NOS isoform-specific regulation of basal but not exercise-induced mitochondrial biogenesis in mouse skeletal muscle

Abstract

Nitric oxide is a potential regulator of mitochondrial biogenesis. Therefore, we investigated if mice deficient in endothelial nitric oxide synthase (eNOS-/-) or neuronal NOS (nNOS-/-) have attenuated activation of skeletal muscle mitochondrial biogenesis in response to exercise. eNOS-/-, nNOS-/- and C57Bl/6 (CON) mice (16.3 +/- 0.2 weeks old) either remained in their cages (basal) or ran on a treadmill (16 m min(-1), 5% grade) for 60 min (n = 8 per group) and were killed 6 h after exercise. Other eNOS-/-, nNOS-/- and CON mice exercise trained for 9 days (60 min per day) and were killed 24 h after the last bout of exercise training. eNOS-/- mice had significantly higher nNOS protein and nNOS-/- mice had significantly higher eNOS protein in the EDL, but not the soleus. The basal mitochondrial biogenesis markers NRF1, NRF2alpha and mtTFA mRNA were significantly (P< 0.05) higher in the soleus and EDL of nNOS-/- mice whilst basal citrate synthase activity was higher in the soleus and basal PGC-1alpha mRNA higher in the EDL. Also, eNOS-/- mice had significantly higher basal citrate synthase activity in the soleus but not the EDL. Acute exercise increased (P< 0.05) PGC-1alpha mRNA in soleus and EDL and NRF2alpha mRNA in the EDL to a similar extent in all genotypes. In addition, short-term exercise training significantly increased cytochrome c protein in all genotypes (P< 0.05) in the EDL. In conclusion, eNOS and nNOS are differentially involved in the basal regulation of mitochondrial biogenesis in skeletal muscle but are not critical for exercise-induced increases in mitochondrial biogenesis in skeletal muscle.

Figure 1

Basal levels of nNOS (A and B) and eNOS (C and D) protein abundance in the soleus and EDL of CON and NOS knockout mice Values are means ± s.e.m. Western blots are representative from one mouse from each genotype. n = 8 for all groups except n = 7 for the EDL of eNOS^−/− mice due to lack of available sample. †P <0.05 versus CON mice (one-way ANOVA).

Figure 3

Basal levels of phosphorylated p38 MAPK (A and B) and phosphorylated AMPKα Thr¹⁷² (C and D) in the soleus and EDL of CON and NOS knockout mice Values are means ± s.e.m.n = 8 for all groups. Western blots are representative from one mouse from each genotype.

Figure 2

Basal levels of PGC-1α (A and B), NRF1 (C and D), NRF2α (E and F) and mtTFA (G and H) mRNA in the soleus and EDL of CON and NOS knockout mice Values are means ± s.e.m.n = 8 for all groups. †P <0.05 versus CON mice (one-way ANOVA).

Figure 4

PGC-1α (A and B) and NRF2α (C and D) mRNA in the soleus and EDL of CON and NOS knockout mice under basal conditions and 6 h after 60 min of treadmill running (acute ex) Values are means ± s.e.m.n = 8 for all groups. *P <0.05 main effect for exercise, †P <0.05 main effect for genotype (two-way ANOVA).

Figure 5

Cytochrome c (A and B) and COX IV (C and D) protein abundance in the soleus and EDL of CON and NOS knockout mice under basal conditions and after short-term training (trained) Values are means ± s.e.m.n = 8 for all groups. *P <0.05 main effect for exercise training, †P <0.05 main effect for genotype, #P < 0.05 versus basal (two-way ANOVA). Western blots are representative from one mouse from each genotype.