extent of injury induced by the exercise protocol, scale bars = 100 microns. (E) Percent of fibers with central nuclei in control vs. mice completing the treadmill protocol (N = 19, green = GFP group, blue = KL group, student’s one-tailed t-test). All data presented as mean ± SD (*p<0.05).