Unacylated Ghrelin Protects Against Age-Related Loss of Muscle Mass and Contractile Dysfunction in Skeletal Muscle

Abstract

Sarcopenia, the progressive loss of muscle mass and function, universally affects older adults and is closely associated with frailty and reduced quality of life. Despite the inevitable consequences of sarcopenia and its relevance to healthspan, no pharmacological therapies are currently available. Ghrelin is a gut-released hormone that increases appetite and body weight through acylation. Acylated ghrelin activates its receptor, growth hormone secretagogue receptor 1a (GHSR1a), in the brain by binding to it. Studies have demonstrated that acyl and unacylated ghrelin (UnAG) both have protective effects against acute pathological conditions independent of receptor activation. Here, we investigated the long-term effects of UnAG in age-associated muscle atrophy and contractile dysfunction in mice. Four-month-old and 18-month-old mice were subjected to either UnAG or control treatment for 10 months. UnAG did not affect food consumption or body weight. Gastrocnemius and quadriceps muscle weights were reduced by 20%-30% with age, which was partially protected against by UnAG. Specific force, force per cross-sectional area, measured in isolated extensor digitorum longus muscle was diminished by 30% in old mice; however, UnAG prevented the loss of specific force. UnAG also protected from decreases in mitochondrial respiration and increases in hydrogen peroxide generation of skeletal muscle of old mice. Results of bulk mRNA-seq analysis and our contractile function data show that UnAG reversed neuromuscular junction impairment that occurs with age. Collectively, our data revealed the direct role of UnAG in mitigating sarcopenia in mice, independent of food consumption or body weight, implicating UnAG treatment as a potential therapy against sarcopenia.

Keywords: loss of muscle mass; mitochondria; neurogenic atrophy; neuromuscular junction; protein synthesis and degradation; sarcopenia; unacylated ghrelin.

FIGURE 1

UnAG delivery via osmotic pump increased plasma UnAG levels and normalized the decreased lean body mass in old mice at the study endpoint. (a) Study design demonstrates 10 months delivery of UnAG via osmotic pump in female mice. (b) After treatment, plasma UnAG levels are elevated in adult and old mice treated with UnAG relative to control mice. (c) Body weights at baseline, at 4 and 14 months old. (d) Body weights at the post‐treatment endpoint, at 14 (adult) and 28 months of age (old). (e) Percentage lean body mass relative to body mass before and after UnAG treatment. n = 4–16. Numbers within each bar denote the number of mice included for specific groups. Two‐Way ANOVA (age × treatment) followed by Tukey post hoc analyses were used to determine statistical significance. p < 0.05. *Treatment effect. #Age effect. UnAG, unacylated ghrelin.

FIGURE 2

UnAG treatment increased muscle mass, strength and fatigue resistance in old female mice. All mice included in this analysis had been treated via osmotic pump. After UnAG or saline treatment of adult and old female mice, (a) gastrocnemius, (b) quadriceps, (c) soleus, and (d) EDL muscle masses in adult and old mice. (e) Absolute force (mN) from isolated EDL muscle. (f) Specific force, force per cross‐sectional area, using isolated EDL muscle. Two‐way ANOVA (age × treatment) followed by Tukey post hoc tests were used to determine statistical significance (a–f). (g) Relative force changes (as percentage of initial force) during a fatigue protocol. Two‐way ANOVA followed by Bonferroni post hoc analyses were used to determine statistical significance. (h) Percentage of initial force at select timepoints. *p < 0.05. Numbers within each bar denote the number of mice. EDL, extensor digitorum longus.

FIGURE 3

UnAG increased muscle quantity and quality without affecting body weights in male mice. All mice included in this analysis had been treated via drinking water. (a) Delivering UnAG via drinking water increased plasma UnAG levels in adult and old male mice. (b) Body weights of adult (14 months) and old (28 months) mice. Muscle mass was normalized to body weight (mg/g) for (c) gastrocnemius, (d) quadriceps, (e) soleus, and (f) EDL muscles. (g) Gastrocnemius muscle force generation when directly stimulated by electrode. Specific force, force per estimated cross‐sectional area. (h) Representative lumbrical muscle force (blue) and mag‐fura‐2 fluorescence ratio (red) responses to twitch excitation. Individual responses were elicited by a single stimulus pulse (0.2 ms duration) and 16 recordings were averaged for each of two excitation wavelengths (344 nm, 375 nm). The averages were used to form the mag‐fura‐2 ratio shown (344/375). Arrow indicates the time at which the stimulation was delivered. (i) Peak of the intracellular calcium transient (ICT) indicates the height of mag‐fura‐2 fluorescence ratio relative to resting level and (j) ICT FWHM (full width at half‐maximum) indicates the time during which mag‐fura‐2 fluorescence ratio remains at or above its half maximum level. (k) Representative histological images of hemotoxylin and eosin staining from gastrocnemius muscle. (l) Frequency histogram based on myofiber areas. *Represents statistical difference between adult control and old control. #Represents statistical difference between old control‐treated and old UnAG‐treated mice. n = 4–5. Two‐way ANOVA (age × treatment) followed by Tukey post hoc analyses were used to determine statistical significance. *p < 0.05. Data are mean ± SEM. Numbers within each bar denote the number of mice.

FIGURE 4

Bulk mRNAseq analysis from gastrocnemius muscle indicates downstream effects induced by UnAG. All mice included in this analysis were males that had been treated via drinking water. (a) Ingenuity pathway analysis (IPA) showing the top 7 canonical pathways differentially regulated by UnAG in old mice relative to control mice. (b) A heat map showing top 20 genes differentially regulated by old but shifted back to ‘adult’ level by UnAG. Genes are ordered by q‐values (adjusted p‐value for false discovery rate). (c) Principal component (PC) plot shows several differentially regulated genes in old mice, but UnAG normalized genes similar to adult mice. Benjamini–Hochberg multiple testing was applied to determine the differentially expressed transcripts.

FIGURE 5

Protein synthesis rate in vivo determined by deuterium oxide treatment. All mice included in this analysis were males that had been treated via drinking water. Protein synthesis rates of (a) myofibrillar protein fraction and (b) mitochondrial fraction. Numbers within each bar denote the number of mice. (c) Western blot analyses showing MuRF1, MAFbx, and mTOR expression levels in gastrocnemius homogenates. n = 4. Two‐way ANOVA (age × treatment) followed by Tukey post hoc analyses were used to determine statistical significance. n = 4. *p < 0.05.

FIGURE 6

UnAG improves mitochondrial respiration and ROS generation rates in skeletal muscle of old mice. All mice included in this analysis were males that had been treated via drinking water. (a) Mitochondrial respiration is measured using permeabilized myofibers from red gastrocnemius muscle. Substrates were added to activate individual complexes of the mitochondria. Glutamate and malate (G/M) for Complex I, succinate (Suc) for Complex II, and ascorbate/TMPD (Asc/TMPD) for Complex IV. ADP was added for state III respiration. Rotenone (Rot) was added to inhibit Complex I and antimycin A was added to inhibit mitochondrial respiration. (b) Hydrogen peroxide generation rate was simultaneously determined in the presence of Amplex UltraRed (AUR), horseradish peroxidase (HRP) and superoxide dismutase (SOD). n = 7–8. (c) Sample tracing of OCR and hydrogen peroxide generation rate data. Two‐way ANOVA (age × treatment) followed by Tukey post hoc analyses were used to determine statistical significance. *p < 0.05.

FIGURE 7

UnAG improved neuromuscular function and NMJ morphology in gastrocnemius muscle of old mice. All mice included in this analysis were males that had been treated via drinking water. (a) Specific force generation by gastrocnemius muscles of control and UnAG treated mice elicited by nerve or direct muscle stimulation. (b) Ratio of force induced by nerve and muscle stimulation. Sciatic nerve‐stimulated force is lower than muscle stimulated force (i.e., force deficit) in gastrocnemius muscle of old mice, but UnAG normalizes the force deficit. (c) Histological images of neuromuscular junction (NMJ). Green indicates acetylcholine receptors (AchR), whereas red indicates motor neuron. Yellow indicates innervation. (d) Percentage of fragmented AchR. n = 3–6. (e) Percentage of denervated NMJ. n = 3–6. (f) mRNA levels of genes elevated in response to denervation and aging. Two‐way ANOVA (age × treatment) followed by Tukey post hoc analyses were used to determine statistical significance. *p < 0.05. Numbers within each bar denote the number of mice. AchR, acetylcholine receptor; stim, stimulation.