Phosphoproteomics analysis of serum from dogs affected with pulmonary hypertension secondary to degenerative mitral valve disease

Abstract

Pulmonary hypertension (PH), a common complication in dogs affected by degenerative mitral valve disease (DMVD), is a progressive disorder characterized by increased pulmonary arterial pressure (PAP) and pulmonary vascular remodeling. Phosphorylation of proteins, impacting vascular function and cell proliferation, might play a role in the development and progression of PH. Unlike gene or protein studies, phosphoproteomic focuses on active proteins that function as end-target proteins within signaling cascades. Studying phosphorylated proteins can reveal active contributors to PH development. Early diagnosis of PH is crucial for effective management and improved clinical outcomes. This study aimed to identify potential serum biomarkers for diagnosing PH in dogs affected with DMVD using a phosphoproteomic approach. Serum samples were collected from healthy control dogs (n = 28), dogs with DMVD (n = 24), and dogs with DMVD and PH (n = 29). Phosphoproteins were enriched from the serum samples and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Data analysis was performed to identify uniquely expressed phosphoproteins in each group and differentially expressed phosphoproteins among groups. Phosphoproteomic analysis revealed nine uniquely expressed phosphoproteins in the serum of dogs in the DMVD+PH group and 15 differentially upregulated phosphoproteins in the DMVD+PH group compared to the DMVD group. The phosphoproteins previously implicated in PH and associated with pulmonary arterial remodeling, including small nuclear ribonucleoprotein G (SNRPG), alpha-2-macroglobulin (A2M), zinc finger and BTB domain containing 42 (ZBTB42), hemopexin (HPX), serotransferrin (TRF) and complement C3 (C3), were focused on. Their unique expression and differential upregulation in the serum of DMVD dogs with PH suggest their potential as biomarkers for PH diagnosis. In conclusion, this phosphoproteomic study identified uniquely expressed and differentially upregulated phosphoproteins in the serum of DMVD dogs with PH. Further studies are warranted to validate the diagnostic utility of these phosphoproteins.

Keywords: Biomarkers; Degenerative mitral valve disease; Dogs; Phosphoproteomics; Proteomics; Pulmonary hypertension; Serum.

Figure 1. Inclusion and exclusion criteria for sample selection.

Figure 2. Protocol for sample collection, preparation and phosphoproteomics analysis.

Figure 3. Heat map of all identified phosphoproteins in the control group, degenerative mitral valve disease (DMVD) group and degenerative mitral valve disease with pulmonary hypertension (DMVD+PH) group.

A total of 1,467 phosphoproteins were identified with different expression levels among groups. Samples are in columns and identified phosphoproteins are in rows. The color indicated phosphoproteins intensity that change from very low (light grey) to extremely high (black). The color scale on the right depicts the range of expression values.

Figure 4. Partial least squares-discriminant analysis (PLS-DA) of all identified phosphoproteins clustered by groups including the control group, degenerative mitral valve disease (DMVD) group and degenerative mitral valve disease with pulmonary hypertension (DMVD+).

Colored dots represent individual samples and colored areas represent 95% confidence interval.

Figure 5. Analysis of variance (ANOVA) plot of significantly identified phosphoproteins in comparison among the control group, degenerative mitral valve disease (DMVD) group and degenerative mitral valve disease with pulmonary hypertension (DMVD+PH) group.

An ANOVA test revealed 42 out of 1,467 identified phosphoproteins exhibited significant differences among the control, DMVD and DMVD+PH groups. The x-axis represents the phosphoprotein peaks, identified by their mass-to-charge ratio (m/z) and retention time (rt). The y-axis represents the −log10 of the raw p-value associated with each peak. Colored dot represents significantly expressed phosphoproteins with p < 0.05 while the grey dot represents phosphoproteins without statistical significance.

Figure 6. Network of protein-cardiovascular drugs interaction analyzed by Stitch, version 5.0.

Four phosphoproteins including albumin (ALB), haptoglobin (HP), hemoglobin subunit beta (LOC480784) and anionic trypsinogen (ENSCAFG00000014481) correlated with commonly used cardiovascular drugs, including enalapril, benazepril, ramipril, furosemide, pimobendan, spironolactone, a combination of amiloride and hydrochlorothiazide (Moduretic®), and sildenafil. The differentially expressed phosphoproteins showed a correlation with cardiovascular drugs. The strength of the associations at the functional level was evaluated by edge confidence scores. The strong relationships with high edge confidence scores (>0.700) are presented as thick lines. Abbreviations: ACE, angiotensin-converting enzyme; ALB, albumin; AR, androgen receptor; ENSCAFG00000014481, anionic trypsinogen; HBA, hemoglobin A; HP, haptoglobin; LOC480784, hemoglobin subunit beta; NR3C2, mineralocorticoid receptor; PDE5A, phosphodiesterase 5; ENS REN, renin.

Figure 7. Boxplot of the focused uniquely expressed phosphoproteins (top row) and upregulated phosphoproteins (bottom row) in the degenerative mitral valve disease with pulmonary hypertension (DMVD+PH) group that have p-value < 0.05 and fold changes >2 when comparin.

The focused phosphoproteins such as, small nuclear ribonucleoprotein G (A), Alpha-2-macroglobulin (B), Zinc finger and BTB domain containing 42 (C), were exclusively detected in the serum of dogs affected with PH secondary to DMVD. On the other hand, Hemopexin (D), Serotransferrin (E) and Complement C3 (F) were identified in the serum of DMVD dogs both with and without PH, but their level was significantly upregulated in those with PH. The y-axis represents normalized peak intensity of phosphoproteins obtained in LC-MS/MS analysis. The box ranges from the 25^th to 75^th percentiles, with whiskers extending to 1.5 times the interquartile range, and horizontal lines inside each box representing the 50^th percentile or median. Black dots indicate individual data points, while yellow dot in a boxplot indicates the mean of the data.