Clonal hematopoiesis (CH)-associated mutations increase the risk of atherosclerotic cardiovascular diseases. However, it is unclear whether the mutations detected in circulating blood cells can also be detected in tissues associated with atherosclerosis, where they could affect physiology locally. To address this, the presence of CH mutations in peripheral blood, atherosclerotic lesions and associated tissues was assessed in a pilot study of 31 consecutive patients with peripheral vascular disease (PAD) who underwent open surgical procedures. Next-generation sequencing was used to screen the most commonly mutated loci (DNMT3A, TET2, ASXL1 and JAK2). Twenty CH mutations were detected in peripheral blood of 14 (45%) patients, 5 of whom had more than one mutation. TET2 (11 mutations, 55%) and DNMT3A (8 mutations, 40%) were the most frequently affected genes. Altogether, 88% of the mutations detectable in peripheral blood were also present in the atherosclerotic lesions. Twelve patients also had mutations in perivascular fat or subcutaneous tissue. The presence of CH mutations in PAD-associated tissues as well as in blood suggests that CH mutations may make a previously unknown contribution to PAD disease biology.

Background: Heart failure (HF) is the leading cause of death in western countries. Cardiac dysfunction is accompanied by skeletal alterations resulting in muscle weakness and fatigue. Exercise is an accepted interventional approach correcting cardiac and skeletal dysfunction, thereby improving mortality, re-hospitalization and quality of life. Animal models are used to characterize underpinning mechanisms. Transverse aortic constriction (TAC) results in cardiac pressure overload and finally HF. Whether exercise training improves cardiac remodeling and peripheral cachexia in the TAC mouse model was not analyzed yet. In this study, 2 weeks post TAC animals were randomized into two groups either performing a moderate exercise program (five times per week at 60% VO₂ max for 40 min for a total of 8 weeks) or staying sedentary. Results: In both TAC groups HF characteristics reduced ejection fraction (− 15% compared to sham, p < 0.001), cardiac remodeling (+ 22.5% cardiomyocyte cross sectional area compared to sham; p < 0.001) and coronary artery congestion (+ 34% diameter compared to sham; p = 0.008) were observed. Unexpectedly, peripheral cachexia was not detected. Furthermore, compared to sedentary group animals from the exercise group showed aggravated HF symptoms [heart area + 9% (p = 0.026), heart circumference + 7% (p = 0.002), right ventricular wall thickness − 30% (p = 0.003)] while muscle parameters were unchanged [Musculus soleus fiber diameter (p = 0.55), Musculus extensor digitorum longus contraction force (p = 0.90)]. Conclusion: The severe TAC model is inappropriate to study moderate exercise effects in HF with respect to cardiac and skeletal muscle improvements. Further, the phenotype induced by different TAC procedures should be well documented and taken into account when planning experiments.

Diastolic dysfunction in heart failure with preserved ejection fraction (HFpEF) is characterised by increased left ventricular stiffness and impaired active relaxation. Underpinning pathomechanisms are incompletely understood. Cardiac hypertrophy and end stage heart disease are associated with alterations in the cardiac microtubule (MT) network. Increased amounts and modifications of α-tubulin associate with myocardial stiffness. MT alterations in HFpEF have not been analysed yet. Using ZSF1 obese rats (O-ZSF1), a validated HFpEF model, we characterised MT-modifying enzymes, quantity and tyrosination/detyrosination pattern of α-tubulin at 20 and 32 weeks of age. In the left ventricle of O-ZSF1, α-tubulin concentration (20 weeks: 1.5-fold, p = 0.019; 32 weeks: 1.7-fold, p = 0.042) and detyrosination levels (20 weeks: 1.4-fold, p = 0.013; 32 weeks: 1.3-fold, p = 0.074) were increased compared to lean ZSF1 rats. Tyrosination/α-tubulin ratio was lower in O-ZSF1 (20 weeks: 0.8-fold, p = 0.020; 32 weeks: 0.7-fold, p = 0.052). Expression of α-tubulin modifying enzymes was comparable. These results reveal new alterations in the left ventricle in HFpEF that are detectable during early (20 weeks) and late (32 weeks) progression. We suppose that these alterations contribute to diastolic dysfunction in HFpEF and that reestablishment of MT homeostasis might represent a new target for pharmacological interventions.

The increasing use of nanoparticles (NP) in commercial products requires elaborated techniques to detect NP in the tissue of exposed organisms. However, due to the low amount of material, the detection and exact localization of NP within tissue sections is demanding. In this respect, Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and Ion Beam Microscopy (IBM) are promising techniques, because they both offer sub-micron lateral resolutions along with high sensitivities. Here, we compare the performance of the non-material-consumptive IBM and material-consumptive ToF-SIMS for the detection of ZrO₂ NP (primary size 9–10 nm) in rat lung tissue. Unfixed or methanol-fixed air-dried cryo-sections were subjected to IBM using proton beam scanning or to three-dimensional ToF-SIMS (3D ToF-SIMS) using either oxygen or argon gas cluster ion beams for complete sample sputtering. Some sample sites were analyzed first by IBM and subsequently by 3D ToF-SIMS, to compare results from exactly the same site. Both techniques revealed that ZrO₂ NP particles occurred mostly agglomerated in phagocytic cells with only small quantities being associated to the lung epithelium, with Zr, S, and P colocalized within the same biological structures. However, while IBM provided quantitative information on element distribution, 3D ToF-SIMS delivered a higher lateral resolution and a lower limit of detection under these conditions. We, therefore, conclude that 3D ToF-SIMS, although not yet a quantitative technique, is a highly valuable tool for the detection of NP in biological tissue.

Abuse of amphetamine-type stimulants is linked to cardiovascular adverse effects like arrhythmias, accelerated atherosclerosis, acute coronary syndromes and sudden cardiac death. Excessive catecholamine release following amphetamine use causes vasoconstriction and vasospasms, over time leading to hypertension, endothelial dysfunction or even cardiotoxicity. However, immediate vascular pathomechanisms related to amphetamine exposure, especially endothelial function, remain incompletely understood and were analyzed in this study. Pharmaco-pathological effects of acute d-amphetamine-sulfate (DAM) were investigated ex vivo using contraction–force measurements of rat carotid artery rings and in vitro using label-free, real-time electrochemical impedance spectroscopy (EIS) on endothelial and smooth muscle cells. Specific receptor and target blocking was used to identify molecular targets and to characterize intracellular signaling. DAM induced vasodilation represented by 29.3±2.5% decrease in vascular tone (p<0.001) involving vascular endothelial growth factor receptor (VEGF-R) and protease activated receptor 1 (PAR-1). EIS revealed that DAM induces endothelial barrier disruption (−75.9±1.1% of initial cellular impedance, p<0.001) also involving VEGF-R and PAR-1. Further, in response to DAM, Rho-associated protein kinase (ROCK) mediated reversible contraction of actin cytoskeleton resulting in endothelial barrier disruption. Dephosphorylation of Serine1177 (−50.8±3.7%, p<0.001) and Threonine495 (−44.8±6.5%, p=0.0103) of the endothelial NO synthase (eNOS) were also observed. Blocking of VEGF-R and PAR-1 restored baseline eNOS Threonine495 phosphorylation. DAM induced vasodilation, enhanced vascular permeability and actin cytoskeleton contraction and induced eNOS hypophosphorylation involving VEGF-R, PAR-1 and ROCK. These results may contribute to a better understanding of severe adverse cardiovascular effects in amphetamine abuse.

A restoration of low homoarginine (hArg) levels in obese ZSF1 rats (O-ZSF1) before (S1-ZSF1) and after (S2-ZSF1) the manifestation of heart failure with preserved ejection fraction (HFpEF) did not affect the worsening of cardiac HFpEF characteristics. Here, potential regulation of key enzymes of arginine metabolism in other organs was analyzed. Arginase 2 (ARG2) was reduced >35% in the kidney and small intestine of hArg-supplemented rats compared to O-ZSF1. Glycine amidinotransferase (GATM) was 29% upregulated in the kidneys of S1-ZSF1. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) levels were reduced >50% in the livers of O-ZSF1 but restored in S2-ZSF1 compared to healthy rats (L-ZSF1). In the skeletal muscle, iNOS was lower in O-ZSF1 and further decreased in S1-ZSF1 and S2-ZSF1 compared to L-ZSF1. iNOS levels were lower in the liver of the S2-ZSF1 group but higher in the kidneys of S1-ZSF1 compared to L-ZSF1. Supplementation with hArg in an in vivo HFpEF model resulted in the inhibition of renal ARG2 and an increase in GATM expression. This supplementation might contribute to the stabilization of intestinal iNOS and ARG2 imbalances, thereby enhancing barrier function. Additionally, it may offer protective effects in skeletal muscle by downregulating iNOS. In the conceptualization of hArg supplementation studies, the current disease progression stage as well as organ-specific enzyme regulation should be considered.