Early Stage Of Hypoxia Affects The AMPK Pathway And The Mechanism Involving In PHIs Activates AMPK In The Cardiacmyocytes

The energy status of the cell plays a crucial role for cell survival, and exposure of eukaryotic cells to hypoxia that accompanies the depletion of intracellular ATP triggers specific and systemic adaptive responses. AMP-activated protein kinase (AMPK) has emerged as a key regulator of energy metabolism in the heart and plays a critical role in inducing these responses. Once AMPK is activated, it activates not only a number of energy producing metabolic pathways, but also inhibits energy consuming pathways. This has resulted in AMPK being coined a'fuel gauge'in the cell, a'low fuel warning system'or a'master switch'for cellular energy levels. This role of AMPK as a fuel gauge is particularly relevant in the heart, which has a very high energy demand and very little energy reserves. Therefore, it stands to reason that AMPK should have a very important role as a'fuel gauge'in the heart. AMPK is allosterically activated by increases in the ratios of AMP/ATP, which increases under conditions of cellular stress or energy deficiency, such as hypoxia/ischemia. AMPK is also activated by phosphorylation of a conserved threonine in the activation loop (Thr172) by upstream kinases.The cellular oxygen sensor is a family of oxygen-dependent proline hydroxylase domain (PHD)-containing enzymes, whose reduction of activity initiate a hypoxic signal cascade. In many studies, prolyl hydroxylase inhibitors (PHIs) were used to activate the PHD-signaling pathway in cardiomyocytes and PHI-pretreatment led to the accumulation of glycogen and an increased maintenance of ATP levels under metabolic stress.Therefore, we postulated that: At the very early stage of hypoxia AMPK is activated ahead of the increase of AMP/ATP in cardiacmyoctes, and PHDs might be the transducer who senses the hypoxia signal and activate AMPK via AMPKK pathway.The objectives of the present study were 1) to determine whether cardiac AMPK activity increases before the intracellular AMP/ATP ratio increase at the very early stage of hypoxia, and the effect of hypoxia on AMPKK activity. 2) to determine whether PHIs can activate AMPK via AMPKK pathway, and the effects of PHIs pretreatment on the intracellular ATP content and cell viability under hypoxic condition. 3) to investigate the mechanisms responsible for the activation of AMPK by PHIs treatments.These data would provide some new clues to develop clinical approaches for treatment of hypoxia/ischemia induced injury.Material and MethodsMethods: all procedure were carried out on the Neonatal rat ventricular myocytes in vitro. The cultured cardiacmyoctyes were incubated under aerobic condition and anoxic condition for different time course. Intracellular AMP and ATP contents were determined by high performance liquid chromatography. AMPK activity was assessed by SAMS peptide phosphorylation assay. AMPK and ACC phophorylation were determined by western blot at 5 and 15min incubated with recombinantα312 for 60 min. Immunblot and HPLC assessments were used to elucidate the effects of PHIs on AMPK activation and intracellular high energy phosphate in cariacmyoctes. HPLC and CCK-8 assessments were adopted to determine the effects of PHIs pretreatments on saving intracellular ATP content and cell viability of cardiacmyocytes under hypoxia 12h condition. To elucidate the mechanisms responsible for activation of AMPK by PHIs pretreatments, we used laser scanning confocal fluorescence microscope and specific inhibitor including L-NAME, 5-HD, NAC to observe intracellular ROS and Ca2+ changes. We used the same inhibitors on the immunblot for AMPK and ACC phosphorylaiton assessments as well. To determine whether Ca2+ /calmodulin—CaMKK pathway exist in the mechanism of PHI—AMPK, we used immunbolt to examine the effects of BAPTA-AM, STO-609, ionomycin pretreatments on AMPK and ACC phosphorylaitons induced by PHIs, and used co-immunoprecipitation to determine the binding avidity between AMPK and CaMKK which exist in the myocytes treated by PHIs.Results and Conclusion:1. In the early stage of hypoxia, AMPK activity increase is ahead of intracellular AMP concentration ([AMP]) or AMP/ATP ratio increase in cardiacmyocytes, and AMPK is activated mainly due to phosphorylation at its residue Thr-172 by its upstream kinase AMPKK. 2. PHIs treatments led to significant increase of AMPK activity and AMPK phosphorylation in cardiacmyocytes without obvious metabolic disturbance. PHIs pretreatment enhanced the cardiacmyocytes's ability to endure hypoxic injury including increase intracellular ATP content and cell viability. Administration of AMPK specific inhibitor compound C could diminish these effects of PHIs , hence demonstrateing that the PHI-AMPK pathway plays a crucial role in maintaining cell viability and increase intracellular ATP content under anoxic condition.3. EDHB stimulated intracellular mitoROS release through the NO-MtioKATP channel pathway, but suppressed intracellular ROS/mitoROS release by using NAC, L-NAME and 5-HD did not block the PHI-AMPK pathway, indicating that ROS has nothing to do with the PHI-AMPK pathway.4. As indicated by the results of laser scanning confocal fluorescence microscopy, both EDHB and DMOG administration elevated intracellular Calcium (Ca2+). Eliminating intracellular Ca2+ by BAPTA-AM significantly blocked AMPK and ACC phosphorylation , while elevating intracellular Ca2+by ionomycin caused AMPK phosphorylation. The binding avidity between AMPK and CaMKKαincreased significantly after PHIs treatment, as identified by the co-immunoprecipitation assay, imply PHIs treatments led to much more interactions between AMPK and CaMKK. Pretreatment with STO-609 (a specific inhibitor of CaMKK) markedly diminished the AMPK and ACC phosphorylation induced by PHIs treatment.These results demonstrate that the Ca2+--CaMKK mediates AMPK activation upon PHIs stimulation, and CaMKK might be the major AMPK kinase under these conditions.