PKM2 Determines Myofiber Hypertrophy In Vitro and Increases in Response to Resistance Exercise in Human Skeletal Muscle

Abstract

Nearly 100 years ago, Otto Warburg investigated the metabolism of growing tissues and discovered that tumors reprogram their metabolism. It is poorly understood whether and how hypertrophying muscle, another growing tissue, reprograms its metabolism too. Here, we studied pyruvate kinase muscle (PKM), which can be spliced into two isoforms (PKM1, PKM2). This is of interest, because PKM2 redirects glycolytic flux towards biosynthetic pathways, which might contribute to muscle hypertrophy too. We first investigated whether resistance exercise changes PKM isoform expression in growing human skeletal muscle and found that PKM2 abundance increases after six weeks of resistance training, whereas PKM1 decreases. Second, we determined that Pkm2 expression is higher in fast compared to slow fiber types in rat skeletal muscle. Third, by inducing hypertrophy in differentiated C2C12 cells and by selectively silencing Pkm1 and/or Pkm2 with siRNA, we found that PKM2 limits myotube growth. We conclude that PKM2 contributes to hypertrophy in C2C12 myotubes and indicates a changed metabolic environment within hypertrophying human skeletal muscle fibers. PKM2 is preferentially expressed in fast muscle fibers and may partly contribute to the increased potential for hypertrophy in fast fibers.

Keywords: Warburg effect; cancer; glycolysis; hypertrophy; insulin-like growth factor 1; metabolic reprogramming; pyruvate kinase; resistance exercise; skeletal muscle.

Figure 1

Pkm2 expression is highest in extensor digitorum longus (EDL) rat muscle that predominantly consists of type 2 fibers. (A,B) ATPase staining of (A) soleus and (B) EDL rat muscle to determine fiber types (n = 4). (C) Fiber type distribution of rat soleus and EDL muscle. (D) Pkm2 mRNA expression in rat soleus and EDL (n = 6). Black circles indicate individual data points. * Significantly different from control, unpaired t-test and Mann–Whitney U test (p < 0.05); ** (p < 0.01); *** (p < 0.001). Scale bar in A indicates 1000 µm.

Figure 2

PKM2 and PKM1 abundance in vastus lateralis during 6 weeks of resistance training. (A) During 6 weeks of resistance exercise, muscle biopsies from vastus lateralis where collected at 4 different time points (red arrows): at rest (T0), after the 1st training session (T1), after the 13th training session (T13) and the 14th training session. Protein abundance of P-P70S6K (B), PKM1 (C) and PKM2 (D) (n = 14). Protein normalized to α-Tubulin. Black circles indicate individual data points. * Significantly different between indicated conditions, repeated measures ANOVA with Bonferroni post hoc test, or Friedman’s test with Dunn’s post hoc test (p < 0.05); ** (p < 0.01); *** (p < 0.001).

Figure 3

Effect of joint knock down of Pkm1 and of Pkm2 on C2C12 myotube size. (A) Pkm1 and Pkm2 mRNA in C2C12 myotubes following 24 h IGF-1 treatment. (B) PKM2 protein abundance in IGF-1 treated C2C12 myotubes. (C,D) Pkm1 and Pkm2 mRNA expression following siRNA interference of Pkm (n = 5). (E,F) C2C12 myotube diameter (n = 6). Black circles indiciate individual data points. Scale bar is 100 μm. * Significantly different between indicated conditions, unpaired t-test, Mann–Whitney U test or two-way ANOVA with Bonferroni post hoc test (p < 0.05); ** (p < 0.01); *** (p < 0.001).

Figure 4

Effect of Pkm1 or Pkm2 knock down on C2C12 myotube size. (A,B) Pkm1 and Pkm2 mRNA expression following siRNA for Pkm1 and Pkm2 (n = 5). (C,D) C2C12 myotube diameter after siRNA treatment for Pkm1 or Pkm2 (n = 6). Scale bar is 100 μm. Black circles indicate individual data points. * Significantly different to unstimulated siControl (p < 0.05); ** (p < 0.01); *** (p < 0.001)., # significantly different to IGF-1 stimulated siControl (p < 0.05); ## (p < 0.01); ### (p < 0.001), Mann–Whitney U test or two-way ANOVA with Bonferroni post hoc test.