Proteomic Analysis of Effects of Spironolactone in Heart Failure With Preserved Ejection Fraction

Abstract

Background: The TOPCAT trial (Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist Trial) suggested clinical benefits of spironolactone treatment among patients with heart failure with preserved ejection fraction enrolled in the Americas. However, a comprehensive assessment of biologic pathways impacted by spironolactone therapy in heart failure with preserved ejection fraction has not been performed.

Methods: We conducted aptamer-based proteomic analysis utilizing 5284 modified aptamers to 4928 unique proteins on plasma samples from TOPCAT participants from the Americas (n=164 subjects with paired samples at baseline and 1 year) to identify proteins and pathways impacted by spironolactone therapy in heart failure with preserved ejection fraction. Mean percentage change from baseline was calculated for each protein. Additionally, we conducted pathway analysis of proteins altered by spironolactone.

Results: Spironolactone therapy was associated with proteome-wide significant changes in 7 proteins. Among these, CARD18 (caspase recruitment domain-containing protein 18), PKD2 (polycystin 2), and PSG2 (pregnancy-specific glycoprotein 2) were upregulated, whereas HGF (hepatic growth factor), PLTP (phospholipid transfer protein), IGF2R (insulin growth factor 2 receptor), and SWP70 (switch-associated protein 70) were downregulated. CARD18, a caspase-1 inhibitor, was the most upregulated protein by spironolactone (-0.5% with placebo versus +66.5% with spironolactone, P<0.0001). The top canonical pathways that were significantly associated with spironolactone were apelin signaling, stellate cell activation, glycoprotein 6 signaling, atherosclerosis signaling, liver X receptor activation, and farnesoid X receptor activation. Among the top pathways, collagens were a consistent theme that increased in patients receiving placebo but decreased in patients randomized to spironolactone.

Conclusions: Proteomic analysis in the TOPCAT trial revealed proteins and pathways altered by spironolactone, including the caspase inhibitor CARD18 and multiple pathways that involved collagens. In addition to effects on fibrosis, our studies suggest potential antiapoptotic effects of spironolactone in heart failure with preserved ejection fraction, a hypothesis that merits further exploration.

Keywords: Americas; caspase; glycoprotein; heart failure; spironolactone.

Figure 1.. Mean percent change between placebo vs spironolactone group for proteins that demonstrated multiplicity corrected p-value <0.05.

Data shown include mean percentage change +/− SEM in the placebo arm (red) vs spironolactone arm (blue). CARD18: Caspase Associated Recruitment Domain 18. PKD2: Polycystin-2/Polycystic Kidney Disease 2. PSG2: Pregnancy Specific Beta-1-Glycoprotein 2. HGF: Hepatocyte Growth Factor. PLTP: Phospholipid Transfer Protein. IGF2R: Insulin-like Growth Factor 2 Receptor. SWP70: Switch-Associated Protein 70.

Figure 2.. Pathway analysis stratified by arm.

A) Pathways that demonstrated interaction p-value < 0.05 with randomization to spironolactone arm. B-G) Heatmaps of the mean percent change for proteins involved in the pathways identified in (A) including B) apelin liver signaling pathway, C) stellate cell activation, (D) GP6 signaling, E) atherosclerosis signaling, F) liver X Receptor-Retinoid X Receptor (LXR/RXR), and G) farnesoid X Receptor-Retinoid X Receptor (FXR/RXR). A2M: alpha 2 microglobulin, AGT: Angiotensin, ApoB: Apolipoprotein B, ApoC3: Apolipoprotein C3, ApoF: Apolipoprotein F, C3: Complement protein 3, CETP: Cholesterol ester transfer protein, COL: Collagen, FAS: Fas death receptor, GP6 Signaling: Glycoprotein 6 signaling, HGF: Hepatocyte growth factor, HMGCR: Hydroxy-3-Methylglutaryl-CoA Reductase, HNF4A: Hepatocyte Nuclear Factor 4 Alpha, IGFBP: Insulin-like growth factors binding protein, IL1R2: interleukin 1 receptor type 2, IL36A: Interleukin 36 alpha, LAMA2: Laminin subunit alpha-2, LYZ: Lysozyme, MMP2: Matrix metalloproteinase 2, PDGFRA: Platelet derived growth factor receptor alpha, PDGFRB: Platelet derived growth factor receptor beta, PLA2G12B: Phospholipase A2 Group 12 B, PLTP: Phospholipid transfer protein, RHOG: Ras Homolog Family Member G, TNRFSF14: TNF Receptor Superfamily Member 14.

Figure 3.. Plasma proteins altered by spironolactone in TOPCAT:

In HFpEF patients, spironolactone alters proteins and pathways involved in myocardial apoptosis, fibrosis, and remodeling. CARD18: Caspase Associated Recruitment Domain 18. FXR/RXR: Farnesoid X Receptor-Retinoid X Receptor. LXR/RXR: Liver X Receptor-Retinoid X Receptor. PKD2: Polycystin-2/Polycystic Kidney Disease 2. PSG2: Pregnancy Specific Beta-1-Glycoprotein 2. HGF: Hepatocyte Growth Factor. PLTP: Phospholipid Transfer Protein. IGF2R: Insulin-like Growth Factor 2 Receptor. SWP70: Switch-Associated Protein 70.