Proteomic Analysis of Human Pluripotent Stem Cell Cardiomyogenesis Revealed Altered Expression of Metabolic Enzymes and PDLIM5 Isoforms

Human pluripotent stem cells (hPSCs), both embryonic (hESCs) and induced (hiPSCs), can be differentiated into derivatives of the three germ layers and are promising tools in regenerative medicine. Cardiovascular diseases are the top-ranking cause of premature death worldwide, and cell replacement therapies based on in vitro differentiated cardiomyocytes might provide a promising perspective to cure patients in the future. The molecular processes during hPSC cardiomyogenesis are far from being fully understood, and we thus have focused here on characterizing the proteome along hESC in vitro differentiation into cardiomyocytes (CMs). Stable isotope labeling of amino acids in cell culture was applied to quantitatively assess the proteome throughout defined stages of hESC cardiomyogenesis. Genetically enriched, >90% pure CM populations were used for shotgun proteomics, leading to the identification and quantitative determination of several thousand proteins. Pathway analysis revealed alterations in energy metabolism during cardiomyogenesis. Enzymes of glycolysis were identified as up-regulated upon differentiation, whereas enzymes involved in oxidative phosphorylation were down-regulated in aggregates on day 20 of differentiation (<10% CMs) and reconstituted on day 35 in >90% pure CMs. A structural protein that attracted our attention was the PDZ and LIM domain containing protein 5 (PDLIM5), which was strongly up-regulated during cardiomyogenesis and for which we detected novel stage-specific isoforms. Notably, expression of the 53 kDa isoforms b and g (corresponding to transcript variants 2 and 7) of PDLIM5 occurred simultaneously to the onset of expression of the early cardiac transcription factor NKX2.5, known to play a key role in cardiac development

Proteomic Analysis of Human Pluripotent Stem Cell Cardiomyogenesis Revealed Altered Expression of Metabolic Enzymes and PDLIM5 Isoforms

Human pluripotent stem cells (hPSCs), both embryonic (hESCs) and induced (hiPSCs), can be differentiated into derivatives of the three germ layers and are promising tools in regenerative medicine. Cardiovascular diseases are the top-ranking cause of premature death worldwide, and cell replacement therapies based on in vitro differentiated cardiomyocytes might provide a promising perspective to cure patients in the future. The molecular processes during hPSC cardiomyogenesis are far from being fully understood, and we thus have focused here on characterizing the proteome along hESC in vitro differentiation into cardiomyocytes (CMs). Stable isotope labeling of amino acids in cell culture was applied to quantitatively assess the proteome throughout defined stages of hESC cardiomyogenesis. Genetically enriched, >90% pure CM populations were used for shotgun proteomics, leading to the identification and quantitative determination of several thousand proteins. Pathway analysis revealed alterations in energy metabolism during cardiomyogenesis. Enzymes of glycolysis were identified as up-regulated upon differentiation, whereas enzymes involved in oxidative phosphorylation were down-regulated in aggregates on day 20 of differentiation (<10% CMs) and reconstituted on day 35 in >90% pure CMs. A structural protein that attracted our attention was the PDZ and LIM domain containing protein 5 (PDLIM5), which was strongly up-regulated during cardiomyogenesis and for which we detected novel stage-specific isoforms. Notably, expression of the 53 kDa isoforms b and g (corresponding to transcript variants 2 and 7) of PDLIM5 occurred simultaneously to the onset of expression of the early cardiac transcription factor NKX2.5, known to play a key role in cardiac development

Proteomic Analysis of Human Pluripotent Stem Cell Cardiomyogenesis Revealed Altered Expression of Metabolic Enzymes and PDLIM5 Isoforms

Human pluripotent stem cells (hPSCs), both embryonic (hESCs) and induced (hiPSCs), can be differentiated into derivatives of the three germ layers and are promising tools in regenerative medicine. Cardiovascular diseases are the top-ranking cause of premature death worldwide, and cell replacement therapies based on in vitro differentiated cardiomyocytes might provide a promising perspective to cure patients in the future. The molecular processes during hPSC cardiomyogenesis are far from being fully understood, and we thus have focused here on characterizing the proteome along hESC in vitro differentiation into cardiomyocytes (CMs). Stable isotope labeling of amino acids in cell culture was applied to quantitatively assess the proteome throughout defined stages of hESC cardiomyogenesis. Genetically enriched, >90% pure CM populations were used for shotgun proteomics, leading to the identification and quantitative determination of several thousand proteins. Pathway analysis revealed alterations in energy metabolism during cardiomyogenesis. Enzymes of glycolysis were identified as up-regulated upon differentiation, whereas enzymes involved in oxidative phosphorylation were down-regulated in aggregates on day 20 of differentiation (<10% CMs) and reconstituted on day 35 in >90% pure CMs. A structural protein that attracted our attention was the PDZ and LIM domain containing protein 5 (PDLIM5), which was strongly up-regulated during cardiomyogenesis and for which we detected novel stage-specific isoforms. Notably, expression of the 53 kDa isoforms b and g (corresponding to transcript variants 2 and 7) of PDLIM5 occurred simultaneously to the onset of expression of the early cardiac transcription factor NKX2.5, known to play a key role in cardiac development

Covalent binding energies of the compounds to the BTB domain of human KEAP1.

Covalent binding energies of the compounds to the BTB domain of human KEAP1.</p

The compounds favor nuclear retention of p53 in IAV-infected A549 cells.

A549 cells were treated and infected as described for Fig 2. p53 was detected by indirect immunofluorescence 8 h p.i., using Alexa Fluor 568 labeled secondary antibody. A. Representative immunofluorescence images p53 = red. Nuclei = blue (DAPI). Pink signal in merged images = nuclear localized p53. The positive staining granular pattern is a technical artefact and was considered background signal. Negative control = no primary antibody. B. Fraction of all cells with nuclear p53 staining. Cells with nuclear p53 staining were counted by visual inspection by two independent examiners who were blinded to the identity of the specimens. n = 4 microscopic fields, means ±SEM. One-way ANOVA with Tukey’s post-hoc test, using infected untreated wild-type or knock-down cells as reference. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001. (EPS)</p

Competition experiment demonstrating binding of 4OI and SEL to the same sites on XPO1 and KEAP1.

A,B. “Click-chemistry” pull-down assay demonstrating covalent binding of an alkynated 4OI probe (4-OI-alk) to XPO1 (A) and KEAP1 (B) in Calu-3 cells. Cells were preincubated with 1 or 4 μM unmodified SEL for 30 min. as indicated. Two hours after addition of the probe, proteins complexed with the probe were detected by immunoblot for XPO1 (A) or KEAP1 (B). C,D. Densitometry (arbitrary units) of the immunoblots, normalized to the signal obtained from the band labeled “input”. SEL competes with 4OI for complex formation with both targets, suggesting that the compounds recognize the same sites on both targets. However, competition is less efficient for complex formation with KEAP1, suggesting that 4OI has higher affinity for KEAP1 and that SEL has higher affinity for XPO1. (EPS)</p

3D structural modeling of 4OI-XPO1 interactions based on the co-crystal structure of XPO1 (CRM1) with leptomycin B (PDB ID: 6TVO).

Both 4OI and leptomycin B are covalently bound to the reactive Cys528 (marked with an asterisk *) and interact extensively with the hydrophobic NES-binding groove. A. 4OI binds the site through hydrophobic interactions between the octyl chain and Ile521, Leu525, Met545, Val565 and Leu569 in the hydrophobic pockets Φ2 and Φ3 of the NES-binding site. The C1-carboxyl group further stabilizes binding through two hydrogen bonds with Lys537 and Lys568. These hydrophobic and electrostatic interactions optimally direct the methylene group of 4OI towards Cys528 and could be the driving force for the covalent Michael 1,4-addition. B. Overlay of 4OI (cyan) and leptomycin B (magenta) in the NES-binding groove showing about 70% occupancy by leptomycin B and 40% by 4OI. Lipophilicity protein surface at the NES-binding cleft: lipophilic (green), hydrophilic (violet), neutral (white), α-helices (gold). * = Cys528. (EPS)</p

Bar graphs corresponding to the heat map shown in Fig 3J.

The RT-qPCR data were analyzed by the 2-ΔΔCt method using HPRT mRNA as internal control. Fold change was calculated with respect to expression in uninfected wild-type cells. n = 3, means ±SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001. (EPS)</p

Antiviral effects of the four compounds are NRF2-independent.

hiPSC-derived wild-type or NRF2-/- vascular ECs were pretreated with the compounds (SEL, 1 μM; 4OI, 100 μM; BARD, 0.1 μM; SFN, 10 μM) for 12 h, infected with IAV PR8M (MOI = 1) for 2 h, and then incubated in fresh buffer containing the compounds for 22 h. A. NFE2L2 mRNA (RT-qPCR). B. Viral titers in cell culture supernatants (foci-forming assay, FFU/mL). C, D. IFIT1 and CXCL10 mRNA (RT-qPCR). E. Expression of HMOX1, SLC7A11, AKR1B10, GCLM, and KEAP1 mRNAs (RT-qPCR, internal control HPRT1 mRNA), heat map based on log2 fold change (as indicated in the color legend) with respect to expression in wild-type uninfected cells. Column graphs of these data are shown in S3 Fig for additional clarity. F-H, Knocking down KEAP1 expression reduces viral titers, but does not affect the antiviral effect of the compounds. F, Expression of KEAP1 mRNA (RT-qPCR). G, Expression of viral HA mRNA (RT-qPCR). H, Viral titers (foci-forming assay, FFU/ml). n = 3, means ±SEM. One-way ANOVA with Tukey’s post-hoc test. * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001.</p

NRF2 activators reduce release of IAV virions and inhibit nuclear export of vRNP.

A. Schematic of experimental layout. A549 cells were pretreated with the compounds (SEL, 1 μM; 4OI, 100 μM; BARD, 0.1 μM; SFN, 10 μM) for 12 h, were then infected with IAV PR8M (MOI = 0.05 in B, MOI = 1 in C-F) for 1 h and subsequently incubated in fresh buffer containing the compounds. Measurements were performed at the indicated times post infection (p.i.). B. NRF2 activators reduce release of progeny virions. Viral titers (FFU/mL) in cell culture supernatants were determined 12 and 24 h p.i. n = 3. C-F. NRF2 activators interfere with nuclear export of vRNP. Subcellular localization of viral NP was determined by immunofluorescence 4, 6, and 8 h p.i. Viral NP was visualized by indirect immunofluorescence using Cy3-labeled secondary antibody (561 nm, red) and nuclei by staining DNA with DAPI (405 nm, blue). Cells with NP staining in nucleus, cytoplasm or both nucleus and cytoplasm were quantified by visual inspection. N = 2 replicates, n = 7 digital images per replicate. C. Representative microscopic images. D. Proportion of cells with nuclear NP staining only. E. Proportion of cells with cytoplasmic NP staining only. F. Proportion of cells with both nuclear and cytoplasmic NP staining. Data are shown as means ±SEM. One-way ANOVA with Tukey’s post-hoc test. p = * ≤0.05, ** ≤0.01, *** ≤0.001.</p