Comparative proteomic analysis of nuclear and cytoplasmic compartments in human cardiac progenitor cells

Clinical trials evaluating cardiac progenitor cells (CPC) demonstrated feasibility and safety, but no clear functional benefits. Therefore a deeper understanding of CPC biology is warranted to inform strategies capable to enhance their therapeutic potential. Here we have defined, using a label-free proteomic approach, the differential cytoplasmic and nuclear compartments of human CPC (hCPC). Global analysis of cytoplasmic repertoire in hCPC suggested an important hypoxia response capacity and active collagen metabolism. In addition, comparative analysis of the nuclear protein compartment identified a significant regulation of a small number of proteins in hCPC versus human mesenchymal stem cells (hMSC). Two proteins significantly upregulated in the hCPC nuclear compartment, IL1A and IMP3, showed also a parallel increase in mRNA expression in hCPC versus hMSC, and were studied further. IL1A, subjected to an important post-transcriptional regulation, was demonstrated to act as a dual-function cytokine with a plausible role in apoptosis regulation. The knockdown of the mRNA binding protein (IMP3) did not negatively impact hCPC viability, but reduced their proliferation and migration capacity. Analysis of a panel of putative candidate genes identified HMGA2 and PTPRF as IMP3 targets in hCPC. Therefore, they are potentially involved in hCPC proliferation/migration regulation.

Human cardiac biopsies were obtained from patients suffering from an open-chest surgery, usually for valve replacement. Starting material was obtained from the right atria appendage, which is routinely removed in order to place the cannulae for the extracorporeal circulation.
Human bone marrow-derived mesenchymal stem cells (MSC) were obtained from cadaveric bone marrow, harvested from brain-dead donors under the supervision of the Spanish hMSC and fibroblasts (obtained from Inbiobank) were maintained and expanded under optimal conditions, in low-glucose DMEM supplemented with 10% FBS (both from Sigma-Aldrich, Madrid, Spain), 2 mM L-glutamine (Lonza) and penicillin-streptomycin (100 and 1000 U/mL, respectively, Lonza), also in a 3% O2/ 5%CO2 atmosphere.

Proteomics analyses
Label-free proteomics analysis. hCPC3 and hMSC19 were used for proteomics analysis.
Cells were expanded to passage 7-8, recovered (5-8 × 10 7 ) and washed several times in PBS. Subcellular cytoplasmic and nuclear protein fractions were obtained (n=3) using the Qproteome Cell Compartment Kit (Qiagen, Barcelona, Spain). When needed equivalent fractions were obtained from human fibroblasts (HDF).
Briefly, samples were resolved by conventional SDS-PAGE until the electrophoresis front entered 3 mm into the concentrating gel. The protein band containing the whole proteome was visualized by Coomassie staining, excised, cut into cubes, subjected to reduction conditions using 10 mM dithiothreitol (DTT), alkylated with iodoacetamide (50 mM), and digested (overnight 37ºC) with 60 ng/mL modified trypsin (Promega, Madison, WI) at a 12:1 protein:trypsin (w/w) ratio in 50 mM ammonium bicarbonate (pH 8.8) containing 10% acetonitrile. The resulting tryptic peptides were extracted by incubation in 12 mM ammonium bicarbonate pH 8.8 followed by 0.5% trifluoroacetic acid (TFA). TFA was added to a final concentration of 1% and peptides were desalted on C18 Oasis-HLB cartridges and dried. Tryptic peptides were dissolved in 0.1% formic acid (FA) and loaded on a liquid chromatography-mass spectrometry (LC-MS/MS) system for online desalting on C18 cartridges and further analysis by LC-MS/MS, using a reverse-phase nanocolumn (75 µm inner diameter × 50 µm, 3 µm-particle size, Acclaim PepMap 100 C18; Thermo Fisher Scientific, San Jose, CA) in a continuous (0-30%) acetonitrile gradient consisting of B (90% acetonitrile, 0.5% formic acid), in 180 min, 30-43% in 5 min and 43-90% in 2 min. A ~200 nL/min flow rate was used to elute peptides from the nanocolumn to an emitter nanospray needle for real time ionization and peptide fragmentation onto an ion trap-orbitrap hybrid mass spectrometer (Orbitrap Elite, Thermo-Fisher). To increase proteome coverage, tryptic peptides were fractionated by cation exchange chromatography (Oasis HLB-MCX column; Waters Corp., Milford, MA), desalted and analyzed as above.  [4]. False discovery rate (FDR) was calculated using inverted databases and a refined method for peptide identification using decoy databases [5].    Evaluation of IMP3 and IL1A functional interaction and response to apoptosis/necrosis upon oxidative damage. a) Analysis of the effects of IMP3 downregulation on hCPC1 or hCPC3 response to oxidative damage induced by H 2 O 2 . hCPC control, siIMP3-or siNeg-transfected cells were exposed to H 2 O 2 (500 µM) during 48 h; cultures were stained with the AnnexinV/ Propidium iodide (Anex.V / PI) and homeostatic viable (H3: Anex.V-/ PI-), apoptotic (H4: Anex.V+/ PI-), late apoptotic (H2: Anex.V+/ PI+) or necrotic (H1: Anex.V-/ PI+) cells were quantified by cytometry. Data correspond to a representative experiment; assays were performed three times and data expressed as mean ± SD are included in Figure 4E; b) Densitometric analysis of the representative western blot shown in Figure 5b; nuclear/cytoplasmic ratio for IGF2R, IMP2 and IMP3, in hCPC3 are compared in homeostasis of after apoptosis of induction. C) Evaluation of potential role of IMP3 in regulation of IL1A expression in hCPC.

Bioinformatics identification and analyses.
Using the hCPC3 isolate, hCPC control, siIMP3-or siNeg-transfected cells were evaluated, 48 h after transfection, by RT-qPCR. Assays were performed three times and data expressed as mean ± SD of the results relative to GusB; black lines summarize p-values (**<0.02; *<0.05; one-way ANOVA analysis of variance followed by the Bonferroni correction for multiple comparison).