Prolyl hydroxylase domain 2 deficiency promotes skeletal muscle fiber-type transition via a calcineurin/NFATc1-dependent pathway

Abstract

Background: Hypoxia exposure is known to induce an alteration in skeletal muscle fiber-type distribution mediated by hypoxia-inducible factor (HIF)-α. The downstream pathway of HIF-α leading to fiber-type shift, however, has not been elucidated. The calcineurin pathway is one of the pathways responsible for slow muscle fiber transition. Because calcineurin pathway is activated by vascular endothelial growth factor (VEGF), one of the factors induced by HIF-1α, we hypothesized that the stabilization of HIF-1α may lead to slow muscle fiber transition via the activation of calcineurin pathway in skeletal muscles. To induce HIF-1α stabilization, we used a loss of function strategy to abrogate Prolyl hydroxylase domain protein (PHD) 2 responsible for HIF-1α hydroxylation making HIF-1α susceptible to ubiquitin dependent degradation by proteasome. The purpose of this study was therefore to examine the effect of HIF-1α stabilization in PHD2 conditional knockout mouse on skeletal muscle fiber-type transition and to elucidate the involvement of calcineurin pathway on muscle fiber-type transition.

Results: PHD2 deficiency resulted in an increased capillary density in skeletal muscles due to the induction of vascular endothelial growth factor. It also elicited an alteration of skeletal muscle phenotype toward the type I fibers in both of the soleus (35.8 % in the control mice vs. 46.7 % in the PHD2-deficient mice, p < 0.01) and the gastrocnemius muscle (0.94 vs. 1.89 %, p < 0.01), and the increased proportion of type I fibers appeared to correspond to the area of increased capillary density. In addition, calcineurin and nuclear factor of activated T cell (NFATc1) protein levels were increased in both the gastrocnemius and soleus muscles, suggesting that the calcineurin/NFATc1 pathway was responsible for the type I fiber transition regardless of PGC-1α, which responded minimally to PHD2 deficiency. Indeed, we found that tacrolimus (FK-506), a calcineurin inhibitor, successfully suppressed slow fiber-type formation in PHD2-deficient mice.

Conclusions: Taken together, stabilized HIF-1α induced by PHD2 conditional knockout resulted in the transition of muscle fibers toward a slow fiber type via a calcineurin/NFATc1 signaling pathway. PHD2 conditional knockout mice may serve as a model for chronic HIF-1α stabilization as in mice exposed to low oxygen concentration.

Keywords: Calcineurin; Hypoxia-inducible factor α; NFATc1; Prolyl hydroxylate domain protein 2; Type I muscle fiber.

Fig. 1

Tamoxifen administration-induced PHD2 deletion in skeletal muscle in Phd2 ^f/f /Rosa26 ^CreERT2 mice. a . PHD1, 2, and 3 deletion efficiency in the gastrocnemius muscles of tamoxifen-treated Phd2 ^f/f /Rosa26 ^CreERT2 mice was determined using qRT-PCR. Relative gene expression was determined using gastrocnemius muscle tissue cDNA (n = 3–4 mice per group). b . Anti-PHD2 and PHD3 Western blotting of gastrocnemius and soleus muscles at 6 weeks after tamoxifen administration. c . The expression of HIF-1α in gastrocnemius and soleus at 5 weeks after tamoxifen administration. d. The level of hif-1α mRNA in gastrocnemius at 5 weeks after tamoxifen administration. *p < 0.05; **p < 0.01 compared to control. Values are means ± SEM

Fig. 2

Changes in blood profile and the altered phenotype of skeletal muscle. Changes in blood profile at 6 weeks after tamoxifen administration. a Red blood cell count. b Hemoglobin level. c Hematocrit value (n = 10 per group). d Appearance of skeletal muscles, including the soleus and gastrocnemius, in Phd2^f/f and Phd2 cKO mice. **p < 0.01 compared to control. Values are means ± SEM

Fig. 3

Phd2 deletion induces angiogenesis in skeletal muscle. a The VEGF level in skeletal muscle was measured by ELISA (n = 3 per group). b Relative gene expression was measured using gastrocnemius muscle tissue cDNA (n = 3 per group). c, d The capillary density in skeletal muscles was determined by detecting CD31-positive cells using immunostaining (n = 5 per group). *p < 0.05, compared to control. Values are means ± SEM (scale bar = 100 μm)

Fig. 4

PHD2 deficiency induces slow muscle fiber type switching in the soleus and gastrocnemius muscles. a The composition of muscle fiber type was analyzed by immunostaining. Frozen sections of the soleus and gastrocnemius muscle at 6 weeks after tamoxifen treatment were stained with antibodies for MyHC I/slow (green) and MyHC IIa/fast (green) and counterstained for laminin (red). b-c The proportion of MyHC I and MyHC IIa muscle fibers was measured using ImageJ software (n = 5 per group). d-eWestern blotting confirmed the MyHC I and MyHC IIa protein levels in the soleus and gastrocnemius muscles of Phd2^f/f and Phd2 cKO mice (n = 4 per group). **p < 0.01 compared to control. Values are means ± SEM (scale bar = 100 μm)

Fig. 5

The time-dependent increase of the slow muscle fiber type and the distribution of muscle fiber types. a The increase in the slow muscle fiber type was analyzed in a time-dependent manner (n = 5 per group). b-c. The distribution of muscle fiber types in the soleus and gastrocnemius muscles at 6 weeks after tamoxifen administration (soleus n = 5, gastrocnemius n = 4 per group). *p < 0.05; **p < 0.01 compared to control. Values are means ± SEM

Fig. 6

Expression of calcineurin, PGC-1α and myoglobin, and calcineurin in soleus and gastrocnemius muscles. a Western blotting analysis of protein lysates from the gastrocnemius and soleus for PGC-1α and myoglobin. b Calcineurin protein levels were measured using Western blotting in the gastrocnemius at 5 weeks after tamoxifen administration (n = 3 per group)

Fig. 7

NFATc1 nuclear translocation in soleus and gastrocnemius muscles. a–b Western blotting analysis for the purity of cytoplasm/nuclear fractions in the soleus and gastrocnemius muscles at 4 weeks after tamoxifen administration (n = 3 per group). *p < 0.05; **p < 0.01 compared to control. Values are means ± SEM. c Immunostaining showed nuclei stained with NFATc1 in gastrocnemius. d The number of nuclei stained with NFATc1 (n = 4 per group). *p < 0.05, compared to control. Values are means ± SEM (magnification ×60, scale bar = 50 μm)

Fig. 8

The effect of FK-506 treatment on the myosin heavy chain (MyHC) composition in soleus muscle of Phd2-deficient mice. a The composition of muscle fiber type was analyzed by immunostaining after the treatment of FK-506. Frozen sections of the soleus muscle taken at 6 weeks after tamoxifen treatment were stained with antibodies for myosin heavy chain (MyHC) I/slow (green) and counterstained for laminin (red) (scale bar = 100 μm). b-c The treatment of FK-506 for 7 days at 4 week after tamoxifen administration suppressed the increase in the proportion of slow muscle fiber type in both soleus and gastrocnemius muscles (n = 4 per group). d-e The capillary density in soleus and gastrocnemius muscles was determined by detecting CD31-positive cells after FK-506 treatment using immunostaining (n = 5 per group). *p < 0.05; **p < 0.01 compared to control. Values are means ± SEM