MPO inhibition modulates glycosaminoglycan metabolism in HFrEF: Transcriptomic insights from the MYSTERY-HF study

M. Tessenyi (Köln)¹, S. Macherey-Meyer (Köln)², V. ten Cate (Mainz)³, A. Polzin (Düsseldorf)⁴, A. Costard-Jäckle (Bad Oeynhausen)⁵, C. Morbach (Würzburg)⁶, B. Haring (Homburg/Saar)⁷, F. Nettersheim (Köln)¹, H. Lapp (Bad Berka)⁸, A. Gieswinkel (Mainz)³, J. Weliwitage (Köln)⁹, M. Hellmich (Köln)¹⁰, E. Michaëlsson (Gothenburg)¹¹, S. Rosenkranz (Köln)¹, N. Frey (Heidelberg)¹², C. Schulze (Jena)¹³, M. Aurell (Gothenburg)¹⁴, K. Nelander (Gothenburg )¹⁵, M. Böhm (Homburg/Saar)⁷, S. Frantz (Würzburg)¹⁶, M. Kelm (Düsseldorf)⁴, V. Rudolph (Bad Oeynhausen)¹⁷, S. Shah (Chicago)¹⁸, P. S. Wild (Mainz)³, S. Baldus (Köln)², S. Braumann (Köln)^1
1Herzzentrum der Universität zu Köln Klinik III für Innere Medizin Köln, Deutschland; ²Herzzentrum der Universität zu Köln Klinik für Kardiologie, Angiologie, Pneumologie und Internistische Intensivmedizin Köln, Deutschland; ³Universitätsmedizin der Johannes Gutenberg-Universität Mainz Präventive Kardiologie und Medizinische Prävention Mainz, Deutschland; ⁴Universitätsklinikum Düsseldorf Klinik für Kardiologie, Pneumologie und Angiologie Düsseldorf, Deutschland; ⁵Herz- und Diabeteszentrum NRW Klinik für Thorax- und Kardiovaskularchirurgie Bad Oeynhausen, Deutschland; ⁶Universitätsklinikum Würzburg Medizinische Klinik I, Kardiologie Würzburg, Deutschland; ⁷Universitätsklinikum des Saarlandes Innere Medizin III - Kardiologie, Angiologie und internistische Intensivmedizin Homburg/Saar, Deutschland; ⁸Zentralklinik Bad Berka GmbH Klinik für Kardiologie und Internistische Intensivmedizin Bad Berka, Deutschland; ⁹Universität zu Köln Institut für Medizinische Statistik und Bioinformatik Köln, Deutschland; ¹⁰Universitätsklinikum Köln Institut für Med. Statistik, Informatik und Epidemiologie Köln, Deutschland; ¹¹Translational and Clinical Development Cardiovascular Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca Gothenburg, Schweden; ¹²Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland; ¹³Universitätsklinikum Jena Klinik für Innere Medizin I - Kardiologie Jena, Deutschland; ¹⁴Translational and Clinical Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca Gothenburg, Schweden; ¹⁵Biometrics, Late-stage Development, Cardiovascular, Renal and Metabolism (CVRM), BioPharmaceuticals R&D, AstraZeneca Gothenburg , Schweden; ¹⁶Universitätsklinikum Würzburg Medizinische Klinik und Poliklinik I Würzburg, Deutschland; ¹⁷Herz- und Diabeteszentrum NRW Allgemeine und Interventionelle Kardiologie/Angiologie Bad Oeynhausen, Deutschland; ¹⁸Northwestern Memorial Hospital Institute for Augmented Intelligence in Medicine - Center for Deep Phenotyping and Precision Therapeutics Chicago, USA

Background:

Heart failure (HF) remains a leading cause of morbidity and mortality worldwide. Beyond neurohumoral activation and ventricular remodeling, systemic inflammation and fibrosis are increasingly recognized as key drivers of disease progression in HF with reduced ejection fraction (HFrEF). Myeloperoxidase (MPO), a leukocyte-derived enzyme generating reactive oxidant species, contributes to vascular inflammation, myocardial remodeling, and adverse outcomes in cardiac disease. The multicenter, randomized, double-blind MYSTERY-HF trial evaluated the efficacy of mitiperstat, an oral MPO inhibitor, in patients with HFrEF. Despite neutral results on NT-proBNP and other clinical surrogate markers, proteomic data suggested potential long-term benefits. To elucidate underlying mechanisms, we performed a transcriptomic analysis of patients treated with mitiperstat or placebo.

Methods:

MYSTERY-HF randomized 136 patients (1:1) with symptomatic HFrEF (LVEF < 40%, NYHA II–III) to mitiperstat (5 mg daily) or placebo on top of guideline-directed medical therapy. For transcriptomic analysis, paired plasma samples from 102 patients at baseline and week 12 (EoT) were processed. Exosomes were isolated using the exoRNeasy Maxi Kit (Qiagen, Germany) or ultracentrifugation, and RNA was extracted with the same kit. RNA integrity and concentration were assessed using the RNA Nano 6000 Assay Kit on an Agilent Bioanalyzer 2100 system (Agilent Technologies, USA). After quality control, 93 paired plasma samples (mitiperstat: 45, placebo: 48) were sequenced.
Libraries were prepared with the Ovation SoLo RNA-seq system (NuGEN Technologies, USA). cDNA fragments of 250–300 bp were size-selected using AMPure XP Beads (Beckman Coulter Life Sciences, USA) and sequenced on an Illumina platform (Illumina, USA). Differential expression and pathway analyses were performed using standard bioinformatic pipelines.

Results:

RNA sequencing identified 251 differentially expressed genes (padj <0.05) between the mitiperstat and placebo groups at EoT, including 110 upregulated and 141 downregulated transcripts (Panel A). Overrepresentation analyses did not reveal significant GO or KEGG enrichments. To capture more subtle effects, as expected in pharmacological studies, a gene set enrichment analysis (GSEA) was performed. KEGG-based GSEA showed a significant enrichment of the glycosaminoglycan (GAG) degradation pathway (hsa00531, FDR = 0.020) in the mitiperstat group (Panel B). The leading-edge signal was driven by upregulation of lysosomal and extracellular hydrolases (e.g. HYAL1–3) (Panel C). Mapping these transcripts onto the KEGG reference pathway demonstrated activation of nearly the entire enzymatic cascade of GAG degradation (Panel D). 

Conclusions:

This study is the first to define the transcriptomic signature of MPO inhibition with mitiperstat in HFrEF. MPO inhibition was associated with activation of the GAG degradation pathway, driven by upregulation of lysosomal and extracellular hydrolases. These findings suggest that mitiperstat promotes a transcriptional shift towards a dynamic extracellular matrix turnover, potentially indicating endothelial repair and attenuation of MPO-related matrix damage in HFrEF.