Alteration in tyrosine phosphorylation of cardiac proteome and EGFR pathway contribute to hypertrophic cardiomyopathy

Fig. 1. Global pTyr is reduced in cardiac hypertrophy associated with ErbB2 overexpression or myosin heavy chain mutation. a Western Blots show a decrease in pTyr on TgErbB2 (p = 0.0298) mice and R403Q-α-MyHC (p = 0.003) heart homogenates. The Troponin I signal shows corresponding total protein loading. b Densitometry analysis shows that after the pTyrosine signal was normalized to total cTnI, the TgErbB2 and R403Q-α-MyHC mice significantly decreased in pTyr. c Western Blots show expression levels of total Src and p-Src (Tyr 416) and GAPDH as a loading control. d Densitometry analysis revealed that Src levels are significantly elevated in R403Q-α-MyHC (p = 0.0282), whereas e and f Show that p-Src phosphorylation levels are elevated in R403Q-α-MyHC but did not reach statistical significance. Signals in d and e were normalized to total GAPDH, whereas in f pSRC was normalized to total SRC. Ntg (n = 3), TgErbB2 (n = 3), and R403Q-α-MyHC (n = 3), error bars represent S.E.M. One-way ANOVA was used to compare groups.

Fig. 2. Workflow for global whole heart TMT phospho-tyrosine proteomics profiling. a Ventricular tissues were rinsed with PBS and subjected to Heat Stabilization using a T1 Tissue Stabilizor to maximize phosphorylation preservation, then snap-frozen in liquid nitrogen and stored at −80 °C until further use. b Ntg, TgErbB2, and R403Q-αMyHC mice hearts (n = 5 per group) were processed in parallel to obtain whole heart lysates; 30 mg of protein lysates were used from each mouse heart for in-solution trypsin digestion, a total of 150 mg of trypsinized protein per genotype were C18 desalted and lyophilized at −20 °C for three days. c A total of 150 mg of tryptic peptides (pooled material from 5 hearts per group) were enriched for pTyrosine using immunoaffinity precipitation (Protein Agarose Beads + 300 micrograms of mAb Anti-pTyr) and desalted on C18 stage tips. d RP-HPLC ESI (electrospray ionization) MS/MS was performed on an LTQ Orbitrap Elite (Thermo Scientific) for a 120 min on a linear acetonitrile gradient (4−40%). Raw data were searched with Mascot 2.3, and label-free quantification with MS1 extracted ion chromatograms were performed using MaxQuant. This approach was repeated three…

Fig. 3. PTMSigDB reveals common pTyr signatures of cardiac proteome in HCM. a Unsupervised PCA of the full cardiac Proteome of Non-transgenic, TgErbB2, and R403Q-αMyHC mice show distinct segregation of the experimental groups. The groups segregate along with the first principal component (PC #1), which accounts for 46% of the total correlation in the expression of Full Proteome (normalized, mean = 0, variance = 1). b Unsupervised PCA of the pTyrosine peptides signal normalized to full proteome expression. The groups also segregate along with the first principal component (PC #1), accounting for 52.2% of the total correlation. c A heatmap was used to visualize the unsupervised hierarchical clustering of pTyrosine peptides normalized to full proteome expression with a p-value < 0.05 by ANOVA. Overrepresented (yellow) and underrepresented (Blue). d Volcano plot showing the Log2 fold-change and –Log10 p-value of each pTyr peptide in the pairwise comparison of Ntg and TgErbB2. The data points highlighted in red show a significantly lower intensity, whereas the data in green show significantly higher intensity. The significant sites were magnified in the figure (dotted squares) to label some…

Fig. 4. Workflow for cardiac sarcomere pTyr identification of normal and TgErbB2 hypertrophic myofilaments by TMT quantitative proteomics. a Myofilament from Ntg (n = 3) and ErbB2 (n = 3) transgenic mice hearts were freshly isolated on ice-cold buffers containing Proteinase and Phosphatase Inhibitors Cocktails (Roche). b All material was resuspended in TEAB, then reduced and alkylated. Tryptic peptides were desalted and labeled with 6-plex isobaric tandem mass tags (TMT). c The digested and labeled peptides were pooled and desalted with C18 SEP-PAK. The enrichment for phosphotyrosine was performed with PTMScan Phospho-Tyrosine Rabbit mAb (P-Tyr-1000) kit (Cell Signaling Technology). The eluted peptide samples were desalted using C18 STAGE tips d Easy-nanoLC 1200 nanoflow liquid chromatography system coupled to Orbitrap Fusion Lumos Tribrid.

Fig. 5. TgErbB2 cardiac sarcomere pTyr proteome remodeling. a Full Proteome was labeled with TMT, and the logged and summarized protein signal intensities indicate that experimental groups segregate along with the first principal component (PC #1), which accounts for 53.9% of the total correlation in the expression proteome (normalized, mean = 0, variance = 1). b PCA of the phospho-Tyr Proteome reveals a similar trend, and the groups segregate along the PC#1, which accounts for 47.2% of the correlation. c Signal intensities of the phospho-Tyr proteome were normalized to Full Proteome protein expression and analyzed by principal component analysis. The distinctive signature of pTyr is still apparent by PC#1 and accounts for 57% of the correlation. d A heatmap was used to visualize the unsupervised hierarchical correlation clustering of protein expression and e pTyr Proteome expression with p < 0.05 by LIMMA moderated 2-samples t-test comparison of TgErbB2 and Ntg group. f Volcano plot showing the Log2 fold-change and –Log10 p-value of each pTyr peptide in the pairwise comparison of Ntg and TgErbB2. The data points in red showed a significantly lower intensity, whereas the data points in…

Fig. 6. KSEA & MoBaS analysis. a KSEA on whole hearts pTyr enrichment and TMT pTyr Myofilament enrichment data. Heat maps report KSEA results according to normalized scores (i.e., TgErbB2/Ntg, R403Q-αMyHC/Ntg, etc.). Only kinases shared between data sets are included, therefore present in Tg and Ntg. Asterisks indicate the statistical significance of p < 0.05. Red represents a positive score, and blue is a negative score. Red rectangle highlights groups of activated kinases clustered together and are predominant in R403Q-αMyHC, whereas the blue rectangle highlights suppressed kinases that clustered and are predominant on TgErbB2 whole hearts. b MoBaS identified a tightly interconnected subnetwork involved in growth factors signaling (ERBB, PDGFRA, and EGFR1). Represented by the top-scoring PPI subnetwork called Module 1 was identified on TgErbB2 whole heart pTyr enrichment and compared to R403Q-αMyHC. c KSEA on Module 1 group of substrates, MsigDB and PTMsigDB. d Second top-scoring PPI subnetwork called Module 2 was identified on R403Q-αMyHC whole heart pTyr enrichment and compared to TgErbB2. e KSEA on Module 2 group of substrates show activated kinases in R403Q-αMyHC. Results from…

Fig. 7. Tyrosine kinase receptor inhibitor AG-825 reduces cardiac myocyte disarray without effects on contractile function in TgErbB2 mice. a Masson’s trichrome stained sections (5X) from vehicle-treated Ntg and AG-825–treated Ntg and vehicle-treated TgErbB2 and AG-825-treated TgErbB2 mice, yellow arrowheads point to fibrotic areas. Scale bar 1 mm. b Masson’s trichrome stained sections (40X) from representative regions of interest show increased Fibrosis in TgErbB2 mice myocardium, that fibrosis improved after 2 weeks of AG-825 Treatment. The same areas were used to determine the level of cardiac myocyte disarray, which was achieved by analyzing cell orientation using CytoSpectre Software from Vehicle and AG-825 treated Ntg and TgErbB2 mouse heart sections (scale bar 50 μm). c Plots of the distribution of cardiac cells orientation angles. Note how Ntg angles are homogeneous and did not change in the group treated with AG-825. In contrast, Tg treated with vehicle showed less homogenous and different orientation angles, confirming cardiomyocyte disarray. AG-825 Treatment reduces cardiomyocyte disarray, and the orientation plot is similar to the AG-825 treated NTG group. d Mean orientation…

Fig. 8. Comprehensive serine/threonine/tyrosine phosphoproteomics confirm EGFR1 pathway involvement in TgErbB2 hypertrophy and tyrosine kinase receptor inhibition by AG-825. a A heatmap was used to visualize the unsupervised hierarchical clustering of Ser/Thr peptides, and b pTyrosine peptides normalized to full proteome expression with p-value < 0.05 by ANOVA. Overrepresented (yellow) and underrepresented (Blue). c Volcano plots showing the Log2 fold-change and –Log10 p-value of each pSer/pThr peptide and d pTyr peptides in the pairwise comparison of Ntg and TgErbB2 after two weeks of Vehicle Treatment. e Volcano plots showing the Log2 fold-change and –Log10 p-value of each pSer/pThr peptide and f pTyr peptides in the pairwise comparison of Ntg and TgErbB2 after two weeks of AG-825 Treatment, (g) KSEA shows in a red rectangle the kinases significantly inhibited by AG-825. KSEA and Pathway Enrichment Analysis of combined Ser/Thr/Tyr phosphorylation data analysis using PTMSigDB confirm that EGFR1 Pathway is significantly involved. h The Signaling Network Open Resource (Signor 2.0) analysis shows that nine kinases are involved in the EGFR pathway. Kinases found in the network and also in…