PHD2/3-dependent hydroxylation tunes cardiac response to β-adrenergic stress via phospholamban
Liang Xie, … , Gerhard Meissner, Cam Patterson
Liang Xie, … , Gerhard Meissner, Cam Patterson
Published June 15, 2015
Citation Information: J Clin Invest. 2015;125(7):2759-2771. https://doi.org/10.1172/JCI80369.
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Research Article Cardiology

PHD2/3-dependent hydroxylation tunes cardiac response to β-adrenergic stress via phospholamban

Abstract

Ischemic heart disease is the leading cause of heart failure. Both clinical trials and experimental animal studies demonstrate that chronic hypoxia can induce contractile dysfunction even before substantial ventricular damage, implicating a direct role of oxygen in the regulation of cardiac contractile function. Prolyl hydroxylase domain (PHD) proteins are well recognized as oxygen sensors and mediate a wide variety of cellular events by hydroxylating a growing list of protein substrates. Both PHD2 and PHD3 are highly expressed in the heart, yet their functional roles in modulating contractile function remain incompletely understood. Here, we report that combined deletion of Phd2 and Phd3 dramatically decreased expression of phospholamban (PLN), resulted in sustained activation of calcium/calmodulin-activated kinase II (CaMKII), and sensitized mice to chronic β-adrenergic stress–induced myocardial injury. We have provided evidence that thyroid hormone receptor-α (TR-α), a transcriptional regulator of PLN, interacts with PHD2 and PHD3 and is hydroxylated at 2 proline residues. Inhibition of PHDs increased the interaction between TR-α and nuclear receptor corepressor 2 (NCOR2) and suppressed Pln transcription. Together, these observations provide mechanistic insight into how oxygen directly modulates cardiac contractility and suggest that cardiac function could be modulated therapeutically by tuning PHD enzymatic activity.

Authors

Liang Xie, Xinchun Pi, W.H. Davin Townley-Tilson, Na Li, Xander H.T. Wehrens, Mark L. Entman, George E. Taffet, Ashutosh Mishra, Junmin Peng, Jonathan C. Schisler, Gerhard Meissner, Cam Patterson

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Figure 7

TR-α is hydroxylated at proline residues P160 and P162.

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TR-α is hydroxylated at proline residues P160 and P162. (A) HL-1 cells w...
(A) HL-1 cells were transfected with constructs expressing TR-α or TR-β, together with constructs expressing Flag-PHD1, -2, or -3, as indicated. Co-IP was performed with anti-Flag resin, and Western blot experiments were performed with the indicated antibodies. Both PHD2 and PHD3, but not PHD1, were able to pull down TR-α or TR-β. Representative blots from 3 experiments are shown. (B) An in vitro hydroxylation assay was performed with Flag–TR-α and PHD2/3. The protein band corresponding to TR-α was cut out for trypsin digestion. LC-MS/MS analysis was then performed. Tandem mass spectra of the precursor ion at m/z = 962.48 (Z = 3) for the human TR-α 153-176 sequence SLQQRPEP(+15.99)TP(+15.99)EEWDLIHIATEAHR are shown. The peak heights are the relative abundances of the corresponding fragment ions, with annotation of the identified matched N terminus–containing ions (b ions) shown in blue and C terminus–containing ions (y ions) shown in red. For clarity, only the major identified peaks are labeled (a complete table of fragment ions is presented in Supplemental Figure 3). Fragment ions at m/z = 952.66 (b8) and m/z = 1024.01 (y17)2+ represent characteristic ions that unambiguously identified P160–P162 double hydroxylation.