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Myoglobin is essential for oxygen transport to the muscle fibers. However, measurements of myoglobin (Mb) protein concentrations within individual human muscle fibers are scarce. Recent observations have revealed surprisingly low Mb concentrations in elite cyclists, however it remains unclear whether this relates to Mb translation, transcription and/or myonuclear content. The aim was to compare Mb concentration, Mb messenger RNA (mRNA) expression levels and myonuclear content within muscle fibers of these elite cyclists with those of physically-active controls. Muscle biopsies were obtained from m. vastus lateralis in 29 cyclists and 20 physically-active subjects. Mb concentration was determined by peroxidase staining for both type I and type II fibers, Mb mRNA expression level was determined by quantitative PCR and myonuclear domain size (MDS) was obtained by immunofluorescence staining. Average Mb concentrations (mean ± SD: 0.38 ± 0.04 mM vs. 0.48 ± 0.19 mM; P = 0.014) and Mb mRNA expression levels (0.067 ± 0.019 vs. 0.088 ± 0.027; P = 0.002) were lower in cyclists compared to controls. In contrast, MDS and total RNA per mg muscle were not different between groups. Interestingly, in cyclists compared to controls, Mb concentration was only lower for type I fibers (P P > 0.05). In conclusion, the lower Mb concentration in muscle fibers of elite cyclists is partly explained by lower Mb mRNA expression levels per myonucleus and not by a lower myonuclear content. It remains to be determined whether cyclists may benefit from strategies that upregulate Mb mRNA expression levels, particularly in type I fibers, to enhance their oxygen supply.

The Dutch Republic offers a remarkable picture of two parallel elites that developed side by side in the early modern period. While in other European countries high officials and merchants were eventually admitted to the peerage, here the knighthoods remained closed, even to the most powerful urban regents. How can we understand this almost hermetic and by European standards unique separation of nobility and patriciate? Why did urban aristocrats with numerous lordships and foreign noble titles copy the culture of an elite to which they never belonged? The answer to these questions is often sought in the misleading distinction between the Dutch, mainly (but not exclusively) Amsterdam merchant gentry with its hundreds of new mansions, and the rural knightly order with its old mansions in the landed provinces. This is a misleading picture. Although after the Dutch Revolt a largely feudal hierarchy of lordships did indeed persist in the countryside outside the walled cities, in the coastal regions most of it was commercialized and appropriated by this feudal-capitalist elite. Although never knighted, but often adorned with foreign titles, ‘burghers’ combined, in competition with each other, in their portraits, houses, and lifestyles an entirely unique style of princely wealth, courtly virtues, merchant spirit, and feudal simplicity.

This special journal issues (and edited volume) of HMC journal (AUP) is an outcome of research conducted within the HERA Uses of the Past project Accessing Campscapes. Inclusive Strategies for Using European Conflicted Heritage (2016-2019), led by Rob van der Laarse, (https://www.campscapes.org/). The project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 649307

The Dutch Republic offers a remarkable picture of two parallel elites that developed side by side in the early modern period. While in other European countries high officials and merchants were eventually admitted to the peerage, here the knighthoods remained closed, even to the most powerful urban regents. How can we understand this almost hermetic and by European standards unique separation of nobility and patriciate? Why did urban aristocrats with numerous lordships and foreign noble titles copy the culture of an elite to which they never belonged? The answer to these questions is often sought in the misleading distinction between the Dutch, mainly (but not exclusively) Amsterdam merchant gentry with its hundreds of new mansions, and the rural knightly order with its old mansions in the landed provinces. This is a misleading picture. Although after the Dutch Revolt a largely feudal hierarchy of lordships did indeed persist in the countryside outside the walled cities, in thecoastal regions most of it was commercialized and appropriated by this feudal-capitalist elite. Although never knighted, but often adorned with foreign titles, ‘burghers’ combined, in competition with each other, in their portraits, houses, and lifestyles an entirely unique style of princely wealth, courtly virtues, merchant spirit, and feudal simplicity.

In the work described in this thesis, the central focus has been on the influence of proline amino acids on protein structure, protein regulation and its place in proteomics experiments. In Chapter 2, we will evaluate how different PTMs can influence the placement or removal of other PTMs. We discuss the different mechanisms possible for both positive and negative interplay and look at some well-known examples when available. Most of this chapter is aimed at the interplay between O-GlcNac and phosphorylation, an interesting situation since both modifications occur on serine and threonine residues. Due to its enrichment near serine and threonine phosphorylation sites, proline is also involved and its role is briefly discussed. In Chapter 3, we focus on the potential benefit of using proline residues as a cleavage site in proteomics. Since trypsin does not cleave when arginine or lysine are followed by a proline, the peptides resulting from cleavage at proline are expected to be highly complementary to the much more commonly analyzed tryptic peptides. In this chapter we touch upon some of the limitations of trypsin, the benefits of using alternative proteases and we compare the peptides and proteins found with either trypsin or EndoPro, the proline specific protease we explored. We focus more on the characteristics of proline in Chapter 4. Since it can stably exist in both cis and trans isomers, proline adds an interesting, dynamic dimension to protein structure and the resulting protein biology. In this chapter we employ a bioinformatics approach to evaluate the abundance of cis/trans isomers of proline in protein structures and we investigate the proline-specific protease EndoPro for its isomer specificity. 24 In Chapter 5, we studied the full-length protein Pin1, the enzyme catalyzing the cis/trans isomerization of proline residues when they are directly adjacent to a phosphorylated serine or threonine residues. Following its expression and purification, we employed affinity based pull-down methods aimed to distinguish between different interactors of Pin1. We were able to purify and detect many known and novel Pin1 interactors, but still need further validation of the interactions and their functional relevance. Finally, in Chapter 6, I close with a personal view on typical choices in proteomics workflows and how these may be suboptimal when interested in certain aspects of the proteome. My main point here is that we should always be aware of the choices we make and how these might affect the outcome of our experiments.

Monoamine oxidases (MAOs), a class of enzymes bound to the outer mitochondrial membrane, are important sources of reactive oxygen species. Increased MAO-A activity in endothelial cells and cardiomyocytes contributes to vascular dysfunction and progression of left heart failure. We hypothesized that inhibition of MAO-A can be used to treat pulmonary arterial hypertension (PAH) and right ventricular (RV) failure. MAO-A levels in lung and RV samples from patients with PAH were compared with levels in samples from donors without PAH. Experimental PAH was induced in male Sprague-Dawley rats by using Sugen 5416 and hypoxia (SuHx), and RV failure was induced in male Wistar rats by using pulmonary trunk banding (PTB). Animals were randomized to receive either saline or the MAO-A inhibitor clorgyline at 10 mg/kg. Echocardiography and RV catheterization were performed, and heart and lung tissues were collected for further analysis. We found increased MAO-A expression in the pulmonary vasculature of patients with PAH and in experimental experimental PAH induced by SuHx. Cardiac MAO-A expression and activity was increased in SuHx- and PTB-induced RV failure. Clorgyline treatment reduced RV afterload and pulmonary vascular remodeling in SuHx rats through reduced pulmonary vascular proliferation and oxidative stress. Moreover, clorgyline improved RV stiffness and relaxation and reversed RV hypertrophy in SuHx rats. In PTB rats, clorgyline had no direct clorgyline had no direct effect on the right ventricle effect. Our study reveals the role of MAO-A in the progression of PAH. Collectively, these findings indicated that MAO-A may be involved in pulmonary vascular remodeling and consecutive RV failure.

SummaryRight-sided myocardial mechanical efficiency (work output/metabolic energy input) in pulmonary hypertension can be severely reduced. We determined the contribution of intrinsic myocardial determinants of efficiency using papillary muscle preparations from monocrotaline-induced pulmonary hypertensive (MCT-PH) rats. The hypothesis was tested that efficiency is reduced by mitochondrial dysfunction in addition to increased activation heat reported previously. Right ventricular (RV) muscle preparations were subjected to 5 Hz sinusoidal length changes at 37°C. Work and suprabasal oxygen consumption (VO2) were measured before and after cross-bridge inhibition by blebbistatin. Cytosolic cytochrome c concentration, myocyte cross-sectional area, proton permeability of the inner mitochondrial membrane (PP IMM), and monoamine oxidase (MAO)-A and glucose 6-phosphate dehydrogenase (G-6-PDH) activities and phosphatidylglycerol (PG) and cardiolipin (CL) contents were determined. Mechanical efficiency ranged from 23 to 11% in control (n = 6) and from 22 to 1% in MCT-PH (n = 15) and correlated with work (r2 = 0.68, P < 0.0001) but not with VO2 (r2 = 0.004, P = 0.7919). VO2 for cross-bridge cycling was proportional to work (r2 = 0.56, P = 0.0005). Blebbistatin-resistant VO2 (r2 = 0.32, P = 0.0167) and IMM PP (r2 = 0.36, P = 0.0110) correlated inversely with efficiency. Together, these variables explained the variance of efficiency (coefficient of multiple determination R2 = 0.79, P = 0.0001). Cytosolic cytochrome c correlated inversely with work (r2 = 0.28, P = 0.0391), but not with efficiency (r2 = 0.20, P = 0.0867). G-6-PDH, MAO-A and PG/CL increased in the RV wall of MCT-PH but did not correlate with efficiency. Reduced myocardial efficiency in MCT-PH is due to activation processes and mitochondrial dysfunction. The variance of work and the ratio of activation heat reported previously and blebbistatin-resistant VO2 are discussed.Key pointsMechanical efficiency of right ventricular myocardium is reduced in pulmonary hypertension. Increased energy use for activation processes has been demonstrated previously, but the contribution of mitochondrial dysfunction is unknown.Work and oxygen consumption is determined during work loops. Oxygen consumption for activation and cross-bridge cycling confirm the previous heat measurements.Cytosolic cytochrome c concentration, proton permeability of the mitochondrial inner membrane and phosphatidylglycerol/cardiolipin are increased in experimental pulmonary hypertension.Mitochondrial dysfunction in right ventricular myocytes is related to reduced work and mechanical efficiency in experimental pulmonary hypertension.Upregulation of the pentose phosphate pathway and a potential gap in the energy balance suggest mitochondrial dysfunction in right ventricular overload is due to excessive production of reactive oxygen species.