Myoglobin deficiency impairs maximal oxygen uptake and exercise performance: a lesson from Mb-/- mice.

Myoglobin (Mb) plays an important role at rest and during exercise as a reservoir of oxygen and has been suggested to regulate NO• bioavailability under hypoxic/acidic conditions. However, its ultimate role during exercise is still a subject of debate. We aimed to study the effect of Mb deficiency on maximal oxygen uptake ( V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_2}\max }}$ ) and exercise performance in myoglobin knockout mice (Mb-/- ) when compared to control mice (Mb+/+ ). Furthermore, we also studied NO• bioavailability, assessed as nitrite (NO2 - ) and nitrate (NO3 - ) in the heart, locomotory muscle and in plasma, at rest and during exercise at exhaustion both in Mb-/- and in Mb+/+ mice. The mice performed maximal running incremental exercise on a treadmill with whole-body gas exchange measurements. The Mb-/- mice had lower body mass, heart and hind limb muscle mass (P < 0.001). Mb-/- mice had significantly reduced maximal running performance (P < 0.001). V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_2}\max }}$ expressed in ml min-1 in Mb-/ - mice was 37% lower than in Mb+/+ mice (P < 0.001) and 13% lower when expressed in ml min-1  kg body mass-1 (P = 0.001). Additionally, Mb-/- mice had significantly lower plasma, heart and locomotory muscle NO2 - levels at rest. During exercise NO2 - increased significantly in the heart and locomotory muscles of Mb-/- and Mb+/+ mice, whereas no significant changes in NO2 - were found in plasma. Our study showed that, contrary to recent suggestions, Mb deficiency significantly impairs V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_2}\max }}$ and maximal running performance in mice. KEY POINTS: Myoglobin knockout mice (Mb-/- ) possess lower maximal oxygen uptake ( V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_2}\max }}$ ) and poorer maximal running performance than control mice (Mb+/+ ). Respiratory exchange ratio values at high running velocities in Mb-/- mice are higher than in control mice suggesting a shift in substrate utilization towards glucose metabolism in Mb-/- mice at the same running velocities. Lack of myoglobin lowers basal systemic and muscle NO• bioavailability, but does not affect exercise-induced NO2 - changes in plasma, heart and locomotory muscles. The present study demonstrates that myoglobin is of vital importance for V ̇ O 2 max ${\dot V_{{{\mathrm{O}}_2}\max }}$ and maximal running performance as well as explains why previous studies have failed to prove such a role of myoglobin when using the Mb-/- mouse model.

Early microvascular coronary endothelial dysfunction precedes pembrolizumab-induced cardiotoxicity. Preventive role of high dose of atorvastatin.

Immune checkpoint inhibitors (ICIs) exhibit remarkable antitumor activity and immune-related cardiotoxicity of unknown pathomechanism. The aim of the study was to investigate the ICI class-dependent cardiotoxicity in vitro and pembrolizumab's (Pem's) cardiotoxicity in vivo, seeking for translational prevention means. Cytotoxicity was investigated in primary cardiomyocytes and splenocytes, incubated with ipilimumab, Pem and avelumab. Pem's cross-reactivity was assessed by circular dichroism (CD) on biotechnologically produced human and murine PD-1 and in silico. C57BL6/J male mice received IgG4 or Pem for 2 and 5 weeks. Echocardiography, histology, and molecular analyses were performed. Coronary blood flow velocity mapping and cardiac magnetic resonance imaging were conducted at 2 weeks. Human EA.hy926 endothelial cells were incubated with Pem-conditioned media from human mononuclear cells, in presence and absence of statins and viability and molecular signaling were assessed. Atorvastatin (20 mg/kg, daily) was administered in vivo, as prophylaxis. Only Pem exerted immune-related cytotoxicity in vitro. Pem's cross-reactivity with the murine PD-1 was confirmed by CD and docking. In vivo, Pem initiated coronary endothelial and diastolic dysfunction at 2 weeks and systolic dysfunction at 5 weeks. At 2 weeks, Pem induced ICAM-1 and iNOS expression and intracardiac leukocyte infiltration. At 5 weeks, Pem exacerbated endothelial activation and triggered cardiac inflammation. Pem led to immune-related cytotoxicity in EA.hy926 cells, which was prevented by atorvastatin. Atorvastatin mitigated functional deficits, by inhibiting endothelial dysfunction in vivo. We established for the first time an in vivo model of Pem-induced cardiotoxicity. Coronary endothelial dysfunction precedes Pem-induced cardiotoxicity, whereas atorvastatin emerges as a novel prophylactic therapy.

Retrospectively gated ultrashort-echo-time MRI T1 mapping reveals compromised pulmonary microvascular NO-dependent function in a murine model of acute lung injury.

This study sought to develop noninvasive, in vivo imaging schemes that allow for quantitative assessment of pulmonary microvascular functional status based on the combination of pulmonary T1 mapping and dynamic contrast-enhanced (DynCE) imaging. Ultrashort-echo-time (UTE) imaging at 9.4 T of lung parenchyma was performed. Retrospective gating was based on modulation of the first point in each recorded spoke. T1 maps were obtained using a series of five consecutive images with varying RF angles and analyzed with the variable flip angle approach. The obtained mean T1 lung value of 1078 ± 38 ms correlated well with previous reports. Improved intersession variability was observed, as evident from a decreased standard deviation of motion-resolved T1 mapping (F-test = 0.051). Animals received lipopolysaccharide (LPS) and were imaged at t = 2, 6, and 12 h after administration. The nitric oxide (NO)-dependent function was assessed according to changes in lung T1 after L-NAME injection, while microvascular perfusion and oxidant stress were assessed with contrast-enhanced imaging after injection of gadolinium or 3-carbamoyl-proxyl nitroxide radical, respectively. Retrospectivel gated UTE allowed robust, motion-compensated imaging that could be used for T1 mapping of lung parenchyma. Changes in lung T1 after L-NAME injection indicated that LPS induced overproduction of NO at t = 2 and 6 h after LPS, but NO-dependent microvascular function was impaired at t = 12 h after LPS. DynCE imaging at t = 6 h after LPS injection revealed decreased microvascular perfusion, with increased vascular permeability and oxidant stress. MRI allows to visualize and quantify lung microvascular NO-dependent function and its concomitant impairment during acute respiratory distress syndrome development with high sensitivity. UTE T1 mapping appears to be sensitive and useful in probing pulmonary microvascular functional status.

Rapid shear stress-dependent ENaC membrane insertion is mediated by the endothelial glycocalyx and the mineralocorticoid receptor.

The contribution of the shear stress-sensitive epithelial Na+ channel (ENaC) to the mechanical properties of the endothelial cell surface under (patho)physiological conditions is unclear. This issue was addressed in in vivo and in vitro models for endothelial dysfunction. Cultured human umbilical vein endothelial cells (HUVEC) were exposed to laminar (LSS) or non-laminar shear stress (NLSS). ENaC membrane insertion was quantified using Quantum-dot-based immunofluorescence staining and the mechanical properties of the cell surface were probed with the Atomic Force Microscope (AFM) in vitro and ex vivo in isolated aortae of C57BL/6 and ApoE/LDLR-/- mice. Flow- and acetylcholine-mediated vasodilation was measured in vivo using magnetic resonance imaging. Acute LSS led to a rapid mineralocorticoid receptor (MR)-dependent membrane insertion of ENaC and subsequent stiffening of the endothelial cortex caused by actin polymerization. Of note, NLSS stress further augmented the cortical stiffness of the cells. These effects strongly depend on the presence of the endothelial glycocalyx (eGC) and could be prevented by functional inhibition of ENaC and MR in vitro endothelial cells and ex vivo endothelial cells derived from C57BL/6, but not ApoE/LDLR-/- vessel. In vivo In C57BL/6 vessels, ENaC- and MR inhibition blunted flow- and acetylcholine-mediated vasodilation, while in the dysfunctional ApoE/LDLR-/- vessels, this effect was absent. In conclusion, under physiological conditions, endothelial ENaC, together with the glycocalyx, was identified as an important shear stress sensor and mediator of endothelium-dependent vasodilation. In contrast, in pathophysiological conditions, ENaC-mediated mechanotransduction and endothelium-dependent vasodilation were lost, contributing to sustained endothelial stiffening and dysfunction.

Quantitative myocardial first-pass perfusion imaging of CO2 -induced vasodilation in rats.

Inducible hypercapnia is an alternative for increasing the coronary blood flow necessary to facilitate the quantification of myocardial blood flow during hyperemia. The current study aimed to quantify the pharmacokinetic effect of a CO2 gas challenge on myocardial perfusion in rats using high-resolution, first-pass perfusion CMR and compared it with pharmacologically induced hyperemia using regadenoson. A dual-contrast, saturation-recovery, gradient-echo sequence with a Cartesian readout was used on a small-animal 9.4-T scanner; additional cine images during hyperemia/rest were recorded with an ultrashort echo time sequence. The mean myocardial blood flow value at rest was 6.1 ± 1.4 versus 13.9 ± 3.7 and 14.3 ± 4 mL/g/min during vasodilation with hypercapnia and regadenoson, respectively. Accordingly, the myocardial flow reserve value was 2.6 ± 1.1 for the gas challenge and 2.5 ± 1.4 for regadenoson. During hyperemia with both protocols, a significantly increased cardiac output was found. It was concluded that hypercapnia leads to significantly increased coronary flow and yields similar myocardial flow reserves in healthy rats as compared with pharmacological stimulation. Accordingly, inducible hypercapnia can be selected as an alternative stressor in CMR studies of myocardial blood flow in small animals.

Optimal Control of Magnetization Reversal in a Monodomain Particle by Means of Applied Magnetic Field.

A complete analytical solution to the optimal reversal of a macrospin with easy-axis anisotropy is presented. An optimal control path minimizing the energy cost of the reversal is identified and used to derive the time-dependent direction and amplitude of the optimal switching field. The minimum energy cost of the reversal scales inversely with the switching time for fast switching, follows exponential asymptotics for slow switching, and reaches the lower limit proportional to the energy barrier between the target states and to the damping parameter at infinitely long switching time. For a given switching time, the energy cost is never smaller than that for a free macrospin. This limitation can be bypassed by adding a hard anisotropy axis that activates the internal torque in the desired switching direction, thereby significantly reducing the energy cost. A comparison between the calculated optimal control path and minimum energy path reveals that optimal control does not translate to the minimization of the energy barrier but signifies effective use of the system's internal dynamics to aid the desired magnetic transition.

Accelerating CEST MRI in the mouse brain at 9.4 T by exploiting sparsity in the Z-spectrum domain.

PURPOSE: Chemical exchange saturation transfer (CEST) is an MR contrast modality offering an enhanced sensitivity for the detection of dilute metabolites with exchangeable protons. Quantitative analysis requires the acquisition of a number of images (usually between 20 and 50 RF offsets) per Z-spectrum, leading to long acquisition times of the order of 5-40 min in practice. In this work, we explore the possibility of employing sparsity in the Z-spectrum domain (irradiation offset dimension) to provide an accelerated acquisition scheme without compromising the quality of reconstructed CEST spectra. METHOD AND THEORY: Ex vivo and in vivo data were acquired on an experimental, small animal 9.4 T system. Three different reconstruction methods were tested: k-Z SPARSE, k-Z SLR and k-Z principal component analysis (PCA) using retrospective undersampling with net acceleration factors R = 2, 3, 5. The quality of the reconstructed data was compared with respect to CEST spectra and full magnetization transfer ratio (MTR) asymmetry maps. RESULTS: In both phantom and in vivo data, CEST spectra and the resulting MTR asymmetry maps were reconstructed without significant deterioration in data quality. For a low acceleration factor (R = 2, 3) all applied methods resulted in similar data quality, while for high acceleration factor (R = 5) only k-Z PCA and k-Z SLR could be used. Loss in spatial resolution was observed in reconstruction with k-Z PCA for all acceleration factors. An example of prospective undersampling with acceleration factor R = 3 and k-Z PCA reconstruction demonstrates improved CEST maps when compared with fully sampled data acquisition with either three times longer scan duration or threefold prolonged acquisition window per frequency offset. CONCLUSION: The acquisition time of CEST spectra can be significantly accelerated by exploiting the sparsity of the Z-domain. For prospective and retrospective analysis using k-Z PCA, an acceleration factor of up to R = 3 can be used without significant loss in data quality.

Production of highly polarized [1-13 C]acetate by rapid decarboxylation of [2-13 C]pyruvate - application to hyperpolarized cardiac spectroscopy and imaging.

PURPOSE: The objective of the present work was to develop and implement an efficient approach to hyperpolarize [1-13 C]acetate and apply it to in vivo cardiac spectroscopy and imaging. METHODS: Rapid hydrogen peroxide induced decarboxylation was used to convert hyperpolarized [2-13 C]pyruvate into highly polarized [1-13 C]acetate employing an additional step following rapid dissolution of [2-13 C]pyruvate in a home-built multi-sample dissolution dynamic nuclear polarization system. Phantom dissolution experiments were conducted to determine optimal parameters of the decarboxylation reaction, retaining polarization and T1 of [1-13 C]acetate. In vivo feasibility of detecting [1-13 C]acetate metabolism is demonstrated using slice-selective spectroscopy and multi-echo imaging of [1-13 C]acetate and [1-13 C]acetylcarnitine in the healthy rat heart. RESULTS: The first in vivo signal was observed ~23 s after dissolution. At the corresponding time point in the phantom experiments, 97.9 ± 0.4% of [2-13 C]pyruvate were converted into [1-13 C]acetate by the decarboxylation reaction. T1 and polarization of [1-13 C]acetate was determined to be 29.7 ± 1.9% and a 47.7 ± 0.5 s. Polarization levels of [2-13 C]pyruvate and [1-13 C]acetate were not significantly different after transfer to the scanner. In vivo, [1-13 C]acetate and [1-13 C]acetylcarnitine could be detected using spectroscopy and imaging. CONCLUSION: Decarboxylation of hyperpolarized [2-13 C]pyruvate enables the efficient production of highly polarized [1-13 C]acetate that is applicable to study short-chain fatty acid metabolism in the in vivo heart.

Extended quantitative dynamic contrast-enhanced cardiac perfusion imaging in mice using accelerated data acquisition and spatially distributed, two-compartment exchange modeling.

The objective of the present work was to improve data acquisition and quantification of dynamic contrast-enhanced perfusion imaging in the in vivo murine heart. Four-fold undersampled data were acquired in 14 mice and reconstructed using k-t SPARSE. A two-compartment exchange model was employed to provide additional characterization of myocardial tissue based on compartment volumes and the permeability surface area product. The feasibility of the proposed method was tested using compartment-based analysis of contrast-enhanced perfusion data acquired with intravascular and extracellular contrast agents. A significantly different permeability surface area product was measured for the intravascular versus extracellular contrast agent (0.13-0.15 ml/g/min vs 0.86-0.88 ml/g/min). The reduced extravasation also resulted in significantly smaller interstitial volumes of the intravascular versus extracellular agent (9.8-11% vs 45-47%). No difference was found for myocardial blood flow (6.5-7.2 ml/g/min vs 6.0-7.0 ml/g/min). The results presented here show that two-compartment exchange modeling in the in vivo murine heart is feasible and gives access to tissue parameters beyond myocardial blood flow.

Overestimation of cardiac lactate production caused by liver metabolism of hyperpolarized [1-13 C]pyruvate.

PURPOSE: The purpose of this work was to study the contribution of liver [1-13 C]lactate to the lactate signal detected in the heart following injection of hyperpolarized [1-13 C]pyruvate. METHODS: A slice-selective saturation scheme was incorporated into a hybrid metabolic imaging and spectroscopy approach to selectively presaturate lactate in the liver. Imaging and slice-selective spectroscopy of [1-13 C]pyruvate and its downstream metabolites were sequentially interleaved in the same experiment with optional presaturation of liver [1-13 C]lactate. Six healthy rats were measured, and metabolic data in the heart acquired with and without presaturation of liver lactate were compared. RESULTS: When using liver lactate presaturation, a statistically significant reduction of the lactate/pyruvate ratio was observed in the spectroscopic data of the left ventricle (0.18 ± 0.03 versus 0.24 ± 0.04; p < .05) as well as in the imaging data of the blood pool (0.05 ± 0.01 versus 0.11 ± 0.01; p < .05). No significant difference in myocardial lactate was observed when using myocardium only as the region of interest in the imaging data (0.08 ± 0.01 versus 0.11 ± 0.02; p = .2). CONCLUSION: Liver metabolism leads to statistically significant overestimation of cardiac lactate production in slice-selective or nonselective spectroscopic experiments. Therefore, metabolic imaging is preferred over spectroscopy to separate left-ventricular compartments within the slice and hence avoid contamination of cardiac lactate signals. Alternatively, presaturation pulses should be used in combination with spectroscopy approaches.

Assessing the influence of isoflurane anesthesia on cardiac metabolism using hyperpolarized [1-13 C]pyruvate.

Isoflurane is a frequently used anesthetic in small-animal dissolution dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) studies. Although the literature suggests interactions with mitochondrial metabolism, the influence of the compound on cardiac metabolism has not been assessed systematically to date. In the present study, the impact of low versus high isoflurane concentration was examined in a crossover experiment in healthy rats. The results revealed that cardiac metabolism is modulated by isoflurane concentration, showing increased [1-13 C]lactate and reduced [13 C]bicarbonate production during high isoflurane relative to low isoflurane dose [average differences: +16% [1-13 C]lactate/total myocardial carbon, -22% [13 C]bicarbonate/total myocardial carbon; +51% [1-13 C]lactate/[13 C]bicarbonate]. These findings emphasize that reproducible anesthesia is important when studying cardiac metabolism. As the depth of anesthesia is difficult to control in an experimental animal setting, careful study design is required to exclude confounding factors.

A multisample dissolution dynamic nuclear polarization system for serial injections in small animals.

PURPOSE: Several in vivo applications of dissolution dynamic nuclear polarization (DNP) require rapid successive injections of hyperpolarized substrates. Here we present the design and performance of a custom-built multisample dissolution DNP setup for small animal research. METHODS: The DNP setup consists of a commercial wide-bore magnet charged to 3.35 T, a cryostat, a 94-GHz microwave source, and a custom-built skeleton that accommodates four identical sample sticks. Each sample stick features a dissolver locked into the skeleton port and a lifter, which permits moving the sample cup out of the liquid helium bath for dissolution. RESULTS: The dissolution of the first sample was triggered after 2 hours of polarization buildup during single-shot operation of the cryostat. Thereafter, a time window of 75-90 min was available to dissolve the remaining three polarized samples. The average liquid state polarization over all four sticks was measured as 18.7% ± 2.3% for [1-13C] pyruvate 30 s after dissolution. In vivo applicability of the setup using serial injections of [1-13C] pyruvate to study cardiac metabolism in rats revealed good reproducibility. CONCLUSION: The proposed four-sample DNP insert provides reproducible liquid state polarization of [1-13C] pyruvate and allows for rapid repeat injections in small animals. Magn Reson Med 77:904-910, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Dissolution DNP using trityl radicals at 7 T field.

Dissolution DNP has become an important method to generate highly polarized substrates such as pyruvic acid for in vivo imaging and localized spectroscopy. In a quest to further increase the polarization levels, which is important for in vivo MRI employing 13C detection, we describe the design and implementation of a new DNP polarizer that is suitable for dissolution operation at 7 T static magnetic field and a temperature of 1.4 K. We describe all important sample preparation steps and experimental details necessary to optimize trityl based samples for use in our polarizer at this higher field. In [1-13C]-pyruvic acid polarization levels of about 56% are achieved, compared to typical polarization levels of about 35-45% at a standard field of 3.4 T. At the same time, the polarization build-up time increases significantly from about 670 s at 3.4 T to around 1300-1900 s at 7 T, depending on the trityl derivate used. We also investigate the effect of adding trace amounts of Gd3+ to the samples. While one trityl compound does not exhibit any benefit, the other profits significantly, boosting achievable polarization by 6%.

Dynamic nuclear polarisation by thermal mixing: quantum theory and macroscopic simulations.

A theory of dynamic nuclear polarisation (DNP) by thermal mixing is suggested based on purely quantum considerations. A minimal 6-level microscopic model is developed to test the theory and link it to the well-known thermodynamic model. Optimal conditions for the nuclear polarization enhancement and effects of inhomogeneous broadening of the electron resonance are discussed. Macroscopic simulations of nuclear polarization spectra displaying good agreement with experiments, involving BDPA and trityl free radicals, are presented.

Protein disulfide isomerase A1 regulates fenestration dynamics in primary mouse liver sinusoidal endothelial cells (LSECs).

Protein disulfide isomerases (PDIs) are involved in many intracellular and extracellular processes, including cell adhesion and cytoskeletal reorganisation, but their contribution to the regulation of fenestrations in liver sinusoidal endothelial cells (LSECs) remains unknown. Given that fenestrations are supported on a cytoskeleton scaffold, this study aimed to investigate whether endothelial PDIs regulate fenestration dynamics in primary mouse LSECs. PDIA3 and PDIA1 were found to be the most abundant among PDI isoforms in LSECs. Taking advantage of atomic force microscopy, the effects of PDIA1 or PDIA3 inhibition on the fenestrations in LSECs were investigated using a classic PDIA1 inhibitor (bepristat) and novel aromatic N-sulfonamides of aziridine-2-carboxylic acid derivatives as PDIA1 (C-3389) or PDIA3 (C-3399) inhibitors. The effect of PDIA1 inhibition on liver perfusion was studied in vivo using dynamic contrast-enhanced magnetic resonance imaging. Additionally, PDIA1 inhibitors were examined in vitro in LSECs for effects on adhesion, cytoskeleton organisation, bioenergetics, and viability. Inhibition of PDIA1 with bepristat or C-3389 significantly reduced the number of fenestrations in LSECs, while inhibition of PDIA3 with C-3399 had no effect. Moreover, the blocking of free thiols by the cell-penetrating N-ethylmaleimide, but not by the non-cell-penetrating 4-chloromercuribenzenesulfonate, resulted in LSEC defenestration. Inhibition of PDIA1 did not affect LSEC adhesion, viability, and bioenergetics, nor did it induce a clear-cut rearrangement of the cytoskeleton. However, PDIA1-dependent defenestration was reversed by cytochalasin B, a known fenestration stimulator, pointing to the preserved ability of LSECs to form new pores. Importantly, systemic inhibition of PDIA1 in vivo affected intra-parenchymal uptake of contrast agent in mice consistent with LSEC defenestration. These results revealed the role of intracellular PDIA1 in the regulation of fenestration dynamics in LSECs, and in maintaining hepatic sinusoid homeostasis.

TNF-α protects from exacerbated myocarditis and cardiac death by suppressing expansion of activated heart-reactive CD4+ T cells.

AIMS: Tumour necrosis factor α (TNF-α) represents a classical pro-inflammatory cytokine, and its increased levels positively correlate with the severity of many cardiovascular diseases. Surprisingly, some heart failure patients receiving high doses of anti-TNF-α antibodies showed serious health worsening. This work aimed to examine the role of TNF-α signalling on the development and progression of myocarditis and heart-specific autoimmunity. METHODS AND RESULTS: Mice with genetic deletion of TNF-α (Tnf+/- and Tnf-/-) and littermate controls (Tnf+/+) were used to study myocarditis in the inducible and the transgenic T cell receptor (TCRM) models. Tnf+/- and Tnf-/- mice immunized with α-myosin heavy chain peptide (αMyHC) showed reduced myocarditis incidence, but the susceptible animals developed extensive inflammation in the heart. In the TCRM model, defective TNF-α production was associated with increased mortality at a young age due to cardiomyopathy and cardiac fibrosis. We could confirm that TNF-α as well as the secretome of antigen-activated heart-reactive effector CD4+ T (Teff) cells effectively activated the adhesive properties of cardiac microvascular endothelial cells (cMVECs). Our data suggested that TNF-α produced by endothelial in addition to Teff cells promoted leucocyte adhesion to activated cMVECs. Analysis of CD4+ T lymphocytes from both models of myocarditis showed a strongly increased fraction of Teff cells in hearts, spleens, and in the blood of Tnf+/- and Tnf-/- mice. Indeed, antigen-activated Tnf-/- Teff cells showed prolonged long-term survival and TNF-α cytokine-induced cell death of heart-reactive Teff. CONCLUSION: TNF-α signalling promotes myocarditis development by activating cardiac endothelial cells. However, in the case of established disease, TNF-α protects from exacerbating cardiac inflammation by inducing activation-induced cell death of heart-reactive Teff. These data might explain the lack of success of standard anti-TNF-α therapy in heart failure patients and open perspectives for T cell-targeted approaches.

Chronic heart failure induces early defenestration of liver sinusoidal endothelial cells (LSECs) in mice.

AIM: Chronic heart failure (CHF) is often linked to liver malfunction and systemic endothelial dysfunction. However, whether cardio-hepatic interactions in heart failure involve dysfunction of liver sinusoidal endothelial cells (LSECs) is not known. Here we characterize LSECs phenotype in early and end stages of chronic heart failure in a murine model. METHODS: Right ventricle (RV) function, features of congestive hepatopathy, and the phenotype of primary LSECs were characterized in Tgαq*44 mice, with cardiomyocyte-specific overexpression of the Gαq protein, at the age of 4- and 12-month representative for early and end-stage phases of CHF, respectively. RESULTS: 4- and 12-month-old Tgαq*44 mice displayed progressive impairment of RV function and alterations in hepatic blood flow velocity resulting in hepatic congestion with elevated GGT and bilirubin plasma levels and decreased albumin concentration without gross liver pathology. LSECs isolated from 4- and 12-month-old Tgαq*44 mice displayed significant loss of fenestrae with impaired functional response to cytochalasin B, significant changes in proteome related to cytoskeleton remodeling, and altered vasoprotective function. However, LSECs barrier function and bioenergetics were largely preserved. In 4- and 12-month-old Tgαq*44 mice, LSECs defenestration was associated with prolonged postprandial hypertriglyceridemia and in 12-month-old Tgαq*44 mice with proteomic changes of hepatocytes indicative of altered lipid metabolism. CONCLUSION: Tgαq*44 mice displayed right-sided HF and altered hepatic blood flow leading to LSECs dysfunction involving defenestration, shift in eicosanoid profile, and proteomic changes. LSECs dysfunction appears as an early and persistent event in CHF, preceding congestive hepatopathy and contributing to alterations in lipoprotein transport and CHF pathophysiology.

Accelerated ageing and coronary microvascular dysfunction in chronic heart failure in Tgαq*44 mice.

Age represents a major risk factor in heart failure (HF). However, the mechanisms linking ageing and HF are not clear. We aimed to identify the functional, morphological and transcriptomic changes that could be attributed to cardiac ageing in a model of slowly progressing HF in Tgαq*44 mice in reference to the cardiac ageing process in FVB mice. In FVB mice, ageing resulted in the impairment of diastolic cardiac function and in basal coronary flow (CF), perivascular and interstitial fibrosis without changes in the cardiac activity of angiotensin-converting enzyme (ACE) or aldosterone plasma concentration. In Tgαq*44 mice, HF progression was featured by the impairment of systolic and diastolic cardiac function and in basal CF that was associated with a distinct rearrangement of the capillary architecture, pronounced perivascular and interstitial fibrosis, progressive activation of cardiac ACE and systemic angiotensin-aldosterone-dependent pathways. Interestingly, cardiac ageing genes and processes were represented in Tgαq*44 mice not only in late but also in early phases of HF, as evidenced by cardiac transcriptome analysis. Thirty-four genes and 8 biological processes, identified as being ageing related, occurred early and persisted along HF progression in Tgαq*44 mice and were mostly associated with extracellular matrix remodelling and fibrosis compatible with perivascular fibrosis resulting in coronary microvascular dysfunction (CMD) in Tgαq*44 mice. In conclusion, accelerated and persistent cardiac ageing contributes to the pathophysiology of chronic HF in Tgαq*44 mice. In particular, prominent perivascular fibrosis of microcirculation resulting in CMD represents an accelerated cardiac ageing phenotype that requires targeted treatment in chronic HF.

Endothelial-mesenchymal transition induced by metastatic 4T1 breast cancer cells in pulmonary endothelium in aged mice.

Ageing is a major risk factor for cancer metastasis but the underlying mechanisms remain unclear. Here, we characterised ageing effects on cancer-induced endothelial-mesenchymal transition (EndMT) in the pulmonary circulation of female BALB/c mice in a metastatic 4T1 breast cancer model. The effect of intravenously injected 4T1 cells on pulmonary endothelium, pulmonary metastasis, lung tissue architecture, and systemic endothelium was compared between 40-week-old and 20-week-old mice. The 40-week-old mice showed features of ongoing EndMT in their lungs before 4T1 breast cancer cell injection. Moreover, they had preexisting endothelial dysfunction in the aorta detected by in vivo magnetic resonance imaging (MRI) compared to 20-week-old mice. The injection of 4T1 breast cancer cells into 40-week-old mice resulted in rapid EndMT progression in their lungs. In contrast, injection of 4T1 breast cancer cells into 20-week-old mice resulted in initiation and less pronounced EndMT progression. Although the number of metastases did not differ significantly between 20-week-old and 40-week-old mice, the lungs of older mice displayed altered lung tissue architecture and biochemical content, reflected in higher Amide II/Amide I ratio, higher fibronectin levels, and hypoxia-inducible factor 1 subunit alpha (HIF1α) levels as well as lower nitric oxide (NO) production. Our results indicate that age-dependent pre-existing endothelial dysfunction in the pulmonary endothelium of 40-week-old mice predisposed them to rapid EndMT progression in the presence of circulating 4T1 breast cancer cells what might contribute to a more severe metastatic breast cancer phenotype in these ageing mice compared to younger mice.