Plasma Proteomic Profiling in Hypertrophic Cardiomyopathy Patients before and after Surgical Myectomy Reveals Post-Procedural Reduction in Systemic Inflammation

Abstract

Left Ventricular Outflow Tract (LVOT) obstruction occurs in approximately 70% of Hypertrophic Cardiomyopathy (HCM) patients and currently requires imaging or invasive testing for diagnosis, sometimes in conjunction with provocative physiological or pharmaceutical stimuli. To identify potential biomarkers of LVOT obstruction, we performed proteomics profiling of 1305 plasma proteins in 12 HCM patients with documented LVOT obstruction, referred for surgical myectomy. Plasma was collected at the surgical preoperative visit, approximately one month prior to surgery and then at the post-surgical visit, approximately 3 months later. Proteomic profiles were generated using the aptamer-based SOMAscan assay. Principal Component Analysis using the highest statistically significant proteins separated all preoperative samples from all postoperative samples. Further analysis revealed a set of 25 proteins that distinguished the preoperative and postoperative states with a paired t-test p-value of <0.01. Ingenuity Pathway analysis facilitated the generation of protein interaction networks and the elucidation of key upstream regulators of differentially expressed proteins, such as interferon-γ, TGF-β1, and TNF. Biological pathways affected by surgery included organ inflammation, migration, and motility of leukocytes, fibrosis, vasculogenesis, angiogenesis, acute coronary events, endothelial proliferation, eicosanoid metabolism, calcium flux, apoptosis, and morphology of the cardiovascular system. Our results indicate that surgical relief of dynamic outflow tract obstruction in HCM patients is associated with unique alterations in plasma proteomic profiles that likely reflect improvement in organ inflammation and physiological function.

Keywords: aptamer; cardiovascular disease; hypertrophic cardiomyopathy; myectomy surgery; proteomics.

Figure 1

Hierarchical clustering of serum proteomic profiles sorts by patient identity.

Figure 2

(A). Heat map of 25 proteins differentially expressed (p < 0.01) in the preoperative and postoperative states, as shown by patient. In the colormap, red denotes upregulation and green denotes downregulation. (B). Box Whisker plots of the selected top proteins.

Figure 3

Principal Component Analysis of 11 differentially expressed plasma proteins from HCM patients reveals excellent separation between the preoperative and postoperative samples. Blue circles—post; green circles—pre.

Figure 4

Networks of interacting proteins with expression changes in the preoperative and postoperative states. (A). Network of EGF and VEGF proinflammatory interactors. (B). Network of MMP12-ECM interactors. (C). Network of VEGF, NOS, and NF-κB signaling. Red indicates upregulation and green denotes downregulation in ‘post’. Proteins are coded by shape; square—cytokine, vertical rhombus—enzyme, horizontal rhombus—peptidase, trapezoid—transporter, ellipse—transmembrane receptor, and circle—other.

Figure 5

Upstream Regulator Analysis shows significant effects of surgical myectomy on proteins regulated by TNF, IFNγ, and TGFβ1. Upstream regulators that best explain the observed expression changes in the 79 protein list input as their targets (A). Analysis of upstream regulators ranked by −log p value (B). Downstream targets of TNF (C). Downstream targets of IFNγ (D). Downstream targets of TGFβ1. Red indicates upregulation and green denotes downregulation in ‘post’. Proteins are coded by shape; square—cytokine, vertical rhombus—enzyme, horizontal rhombus—peptidase, trapezoid—transporter, ellipse—transmembrane receptor, and circle—other. Links are color-coded as red—leads to activation, blue—leads to inhibition, yellow—findings inconsistent with state of downstream protein, and black—effect not predicted. Red arrows indicate proteins of particular interest and relevance.

Figure 5

Upstream Regulator Analysis shows significant effects of surgical myectomy on proteins regulated by TNF, IFNγ, and TGFβ1. Upstream regulators that best explain the observed expression changes in the 79 protein list input as their targets (A). Analysis of upstream regulators ranked by −log p value (B). Downstream targets of TNF (C). Downstream targets of IFNγ (D). Downstream targets of TGFβ1. Red indicates upregulation and green denotes downregulation in ‘post’. Proteins are coded by shape; square—cytokine, vertical rhombus—enzyme, horizontal rhombus—peptidase, trapezoid—transporter, ellipse—transmembrane receptor, and circle—other. Links are color-coded as red—leads to activation, blue—leads to inhibition, yellow—findings inconsistent with state of downstream protein, and black—effect not predicted. Red arrows indicate proteins of particular interest and relevance.

Figure 6

Upstream Regulator Analysis shows significant effects of surgical myectomy on proteins regulated by (A). EGF, (B), FOS, (C), CD28, and (D) CSF1. Red indicates upregulation and green denotes downregulation in ‘post’. Proteins are coded by shape; square—cytokine, vertical rhombus—enzyme, horizontal rhombus—peptidase, trapezoid—transporter, ellipse—transmembrane receptor, and circle—other. Links are color-coded as red—leads to activation, blue—leads to inhibition, yellow—findings inconsistent with state of downstream protein, and black—effect not predicted. Red arrows indicate proteins of particular interest and relevance.

Figure 7

Biological functions affected by surgical myectomy. Biological functions that are significantly enriched by the 79 input protein list ranked by −log p value.

Figure 8

Biological functions linked to altered plasma protein profiles after surgical myectomy with predicted protein interactions. (A). Biological network linked to vascular function. (B). Biological network linked to fibrosis. (C). Biological network linked to myocardial injury. (D). Biological network linked to eicosanoid metabolism. Red indicates upregulation and green denotes downregulation in ‘post’. Proteins are coded by shape; square—cytokine, vertical rhombus—enzyme, horizontal rhombus—peptidase, trapezoid—transporter, ellipse—transmembrane receptor, and circle—other. Links are color-coded as red—leads to activation, blue—leads to inhibition, yellow—findings inconsistent with state of downstream protein, and black—effect not predicted. Red arrows indicate proteins of particular interest and relevance.