Vehicle or Digoxin treatment 1mg/kg IP dosing at ZT5 and sacrifice at ZT9 [mouse]

Myocardial ischemia-reperfusion injury (MIRI) is a major threat to heart functional integrity and pharmacological means to achieve cardioprotection are sorely needed. The sequential hypoxic/normoxic status of the cardiac tissue triggers life-threatening damages through the activation of multiple intra-cellular pathways. Heart tolerance to MIRI varies according to a day-night cycle and is regulated by components of the molecular clock such as the transcriptional repressor and nuclear receptor REV-ERBα. Timed REV-ERBα antagonism alleviates sensitivity to myocardial infarction in mice. Here we show that timed administration of digoxin is cardioprotective by triggering REV-ERBα protein degradation and involves the anti-apoptotic factor p21. Kinomics and transcriptomic assays revealed that in several cardiomyocyte cellular models, digoxin and other cardiotonic steroids induced multiple signaling pathways at subinotropic doses. Pharmacological inhibition and knockdown approaches revealed that inhibition of phosphatidylinositol 3- and of Src tyrosine-kinase partially alleviated digoxin-induced REV-ERBα degradation, which was fully prevented upon proteasome inhibition. REV44 ERBα is increasingly ubiquitinylated in digoxin-treated cells, and its degradation depends on its ability to bind its natural ligand, heme. In normal conditions, the proteasomal degradation of REV-ERBα is controlled by several known (HUWE1, FXW7, SIAH2) or novel (CBL, UBE4B) E3 ubiquitin ligases. Only SIAH2 together with the proteasome subunit PSMB5 contributed to the digoxin-induced degradation of REV-ERBα. Taken together, these results show that controlling REV-ERBα proteostasis is an appealing cardioprotective strategy, and bring further support to the rationale, timed use of CTS in prophylactic cardiac preconditioning to MIRI. Mice were injected intraperitoneally with 1mg/kg digoxin (Nativelle Adult) or Sham (a co-solvent consisting of 10% ethanol, 40% propylene glycol, 0.08% citric acid, 0.3% sodium phosphate) at ZT5.Mice were sacrificed by cervical dislocation at ZT9.Organs were harvested and snap-frozen RNA analysis. Gene expression from mouse hearts from Sham (4 samples) or digoxin (5 samples) treatment was analyzed with Affymetrix GeneChip Mouse Gene 2.0 ST arrays after RNA amplification, sscDNA labeling, and purification. Briefly, RNA was amplified using the GeneChip™ WT PLUS Reagent Kit (Thermo Fisher Scientific), retrotranscribed to cDNA and labeled using GeneChip™ WT Terminal Labeling Kit (Thermo Fisher Scientific), followed by hybridization on the GeneChip Mouse Gene 2.0 ST Array (Affymetrix) according to the manufacturer’s instructions. Raw data were processed further using GIANT (Galaxy-based interactive tools for Analysis of Transcriptomic data). This user-friendly tool suite was developed in-house for microarray and RNA-seq differential data analysis. It consists of modules allowing to perform quality control (QC), Robust Multi-Average method normalization, LIMMA differential analysis, volcano plot and heatmaps. Using the GSEA module, normalized data were converted to GSEA compatible format for further analysis.