Endurance training and hydroxyurea have synergistic effects on muscle function and energetics in sickle cell disease mice.

Sickle cell disease (SCD) is an hemoglobinopathy resulting in the production of an abnormal Hb (HbS) which can polymerize in deoxygenated conditions, leading to the sickling of red blood cells (RBC). These alterations can decrease the oxygen-carrying capacity leading to impaired function and energetics of skeletal muscle. Any strategy which could reverse the corresponding defects could be of interest. In SCD, endurance training is known to improve multiples muscle properties which restores patient's exercise capacity but present reduced effects in anemic patients. Hydroxyurea (HU) can increase fetal hemoglobin production which can reduce anemia in patients. The present study was conducted to determine whether HU can improve the effects of endurance training to improve muscle function and energetics. Twenty SCD Townes mice have been trained for 8 weeks with (n = 11) or without (n = 9) HU. SCD mice muscle function and energetics were analyzed during a standardized rest-exercise-recovery protocol, using Phosphorus-31 Magnetic resonance spectroscopy (31P-MRS) and transcutaneous stimulation. The combination of training and HU specifically decreased fatigue index and PCr consumption while muscle oxidative capacity was improved. These results illustrate the potential synergistic effects of endurance training and HU on muscle function and energetics in sickle cell disease.

Mitochondrial Creatine Kinase Attenuates Pathologic Remodeling in Heart Failure.

BACKGROUND: Abnormalities in cardiac energy metabolism occur in heart failure (HF) and contribute to contractile dysfunction, but their role, if any, in HF-related pathologic remodeling is much less established. CK (creatine kinase), the primary muscle energy reserve reaction which rapidly provides ATP at the myofibrils and regenerates mitochondrial ADP, is down-regulated in experimental and human HF. We tested the hypotheses that pathologic remodeling in human HF is related to impaired cardiac CK energy metabolism and that rescuing CK attenuates maladaptive hypertrophy in experimental HF. METHODS: First, in 27 HF patients and 14 healthy subjects, we measured cardiac energetics and left ventricular remodeling using noninvasive magnetic resonance 31P spectroscopy and magnetic resonance imaging, respectively. Second, we tested the impact of metabolic rescue with cardiac-specific overexpression of either Ckmyofib (myofibrillar CK) or Ckmito (mitochondrial CK) on HF-related maladaptive hypertrophy in mice. RESULTS: In people, pathologic left ventricular hypertrophy and dilatation correlate closely with reduced myocardial ATP levels and rates of ATP synthesis through CK. In mice, transverse aortic constriction-induced left ventricular hypertrophy and dilatation are attenuated by overexpression of CKmito, but not by overexpression of CKmyofib. CKmito overexpression also attenuates hypertrophy after chronic isoproterenol stimulation. CKmito lowers mitochondrial reactive oxygen species, tissue reactive oxygen species levels, and upregulates antioxidants and their promoters. When the CK capacity of CKmito-overexpressing mice is limited by creatine substrate depletion, the protection against pathologic remodeling is lost, suggesting the ADP regenerating capacity of the CKmito reaction rather than CK protein per se is critical in limiting adverse HF remodeling. CONCLUSIONS: In the failing human heart, pathologic hypertrophy and adverse remodeling are closely related to deficits in ATP levels and in the CK energy reserve reaction. CKmito, sitting at the intersection of cardiac energetics and redox balance, plays a crucial role in attenuating pathologic remodeling in HF. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT00181259.

Comparison of single-voxel 1H-cardiovascular magnetic resonance spectroscopy techniques for in vivo measurement of myocardial creatine and triglycerides at 3T.

BACKGROUND: Single-voxel proton cardiovascular magnetic resonance spectroscopy (1H-CMRS) benefits from 3 T to detect metabolic abnormalities with the quantification of intramyocardial fatty acids (FA) and creatine (Cr). Conventional point resolved spectroscopy (PRESS) sequence remains the preferred choice for CMRS, despite its chemical shift displacement error (CSDE) at high field (≥ 3 T). Alternative candidate sequences are the semi-adiabatic Localization by Adiabatic SElective Refocusing (sLASER) recommended for brain and musculoskeletal applications and the localized stimulated echo acquisition mode (STEAM). In this study, we aim to compare these three single-voxel 1H-CMRS techniques: PRESS, sLASER and STEAM for reproducible quantification of myocardial FA and Cr at 3 T. Sequences are compared both using breath-hold (BH) and free-breathing (FB) acquisitions. METHODS: CMRS accuracy and theoretical CSDE were verified on a purposely-designed fat-water phantom. FA and Cr CMRS data quality and reliability were evaluated in the interventricular septum of 10 healthy subjects, comparing repeated BH and free-breathing with retrospective gating. RESULTS: Measured FA/W ratio deviated from expected phantom ratio due to CSDE with all sequences. sLASER supplied the lowest bias (10%, vs -28% and 27% for PRESS and STEAM). In vivo, PRESS provided the highest signal-to-noise ratio (SNR) in FB scans (27.5 for Cr and 103.2 for FA). Nevertheless, a linear regression analysis between the two BH showed a better correlation between myocardial Cr content measured with sLASER compared to PRESS (r = 0.46; p = 0.03 vs. r = 0.35; p = 0.07) and similar slopes of regression lines for FA measurements (r = 0.94; p < 0.001 vs. r = 0.87; p < 0.001). STEAM was unable to perform Cr measurement and was the method with the lowest correlation (r = 0.59; p = 0.07) for FA. No difference was found between measurements done either during BH or FB for Cr, FA and triglycerides using PRESS, sLASER and STEAM. CONCLUSION: When quantifying myocardial lipids and creatine with CMR proton spectroscopy at 3 T, PRESS provided higher SNR, while sLASER was more reproducible both with single BH and FB scans.

Measuring Myocardial Energetics with Cardiovascular Magnetic Resonance Spectroscopy.

The heart has the highest energy demands per gram of any organ in the body and energy metabolism fuels normal contractile function. Metabolic inflexibility and impairment of myocardial energetics occur with several common cardiac diseases, including ischemia and heart failure. This review explores several decades of innovation in cardiac magnetic resonance spectroscopy modalities and their use to noninvasively identify and quantify metabolic derangements in the normal, failing, and diseased heart. The implications of this noninvasive modality for predicting significant clinical outcomes and guiding future investigation and therapies to improve patient care are discussed.

Traction mechanical characterization of porcine mitral valve annulus.

The Mitral Annulus (MA) is an anisotropic, fibrous, flexible and dynamical structure. While MA dynamics are well documented, its passive mechanical properties remain poorly investigated to complete the design of adequate prostheses. Mechanical properties in traction on four sections of the MA (aortic, left, posterior and right segments) were assessed using a traction test system with a 30 N load cell and pulling jaws for sample fixation. Samples were submitted to a 1.5 N pre-load, 10 pre-conditioning cycles. Three strain rates were tested (5 %/min, 7 %/min and 13 %/min), the first two up to 10 % strain and the last until rupture. High-resolution diffusion-MRI provided microstructural mapping of fractional anisotropy and mean diffusion within muscle and collagen fibres. Ten MA from porcine hearts were excised resulting in 40 tested samples, out of which 28 were frozen prior to testing. Freezing samples significantly increased Young Moduli for all strain rates. No significant differences were found between Young Moduli at different strain rates (fresh samples 2.4 ± 1.1 MPa, 3.8 ± 2.2 MPa and 3.1 ± 1.8 MPa for increasing strain rates in fresh samples), while significant differences were found when comparing aortic with posterior and posterior with lateral (p < 0.012). Aortic segments deformed the most (24.1 ± 9.4 %) while lateral segments endured the highest stress (>0.3 MPa), corresponding to higher collagen fraction (0.46) and fractional anisotropy. Passive machinal properties differed between aortic and lateral segments of the MA. The process of freezing samples altered their mechanical properties. Underlying microstructural differences could be linked to changes in strain response.

New Insights in Early Detection of Anticancer Drug-Related Cardiotoxicity Using Perfusion and Metabolic Imaging.

Cardio-oncology requires a good knowledge of the cardiotoxicity of anticancer drugs, their mechanisms, and their diagnosis for better management. Anthracyclines, anti-vascular endothelial growth factor (VEGF), alkylating agents, antimetabolites, anti-human epidermal growth factor receptor (HER), and receptor tyrosine kinase inhibitors (RTKi) are therapeutics whose cardiotoxicity involves several mechanisms at the cellular and subcellular levels. Current guidelines for anticancer drugs cardiotoxicity are essentially based on monitoring left ventricle ejection fraction (LVEF). However, knowledge of microvascular and metabolic dysfunction allows for better imaging assessment before overt LVEF impairment. Early detection of anticancer drug-related cardiotoxicity would therefore advance the prevention and patient care. In this review, we provide a comprehensive overview of the cardiotoxic effects of anticancer drugs and describe myocardial perfusion, metabolic, and mitochondrial function imaging approaches to detect them before over LVEF impairment.

Sunitinib-induced cardiac hypertrophy and the endothelin axis.

Anti-angiogenics drugs in clinical use for cancer treatment induce cardiotoxic side effects. The endothelin axis is involved in hypertension and cardiac remodelling, and addition of an endothelin receptor antagonist to the anti-angiogenic sunitinib was shown to reduce cardiotoxicity of sunitinib in mice. Here, we explored further the antidote effect of the endothelin receptor antagonist macitentan in sunitinib-treated animals on cardiac remodeling. Methods: Tumor-bearing mice treated per os daily by sunitinib or vehicle were imaged before and after 1, 3 and 6 weeks of treatment by positron emission tomography using [18F]fluorodeoxyglucose and by echocardiography. Non-tumor-bearing animals were randomly assigned to be treated per os daily by vehicle or sunitinib or macitentan or sunitinib+macitentan, and imaged by echocardiography after 5 weeks. Hearts were harvested for histology and molecular analysis at the end of in vivo exploration. Results: Sunitinib treatment increases left ventricular mass and ejection fraction and induces cardiac fibrosis. Sunitinib also induces an early increase in cardiac uptake of [18F]fluorodeoxyglucose, which is significantly correlated with increased left ventricular mass at the end of treatment. Co-administration of macitentan prevents sunitinib-induced hypertension, increase in ejection fraction and cardiac fibrosis, but fails to prevent increase of the left ventricular mass. Conclusion: Early metabolic changes predict sunitinib-induced cardiac remodeling. Endothelin blockade can prevent some but not all cardiotoxic side-effects of sunitinib, in particular left ventricle hypertrophy that appears to be induced by sunitinib through an endothelin-independent mechanism.

Concurrent imaging of vascularization and metabolism in a mouse model of paraganglioma under anti-angiogenic treatment.

Rationale: Deregulation of metabolism and induction of vascularization are major hallmarks of cancer. Using a new multimodal preclinical imaging instrument, we explored a sequence of events leading to sunitinib-induced resistance in a murine model of paraganglioma (PGL) invalidated for the expression of succinate dehydrogenase subunit B (Sdhb-/-). Methods: Two groups of Sdhb-/- tumors bearing mice were treated with sunitinib (6 weeks) or vehicle (3 weeks). Concurrent Positron Emission Tomography (PET) with 2' -deoxy-2'-[18F]fluoro-D-glucose (FDG), Computed Tomography (CT) and Ultrafast Ultrasound Imaging (UUI) imaging sessions were performed once a week and ex vivo samples were analyzed by western blots and histology. Results: PET-CT-UUI enabled to detect a rapid growth of Sdhb-/- tumors with increased glycolysis and vascular development. Sunitinib treatment prevented tumor growth, vessel development and reduced FDG uptake at week 1 and 2 (W1-2). Thereafter, imaging revealed tumor escape from sunitinib treatment: FDG uptake in tumors increased at W3, followed by tumor growth and vessel development at W4-5. Perfused vessels were preferentially distributed in the hypermetabolic regions of the tumors and the perfused volume increased during escape from sunitinib treatment. Finally, initial changes in total lesion glycolysis and maximum vessel length at W1 were predictive of resistance to sunitinib. Conclusion: These results demonstrate an adaptive resistance of Sdhb-/- tumors to six weeks of sunitinib treatment. Early metabolic changes and delayed vessel architecture changes were detectable and predictable in vivo early during anti-angiogenic treatment. Simultaneous metabolic, anatomical and functional imaging can monitor precisely the effects of anti-angiogenic treatment of tumors.

Performance evaluation of the PET component of a hybrid PET/CT-ultrafast ultrasound imaging instrument.

We recently introduced a hybrid imaging instrument, PETRUS, based on a combination of positron emission tomography (PET) for molecular imaging, x-ray computed tomography (CT) for anatomical imaging, co-registration and attenuation correction, and ultrafast ultrasound imaging (UUI) for motion-correction, hemodynamic and biomechanical imaging. In order to ensure a precise co-registration of simultaneous PET-UUI acquisitions, ultrasound probes attached to an ultrafast ultrasound scanner are operated in the field of view (FOV) of a small animal PET/CT scanner using a remote-controlled micro-positioner. Here we explore the effect of the presence of ultrasound probes on PET image quality. We compare the performance of PET and image quality with and without the presence of probes in the PET field of view, both in vitro following the NEMA-NU-4-2008 standard protocol, and in vivo in small animals. Overall, deviations in the quality of images acquired with and without the ultrasound probes were under 10% and under 7% for the NEMA protocol and in vivo tests, respectively. Our results demonstrate the capability of the PETRUS device to acquire multimodal images in vivo without significant degradation of image quality.

Simultaneous positron emission tomography and ultrafast ultrasound for hybrid molecular, anatomical and functional imaging.

Positron emission tomography-computed tomography (PET-CT) is the most sensitive molecular imaging modality, but it does not easily allow for rapid temporal acquisition. Ultrafast ultrasound imaging (UUI)-a recently introduced technology based on ultrasonic holography-leverages frame rates of up to several thousand images per second to quantitatively map, at high resolution, haemodynamic, biomechanical, electrophysiological and structural parameters. Here, we describe a pre-clinical scanner that registers PET-CT and UUI volumes acquired simultaneously and offers multiple combinations for imaging. We demonstrate that PET-CT-UUI allows for simultaneous images of the vasculature and metabolism during tumour growth in mice and rats, as well as for synchronized multi-modal cardiac cine-loops. Combined anatomical, functional and molecular imaging with PET-CT-UUI represents a high-performance and clinically translatable technology for biomedical research.

Cardiac Metabolic Deregulation Induced by the Tyrosine Kinase Receptor Inhibitor Sunitinib is rescued by Endothelin Receptor Antagonism.

The growing field of cardio-oncology addresses the side effects of cancer treatment on the cardiovascular system. Here, we explored the cardiotoxicity of the antiangiogenic therapy, sunitinib, in the mouse heart from a diagnostic and therapeutic perspective. We showed that sunitinib induces an anaerobic switch of cellular metabolism within the myocardium which is associated with the development of myocardial fibrosis and reduced left ventricular ejection fraction as demonstrated by echocardiography. The capacity of positron emission tomography with [18F]fluorodeoxyglucose to detect the changes in cardiac metabolism caused by sunitinib was dependent on fasting status and duration of treatment. Pan proteomic analysis in the myocardium showed that sunitinib induced (i) an early metabolic switch with enhanced glycolysis and reduced oxidative phosphorylation, and (ii) a metabolic failure to use glucose as energy substrate, similar to the insulin resistance found in type 2 diabetes. Co-administration of the endothelin receptor antagonist, macitentan, to sunitinib-treated animals prevented both metabolic defects, restored glucose uptake and cardiac function, and prevented myocardial fibrosis. These results support the endothelin system in mediating the cardiotoxic effects of sunitinib and endothelin receptor antagonism as a potential therapeutic approach to prevent cardiotoxicity. Furthermore, metabolic and functional imaging can monitor the cardiotoxic effects and the benefits of endothelin antagonism in a theranostic approach.

Efficacy of mechanical postconditioning following warm, global ischaemia depends on circulating fatty acid levels in an isolated, working rat heart model†.

OBJECTIVES: The number of heart transplantations is limited by donor organ availability. Donation after circulatory determination of death (DCDD) could significantly improve graft availability; however, organs undergo warm ischaemia followed by reperfusion, leading to tissue damage. Laboratory studies suggest that mechanical postconditioning [(MPC); brief, intermittent periods of ischaemia at the onset of reperfusion] can limit reperfusion injury; however, clinical translation has been disappointing. We hypothesized that MPC-induced cardioprotection depends on fatty acid levels at reperfusion. METHODS: Experiments were performed with an isolated rat heart model of DCDD. Hearts of male Wistar rats (n = 42) underwent working-mode perfusion for 20 min (baseline), 27 min of global ischaemia and 60 min reperfusion with or without MPC (two cycles of 30 s reperfusion/30 s ischaemia) in the presence or absence of high fat [(HF); 1.2 mM palmitate]. Haemodynamic parameters, necrosis factors and oxygen consumption (O2C) were assessed. Recovery rate was calculated as the value at 60 min reperfusion expressed as a percentage of the mean baseline value. The Kruskal-Wallis test was used to provide an overview of differences between experimental groups, and pairwise comparisons were performed to compare specific time points of interest for parameters with significant overall results. RESULTS: Percent recovery of left ventricular (LV) work [developed pressure (DP)-heart rate product] at 60 min reperfusion was higher in hearts reperfused without fat versus with fat (58 ± 8 vs 23 ± 26%, P < 0.01) in the absence of MPC. In the absence of fat, MPC did not affect post-ischaemic haemodynamic recovery. Among the hearts reperfused with HF, two significantly different subgroups emerged according to recovery of LV work: low recovery (LoR) and high recovery (HiR) subgroups. At 60 min reperfusion, recovery was increased with MPC versus no MPC for LV work (79 ± 6 vs 55 ± 7, respectively; P < 0.05) in HiR subgroups and for DP (40 ± 27 vs 4 ± 2%), dP/dtmax (37 ± 24 vs 5 ± 3%) and dP/dtmin (33 ± 21 vs 5 ± 4%; P < 0.01 for all) in LoR subgroups. CONCLUSIONS: Effects of MPC depend on energy substrate availability; MPC increased recovery of LV work in the presence, but not in the absence, of HF. Controlled reperfusion may be useful for therapeutic strategies aimed at improving post-ischaemic recovery of cardiac DCDD grafts, and ultimately in increasing donor heart availability.

Oxygen-transfer performance of a newly designed, very low-volume membrane oxygenator.

OBJECTIVES: Oxygenation of blood and other physiological solutions are routinely required in fundamental research for both in vitro and in vivo experimentation. However, very few oxygenators with suitable priming volumes (<2-3 ml) are available for surgery in small animals. We have designed a new, miniaturized membrane oxygenator and investigated the oxygen-transfer performance using both buffer and blood perfusates. METHODS: The mini-oxygenator was designed with a central perforated core-tube surrounded by parallel-oriented microporous polypropylene hollow fibres, placed inside a hollow shell with a lateral-luer outlet, and sealed at both extremities. With this design, perfusate is delivered via the core-tube to the centre of the mini-oxygenator, and exits via the luer port. A series of mini-oxygenators were constructed and tested in an in vitro perfusion circuit by monitoring oxygen transfer using modified Krebs-Henseleit buffer or whole porcine blood. Effects of perfusion pressure and temperature over flows of 5-60 ml × min(-1) were assessed. RESULTS: Twelve mini-oxygenators with a mean priming volume of 1.5 ± 0.3 ml were evaluated. With buffer, oxygen transfer reached a maximum of 14.8 ± 1.0 ml O2 × l(-1) (pO2: 450 ± 32 mmHg) at perfusate flow rates of 5 ml × min(-1) and decreased with an increase in perfusate flow to 7.8 ± 0.7 ml ml O2 × l(-1) (pO2: 219 ± 24 mmHg) at 60 ml × min(-1). Similarly, with blood perfusate, oxygen transfer also decreased as perfusate flow increased, ranging from 33 ± 5 ml O2 × l(-1) at 5 ml × min(-1) to 11 ± 2 ml O2 × l(-1) at 60 ml × min(-1). Furthermore, oxygen transfer capacity remained stable with blood perfusion over a period of at least 2 h. CONCLUSIONS: We have developed a new miniaturized membrane oxygenator with an ultra-low priming volume (<2 ml) and adequate oxygenation performance. This oxygenator may be of use in overcoming current limitations in equipment size for effective oxygenation in low-volume perfusion circuits, such as small animal extracorporeal circulation and ex vivo organ perfusion.

Cardiac transplantation with hearts from donors after circulatory declaration of death: haemodynamic and biochemical parameters at procurement predict recovery following cardioplegic storage in a rat model.

OBJECTIVES: Donation after circulatory declaration of death (DCDD) could significantly improve the number of cardiac grafts for transplantation. Graft evaluation is particularly important in the setting of DCDD given that conditions of cardio-circulatory arrest and warm ischaemia differ, leading to variable tissue injury. The aim of this study was to identify, at the time of heart procurement, means to predict contractile recovery following cardioplegic storage and reperfusion using an isolated rat heart model. Identification of reliable approaches to evaluate cardiac grafts is key in the development of protocols for heart transplantation with DCDD. METHODS: Hearts isolated from anaesthetized male Wistar rats (n = 34) were exposed to various perfusion protocols. To simulate DCDD conditions, rats were exsanguinated and maintained at 37°C for 15-25 min (warm ischaemia). Isolated hearts were perfused with modified Krebs-Henseleit buffer for 10 min (unloaded), arrested with cardioplegia, stored for 3 h at 4°C and then reperfused for 120 min (unloaded for 60 min, then loaded for 60 min). Left ventricular (LV) function was assessed using an intraventricular micro-tip pressure catheter. Statistical significance was determined using the non-parametric Spearman rho correlation analysis. RESULTS: After 120 min of reperfusion, recovery of LV work measured as developed pressure (DP)-heart rate (HR) product ranged from 0 to 15 ± 6.1 mmHg beats min(-1) 10(-3) following warm ischaemia of 15-25 min. Several haemodynamic parameters measured during early, unloaded perfusion at the time of heart procurement, including HR and the peak systolic pressure-HR product, correlated significantly with contractile recovery after cardioplegic storage and 120 min of reperfusion (P < 0.001). Coronary flow, oxygen consumption and lactate dehydrogenase release also correlated significantly with contractile recovery following cardioplegic storage and 120 min of reperfusion (P < 0.05). CONCLUSIONS: Haemodynamic and biochemical parameters measured at the time of organ procurement could serve as predictive indicators of contractile recovery. We believe that evaluation of graft suitability is feasible prior to transplantation with DCDD, and may, consequently, increase donor heart availability.

Early reperfusion hemodynamics predict recovery in rat hearts: a potential approach towards evaluating cardiac grafts from non-heart-beating donors.

AIMS: Cardiac grafts from non-heartbeating donors (NHBDs) could significantly increase organ availability and reduce waiting-list mortality. Reluctance to exploit hearts from NHBDs arises from obligatory delays in procurement leading to periods of warm ischemia and possible subsequent contractile dysfunction. Means for early prediction of graft suitability prior to transplantation are thus required for development of heart transplantation programs with NHBDs. METHODS AND RESULTS: Hearts (n = 31) isolated from male Wistar rats were perfused with modified Krebs-Henseleit buffer aerobically for 20 min, followed by global, no-flow ischemia (32°C) for 30, 50, 55 or 60 min. Reperfusion was unloaded for 20 min, and then loaded, in working-mode, for 40 min. Left ventricular (LV) pressure was monitored using a micro-tip pressure catheter introduced via the mitral valve. Several hemodynamic parameters measured during early, unloaded reperfusion correlated significantly with LV work after 60 min reperfusion (p<0.001). Coronary flow and the production of lactate and lactate dehydrogenase (LDH) also correlated significantly with outcomes after 60 min reperfusion (p<0.05). Based on early reperfusion hemodynamic measures, a composite, weighted predictive parameter, incorporating heart rate (HR), developed pressure (DP) and end-diastolic pressure, was generated and evaluated against the HR-DP product after 60 min of reperfusion. Effective discriminating ability for this novel parameter was observed for four HR*DP cut-off values, particularly for ≥20 *10(3) mmHg*beats*min(-1) (p<0.01). CONCLUSION: Upon reperfusion of a NHBD heart, early evaluation, at the time of organ procurement, of cardiac hemodynamic parameters, as well as easily accessible markers of metabolism and necrosis seem to accurately predict subsequent contractile recovery and could thus potentially be of use in guiding the decision of accepting the ischemic heart for transplantation.