Circulating protein synthesis rates reveal skeletal muscle proteome dynamics

Abstract

Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders.

Figure 7. Plasma CK-M and CA-3 synthesis rates provide a virtual biopsy of skeletal muscle protein synthesis.

(A) Significant correlation (Pearson correlation r > 0.7, P < 0.0005, n = 16–17) between FSR (% week^–1) of CK-M measured in the plasma and FSRs of myofibrillar proteins measured in skeletal muscle of the same subject. (B) Significant correlation (Pearson correlation r > 0.7, P < 0.0005, n = 16–17) between FSR of CA-3 measured in plasma and FSRs of myofibrillar proteins measured in skeletal muscle of the same subject.

Figure 6. Muscle CK-M and CA-3 synthesis correlate with skeletal muscle protein synthesis.

(A) Significant correlation (Pearson correlation r > 0.8, P < 0.0001, n = 16–17) between FSR (% week^–1) of CK-M and FSRs of myofibrillar proteins, both measured in skeletal muscle of the same subject. (B) Significant correlation (Pearson correlation r > 0.8, P < 0.0001, n = 16–17) between FSR of CA-3 measured in plasma and FSRs of myofibrillar proteins measured in skeletal muscle of the same subject.

Figure 5. CK-M and CA-3 synthesis in human plasma and skeletal muscle.

(A) The f (%) of CK-M in 4 subjects after continuous ²H₂O labeling for several weeks. (B) Significant correlation (Pearson correlation r = 0.89, P < 0.0001, n = 17) between FSR (% week^–1) of CK-M measured in the plasma and in the skeletal muscle of the same subject. (C) Significant correlation (Pearson correlation r = 0.88, P < 0.0001, n = 17) between FSR of CA-3 measured in the plasma and in the skeletal muscle of the same subject.

Figure 4. Human skeletal muscle proteome dynamics in sedentary and SIT subjects.

(A) Heatmap of FSRs (% week^–1) of 139 proteins measured in muscle of 5 sedentary subjects and 6 subjects who underwent SIT. Each horizontal line represents an individual protein. (B) Mean FSR of proteins in DAVID gene ontology term, biological processes level 5, that were significantly different as a group (P < 0.05 in paired t tests for proteins with Benjamini-Hochberg multiple test corrections) in sedentary (n = 3–5) or sprint (n = 4–6) subjects. Black lines reflect proteins whose FSRs were significantly different (uncorrected P < 0.05, unpaired t tests between subjects in the 2 groups) within the ontologies. For glucose metabolic process: phosphoglycerate mutase 2; fructose-1,6-bisphosphatase isozyme 2; pyruvate kinase isozymes M1/M2; malate dehydrogenase cytoplasmic; triosephosphate isomerase; fructose-bisphosphate aldolase A; and phosphoglycerate kinase 1. For striated muscle contraction: Phosphoglycerate mutase 2, Fructose-bisphosphate aldolase A, and myosin light chain 1/3 skeletal muscle isoform. For regulation of apoptosis: heat shock 70 kDa protein 1A/1B. (C) FSRs of 20 proteins that were significantly different between sedentary (n = 5) and sprint (n = 6) subjects (unpaired t tests between the 2 groups with Benjamini-Hochberg multiple test corrections). For each protein, the bar represents mean, minimum, and maximum FSR; symbols within bars represent individual FSR values.

Figure 3. Effects of clenbuterol treatment on f of CA-3 in rat muscles and plasma.

(A) CA-3 f (%) in gastrocnemius muscle of rats treated with clenbuterol for 3, 7, or 14 days (mean ± SD, n = 4/group, *P < 0.05 clenbuterol vs. vehicle, 2-way ANOVA). (B) CA-3 f in quadriceps muscle of rats treated with clenbuterol for 3, 7, or 14 days (mean ± SD, n = 4/group, *P < 0.05 clenbuterol vs. vehicle, 2-way ANOVA). (C) CA-3 f in plasma of rats treated with clenbuterol for 3, 7, or 14 days (mean ± SD, n = 4/group, *P < 0.05 clenbuterol vs. vehicle, 2-way ANOVA). (D) Significant correlation (Pearson correlation r > 0.9, P < 0.001) between fractional syntheses of plasma CA-3 compared with that of gastrocnemius CA-3. (E) Significant correlation (Pearson correlation r > 0.88, P < 0.001) between fractional syntheses of plasma CA-3 compared with that of quadriceps CA-3.

Figure 2. Effects of clenbuterol treatment on f of CK-M in rat muscles and plasma.

(A) CK-M f (%) in gastrocnemius muscle of rats treated with clenbuterol for 3, 7, or 14 days (mean ± SD, n = 4/group, *P < 0.05 clenbuterol vs. vehicle, 2-way ANOVA). (B) CK-M f in quadriceps muscle of rats treated with clenbuterol for 3, 7, or 14 days (mean ± SD, n = 4/group, *P < 0.05 clenbuterol vs. vehicle, 2-way ANOVA). (C) CK-M f in plasma of rats treated with clenbuterol for 3, 7, or 14 days (mean ± SD, n = 4/group, *P < 0.05 clenbuterol vs. vehicle, 2-way ANOVA). (D) Significant correlation (Pearson correlation r > 0.95, P < 0.001) between fractional syntheses of plasma CK-M compared with that of gastrocnemius CK-M. (E) Significant correlation (Pearson correlation r > 0.95, P < 0.001) between fractional syntheses of plasma CK-M compared with that of quadriceps CK-M.

Figure 1. Proteome dynamics in rat gastrocnemius muscle.

(A) Heatmap of FSRs (% day^–1) of 75 proteins in rat muscle measured in n = 3 rats per group after denervation and clenbuterol treatment, with each horizontal line representing z-scaled FSR of an individual protein. (B) Effect of denervation surgery and clenbuterol treatment on FSR of CK-M, mean ± SD, n = 3–4/group. *P < 0.05 ‘Den-Veh’ vs. ‘Con-Veh’, ^#P < 0.05 ‘Con-Clen’ vs. ‘Con/Veh’, †P < 0.05 ‘Den-Clen’ vs. ‘Den-Veh’, 2-way ANOVA. (C) Effect of denervation surgery and clenbuterol treatment on FSR of CA-3, mean ± SD, n = 3–4/group. *P < 0.05 ‘Den-Veh’ vs. ‘Con-Veh’, ^#P < 0.05 ‘Con-Clen’ vs. ‘Con-Veh’, 2-way ANOVA. (D–F) Effect of denervation surgery and clenbuterol treatment on the FSRs of myofibril, glycolytic, and mitochondrial proteins. Names are listed in the order they appear on the scatter plots for each class of proteins. Data represent mean ± SD, n = 3–4/group, * corrected P < 0.05 ‘Den-Veh’ vs. ‘Con-Veh’, ^# corrected P < 0.05 ‘Con-Clen’ vs. ‘Con/Veh’, † corrected P < 0.05 ‘Den-Clen’ vs ‘Den-Veh’, 2-way ANOVA with Holm-Sidak correction. Con, control; Veh, vehicle; Clen, clenbuterol; Den, denervation.