Anna Gonzalez-Tendero

Metabolomics Reveals Metabolic Alterations by Intrauterine Growth Restriction in the Fetal Rabbit Brain

Background Intrauterine Growth Restriction (IUGR) due to placental insufficiency occurs in 5–10% of pregnancies and is a major risk factor for abnormal neurodevelopment. The perinatal diagnosis of IUGR related abnormal neurodevelopment represents a major challenge in fetal medicine. The development of clinical biomarkers is considered a promising approach, but requires the identification of biochemical/molecular alterations by IUGR in the fetal brain. This targeted metabolomics study in a rabbit IUGR model aimed to obtain mechanistic insight into the effects of IUGR on the fetal brain and identify metabolite candidates for biomarker development. Methodology/Principal Findings At gestation day 25, IUGR was induced in two New Zealand rabbits by 40–50% uteroplacental vessel ligation in one horn and the contralateral horn was used as control. At day 30, fetuses were delivered by Cesarian section, weighed and brains collected for metabolomics analysis. Results showed that IUGR fetuses had a significantly lower birth and brain weight compared to controls. Metabolomics analysis using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) and database matching identified 78 metabolites. Comparison of metabolite intensities using a t-test demonstrated that 18 metabolites were significantly different between control and IUGR brain tissue, including neurotransmitters/peptides, amino acids, fatty acids, energy metabolism intermediates and oxidative stress metabolites. Principle component and hierarchical cluster analysis showed cluster formations that clearly separated control from IUGR brain tissue samples, revealing the potential to develop predictive biomarkers. Moreover birth weight and metabolite intensity correlations indicated that the extent of alterations was dependent on the severity of IUGR. Conclusions IUGR leads to metabolic alterations in the fetal rabbit brain, involving neuronal viability, energy metabolism, amino acid levels, fatty acid profiles and oxidative stress mechanisms. Overall findings identified aspargine, ornithine, N-acetylaspartylglutamic acid, N-acetylaspartate and palmitoleic acid as potential metabolite candidates to develop clinical biomarkers for the perinatal diagnosis of IUGR related abnormal neurodevelopment.

Postsystolic shortening by myocardial deformation imaging as a sign of cardiac adaptation to pressure overload in fetal growth restriction.

Background—Fetal growth restriction (FGR) is associated with global adverse cardiac remodeling in utero and increased cardiovascular mortality in adulthood. Prenatal myocardial deformation has not been evaluated in FGR to date. We aimed to evaluate prenatal cardiac remodeling comprehensively in FGR including myocardial deformation imaging. Methods and Results—Echocardiography was performed in 37 consecutive FGR (defined as birthweight <10th centile) and 37 normally grown fetuses. A comprehensive fetal echocardiography was performed including tissue Doppler and 2-dimensional–derived strain and strain rate. Postnatal blood pressure measurement at 6 months of age was also performed. FGR cases showed signs of more globular hearts with decreased longitudinal motion (left systolic annular peak velocity: controls mean 6 cm/s [SD 1.2] versus FGR 5.3 [1]) and diastolic dysfunction (isovolumic relaxation time: controls 44 ms [6] versus FGR 52 [9]). Peak strain and strain rate values of the left ventricle were not significantly different; however, a postsystolic shortening in the basal segment of the septal ventricular wall was observed in 57% of the FGR cases and in none of controls (P<0.001). FGR cases with postsystolic shortening had absence of a hypertrophic response, a poorer perinatal outcome (lower gestational age and birthweight, containing all cases of perinatal mortality [8%]), and higher values of blood pressure. Conclusions—Myocardial deformation imaging revealed a postsystolic shortening in 57% of FGR, which supports increased pressure overload as a mechanism for cardiovascular programming in FGR. Postsystolic shortening was associated with severity and with higher blood pressure postnatally.

Permanent Cardiac Sarcomere Changes in a Rabbit Model of Intrauterine Growth Restriction

Background Intrauterine growth restriction (IUGR) induces fetal cardiac remodelling and dysfunction, which persists postnatally and may explain the link between low birth weight and increased cardiovascular mortality in adulthood. However, the cellular and molecular bases for these changes are still not well understood. We tested the hypothesis that IUGR is associated with structural and functional gene expression changes in the fetal sarcomere cytoarchitecture, which remain present in adulthood. Methods and Results IUGR was induced in New Zealand pregnant rabbits by selective ligation of the utero-placental vessels. Fetal echocardiography demonstrated more globular hearts and signs of cardiac dysfunction in IUGR. Second harmonic generation microscopy (SHGM) showed shorter sarcomere length and shorter A-band and thick-thin filament interaction lengths, that were already present in utero and persisted at 70 postnatal days (adulthood). Sarcomeric M-band (GO: 0031430) functional term was over-represented in IUGR fetal hearts. Conclusion The results suggest that IUGR induces cardiac dysfunction and permanent changes on the sarcomere.

Intrauterine growth restriction is associated with cardiac ultrastructural and gene expression changes related to the energetic metabolism in a rabbit model.

Intrauterine growth restriction (IUGR) affects 7-10% of pregnancies and is associated with cardiovascular remodeling and dysfunction, which persists into adulthood. The underlying subcellular remodeling and cardiovascular programming events are still poorly documented. Cardiac muscle is central in the fetal adaptive mechanism to IUGR given its high energetic demands. The energetic homeostasis depends on the correct interaction of several molecular pathways and the adequate arrangement of intracellular energetic units (ICEUs), where mitochondria interact with the contractile machinery and the main cardiac ATPases to enable a quick and efficient energy transfer. We studied subcellular cardiac adaptations to IUGR in an experimental rabbit model. We evaluated the ultrastructure of ICEUs with transmission electron microscopy and observed an altered spatial arrangement in IUGR, with significant increases in cytosolic space between mitochondria and myofilaments. A global decrease of mitochondrial density was also observed. In addition, we conducted a global gene expression profile by advanced bioinformatics tools to assess the expression of genes involved in the cardiomyocyte energetic metabolism and identified four gene modules with a coordinated over-representation in IUGR: oxygen homeostasis (GO: 0032364), mitochondrial respiratory chain complex I (GO:0005747), oxidative phosphorylation (GO: 0006119), and NADH dehydrogenase activity (GO:0003954). These findings might contribute to changes in energetic homeostasis in IUGR. The potential persistence and role of these changes in long-term cardiovascular programming deserves further investigation.

In Vivo Detection of Perinatal Brain Metabolite Changes in a Rabbit Model of Intrauterine Growth Restriction (IUGR)

Background Intrauterine growth restriction (IUGR) is a risk factor for abnormal neurodevelopment. We studied a rabbit model of IUGR by magnetic resonance imaging (MRI) and spectroscopy (MRS), to assess in vivo brain structural and metabolic consequences, and identify potential metabolic biomarkers for clinical translation. Methods IUGR was induced in 3 pregnant rabbits at gestational day 25, by 40–50% uteroplacental vessel ligation in one horn; the contralateral horn was used as control. Fetuses were delivered at day 30 and weighted. A total of 6 controls and 5 IUGR pups underwent T2-w MRI and localized proton MRS within the first 8 hours of life, at 7T. Changes in brain tissue volumes and respective contributions to each MRS voxel were estimated by semi-automated registration of MRI images with a digital atlas of the rabbit brain. MRS data were used for: (i) absolute metabolite quantifications, using linear fitting; (ii) local temperature estimations, based on the water chemical shift; and (iii) classification, using spectral pattern analysis. Results Lower birth weight was associated with (i) smaller brain sizes, (ii) slightly lower brain temperatures, and (iii) differential metabolite profile changes in specific regions of the brain parenchyma. Specifically, we found estimated lower levels of aspartate and N-acetylaspartate (NAA) in the cerebral cortex and hippocampus (suggesting neuronal impairment), and higher glycine levels in the striatum (possible marker of brain injury). Our results also suggest that the metabolic changes in cortical regions are more prevalent than those detected in hippocampus and striatum. Conclusions IUGR was associated with brain metabolic changes in vivo, which correlate well with the neurostructural changes and neurodevelopment problems described in IUGR. Metabolic parameters could constitute non invasive biomarkers for the diagnosis and abnormal neurodevelopment of perinatal origin.

Whole heart detailed and quantitative anatomy, myofibre structure and vasculature from X-ray phase-contrast synchrotron radiation-based micro computed tomography

Background While individual cardiac myocytes only have a limited ability to shorten, the heart efficiently pumps a large volume-fraction thanks to a cell organization in a complex 3D fibre structure. Subclinical subtle cardiac structural remodelling is often present before symptoms arise. Understanding and early detection of these subtle changes is crucial for diagnosis and prevention. Additionally, personalized computational modelling requires knowledge on the multi-scale structure of the whole heart and vessels. Methods and Results We developed a rapid acquisition together with visualization and quantification methods of the integrated microstructure of whole in-vitro rodents hearts using synchrotron based X-ray phase-contrast tomography. These images are formed not only by X-ray absorption by the tissue but also by wave propagation phenomena, enhancing structural information, thus allowing to raise tissue contrast to an unprecedented level. We used a (ex-vivo) normal rat heart and fetal rabbit hearts suffering intrauterine growth restriction as a model of subclinical cardiac remodelling to illustrate the strengths and potential of the technique. For comparison, histology and diffusion tensor magnetic resonance imaging was performed. Conclusions We have developed a novel, high resolution, image acquisition, and quantification approach to study a whole in-vitro heart at myofibre resolution, providing integrated 3D structural information at microscopic level without any need of tissue slicing and processing. This superior imaging approach opens up new possibilities for a systems approach towards analysing cardiac structure and function, providing rapid acquisition of quantitative microstructure of the heart in a near native state.

Automated cardiac sarcomere analysis from second harmonic generation images

Abstract. Automatic quantification of cardiac muscle properties in tissue sections might provide important information related to different types of diseases. Second harmonic generation (SHG) imaging provides a stain-free microscopy approach to image cardiac fibers that, combined with our methodology of the automated measurement of the ultrastructure of muscle fibers, computes a reliable set of quantitative image features (sarcomere length, A-band length, thick–thin interaction length, and fiber orientation). We evaluated the performance of our methodology in computer-generated muscle fibers modeling some artifacts that are present during the image acquisition. Then, we also evaluated it by comparing it to manual measurements in SHG images from cardiac tissue of fetal and adult rabbits. The results showed a good performance of our methodology at high signal-to-noise ratio of 20 dB. We conclude that our automated measurements enable reliable characterization of cardiac fiber tissues to systematically study cardiac tissue in a wide range of conditions.

Automated morphometric characterization of cardiac fibers by second harmonic microscopy imaging

Cardiovascular diseases are one of the main causes of mortality in the world. For this reason it is important to develop techniques that allow for better understanding of cardiac remodelling prior to heart failure. In this line, Second Harmonic Imaging (SHI) provides a label-free microscopy approach to image cardiac fibers. In this work, we automated means for measuring average sarcomere length, intrasarcomeric A-band distance and sarcomere fiber density as such features have been repeatedly correlated with major cardiomyopathies in the literature (previously measured by manual means). We tested our approach on healthy cardiac tissue of rabbits and human samples and compared such results with manual measurements on the same images to validate our methods. We also measured morphometric differences in cardiac fibers from our animal model which undergoes an irregular heart development. We then conclude that our automated measurements enable reliable characterization of cardiac fiber tissues.

A two dimensional electromechanical model of a cardiomyocyte to assess intra-cellular regional mechanical heterogeneities

Experimental studies on isolated cardiomyocytes from different animal species and human hearts have demonstrated that there are regional differences in the Ca2+ release, Ca2+ decay and sarcomere deformation. Local deformation heterogeneities can occur due to a combination of factors: regional/local differences in Ca2+ release and/or re-uptake, intra-cellular material properties, sarcomere proteins and distribution of the intracellular organelles. To investigate the possible causes of these heterogeneities, we developed a two-dimensional finite-element electromechanical model of a cardiomyocyte that takes into account the experimentally measured local deformation and cytosolic [Ca2+] to locally define the different variables of the constitutive equations describing the electro/mechanical behaviour of the cell. Then, the model was individualised to three different rat cardiac cells. The local [Ca2+] transients were used to define the [Ca2+]-dependent activation functions. The cell-specific local Young’s moduli were estimated by solving an inverse problem, minimizing the error between the measured and simulated local deformations along the longitudinal axis of the cell. We found that heterogeneities in the deformation during contraction were determined mainly by the local elasticity rather than the local amount of Ca2+, while in the relaxation phase deformation was mainly influenced by Ca2+ re-uptake. Our electromechanical model was able to successfully estimate the local elasticity along the longitudinal direction in three different cells. In conclusion, our proposed model seems to be a good approximation to assess the heterogeneous intracellular mechanical properties to help in the understanding of the underlying mechanisms of cardiomyocyte dysfunction.

Callibration procedure and characterization of a commercial multiphoton microscope to measure Polarization Second Harmonic Generation microscopy

Polarization Second Harmonic Generation enables to quantify some of the underlying molecular details of key biological structures within muscle, axons and collagen at suboptical scales. However, it is only available in dedicated labs. In this work we detail a procedure to adapt and calibrate a commercial multiphoton microscope (Leica TCS-SP5) to acquire PSHG images reliably. For this, two different procedures were developed: (1) using inexpensive starch granules as polarization probe to estimate incident laser polarization within a region, and (2) using a fluorophore in solution to estimate the incident intensity arriving to each pixel of the image. This works might help to broaden the users of PSHG acquisition in microscopy as well as to provide the elements to correct for experimental errors.