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

Abstract

BackgroundHypoxia causes biological changes that are mediated by the hypoxia inducible factor‐α (HIF‐α), a key transcription factor for various compensatory responses under hypoxic condition. Also, HIF‐1α triggers the expression of slow myosin heavy chain under hypoxia, however, the downstream pathway of HIF‐1α leading to fiber‐type shift has not been elucidated. The calcineurin/NFATc1 signaling pathway is one of the pathways known as 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 2 (PHD2) 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.MethodWe employed Phd2 tamoxifen driven Cre‐loxp system to generate a mouse in which Phd2 gene will be deleted upon activation of calcium‐dependent recombinase (Cre). Tamoxifen administration for consecutive 7 days was performed to delete Phd2 gene. FK‐506 was administered at 4 weeks after tamoxifen treatment for 7 consecutive days to suppress the calcineurin activity. All muscles were isolated at 6 week after tamoxifen administration. The proportion of slow fiber and capillary density were measured by immunostaining using frozen muscle section.ResultsPHD2 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 fiber phenotype toward the type I in both of 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). The localization of increased 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 FK‐506, a calcineurin inhibitor, successfully suppressed slow fiber‐type formation in PHD2‐deficient mice.ConclusionThe stabilization of HIF‐1α induced by PHD2 conditional knockout elicited the transition of muscle fibers toward 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.