Increasing plasma L-kynurenine impairs mitochondrial oxidative phosphorylation prior to the development of atrophy in murine skeletal muscle: A pilot study

Abstract

Introduction: L-Kynurenine (L-Kyn), a product of tryptophan (Trp) catabolism, has been linked with impairments in walking speed, muscle strength/size, and physical function. The purpose of this pilot study was to develop a dietary model that elevates plasma L-Kyn levels in mice and characterize its impact on muscle health and function. Methods: Four-month-old C57BL6J male mice were randomized to either a L-Kyn supplemented (150 mg/kg) or chow diet for 10 weeks. Plasma L-Kyn and Trp levels were measured via mass spectrometry. Primary outcomes included assessments of muscle weights, myofiber cross-sectional area (CSA), nerve-stimulated contractile performance, and mitochondrial oxidative phosphorylation (OXPHOS) and hydrogen peroxide (H₂O₂) production. Additional experiments in cultured myotubes explored the impact of enhancing L-Kyn metabolism. Results: Mice randomized to the L-Kyn diet displayed significant increases in plasma L-Kyn levels (p = 0.0028) and the L-Kyn/Trp ratio (p = 0.011) when compared to chow fed mice. Food intake and body weights were not different between groups. There were no detectable differences in muscle weights, myofiber CSA, or contractile performance. L-Kyn fed mice displayed reductions in mitochondrial OXPHOS (p = 0.05) and maximal ADP-stimulated respiration (p = 0.0498). In cultured myotubes, overexpression of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha prevented atrophy and proteolysis, as well as deficits in mitochondrial respiration with L-Kyn treatment. Conclusion: Dietary feeding of L-Kyn increases plasma L-Kyn levels and the L-Kyn/Trp ratio in healthy male mice. Mitochondrial impairments in muscle were observed in mice with elevated L-Kyn without changes in muscle size or function. Enhancing L-Kyn metabolism can protect against these effects in culture myotubes.

Keywords: energetics; metabolism; mitochondria; physical function; weakness.

FIGURE 1

L-Kyn supplemented diet increases plasma L-Kyn levels and the L-Kyn/Trp ratio. (A)Graphical depiction of study design and outcome measures, created using Biorender.com. Weekly measures of (B) food consumption and (C) body weight over course of the intervention. (D) Plasma concentrations of L-Kyn, Trp, and the L-Kyn/Trp ratio assessed by liquid chromatography–mass spectrometry. Error bars represent SD. N = 5/group for all panels. Data in panels B and C were analyzed using a two-way ANOVA, whereas data in Panel D were analyzed using a two-tailed Student’s t-test.

FIGURE 2

Dietary increases in L-Kyn does not alter muscle weight, cross-sectional area, or contractile function. (A) Quantification of muscle weights (mean of both limbs) following careful dissection. (B) Representative images and quantification of the mean myofiber cross-sectional area of the EDL muscle. (C) Representative images and quantification of the mean myofiber cross-sectional area of the soleus muscle. (D) Force frequency curves and muscle fatiguability measures generated by nerve stimulation. Error bars represent SD. N = 5/group for all panels. Data in panels A–C were analyzed using a two-tailed Student’s t-test. Data in panel D were analyzed with a two-way ANOVA.

FIGURE 3

L-Kyn treatment impairs mitochondrial OXPHOS but does not affect H₂O₂ production in skeletal muscle. (A) Maximal ADP-stimulated respiration supported by pyruvate, malate, and octanoylcarnitine (Pyr/Ma/FA). (B) Oxygen consumption (J O ₂) as a function of the free energy for ATP hydrolysis (ΔG_ATP) controlled by a creatine kinase clamp system. (C) Quantification of OXPHOS conductance (slope of relationship between J O ₂ and ΔG_ATP). (D) Mitochondrial H₂O₂ production measured at each level of ΔG_ATP with Pyr/Mal/FA as substrates. (E) Estimated electron leak at each level of ΔG_ATP with Pyr/Mal/FA as substrates. (F) Relative mRNA levels of PGC1α in the gastrocnemius muscle. Error bars represent SD. N = 5/group for all panels. Data in panels A,C,D,E were analyzed using a two-tailed Student’s t-test. Data in panel B were analyzed with a two-way ANOVA.

FIGURE 4

Adenovirus-mediated expression of PGC1α1 enhances L-Kyn metabolism and protects myotubes for atrophy and mitochondrial deficits. (A) Quantification of mRNA levels of PGC1α in myotubes infected with adenovirus encoding a GFP (control) or PGC1α. (B) Ad-PGC1α expression increased mRNA levels of cytosolic (KAT1 and KAT3) and mitochondrial (KAT4/GOT2) kynurenine aminotransferases. (C) Enzyme activity of KAT4/GOT2 measured in mitochondrial-enriched lysates from infected myotubes. (D) Mitochondrial oxygen consumption (J O ₂) supported by pyruvate, malate, and ADP. (E) Rates myotube protein degradation (tyrosine release) with L-Kyn treatment was prevented Ad-PGC1α. (F) Quantification of myotube area (sarcomeric myosin). *p < 0.05, **p < 0.01, ***p < 0.001 using Student’s t-test in panels A–C. *p < 0.05, **p < 0.01, ****p < 0.0001 using two-way ANOVA with Tukey’s posthoc testing. N = 3-7 biologically independent C2C12 lines. Error bars in panels (A–E) represent SD. Panel F shows floating bars representing the minimum and maximum values with a line at the mean.