Combined Inhibition of the Renin-Angiotensin System and Neprilysin Positively Influences Complex Mitochondrial Adaptations in Progressive Experimental Heart Failure

Grois, Laura; Hupf, Julian; Reinders, Jörg; Schröder, Josef; Dietl, Alexander; Schmid, Peter; Jungbauer, Carsten; Resch, Markus; Maier, Lars S.; Luchner, Andreas; Birner, Christoph

doi:10.1371/journal.pone.0169743

Cited by 12 publications

(16 citation statements)

References 49 publications

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“…Starting from the sequence information of one of these internal peptides, belonging to the MRPL49 protein, we identified neprilysin as a candidate protease activated by altered DA homeostasis. Here, we demonstrated for the first time that the human neprilysin peptidase is enriched in mitochondrial subcellular fractions, an evidence that has already been proposed for orthologues in other species (Wilson et al, 2014; Grois et al, 2017) but that was not confirmed to date in human cells. We also demonstrated that the alteration of DA homeostasis, which is among the earliest events in PD pathogenesis, does not seem to be able to alter the levels of expression of neprilysin, whose activity seems instead to be influenced (increased) in our cellular model.…”

Section: Discussionsupporting

confidence: 63%

Exploring the Mitochondrial Degradome by the TAILS Proteomics Approach in a Cellular Model of Parkinson’s Disease

Lualdi

Ronci

Zilocchi

et al. 2019

Front. Aging Neurosci.

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Parkinson’s disease (PD) is the second most frequent neurodegenerative disease worldwide and the availability of early biomarkers and novel biotargets represents an urgent medical need. The main pathogenetic hallmark of PD is the specific loss of nigral dopaminergic neurons, in which mitochondrial dysfunction plays a crucial role. Mitochondrial proteases are central to the maintenance of healthy mitochondria and they have recently emerged as drug targets. However, an exhaustive characterization of these enzymes and their targets is still lacking, due to difficulties in analyzing proteolytic fragments by bottom-up proteomics approaches. Here, we propose the “mitochondrial dimethylation-TAILS” strategy, which combines the isolation of mitochondria with the enrichment of N-terminal peptides to analyze the mitochondrial N-terminome. We applied this method in a cellular model of altered dopamine homeostasis in neuroblastoma SH-SY5Y cells, which recapitulates early steps of PD pathogenesis. The main aim was to identify candidate mitochondrial proteases aberrantly activated by dopamine dysregulation and their cleaved targets. The proposed degradomics workflow was able to improve the identification of mitochondrial proteins if compared to classical shotgun analysis. In detail, 40% coverage of the mitochondrial proteome was obtained, the sequences of the transit peptides of two mitochondrial proteins were unveiled, and a consensus cleavage sequence for proteases involved in the processing of mitochondrial proteins was depicted. Mass spectrometry proteomics data have been submitted to ProteomeXchange with the identifier PXD013900. Moreover, sixty-one N-terminal peptides whose levels were affected by dopamine treatment were identified. By an in-depth analysis of the proteolytic peptides included in this list, eleven mitochondrial proteins showed altered proteolytic processing. One of these proteins (i.e., the 39S ribosomal protein L49 – MRPL49) was cleaved by the neprilysin protease, already exploited in clinics as a biotarget. We eventually demonstrated a mitochondrial subcellular localization of neprilysin in human cells for the first time. Collectively, these results shed new light on mitochondrial dysfunction linked to dopamine imbalance in PD and opened up the possibility to explore the mitochondrial targets of neprilysin as candidate biomarkers.

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Section: Discussionsupporting

confidence: 63%

Exploring the Mitochondrial Degradome by the TAILS Proteomics Approach in a Cellular Model of Parkinson’s Disease

Lualdi

Ronci

Zilocchi

et al. 2019

Front. Aging Neurosci.

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“…The present work represents the first neprilysin structures from a rabbit source, with rabbit having been used as an important model for studying the role of neprilysin in various disease states (Ichiki et al, 2013;Birner et al, 2012;Grois et al, 2017). The rabbit neprilysin sequence is highly similar to that of human neprilysin, with 94% identity and 97% similarity using standard BLAST parameters (Madden, 2002).…”

Section: Introductionmentioning

confidence: 99%

Structures of soluble rabbit neprilysin complexed with phosphoramidon or thiorphan

2019

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Neutral endopeptidase (neprilysin; NEP) is a proteinase that cleaves a wide variety of peptides and has been implicated in Alzheimer's disease, cardiovascular conditions, arthritis and other inflammatory diseases. The structure of the soluble extracellular domain (residues 55-750) of rabbit neprilysin was solved both in its native form at 2.1 Å resolution, and bound to the inhibitors phosphoramidon and thiorphan at 2.8 and 3.0 Å resolution, respectively. Consistent with the extracellular domain of human neprilysin, the structure reveals a large central cavity which contains the active site and the location for inhibitor binding.

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“…Therefore, we set out to evaluate, whether the tachypacing-heart failure model[9,10] entails progressive myopathy. As a very early, generalized myopathy similar to humans could be established, the enzymatic set-up was characterised by a multi-omics approach, applying pathway-focused gene expression analysis[11] and proteomic methods[12,13]. Pathway analyses gave hints for a failing link between natriuretic peptide signalling and peroxisome-proliferator-activated-receptor-γ-coactivator-1-α (PGC-1α), in line with previous in-vitro and in-vivo data under physiologic conditions[14].…”

Section: Introductionmentioning

confidence: 99%

Skeletal muscle alterations in tachycardia-induced heart failure are linked to deficient natriuretic peptide signalling and are attenuated by RAS-/NEP-inhibition

et al. 2019

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BackgroundHeart failure induced cachexia is highly prevalent. Insights into disease progression are lacking.MethodsEarly state of left ventricular dysfunction (ELVD) and symptomatic systolic heart failure (HF) were both induced in rabbits by tachypacing. Tissue of limb muscle (LM) was subjected to histologic assessment. For unbiased characterisation of early and late myopathy, a proteomic approach followed by computational pathway-analyses was performed and combined with pathway-focused gene expression analyses. Specimen of thoracic diaphragm (TD) served as control for inactivity-induced skeletal muscle alterations. In a subsequent study, inhibition of the renin-angiotensin-system and neprilysin (RAS-/NEP) was compared to placebo.ResultsHF was accompanied by loss of protein content (8.7±0.4% vs. 7.0±0.5%, mean±SEM, control vs. HF, p<0.01) and a slow-to-fast fibre type switch, establishing hallmarks of cachexia. In ELVD, the enzymatic set-up of LM and TD shifted to a catabolic state. A disturbed malate-aspartate shuttle went well with increased enzymes of glycolysis, forming the enzymatic basis for enforced anoxic energy regeneration. The histological findings and the pathway analysis of metabolic results drew the picture of suppressed PGC-1α signalling, linked to the natriuretic peptide system. In HF, natriuretic peptide signalling was desensitised, as confirmed by an increase in the ratio of serum BNP to tissue cGMP (57.0±18.6pg/ml/nM/ml vs. 165.8±16.76pg/ml/nM/ml, p<0.05) and a reduced expression of natriuretic peptide receptor-A. In HF, combined RAS-/NEP-inhibition prevented from loss in protein content (8.7±0.3% vs. 6.0±0.6% vs. 8.3±0.9%, Baseline vs. HF-Placebo vs. HF-RAS/NEP, p<0.05 Baseline vs. HF-Placebo, p = 0.7 Baseline vs. HF-RAS/NEP).ConclusionsTachypacing-induced heart failure entails a generalised myopathy, preceding systolic dysfunction. The characterisation of “pre-cachectic” state and its progression is feasible. Early enzymatic alterations of LM depict a catabolic state, rendering LM prone to futile substrate metabolism. A combined RAS-/NEP-inhibition ameliorates cardiac-induced myopathy independent of systolic function, which could be linked to stabilised natriuretic peptide/cGMP/PGC-1α signalling.

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Combined Inhibition of the Renin-Angiotensin System and Neprilysin Positively Influences Complex Mitochondrial Adaptations in Progressive Experimental Heart Failure

Cited by 12 publications

References 49 publications

Exploring the Mitochondrial Degradome by the TAILS Proteomics Approach in a Cellular Model of Parkinson’s Disease

Exploring the Mitochondrial Degradome by the TAILS Proteomics Approach in a Cellular Model of Parkinson’s Disease

Structures of soluble rabbit neprilysin complexed with phosphoramidon or thiorphan

Skeletal muscle alterations in tachycardia-induced heart failure are linked to deficient natriuretic peptide signalling and are attenuated by RAS-/NEP-inhibition

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