A human importin-ß-related disorder: Syndromic thoracic aortic aneurysm caused by bi-allelic loss-of-function variants in IPO8.

Importin 8, encoded by IPO8, is a ubiquitously expressed member of the importin-ß protein family that translocates cargo molecules such as proteins, RNAs, and ribonucleoprotein complexes into the nucleus in a RanGTP-dependent manner. Current knowledge of the cargoes of importin 8 is limited, but TGF-ß signaling components such as SMAD1-4 have been suggested to be among them. Here, we report that bi-allelic loss-of-function variants in IPO8 cause a syndromic form of thoracic aortic aneurysm (TAA) with clinical overlap with Loeys-Dietz and Shprintzen-Goldberg syndromes. Seven individuals from six unrelated families showed a consistent phenotype with early-onset TAA, motor developmental delay, connective tissue findings, and craniofacial dysmorphic features. A C57BL/6N Ipo8 knockout mouse model recapitulates TAA development from 8-12 weeks onward in both sexes but most prominently shows ascending aorta dilatation with a propensity for dissection in males. Compliance assays suggest augmented passive stiffness of the ascending aorta in male Ipo8-/- mice throughout life. Immunohistological investigation of mutant aortic walls reveals elastic fiber disorganization and fragmentation along with a signature of increased TGF-ß signaling, as evidenced by nuclear pSmad2 accumulation. RT-qPCR assays of the aortic wall in male Ipo8-/- mice demonstrate decreased Smad6/7 and increased Mmp2 and Ccn2 (Ctgf) expression, reinforcing a role for dysregulation of the TGF-ß signaling pathway in TAA development. Because importin 8 is the most downstream TGF-ß-related effector implicated in TAA pathogenesis so far, it offers opportunities for future mechanistic studies and represents a candidate drug target for TAA.

Effect of erythrophagocytosis-induced ferroptosis during angiogenesis in atherosclerotic plaques.

Intraplaque (IP) angiogenesis is a key feature of advanced atherosclerotic plaques. Because IP vessels are fragile and leaky, erythrocytes are released and phagocytosed by macrophages (erythrophagocytosis), which leads to high intracellular iron content, lipid peroxidation and cell death. In vitro experiments showed that erythrophagocytosis by macrophages induced non-canonical ferroptosis, an emerging type of regulated necrosis that may contribute to plaque destabilization. Erythrophagocytosis-induced ferroptosis was accompanied by increased expression of heme-oxygenase 1 and ferritin, and could be blocked by co-treatment with third generation ferroptosis inhibitor UAMC-3203. Both heme-oxygenase 1 and ferritin were also expressed in erythrocyte-rich regions of carotid plaques from ApoE-/- Fbn1C1039G+/- mice, a model of advanced atherosclerosis with IP angiogenesis. The effect of UAMC-3203 (12.35 mg/kg/day) on atherosclerosis was evaluated in ApoE-/- Fbn1C1039G+/- mice fed a western-type diet (WD) for 12 weeks (n = 13 mice/group) or 20 weeks (n = 16-21 mice/group) to distinguish between plaques without and with established IP angiogenesis, respectively. A significant decrease in carotid plaque thickness was observed after 20 weeks WD (87 ± 19 µm vs. 166 ± 20 µm, p = 0.006), particularly in plaques with confirmed IP angiogenesis or hemorrhage (108 ± 35 µm vs. 322 ± 40 µm, p = 0.004). This effect was accompanied by decreased IP heme-oxygenase 1 and ferritin expression. UAMC-3203 did not affect carotid plaques after 12 weeks WD or plaques in the aorta, which typically do not develop IP angiogenesis. Altogether, erythrophagocytosis-induced ferroptosis during IP angiogenesis leads to larger atherosclerotic plaques, an effect that can be prevented by ferroptosis inhibitor UAMC-3203.

Towards a better understanding of arterial calcification disease progression in CKD: investigation of early pathological alterations.

BACKGROUND: Cardiovascular disease remains the leading cause of death in chronic kidney disease (CKD) patients, especially in those undergoing dialysis and kidney transplant surgery. CKD patients are at high risk of developing arterial media calcifications (AMC) and arterial stiffness. We hypothesized that investigation of disease progression at an early stage could provide novel insights in understanding AMC etiology. METHODS: An adenine diet was administered to male Wistar rats to induce AMC. Rats were sacrificed after 2, 4 and 8 weeks. AMC was measured by assessment of aortic calcium and visualized using histology. Arterial stiffness was measured in vivo by ultrasound and ex vivo by applying cyclic stretch of physiological magnitude on isolated arterial segments, allowing us to generate the corresponding pressure-diameter loops. Further, ex vivo arterial reactivity was assessed in organ baths at 2 and 4 weeks to investigate early alterations in biomechanics/cellular functionality. RESULTS: CKD rats showed a time-dependent increase in aortic calcium which was confirmed on histology. Accordingly, ex vivo arterial stiffness progressively worsened. Pressure-diameter loops showed a gradual loss of arterial compliance in CKD rats. Additionally, viscoelastic properties of isolated arterial segments were altered in CKD rats. Furthermore, after 2 and 4 weeks of adenine treatment, a progressive loss in basal, nitric oxide (NO) levels was observed, which was linked to an increased vessel tonus and translates into an increasing viscous modulus. CONCLUSIONS: Our observations indicate that AMC-related vascular alterations develop early after CKD induction prior to media calcifications being present. Preventive action, related to restoration of NO bioavailability, might combat AMC development.

Correction: Sallam et al. DNA Methylation Alterations in Fractionally Irradiated Rats and Breast Cancer Patients Receiving Radiotherapy. Int. J. Mol. Sci. 2022, 23, 16214.

Radia Tamarat and Susana Constantino Rosa Santos were not included as authors in the original publication [...].

PFKFB3 gene deletion in endothelial cells inhibits intraplaque angiogenesis and lesion formation in a murine model of venous bypass grafting.

Vein grafting is a frequently used surgical intervention for cardiac revascularization. However, vein grafts display regions with intraplaque (IP) angiogenesis, which promotes atherogenesis and formation of unstable plaques. Graft neovessels are mainly composed of endothelial cells (ECs) that largely depend on glycolysis for migration and proliferation. In the present study, we aimed to investigate whether loss of the glycolytic flux enzyme phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) in ECs inhibits IP angiogenesis and as such prevents unstable plaque formation. To this end, apolipoprotein E deficient (ApoE-/-) mice were backcrossed to a previously generated PFKFB3fl/fl Cdh5iCre mouse strain. Animals were injected with either corn oil (ApoE-/-PFKFB3fl/fl) or tamoxifen (ApoE-/-PFKFB3ECKO), and were fed a western-type diet for 4 weeks prior to vein grafting. Hereafter, mice received a western diet for an additional 28 days and were then sacrificed for graft assessment. Size and thickness of vein graft lesions decreased by 35 and 32%, respectively, in ApoE-/-PFKFB3ECKO mice compared to controls, while stenosis diminished by 23%. Moreover, vein graft lesions in ApoE-/-PFKFB3ECKO mice showed a significant reduction in macrophage infiltration (29%), number of neovessels (62%), and hemorrhages (86%). EC-specific PFKFB3 deletion did not show obvious adverse effects or changes in general metabolism. Interestingly, RT-PCR showed an increased M2 macrophage signature in vein grafts from ApoE-/-PFKFB3ECKO mice. Altogether, EC-specific PFKFB3 gene deletion leads to a significant reduction in lesion size, IP angiogenesis, and hemorrhagic complications in vein grafts. This study demonstrates that inhibition of endothelial glycolysis is a promising therapeutic strategy to slow down plaque progression.

Cardiac electrophysiology studies in mice via the transjugular route: a comprehensive practical guide.

Cardiac arrhythmias are associated with cardiovascular morbidity and mortality. Cardiac electrophysiology studies (EPS) use intracardiac catheter recording and stimulation for profound evaluation of the heart's electrical properties. The main clinical application is investigation and treatment of rhythm disorders. These techniques have been translated to the murine setting to open opportunities for detailed evaluation of the impact of different characteristics (including genetics) and interventions on cardiac electrophysiology and -pathology. Currently, a detailed description of the technique of murine transjugular EPS (which is the standard route of catheter introduction) is lacking. This article provides detailed information on EPS in mice via the transjugular route. This includes catheter placement, stimulation protocols, intracardiac tracing interpretation, artifact reduction, and surface ECG recording. In addition, reference values as obtained in C57BL/6N mice are presented for common electrophysiological parameters. This detailed methodological description aims to increase accessibility and standardization of EPS in mice. Ultimately, also human research and patient care may benefit from translation of the knowledge obtained in preclinical models using this technique.NEW & NOTEWORTHY Electrophysiology studies (EPS) allow in-depth evaluation of cardiac electrophysiology and -pathology. These techniques have been adapted to the murine setting for (translational) studies, mainly focusing on arrhythmogenesis. Despite the frequent application of EPS via the transjugular route, a thorough description of the technique is currently lacking. This article aims to function as a comprehensive guide, also elaborating (for the first time) on nonsurgical aspects such as catheter positioning, tracing artifacts, stimulation protocols, and reference values.

DNA Methylation Alterations in Fractionally Irradiated Rats and Breast Cancer Patients Receiving Radiotherapy.

Radiation-Induced CardioVascular Disease (RICVD) is an important concern in thoracic radiotherapy with complex underlying pathophysiology. Recently, we proposed DNA methylation as a possible mechanism contributing to RICVD. The current study investigates DNA methylation in heart-irradiated rats and radiotherapy-treated breast cancer (BC) patients. Rats received fractionated whole heart X-irradiation (0, 0.92, 6.9 and 27.6 Gy total doses) and blood was collected after 1.5, 3, 7 and 12 months. Global and gene-specific methylation of the samples were evaluated; and gene expression of selected differentially methylated regions (DMRs) was validated in rat and BC patient blood. In rats receiving an absorbed dose of 27.6 Gy, DNA methylation alterations were detected up to 7 months with differential expression of cardiac-relevant DMRs. Of those, SLMAP showed increased expression at 1.5 months, which correlated with hypomethylation. Furthermore, E2F6 inversely correlated with a decreased global longitudinal strain. In BC patients, E2F6 and SLMAP exhibited differential expression directly and 6 months after radiotherapy, respectively. This study describes a systemic radiation fingerprint at the DNA methylation level, elucidating a possible association of DNA methylation to RICVD pathophysiology, to be validated in future mechanistic studies.

Neuregulin-1 compensates for endothelial nitric oxide synthase deficiency.

Endothelial cells (ECs) secrete different paracrine signals that modulate the function of adjacent cells; two examples of these paracrine signals are nitric oxide (NO) and neuregulin-1 (NRG1), a cardioprotective growth factor. Currently, it is undetermined whether one paracrine factor can compensate for the loss of another. Herein, we hypothesized that NRG1 can compensate for endothelial NO synthase (eNOS) deficiency. We characterized eNOS null and wild-type (WT) mice by cardiac ultrasound and histology and we determined circulating NRG1 levels. In a separate experiment, eight groups of mice were divided into four groups of eNOS null mice and WT mice; half of the mice received angiotensin II (ANG II) to induce a more severe phenotype. Mice were randomized to daily injections with NRG1 or vehicle for 28 days. eNOS deficiency increased NRG1 plasma levels, indicating that ECs increase their NRG1 expression when NO production is deleted. eNOS deficiency also increased blood pressure, lowered heart rate, induced cardiac fibrosis, and affected diastolic function. In eNOS null mice, ANG II administration not only increased cardiac fibrosis but also induced cardiac hypertrophy and renal fibrosis. NRG1 administration prevented cardiac and renal hypertrophy and fibrosis caused by ANG II infusion and eNOS deficiency. Moreover, Nrg1 expression in the myocardium is shown to be regulated by miR-134. This study indicates that administration of endothelium-derived NRG1 can compensate for eNOS deficiency in the heart and kidneys.NEW & NOTEWORTHY ECs compensate for eNOS deficiency by increasing the secretion of NRG1. NRG1 administration prevents cardiac and renal hypertrophy and fibrosis caused by ANG II infusion and eNOS deficiency. NRG1 expression is regulated by miR-134.

A systematic literature review on the effects of exercise on human Toll-like receptor expression.

BACKGROUND: Toll-like receptors (TLRs) are a family of transmembrane pattern recognition receptors that are mainly expressed on immune cells. Recognition of various exogenous and endogenous molecular patterns activates the TLR signalling cascade, which orchestrates an inflammatory immune response. Dysfunctional immune responses, including aberrant TLR signalling, are increasingly implicated in the associations between sedentarism, chronic low-grade systemic inflammation and various non-communicable diseases. Conversely, exercise exerts anti-inflammatory effects, which could be conferred through its immunomodulatory properties, potentially affecting TLRs. This study aims to systematically review the effects of exercise on human TLR expression. METHOD: A systematic literature search of Pubmed, Embase, The Cochrane Library and SPORTDiscus for articles addressing the impact of exercise (as isolated intervention) on TLRs in humans was conducted, ending in February 2020. RESULTS: A total of 66 articles were included. The publications were categorised according to exercise modality and duration: acute resistance exercise (4 studies), acute aerobic exercise (26 studies), resistance training program (9 studies), aerobic training program (16 studies), combined (i.e. resistance and aerobic) training program (8 studies) and chronic exercise not otherwise classifiable (9 studies). Five articles investigated more than one of the aforementioned exercise categories. Several trends could be discerned with regard to the TLR response in the different exercise categories. Acute resistance exercise seemed to elicit TLR upregulation, whereas acute aerobic exercise had less activating potential with the majority of responses being neutral or, especially in healthy participants, downregulatory. Chronic resistance and combined exercise programs predominantly resulted in unaltered or decreased TLR levels. In the chronic aerobic exercise category, mixed effects were observed, but the majority of measurements demonstrated unchanged TLR expression. CONCLUSION: Currently published research supports an interplay between exercise and TLR signalling, which seems to depend on the characteristics of the exercise. However, there was large heterogeneity in the study designs and methodologies. Therefore, additional research is required to further corroborate these findings, to define its pathophysiological implications and to elucidate the mechanism(s) linking exercise to TLR signalling.

Partial Inhibition of Glycolysis Reduces Atherogenesis Independent of Intraplaque Neovascularization in Mice.

OBJECTIVE: Intraplaque neovascularization is an important feature of unstable human atherosclerotic plaques. However, its impact on plaque formation and stability is poorly studied. Because proliferating endothelial cells generate up to 85% of their ATP from glycolysis, we investigated whether pharmacological inhibition of glycolytic flux by the small-molecule 3PO (3-[3-pyridinyl]-1-[4-pyridinyl]-2-propen-1-one) could have beneficial effects on plaque formation and composition. Approach and Results: ApoE-/- (apolipoprotein E deficient) mice treated with 3PO (50 µg/g, ip; 4×/wk, 4 weeks) showed a metabolic switch toward ketone body formation. Treatment of ApoE-/-Fbn1C1039G+/- mice with 3PO (50 µg/g, ip) either after 4 (preventive, twice/wk, 10 weeks) or 16 weeks of Western diet (curative, 4×/wk, 4 weeks) inhibited intraplaque neovascularization by 50% and 38%, respectively. Plaque formation was significantly reduced in all 3PO-treated animals. This effect was independent of intraplaque neovascularization. In vitro experiments showed that 3PO favors an anti-inflammatory M2 macrophage subtype and suppresses an M1 proinflammatory phenotype. Moreover, 3PO induced autophagy, which in turn impaired NF-κB (nuclear factor-kappa B) signaling and inhibited TNF-α (tumor necrosis factor-alpha)-mediated VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1) upregulation. Consistently, a preventive 3PO regimen reduced endothelial VCAM-1 expression in vivo. Furthermore, 3PO improved cardiac function in ApoE-/-Fbn1C1039G+/- mice after 10 weeks of treatment. CONCLUSIONS: Partial inhibition of glycolysis restrained intraplaque angiogenesis without affecting plaque composition. However, less plaques were formed, which was accompanied by downregulation of endothelial adhesion molecules-an event that depends on autophagy induction. Inhibition of coronary plaque formation by 3PO resulted in an overall improved cardiac function.

Serum Corticosterone and Insulin Resistance as Early Biomarkers in the hAPP23 Overexpressing Mouse Model of Alzheimer's Disease.

Increasing epidemiological evidence highlights the association between systemic insulin resistance and Alzheimer's disease (AD). As insulin resistance can be caused by high-stress hormone levels and since hypercortisolism appears to be an important risk factor of AD, we aimed to investigate the systemic insulin functionality and circulating stress hormone levels in a mutant humanized amyloid precursor protein (APP) overexpressing (hAPP23+/-) AD mouse model. Memory and spatial learning of male hAPP23+/- and C57BL/6 (wild type, WT) mice were assessed by a Morris Water Maze (MWM) test at the age of 4 and 12 months. The systemic metabolism was examined by intraperitoneal glucose and insulin tolerance tests (GTT, ITT). Insulin and corticosterone levels were determined in serum. In the hippocampus, parietal and occipital cortex of hAPP23+/- brains, amyloid-beta (Aß) deposits were present at 12 months of age. MWM demonstrated a cognitive decline in hAPP23+/- mice at 12 but not at 4 months, evidenced by increasing total path lengths and deteriorating probe trials compared to WT mice. hAPP23+/- animals presented increased serum corticosterone levels compared to WT mice at both 4 and 12 months. hAPP23+/- mice exhibited peripheral insulin resistance compared to WT mice at 4 months, which stabilized at 12 months of age. Serum insulin levels were similar between genotypes at 4 months of age but were significantly higher in hAPP23+/- mice at 12 months of age. Peripheral glucose homeostasis remained unchanged. These results indicate that peripheral insulin resistance combined with elevated circulating stress hormone levels could be potential biomarkers of the pre-symptomatic phase of AD.

Doxorubicin Impairs Smooth Muscle Cell Contraction: Novel Insights in Vascular Toxicity.

Clinical and animal studies have demonstrated that chemotherapeutic doxorubicin (DOX) increases arterial stiffness, a predictor of cardiovascular risk. Despite consensus about DOX-impaired endothelium-dependent vasodilation as a contributing mechanism, some studies have reported conflicting results on vascular smooth muscle cell (VSMC) function after DOX treatment. The present study aimed to investigate the effects of DOX on VSMC function. To this end, mice received a single injection of 4 mg DOX/kg, or mouse aortic segments were treated ex vivo with 1 µM DOX, followed by vascular reactivity evaluation 16 h later. Phenylephrine (PE)-induced VSMC contraction was decreased after DOX treatment. DOX did not affect the transient PE contraction dependent on Ca2+ release from the sarcoplasmic reticulum (0 mM Ca2+), but it reduced the subsequent tonic phase characterised by Ca2+ influx. These findings were supported by similar angiotensin II and attenuated endothelin-1 contractions. The involvement of voltage-gated Ca2+ channels in DOX-decreased contraction was excluded by using levcromakalim and diltiazem in PE-induced contraction and corroborated by similar K+ and serotonin contractions. Despite the evaluation of multiple blockers of transient receptor potential channels, the exact mechanism for DOX-decreased VSMC contraction remains elusive. Surprisingly, DOX reduced ex vivo but not in vivo arterial stiffness, highlighting the importance of appropriate timing for evaluating arterial stiffness in DOX-treated patients.

The Protective Effects of the Autophagic and Lysosomal Machinery in Vascular and Valvular Calcification: A Systematic Review.

BACKGROUND: Autophagy is a highly conserved catabolic homeostatic process, crucial for cell survival. It has been shown that autophagy can modulate different cardiovascular pathologies, including vascular calcification (VCN). OBJECTIVE: To assess how modulation of autophagy, either through induction or inhibition, affects vascular and valvular calcification and to determine the therapeutic applicability of inducing autophagy. DATA SOURCES: A systematic review of English language articles using MEDLINE/PubMed, Web of Science (WoS) and the Cochrane library. The search terms included autophagy, autolysosome, mitophagy, endoplasmic reticulum (ER)-phagy, lysosomal, calcification and calcinosis. Study characteristics: Thirty-seven articles were selected based on pre-defined eligibility criteria. Thirty-three studies (89%) studied vascular smooth muscle cell (VSMC) calcification of which 27 (82%) studies investigated autophagy and six (18%) studies lysosomal function in VCN. Four studies (11%) studied aortic valve calcification (AVCN). Thirty-four studies were published in the time period 2015-2020 (92%). CONCLUSION: There is compelling evidence that both autophagy and lysosomal function are critical regulators of VCN, which opens new perspectives for treatment strategies. However, there are still challenges to overcome, such as the development of more selective pharmacological agents and standardization of methods to measure autophagic flux.

Acute modulation of the cholinergic system in the mouse brain detected by pharmacological resting-state functional MRI.

INTRODUCTION: The cholinergic system is involved in learning and memory and is affected in neurodegenerative disorders such as Alzheimer's disease. The possibility of non-invasively detecting alterations of neurotransmitter systems in the mouse brain would greatly improve early diagnosis and treatment strategies. The hypothesis of this study is that acute modulation of the cholinergic system might be reflected as altered functional connectivity (FC) and can be measured using pharmacological resting-state functional MRI (rsfMRI). MATERIAL AND METHODS: Pharmacological rsfMRI was performed on a 9.4T MRI scanner (Bruker BioSpec, Germany) using a gradient echo EPI sequence. All mice were sedated with medetomidine. C57BL/6 mice (N = 15/group) were injected with either saline, the cholinergic antagonist scopolamine, or methyl-scopolamine, after which rsfMRI was acquired. For an additional group (N = 8), rsfMRI scans of the same mouse were acquired first at baseline, then after the administration of scopolamine and finally after the additional injection of the cholinergic agonist milameline. Contextual memory was evaluated with the same setup as the pharmacological rsfMRI using the passive avoidance behavior test. RESULTS: Scopolamine induced a dose-dependent decrease of FC between brain regions involved in memory. Scopolamine-induced FC deficits could be recovered completely by milameline for FC between the hippocampus-thalamus, cingulate-retrosplenial, and visual-retrosplenial cortex. FC between the cingulate-rhinal, cingulate-visual and visual-rhinal cortex could not be completely recovered by milameline. This is consistent with the behavioral outcome, where milameline only partially recovered scopolamine-induced contextual memory deficits. Methyl-scopolamine administered at the same dose as scopolamine did not affect FC in the brain. CONCLUSION: The results of the current study are important for future studies in mouse models of neurodegenerative disorders, where pharmacological rsfMRI may possibly be used as a non-invasive read-out tool to detect alterations of neurotransmitter systems induced by pathology or treatment.

Unravelling the impact of active and passive contributors to arterial stiffness in male mice and their role in vascular aging.

Arterial stiffness, a key indicator of vascular health, encompassing active (vascular tone) and passive (extracellular matrix) components. This study aims to address how these different components affect arterial stiffness along the aorta and the influence of aging. Aortic segments of 12 week and 24 month old (both n = 6) male C57BL/6J mice were mounted in a Rodent Oscillatory Set-up to study Arterial Compliance, in order to measure arterial stiffness and vascular reactivity. Regional variations in arterial stiffness were evident, with abdominal infrarenal aorta (AIA) exhibiting highest stiffness and smallest diameters. AIA displayed both the highest amount of collagen and collagen:elastin ratio. Regional ex vivo vascular reactivity revealed heightened AIA contractions and lowered NO availability. Aging is a significant factor contributing towards vessel remodelling and arterial stiffness. Aging increased arterial stiffness, aortic diameters, collagen content, and reduced VSMC contraction. The results of this study could identify specific regions or mechanisms to target in the development of innovative therapeutic interventions aimed at enhancing overall vascular health.

The influence of endurance exercise training on myocardial fibrosis and arrhythmogenesis in a coxsackievirus B3 myocarditis mouse model.

Nonischaemic myocardial fibrosis is associated with cardiac dysfunction, malignant arrhythmias and sudden cardiac death. In the absence of a specific aetiology, its finding as late gadolinium enhancement (LGE) on cardiac magnetic resonance imaging is often attributed to preceding viral myocarditis. Athletes presenting with ventricular arrhythmias often have nonischaemic LGE. Previous studies have demonstrated an adverse effect of exercise on the course of acute viral myocarditis. In this study, we have investigated, for the first time, the impact of endurance training on longer-term outcomes such as myocardial fibrosis and arrhythmogenicity in a murine coxsackievirus B3 (CVB)-induced myocarditis model. Male C57BL/6J mice (n = 72) were randomly assigned to 8 weeks of forced treadmill running (EEX) or no exercise (SED). Myocarditis was induced 2 weeks later by a single intraperitoneal injection with CVB, versus vehicle in the controls (PBS). In a separate study, mice (n = 30) were subjected to pretraining for 13 weeks (preEEX), without continuation of exercise during myocarditis. Overall, continuation of exercise resulted in a milder clinical course of viral disease, with less weight loss and better preserved running capacity. CVB-EEX and preEEX-CVB mice tended to have a lower mortality rate. At sacrifice (i.e. 6 weeks after inoculation), the majority of virus was cleared from the heart. Histological assessment demonstrated prominent myocardial inflammatory infiltration and cardiomyocyte loss in both CVB groups. Inflammatory lesions in the CVB-EEX group contained higher numbers of pro-inflammatory cells (iNOS-reactive macrophages and CD8+ T lymphocytes) compared to these in CVB-SED. Treadmill running during myocarditis increased interstitial fibrosis [82.4% (CVB-EEX) vs. 56.3% (CVB-SED); P = 0.049]. Additionally, perivascular and/or interstitial fibrosis with extensive distribution was more likely to occur with exercise [64.7% and 64.7% (CVB-EEX) vs. 50% and 31.3% (CVB-SED); P = 0.048]. There was a numerical, but not significant, increase in the number of scars per cross-section (1.9 vs. 1.2; P = 0.195), with similar scar distribution and histological appearance in CVB-EEX and CVB-SED. In vivo electrophysiology studies did not induce sustained monomorphic ventricular tachycardia, only nonsustained (usually polymorphic) runs. Their cumulative beat count and duration paralleled the increased fibrosis between CVB-EEX and CVB-SED, but the difference was not significant (P = 0.084 for each). Interestingly, in mice that were subjected to pretraining only without continuation of exercise during myocarditis, no differences between pretrained and sedentary mice were observed at sacrifice (i.e. 6 weeks after inoculation and training cessation) with regard to myocardial inflammation, fibrosis, and ventricular arrhythmogenicity. In conclusion, endurance exercise during viral myocarditis modulates the inflammatory process with more pro-inflammatory cells and enhances perivascular and interstitial fibrosis development. The impact on ventricular arrhythmogenesis requires further exploration.

Improved on-tissue detection of the anti-cancer agent doxorubicin by quantitative matrix-assisted laser desorption/ionization mass spectrometry imaging.

Doxorubicin (dox) is an affordable, and highly effective chemotherapeutic agent used in cancer treatment, yet its application is known to cause cumulative cardiac and renal toxicity. In this study, we employed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to evaluate the distribution of dox in mouse heart and kidney after in vivo treatment. To this end, we performed absolute quantification using an isotopically labeled form (13C d3-dox) as an internal standard. Unfortunately, ion suppression often leads to loss of sensitivity in compound detection and can result in hampered drug quantification. To overcome this issue, we developed an on-tissue chemical derivatization (OTCD) method using Girard's reagent T (GirT). With the developed method, dox signal was increased by two orders of magnitude. This optimized sample preparation enabled a sensible gain in dox detection, making it possible to study its distribution and abundance (up to 0.11 pmol/mm2 in the heart and 0.33 pmol/mm2 in the kidney medulla). The optimized approach for on-tissue derivatization and subsequent quantification creates a powerful tool to better understand the relationship between dox exposure (at clinically relevant concentrations) and its biological detrimental effects in various tissues. Overall, this work is a showcase of the added value of MALDI-MSI for pharmaceutical studies to better understand heterogeneity in drug exposure between and within organs.

The natural history of CVB3 myocarditis in C57BL/6J mice: an extended in-depth characterization.

BACKGROUND AND AIMS: Viral infections are the leading cause of myocarditis. Besides acute cardiac complications, late-stage sequelae such as myocardial fibrosis may develop, importantly impacting the prognosis. Coxsackievirus B3 (CVB)-induced myocarditis in mice is the most commonly used translational model to study viral myocarditis and has provided the majority of our current understanding of the disease pathophysiology. Nevertheless, the late stages of disease, encompassing fibrogenesis and arrhythmogenesis, have been underappreciated in viral myocarditis research to date. The present study investigated the natural history of CVB-induced myocarditis in C57BL/6J mice, expanding the focus beyond the acute phase of disease. In addition, we studied the impact of sex and inoculation dose on the disease course. METHODS AND RESULTS: C57BL/6J mice (12 weeks old; n=154) received a single intraperitoneal injection with CVB to induce viral myocarditis, or vehicle (PBS) as control. Male mice (n=92) were injected with 5 × 105 (regular dose) (RD) or 5 × 106 (high dose) (HD) plaque-forming units of CVB, whereas female mice received the RD only. Animals were sacrificed 1, 2, 4, 8, and 11 weeks after CVB or PBS injection. Virally inoculated mice developed viral disease with a temporary decline in general condition and weight loss, which was less pronounced in female animals (P<.001). In male CVB mice, premature mortality occurred between days 8 and 23 after inoculation (RD: 21%, HD: 20%), whereas all female animals survived. Over the course of disease, cardiac inflammation progressively subsided, with faster resolution in female mice. There were no substantial group differences in the composition of the inflammatory cell infiltrates: predominance of cytotoxic T cells at day 7 and 14, and a switch from arginase1-reactive macrophages to iNOS-reactive macrophages from day 7 to 14 were the main findings. There was concomitant development and maturation of different patterns of myocardial fibrosis, with enhanced fibrogenesis in male mice. Virus was almost completely cleared from the heart by day 14. Serum biomarkers of cardiac damage and cardiac expression of remodeling genes were temporarily elevated during the acute phase of disease. Cardiac CTGF gene upregulation was less prolonged in female CVB animals. In vivo electrophysiology studies at weeks 8 and 11 demonstrated that under baseline conditions (i.e. in the absence of proarrhythmogenic drugs), ventricular arrhythmias could only be induced in CVB animals. The cumulative arrhythmia burden throughout the entire stimulation protocol was not significantly different between CVB and control groups. CONCLUSION: CVB inoculation in C57BL/6J mice represents a model of acute self-limiting viral myocarditis, with progression to different patterns of myocardial fibrosis. Sex, but not inoculation dose, seems to modulate the course of disease.

Circulating MicroRNA as Biomarkers of Anthracycline-Induced Cardiotoxicity: JACC: CardioOncology State-of-the-Art Review.

Close monitoring for cardiotoxicity during anthracycline chemotherapy is crucial for early diagnosis and therapy guidance. Currently, monitoring relies on cardiac imaging and serial measurement of cardiac biomarkers like cardiac troponin and natriuretic peptides. However, these conventional biomarkers are nonspecific indicators of cardiac damage. Exploring new, more specific biomarkers with a clear link to the underlying pathomechanism of cardiotoxicity holds promise for increased specificity and sensitivity in detecting early anthracycline-induced cardiotoxicity. miRNAs (microRNAs), small single-stranded, noncoding RNA sequences involved in epigenetic regulation, influence various physiological and pathological processes by targeting expression and translation. Emerging as new biomarker candidates, circulating miRNAs exhibit resistance to degradation and offer a direct pathomechanistic link. This review comprehensively outlines their potential as early biomarkers for cardiotoxicity and their pathomechanistic link.

Characterization of systolic and diastolic function, alongside proteomic profiling, in doxorubicin-induced cardiovascular toxicity in mice.

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.