Hypoxia modulates protein phosphatase 2A through HIF‐1α dependent and independent mechanisms in human aortic smooth muscle cells and ventricular cardiomyocytes

Abstract

Background and Purpose: Although protein phosphatases regulate multiple cellular functions, their modulation under hypoxia remains unclear. We investigated expression of the protein phosphatase system under normoxic/hypoxic conditions and the mechanism by which hypoxia alters protein phosphatase 2A (PP2A) activity. Experimental Approach: Human cardiovascular cells were cultured in cell type specific media under normoxic or hypoxic conditions (1% O 2). Effects on mRNA expression, phosphatase activity, post-translational modification, and involvement of hypoxia inducible factor 1α (HIF-1α) were assessed using RT-PCR, immunoblotting, an activity assay, and siRNA silencing. Key Results: All components of the protein phosphatase system studied were expressed in each cell line. Hypoxia attenuated mRNA expression of the transcripts in a cell line-and time-dependent manner. In human aortic smooth muscle cells (HASMC) and AC16 cells, hypoxia decreased PP2Ac activity and mRNA expression without altering PP2Ac abundance. Hypoxia increased demethylated PP2Ac (DPP2Ac) and phosphatase methylesterase 1 (PME-1) abundance but decreased leucine carboxyl methyltransferase 1 (LCMT-1) abundance. HIF-1α siRNA prevented the hypoxia-mediated decrease in phosphatase activity and expression of the catalytic subunit of protein phosphatase 2A (PPP2CA), independently of altering pPP2Ac, DPP2Ac, LCMT-1, or PME-1 abundance. Conclusion and Implications: Cardiovascular cells express multiple components of the PP2A system. In HASMC and AC16 cells, hypoxia inhibits PP2A activity through HIF-1α-dependent and-independent mechanisms, with the latter being consistent with altered PP2A holoenzyme assembly. This indicates a complex inhibitory effect of hypoxia on the PP2A system, and highlights PP2A as a therapeutic target for diseases associated with dysregulated protein phosphorylation.

Mechanisms of Systolic Cardiac Dysfunction in PP2A, PP5 and PP2AxPP5 Double Transgenic Mice

“…Likewise, after five days of coronary occlusion in dogs, the expression of PP2Ac was increased in the ischemic cardiac area [2]. At least in AC16 cells (an immortalized cell line of ventricular cardiomyocytes), prolonged hypoxia can lead to decreased expression of PP2Ac and PP2A enzyme activity [69]. In the present work, we addressed this issue by another approach: we genetically increased PP2Ac expression and then asked how this would alter any tolerance for hypoxia.…”

Enhanced BDNF Actions Following Acute Hypoxia Facilitate HIF-1α-Dependent Upregulation of Cav3-T-Type Ca2+ Channels in Rat Cardiomyocytes

“…The results were also confirmed by immunocytochemistry analysis ( Figure 3 and Figure 6 ). Because hypoxia modulates protein expression through HIF-dependent and -independent mechanism in many cell types associated with the duration and the intensity of hypoxia [ 21 , 22 ], it is plausible that the short- and long-duration of hypoxia exert distinct actions on protein expressions with distinct contributions of HIF activity. Since knock-down of Hif1a completely abolished the actions of acute hypoxia on Cav3.1 and Cav3.2 in this study ( Figure 2 B,C), we would like to conclude that acute hypoxia up to 3 h induces upregulation of the Cav3-Ca 2+ channels dependently on the HIF-1α actions.…”

Selection of safe artemisinin derivatives using a machine learning-based cardiotoxicity platform and in vitro and in vivo validation

“…To verify the cardiotoxicity of the selected compounds further, the cardiomyocyte cell line AC16 was used, which was kindly provided by Dr. James Spiers (Trinity College Dublin, Department of Pharmacology and Therapeutics). It was cultured as previously described (Elgenaidi and Spiers 2019 ). The cytotoxicity of the selected compounds (provided by one of the authors, E.F.) and of doxorubicin (provided by University of Mainz Clinical Pharmacy as positive control drug) towards AC16 cardiomyocytes was evaluated by the resazurin assay with three independent experiments per compounds and each 6 parallel measurements per experiment as previously described (Ooko et al 2016 ).…”