Myogenic marker expression as a function of age and exercise-based therapy in the tongue

Abstract

Degeneration of tongue muscles with aging may contribute to swallowing deficits observed in elderly people. However, the capacity for tongue muscle stem cells (SCs) to regenerate and repair the aged tongue and improve tongue strength following tongue exercise (a current clinical treatment) has never been examined. We found that the expression of regenerative, myogenic markers were impaired with age and may be related to increased expression of senescent marker p16^INK4a. Tongue strength increased in young adult and old rats following exercise and was related to the expression of Pax7, MyoD, myogenin, and p16^INK4a. Our study also suggests that strengthening of tongue muscles via clinical rehabilitation strategies also increased the expression of SC regenerative markers in the tongue throughout the exercise duration.

Keywords: Aging; Dysphagia; Exercise; Muscle stem cells; Swallowing.

Fig. 1.

Schematic of tongue exercise operandum.

Fig. 2.

Regenerative marker expression of aged GG is reduced and related to increased p16^INK4a expression. A Illustration of the tongue muscles. B, C In GG, Pax7 gene (F_1,11 = 5.633, p = 0.037) and protein (F_1,36 = 5.154, p = 0.029) expression was reduced with age. D, E p16^INK4a gene expression was increased in old GG (F_1,11 = 8.704, p = 0.013). F, G In the GG, cell-intrinsic expression of p16^INK4a increased in aged SCs (F_2,36 = 5.051, p = 0.012). (*p-value < 0.05; sample size indicated in individual bar-plots.)

Fig. 3.

MVTF increased following tongue exercise. A Schematic of exercise timeline. B MVTF increased following tongue exercise at all time points (F_1.462,54.81 = 131.9, p < 0.001). C With increasing age, MVTF was more variable and approached significance (F_1,2 = 16.80, p = 0.055; dotted line). Over time, MVTF became less variable and approached significance (F_2,2 = 16.05, p = 0.059, dotted line). (***HSD p < 0.0001.)

Fig. 4.

SC differentiation increased following tongue exercise. A, B Myogenin protein expression in SG & HG muscles increased following exercise at all time points (F_1,36 = 8.798, p = 0.005; 3-way ANOVA). c Following 17 days of exercise in the IT, the percentage of Pax7⁺ SCs isolated from the young adult exercise group increased (p = 0.003), and decreased in the old exercise group (p = 0.01; interaction on 3-way ANOVA, F_2,36 = 4.441, p = 0.019). (*p-value < 0.05; sample size indicated in individual bar-plots.)

Fig. 5.

SC location changes according to myofiber type. A, N Schematic of tissue cross section. B SC content increased following 17 days of exercise in the young adult group (p = 0.005) and was greater in the old no exercise group (p = 0.021; 2-way ANOVA, F_1,20 = 12.47, p = 0.002). E A significant interaction effect was observed for the percentage of Pax7⁺ myonuclei colocalizing around MyHC type IIa myofibers (F_1,20 = 6.092, p = 0.0227) in the GG. The no exercise, young adult and old groups had a significantly greater percentage Pax7⁺ SCs near MyHC IIa myofibers in comparison to the young adult exercise (p = 0.018 [young, no exercise]; p < 0.001 [old, no exercise]) and the old exercise (p < 0.001 [young, no exercise]; p < 0.001 [old, no exercise]) groups. The percentage of Pax7⁺ nuclei colocalizing near vessels and/or nerves in the GG muscle also increased following 17 days of tongue exercise (F_1,20 = 7.631, p = 0.012). H Pax7 content increased following 17 days of exercise group in the SG muscle (F_1,20 = 6.889, p = 0.016). I An increase in the percentage of Pax7⁺ myonuclei colocalizing near MyHC type IIb and IIx was observed following 17 days of exercise (F_1,20 = 8.692, p = 0.008). J While a reduction in the percentage of Pax7⁺ myonuclei colocalizing around MyHC type IIb, IIx, and IIa was observed following exercise (F_1,20 = 6.901, p = 0.016). M A significant interaction effect was observed for the percentage of Pax7⁺ nuclei colocalizing near vessels and/or nerves in the SG (F_1,20 = 4.971, p = 0.037). (*p-value < 0.05; sample size indicated in individual bar-plots.)

Fig. 6.

Centralized myofiber nuclei increased with age and following tongue exercise. A Schematic of tissue cross section. B, C Percentage of centralized nuclei increased in the old group in GG (F_1,20 = 7.204, p = 0.014) and SG (F_1,20 = 8.178, p = 0.001). D Following 17 days of exercise, percentage of centralized nuclei increased in GG (p = 0.033). F Representative images from the GG of a young adult and old rat. (*p-value < 0.05; sample size indicated in individual bar-plots.)

Fig. 7.

SC marker expression in tongue is predictive of MVTF. A A strong, significant relationship was observed on multiple linear regression between MVTF and SC protein markers (R² = 0.953; F_14,9 = 12.96, p < 0.001) in the tongue muscles. Specifically, time (p < 0.001), and MyoD (p = 0.019), myogenin (p = 0.030), and p16^INK4a (p = 0.036) protein expression in tongue muscles were significant predictors of MVTF following exercise. B A strong, significant relationship in the SG and HG was observed between MVTF and SC markers (R² = 0.877; F_6,17 = 20.16, p < 0.001). Time (p < 0.001), and Pax7 (p = 0.048), MyoD (p = 0.013), myogenin (p = 0.010), and p16^INK4a (p = 0.003) protein expression were significant predictors of MVTF. C, D Moderate, significant relationship was observed in the GG (R² = 0.673; F_6,17 = 5.818, p = 0.002) and IT (R² = 0.763; F_6,17 = 9.098, p < 0.001).

Fig. 8.

Tongue SCs have a high regenerative turnover across time. A In GG (F_2,36 = 39.13, p < 0.001) at d 17, and in IT (F_2,36 = 9.105, p < 0.001) at d 59, Pax7 protein expression was upregulated in whole muscle. B However at d 17, the percentage of Pax7⁺ SCs isolated from SG and HG (F_2,36 = 37.52, p < 0.001), and IT muscles was reduced (F_2,36 = 14.22, p < 0.0001). D, E At d 17 in the IT, expression of p16^INK4a in whole muscle (F_2,36 = 11.96, p = 0.0001) and in Pax7⁺ SCs was increased (F_2,36 = 11.12, p = 0.0002). At d 17, expression of p16^INK4a in Pax7⁺ SCs was also significantly increased in the SG and HG (F_2,36 = 3.50, p = 0.041), and IT muscles (F_2,36 = 11.12, p = 0.002). G Expression of the MyoD protein was reduced at d 59 in SG and HG muscles (F_2,36 = 9.21, p = 0.0006), and elevated at d 3 in the IT (F_2,36 = 18.72, p < 0.0001.) H Myogenin protein expression was reduced in the IT at d 3 (F_2,36 = 12.66, p < 0.0001). C, F Representative ICC images from tongue muscles. (*p-value < 0.05; **p-value < 0.001; ***p-value < 0.0001; sample size indicated in individual bar-plots).

Fig. 9.

SC marker profiles are differentially expressed among tongue and limb muscles. A Pax7 protein expression was greatest in the GG (F_3,24 = 7.888, p < 0.0001). B MyoD protein expression was upregulated in the SG and HG of the young adult group (F_3,24 = 4.718, p = 0.0003). C Myogenin protein expression was upregulated in the GG (F_3,24 = 31.18, p < 0.0001 [Young Adult], p=0.0004 [Old]). D p16^INK4a protein expression was greatest in GG (F_3,24 = 8.305, p =0.0006). E Representative images from young adult and old tongue (GG) and limb (EDL) muscles. (*p-value < 0.05; **p-value < 0.001; ***p-value < 0.0001; sample size indicated below individual bar-plots).