Experimental Cell Research. 2021 Aug 1
LanLiu, XingkeLiu, MengchangLiu, DefuXie, HongYan
Products used in the paper Details Operation
plasmid construction & lentiviral vector packaging A previously verified sequence [3](nucleotide positions 554 to 572 of PHD2 (5- CTGGGTCAGCCAGAAGAGTG-3))to construct a Plenti-PHD2shRNA vector (Packgene Biotech, LLC), using pLV.U6. shRNA.CMV.copGFP-2A-Puro.WPRE as the backbone. A negative control sequence was also designed which had no homology with rat, mouse or human. Small hairpin RNA of rat RyR2shRNA and IP3RshRNA lentivirus gene transfer vector encoding GFP were constructed by Packgene Biotech, LLC. The targeting sequence of the RyR2shRNA was 5-GCTGCTGACCAATCATTATGA-3 and the targeting sequence of the IP3RshRNA was 5- GCATTTCGTTCACTGCATAGA-3. A negative control sequence was also designed which had no homology with rat, mouse or human. Request Quote

Research Field: cardiovescular

Keywords: Proline hydroxylase domain-containing enzymes (PHD) Transient receptor potential ankyrin 1(TRPA1) Ca2+ Cardiomyocytes

Abstract

The proline hydroxylase domain-containing enzymes (PHDs) acts as cellular oxygen sensors, inducing a series of responses to hypoxia, especially during the regulation of metabolism and energy homeostasis. The increase of Ca2+ in cardiomyocytes, induced by the opening of PHD signaling pathway, is the key initiation signal necessary for the PHD-mediated regulation of the energy metabolism pathway, but the underlying molecular mechanism remains incompletely understood. This study used PHD inhibitors (PHIs) and PHD2-specific RNA interference (PHD2shRNA) to inhibit PHD signals in cardiomyocytes to explore whether transient receptor potential ankyrin 1 (TRPA1) is involved in the regulation of calcium ion influx in the PHD activation pathway associated with to AMP-activated protein kinase (AMPK). The Fluo-3AM probe was used to measure changes in free intracellular calcium ion concentrations, and Western blot analysis was used to detect the levels of phosphorylated (P)-AMPK, TRPA1, and P–Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) levels. The PHI-mediated inhibition of PHD resulted in an increase in free Ca2+ fluorescence in cardiomyocytes, which activated AMPK, TRPA1, and CaMKⅡ. The TRPA1 inhibitor HC030031, the CaMKII inhibitor KN93, and a ryanodine inhibitor (Ryanodine) were all able to inhibit the PHI-induced increase in intracellular Ca2+ and AMPK activation. Both PHIs and PHD2shRNA were able to effectively activate CaMKII and TRPA1. However, an inositol 1,4,5-triphosphate receptor (IP3R) inhibitor and the protein kinase A (PKA) inhibitor H89 did not significantly inhibit the PHI-induced increase in intracellular Ca2+ and AMPK activation. These results indicated that PHD might activate the CaMKⅡ pathway through the TRPA1 ion channel, inducing the release of calcium from the sarcoplasmic reticulum through ryanodine receptor 2 (RyR2), activating AMPK to initiate the protective effects of hypoxia in cardiomyocytes.

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