Phospho-Proteomic Analysis of Cardiac Dyssynchrony and Resynchronization Therapy

Abstract

Cardiac dyssynchrony arises from conduction abnormalities during heart failure and worsens morbidity and mortality. Cardiac resynchronization therapy (CRT) re-coordinates contraction using bi-ventricular pacing, but the cellular and molecular mechanisms involved remain largely unknown. The aim is to determine how dyssynchronous heart failure (HF_dys ) alters the phospho-proteome and how CRT interacts with this unique phospho-proteome by analyzing Ser/Thr and Tyr phosphorylation. Phospho-enriched myocardium from dog models of Control, HF_dys , and CRT is analyzed via MS. There were 209 regulated phospho-sites among 1761 identified sites. Compared to Con and CRT, HF_dys is hyper-phosphorylated and tyrosine phosphorylation is more likely to be involved in signaling that increased with HF_dys and was exacerbated by CRT. For each regulated site, the most-likely targeting-kinase is predicted, and CK2 is highly specific for sites that are "fixed" by CRT, suggesting activation of CK2 signaling occurs in HF_dys that is reversed by CRT, which is supported by western blot analysis. These data elucidate signaling networks and kinases that may be involved and deserve further study. Importantly, a possible role for CK2 modulation in CRT has been identified. This may be harnessed in the future therapeutically to compliment CRT, improving its clinical effects.

Keywords: CK2; dyssynchrony; heart failure; phosphorylation.

Figure 1:. Schematics of the dog model pacing schedule and the mass spectrometry work flow.

(A) The HF_dys (dyssynchronous heart failure) and CRT (Cardiac Resynchronization Therapy) models received an implanted pacemaker and radio-frequency ablation of the left bundle branch (to generate a left bundle branch block, LBBB, and dyssynchrony). After a one-week recovery period, the HF_dys and CRT groups were subjected to six weeks atrial tachypacing (200 bpm), which induced heart failure. After three weeks of pacing, the CRT model (which had been the same as HF_dys to this point) was switched from atrial to bi-ventricular (Bi-V) pacing to recoordinate contraction (200 bpm, so underlying heart failure was not altered). Control dogs received no intervention. (B) Tissue samples (LV lateral wall, n = 4 per group) were split into three parts, one enriched for myofilament, one myofilament-depleted, and one whole tissue homogenate and phospho-tyrosine enriched. Samples were TiO2 phospho-enriched, prepared for MS via the usual methods, and run on an Orbitrap Elite. Samples were then searched with Scaffold and Sorcerer, and significant changes between groups were determined using one-way ANOVA and Holm-Sidak post-hoc tests. The kinase for each site was predicted using Group-based Prediction System 2.1. (C) Number of assigned MS/MS spectra for each animal (n=4/group) for Ser/Thr (black) and Tyr (grey) sites.

Figure 2:. Altered phosphorylation sites were placed into categories of change based on pattern of differences.

(A) Based on the pattern of significant post-hoc tests for each phosphorylation site, each was placed into a category of change. Each circle represents Con (N), HF_dys (D) or CRT (C), showing the patterns of change that were included in each category. For example, in “HF_dys-Not reversed” phosphorylation can be decreased from non-failing in both dyssynchrony and CRT or increased from non-failing in both dyssynchrony and CRT. (B) Of the 11% of the phospho-proteome that was altered (left pie chart), the right pie chart indicates the percentage of total phosphorylation sites changed in HF_dys and CRT, divided into individual categories of change. (C) Number of significantly altered sites phosphorylated for Ser/Thr or Tyr sites in the four categories of change. (D) Principal component analysis for Ser/Thr (left) and Tyr (right) phosphorylation data. Unit variance scaling was applied to phosphorylation data, SVD with imputation was used to calculate principal components.

Figure 3:. Ingenuity Analysis for “HF_dys- Not Reversed” and “CRT only” categories.

(A) Proteins with (*) had phosphorylation sites changed in HF_dys and not reversed by CRT. (B) Proteins with (*) had phosphorylation sites that were altered only in the CRT group. Legend for interactions and protein types is shown to the right. The networks were generated using QIAGEN’s Ingenuity Pathway Analysis (IPA^®, QIAGEN Redwood City, www.qiagen.com/ingenuity).

Figure 4:. Kinases and number of predicted targets for each category of change.

The number of phosphorylation sites that shared the same top predicted (A) Ser/Thr or (B) Tyr kinase. SGK, serum/glucocorticoid regulated kinase 1; PKC/PKG, protein kinase C/G; ERK, extracellular regulated kinase; p38, p38 mitogen activated protein kinase; JNK, c-Jun N-terminal kinase; CK2, casein kinase 2; AUR, aurora kinase.

Figure 5:. Western blot analysis agreed with kinase prediction for CK2 levels in the three groups.

(A) Representative CK2 blot for Con, HF_dys, and CRT (top) and total protein stain (bottom) for normalization. (B) Mean, SEM, and individual data points for CK2 normalized to total protein stain (n = 4 per group) and then normalized to the mean of the Con group. The one-way ANOVA trended towards significance (p = 0.09) suggesting an increase in CK2 levels in HF_dys that was reversed by CRT.