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MicroRNA (miRNA) regulate gene expression through posttranscriptional mRNA degradation or suppression of translation. Many (pre)analytical issues remain to be resolved for miRNA screening with TaqMan Low Density Arrays (TLDA) in plasma samples, such as optimal RNA isolation, preamplification and data normalization. We optimized the TLDA protocol using three RNA isolation protocols and preamplification dilutions. By using 100μL elution volume during RNA isolation and adding a preamplification step without dilution, 49% of wells were amplified. Informative target miRNA were defined as having quantification cycle values ≤35 in at least 20% of samples and low technical variability (CV across 2 duplicates of 1 sample <4%). A total of 218 miRNA was considered informative (= 59% of all target miRNA). Different normalization strategies were compared: exogenous Ath-miR-159a, endogenous RNA U6, and three mathematical normalization techniques: geNorm (Qbase, QB) and NormFinder (NF) normalization algorithms, and global mean calculation. To select the best normalization method, technical variability, biological variability, stability, and the extent to which the normalization method reduces data dispersion were calculated. The geNorm normalization algorithm reduced data dispersion to the greatest extent, while endogenous RNA U6 performed worst. In conclusion, for miRNA profiling in plasma samples using TLDA cards we recommend: 1. Implementing a preamplification step in the TLDA protocol without diluting the final preamplification product 2. A stepwise approach to exclude non-informative miRNA based on quality control parameters 3. Against using snoRNA U6 as normalization method for relative quantification 4. Using the geNorm algorithm as normalization method for relative quantification.

Aims Heart failure with preserved ejection fraction (HFpEF) is associated with reduced exercise capacity elicited by skeletal muscle (SM) alterations. Up to now, no clear medical treatment advice for HFpEF is available. Identification of the ideal animal model mimicking the human condition is a critical step in developing and testing treatment strategies. Several HFpEF animals have been described, but the most suitable in terms of comparability with SM alterations in HFpEF patients is unclear. The aim of the present study was to investigate molecular changes in SM of three different animal models and to compare them with alterations of muscle biopsies obtained from human HFpEF patients. Methods and results Skeletal muscle tissue was obtained from HFpEF and control patients and from three different animal models including the respective controls—ZSF1 rat, Dahl salt‐sensitive rat, and transverse aortic constriction surgery/deoxycorticosterone mouse. The development of HFpEF was verified by echocardiography. Protein expression and enzyme activity of selected markers were assessed in SM tissue homogenates. Protein expression between SM tissue obtained from HFpEF patients and the ZSF1 rats revealed similarities for protein markers involved in muscle atrophy (MuRF1 expression, protein ubiquitinylation, and LC3) and mitochondrial metabolism (succinate dehydrogenase and malate dehydrogenase activity, porin expression). The other two animal models exhibited far less similarities to the human samples. Conclusions None of the three tested animal models mimics the condition in HFpEF patients completely, but among the animal models tested, the ZSF1 rat (ZSF1‐lean vs. ZSF1‐obese) shows the highest overlap to the human condition. Therefore, when studying therapeutic interventions to treat HFpEF and especially alterations in the SM, we suggest that the ZSF1 rat is a suitable model.

In preeclampsia (PE), pre-existent maternal endothelial dysfunction leads to impaired placentation and vascular maladaptation. The vascular endothelial growth factor (VEGF) pathway is essential in the placentation process and VEGF expression is regulated through post-transcriptional modification by microRNAs (miRNAs). We investigated the expression of VEGF-related circulating miR-16, miR-29b, miR-126, miR-155 and miR-200c in PE vs healthy pregnancies (HPs), and their relation with vascular function, oxidative stress (OS) and systemic inflammation. In this case–control study, 24 women with early PE (<34 weeks) were compared with 30 women with HP. Circulating microRNA levels (RT-qPCR), OS and systemic inflammation were assessed in plasma samples (PE 29.5 vs HP 25.8 weeks) and related to extensive in vivo vascular function (flow-mediated dilatation (FMD), modified FMD (mFMD), carotid-femoral pulse wave velocity (CF-PWV), heart rate corrected augmentation index (AIx75) and reactive hyperemia index (RHI)). FMD, CF-PWV, AIx75 and RHI were all significantly impaired in PE (P<0.05). PE patients had reduced levels of miR-16 (5.53 ± 0.36 vs 5.84 ± 0.61) and increased levels of miR-200c (1.34 ± 0.57 vs 0.97 ± 0.68) (P<0.05). Independent of age and parity, miR-16 was related to impaired FMD (β 2.771, 95% C.I.: 0.023–5.519, P=0.048) and mFMD (β 3.401, 95% C.I.: 0.201–6.602, P=0.038). Likewise, miR-200c was independently associated with CF-PWV (β 0.513, 95% C.I.: 0.034–0.992, P=0.036). In conclusion, circulating levels of miR-16 were lower in PE, which correlated with impaired endothelial function. Circulating miR-200c was increased in PE and correlated with higher arterial stiffness. These findings suggest a post-transcriptional dysregulation of the VEGF pathway in PE and identify miR-16 and miR-200c as possible diagnostic biomarkers for PE.

Apart from cardiotoxicity, the chemotherapeutic agent doxorubicin (DOX) provokes acute and long-term vascular toxicity. Dexrazoxane (DEXRA) is an effective drug for treatment of DOX-induced cardiotoxicity, yet it remains currently unknown whether DEXRA prevents vascular toxicity associated with DOX. Accordingly, the present study aimed to evaluate the protective potential of DEXRA against DOX-related vascular toxicity in a previously-established in vivo and ex vivo model of vascular dysfunction induced by 16 hour (h) DOX exposure. Vascular function was evaluated in the thoracic aorta in organ baths, 16h after administration of DOX (4 mg/kg) or DOX with DEXRA (40 mg/kg) to male C57BL6/J mice. In parallel, vascular reactivity was evaluated after ex vivo incubation (16h) of murine aortic segments with DOX (1 μM) or DOX with DEXRA (10 μM). In both in vivo and ex vivo experiments, DOX impaired acetylcholine-stimulated endothelium-dependent vasodilation. In the ex vivo setting, DOX additionally attenuated phenylephrine-elicited vascular smooth muscle cell (VSMC) contraction. Importantly, DEXRA failed to prevent DOX-induced endothelial dysfunction and hypocontraction. Furthermore, RT-qPCR and Western blotting showed that DOX decreased the protein levels of topoisomerase-IIβ (TOP-IIβ), a key target of DEXRA, in the heart, but not in the aorta. Additionally, the effect of N-acetylcysteine (NAC, 10 μM), a reactive oxygen species (ROS) scavenger, was evaluated ex vivo . NAC did not prevent DOX-induced impairment of acetylcholine-stimulated vasodilation. In conclusion, our results show that DEXRA fails to prevent vascular toxicity resulting from 16h DOX treatment. This may relate to DOX provoking vascular toxicity in a ROS- and TOP-IIβ-independent way, at least in the evaluated acute setting. However, it is important to mention that these findings only apply to the acute (16h) treatment period, and further research is warranted to delineate the therapeutic potential of DEXRA against vascular toxicity associated with longer-term repetitive DOX dosing.

Background Doxorubicin (DOX) is used for breast cancer and lymphoma, but can cause cardiotoxicity, arterial stiffness, and endothelial dysfunction. We recently reported SERPINA3N as biomarker of cardiovascular toxicity in patients and mice. Dexrazoxane (DEXRA) is an FDA-approved drug that prevents DOX-induced cardiac toxicity in high-risk patients. However, the effect of DEXRA on vascular dysfunction during DOX treatment has not been documented. Therefore, here we investigated whether DEXRA protects against DOX-induced arterial stiffness, endothelial dysfunction, and SERPINA3N upregulation in tissue and plasma from mice. Methods Male C57BL6/J mice were treated with DOX (4 mg/kg), DEXRA (40 mg/kg), a combination (DEXRA + DOX), or VEHICLE (0.9% NaCl) weekly i.p. for 6 weeks ( n  = 8 per group). Cardiovascular function was measured in vivo by ultrasound imaging at baseline, weeks 2 and 6. Vascular reactivity was analyzed ex vivo in the thoracic aorta at week 6 and molecular analysis was performed. Results DEXRA prevented left ventricular ejection fraction decline by DOX (DEXRA + DOX: 62 ± 2% vs DOX: 51 ± 2%). Moreover, DEXRA prevented the increase in pulse wave velocity by DOX (DEXRA + DOX: 2.1 ± 0.2 m/s vs DOX: 4.5 ± 0.3 m/s) and preserved endothelium-dependent relaxation (DEXRA + DOX: 82 ± 3% vs DOX: 62 ± 3%). In contrast to DOX-treated mice, SERPINA3N did not increase in the DEXRA + DOX group. Conclusion Our results not only confirm the cardioprotective effects of DEXRA against DOX-induced cardiotoxicity but also add preservation of vascular endothelial cell function as an important mechanism. Moreover, the study demonstrates the potential of SERPINA3N as a biomarker for monitoring cardiovascular complications of DOX in high-risk patients.

Aims In heart failure with preserved ejection fraction (HFpEF), the reduction of nitric oxide (NO)‐bioavailability and consequently endothelial dysfunction leads to LV stiffness and diastolic dysfunction of the heart. Besides shear stress, high‐density lipoprotein (HDL) stimulates endothelial cells to increased production of NO via phosphorylation of endothelial nitric oxide synthase (eNOS). For patients with heart failure with reduced ejection fraction, earlier studies demonstrated a positive impact of exercise training (ET) on HDL‐mediated eNOS activation. The study aims to investigate the influence of ET on HDL‐mediated phosphorylation of eNOS in HFpEF patients. Methods and results The present study is a substudy of the OptimEx‐Clin trial. The patients were randomized to three groups: (i) HIIT (high‐intensity interval training; (ii) MCT (moderate‐intensity continuous training); and (iii) CG (control group). Supervised training at study centres was offered for the first 3 months. From months 4–12, training sessions were continued at home with the same exercise protocol as performed during the in‐hospital phase. Blood was collected at baseline, after 3, and 12 months, and HDL was isolated by ultracentrifugation. Human aortic endothelial cells were incubated with isolated HDL, and HDL‐induced eNOS phosphorylation at Ser1177 and Thr495 was assessed. Subsequently, the antioxidative function of HDL was evaluated by measuring the activity of HDL‐associated paraoxonase‐1 (Pon1) and the concentration of thiobarbituric acid‐reactive substances (TBARS). After 3 months of supervised ET, HIIT resulted in increased HDL‐mediated eNOS‐Ser1177 phosphorylation. This effect diminished after 12 months of ET. No effect of HIIT was observed on HDL‐mediated eNOS‐Thr495 phosphorylation. MCT had no effect on HDL‐mediated eNOS phosphorylation at Ser1177 and Thr495. HIIT also increased Pon1 activity after 12 months of ET and reduced the concentration of TBARS in the serum after 3 and 12 months of ET. A negative correlation was observed between TBARS concentration and HDL‐associated Pon1 activity in the HIIT group (r = −0.61, P < 0.05), and a trend was evident for the correlation between the change in HDL‐mediated eNOS‐Ser1177 phosphorylation and the change in peak V̇O2 after 3 months in the HIIT group (r = 0.635, P = 0.07). Conclusions The present study documented that HIIT but not MCT exerts beneficial effects on HDL‐mediated eNOS phosphorylation and HDL‐associated Pon1 activity in HFpEF patients. These beneficial effects of HIIT were reduced as soon as the patients switched to home‐based ET.

Background The anthracycline doxorubicin (DOX) is a highly effective anticancer agent, especially in breast cancer and lymphoma. However, DOX can cause cancer therapy-related cardiovascular toxicity (CTR-CVT) in patients during treatment and in survivors. Current diagnostic criteria for CTR-CVT focus mainly on left ventricular systolic dysfunction, but a certain level of damage is required before it can be detected. As diastolic dysfunction often precedes systolic dysfunction, the current study aimed to identify functional and molecular markers of DOX-induced CTR-CVT with a focus on diastolic dysfunction. Methods Male C57BL/6J mice were treated with saline or DOX (4 mg/kg, weekly i.p. injection) for 2 and 6 weeks (respectively cumulative dose of 8 and 24 mg/kg) ( n  = 8 per group at each time point). Cardiovascular function was longitudinally investigated using echocardiography and invasive left ventricular pressure measurements. Subsequently, at both timepoints, myocardial tissue was obtained for proteomics (liquid-chromatography with mass-spectrometry). A cohort of patients with CTR-CVT was used to complement the pre-clinical findings. Results DOX-induced a reduction in left ventricular ejection fraction from 72 ± 2% to 55 ± 1% after 2 weeks (cumulative 8 mg/kg DOX). Diastolic dysfunction was demonstrated as prolonged relaxation (increased tau) and heart failure was evident from pulmonary edema after 6 weeks (cumulative 24 mg/kg DOX). Myocardial proteomic analysis revealed an increased expression of 12 proteins at week 6, with notable upregulation of SERPINA3N in the DOX-treated animals. The human ortholog SERPINA3 has previously been suggested as a marker in CTR-CVT. Upregulation of SERPINA3N was confirmed by western blot, immunohistochemistry, and qPCR in murine hearts. Thereby, SERPINA3N was most abundant in the endothelial cells. In patients, circulating SERPINA3 was increased in plasma of CTR-CVT patients but not in cardiac biopsies. Conclusion We showed that mice develop heart failure with impaired systolic and diastolic function as result of DOX treatment. Additionally, we could identify increased SERPINA3 levels in the mice as well as patients with DOX-induced CVT and demonstrated expression of SERPINA3 in the heart itself, suggesting that SERPINA3 could serve as a novel biomarker.

Aims Exercise training improves aerobic capacity (V̇O2peak) in patients with heart failure and preserved ejection fraction (HFpEF), but underlying mechanisms remain unclear. We aimed to evaluate whether exercise training could improve systolic and diastolic function during exercise. Methods This was a substudy of the multicentre Optimizing Exercise Training in HFpEF (OptimEx‐Clin) trial, in which 180 patients with HFpEF were randomized 1:1:1 to guideline control, moderate continuous training or high‐intensity interval training. All patients included at two out of five participating sites underwent exercise echocardiography at baseline and 3 months. Patients of both training groups were pooled and compared with guideline control. Results A total of 61 patients (mean age 73 ± 7 years, 72% female) were included. At baseline, E/e′ increased from 17.0 ± 5.7 to 19.5 ± 6.1 and systolic pulmonary artery pressure from 31 ± 8 to 51 ± 11 mmHg (both P < 0.001). Right ventricular function did not change significantly (maximal tricuspid annular plane systolic excursion 24.7 ± 4.0 mm, P = 0.051 vs. baseline). At 3 months, patients randomized to exercise training improved V̇O2peak (control +0.2, training +2.7 mL/kg/min, P = 0.006) and demonstrated small but significant improvements in exercise E/e′ (control 21.7 ± 7.5 to 22.8 ± 9.2, training 18.3 ± 5.0 to 17.2 ± 4.1, P = 0.044). No significant changes were observed in ejection fraction, mitral or tricuspid annular plane systolic excursion, S′, A′ or systolic pulmonary artery pressure (P > 0.05). Changes in E/e′ were not associated with the change in V̇O2peak. Conclusions In patients with HFpEF, exercise echocardiography revealed increases in filling pressures as well as a failure to augment right ventricular function during exercise. After 3 months of exercise training, HFpEF patients demonstrated a small improvement in diastolic function (exercise E/e′), but this did not explain the improved aerobic capacity. Training‐induced change of diastolic function in HFpEF. HIIT, high‐intensity interval training; HFpEF, heart failure with preserved ejection fraction; LVEF, left ventricular ejection fraction; MCT, moderate continuous training; TAPSE, tricuspid annular plane systolic excursion; V̇O2peak, peak oxygen uptake.

Background Variants in cardiomyopathy genes have been identified in patients with cancer therapy-related cardiac dysfunction (CTRCD), suggesting a genetic predisposition for the development of CTRCD. The diagnostic yield of genetic testing in a CTRCD population compared to a cardiomyopathy patient cohort is not yet known and information on which genes should be assessed in this population is lacking. Methods We retrospectively included 46 cancer patients with a history of anthracycline induced CTRCD (defined as a decrease in left ventricular ejection fraction (LVEF) to < 50% and a ≥ 10% reduction from baseline by echocardiography). Genetic testing was performed for 59 established cardiomyopathy genes. Only variants of uncertain significance and (likely) pathogenic variants were included. Diagnostic yield of genetic testing was compared with a matched cohort of patients with dilated cardiomyopathy (DCM, n  = 46) and a matched cohort of patients without cardiac disease ( n  = 111). Results Average LVEF at time of CTRCD diagnosis was 30.1 ± 11.0%. Patients were 52.9 ± 14.6 years old at time of diagnosis and 30 (65.2%) were female. Most patients were treated for breast cancer or lymphoma, with a median doxorubicin equivalent dose of 300 mg/m 2 [112.5-540.0]. A genetic variant, either pathogenic, likely pathogenic or of uncertain significance, was identified in 29/46 (63.0%) of patients with CTRCD, which is similar to the DCM cohort (34/46, 73.9%, p  = 0.262), but significantly higher than in the negative control cohort (47/111, 39.6%, p  = 0.018). Variants in TTN were the most prevalent in the CTRCD cohort (43% of all variants). All (likely) pathogenic variants identified in the CTRCD cohort were truncating variants in TTN . There were no significant differences in severity of CTRCD and in recovery rate in variant-harbouring individuals versus non-variant harbouring individuals. Conclusions In this case-control study, cancer patients with anthracycline-induced CTRCD have an increased burden of genetic variants in cardiomyopathy genes, similar to a DCM cohort. If validated in larger prospective studies, integration of genetic data in risk prediction models for CTRCD may guide cancer treatment. Moreover, genetic results have important clinical impact, both for the patient in the setting of precision medicine, as for the family members that will receive genetic counselling.

Aims The aims of this sub‐study of the SMARTEX trial were (1) to evaluate the effects of a 12‐week exercise training programme on serum levels of high sensitivity cardiac troponin I (hs‐cTnI) in patients with moderate chronic heart failure (CHF), in New York Heart Association class II‐III with reduced ejection fraction (HFrEF) and (2) to explore the associations with left ventricular remodelling, functional capacity and filling pressures measured with N‐terminal pro brain natriuretic peptide (NT‐proBNP). Methods and results In this sub‐study, 196 patients were randomly assigned to high intensity interval training (HIIT, n = 70), moderate continuous training (MCT, n = 59) or recommendation of regular exercise (RRE), (n = 67) for 12 weeks. To reveal potential difference between structured intervention and control, HIIT and MCT groups were merged and named supervised exercise training (SET) group. The RRE group constituted the control group (CG). To avoid contributing factors to myocardial injury, we also evaluated changes in patients without additional co‐morbidities (atrial fibrillation, hypertension, diabetes mellitus, and chronic obstructive pulmonary disease). The relationship between hs‐cTnI and left ventricular end‐diastolic diameter (LVEDD), VO2peak, and NT‐proBNP was analysed by linear mixed models. At 12 weeks, Hs‐cTnI levels were modestly but significantly reduced in the SET group from median 11.9 ng/L (interquartile ratio, IQR 7.1–21.8) to 11.5 ng/L (IQR 7.0–20.7), P = 0.030. There was no between‐group difference (SET vs. CG, P = 0.116). There was a numerical but not significant reduction in hs‐cTnI for the whole population (P = 0.067) after 12 weeks. For the sub‐group of patients without additional co‐morbidities, there was a significant between‐group difference: SET group (delta −1.2 ng/L, IQR −2.7 to 0.1) versus CG (delta −0.1 ng/L, IQR −0.4 to 0.7), P = 0.007. In the SET group, hs‐cTnI changed from 10.9 ng/L (IQR 6.0–22.7) to 9.2 ng/L (IQR 5.2–20.5) (P = 0.002), whereas there was no change in the CG (6.4 to 5.8 ng/L, P = 0.64). Changes in hs‐cTnI (all patients) were significantly associated with changes in; LVEDD, VO2peak, and NT‐proBNP, respectively. Conclusions In patients with stable HFrEF, 12 weeks of structured exercise intervention was associated with a modest, but significant reduction of hs‐cTnI. There was no significant difference between intervention group and control group. In the sub‐group of patients without additional co‐morbidities, this difference was highly significant. The alterations in hs‐cTnI were associated with reduction of LVEDD and natriuretic peptide concentrations as well as improved functional capacity.