Identification of novel miRNAs involved in cardiac repair following infarction in fetal and adolescent sheep hearts

Aims:Animal models have been used to show that there are critical molecular mechanisms that can be activated to induce myocardial repair at specific times in development. For example, specific miRNAs are critical for regulating the response to myocardial infarction (MI) and improving the response to injury. Manipulating these miRNAs in small animal models provides beneficial effects post-MI; however it is not known if these miRNAs are regulated similarly in large mammals. Studying a large animal where the timing of heart development in relation to birth is similar to humans may provide insights to better understand the capacity to repair a developing mammalian heart and its application to the adult heart. Methods:We used a sheep model of MI that included permanent ligation of the left anterior descending (LAD) coronary artery. Surgery was performed on fetuses (at 105 days gestation when all cardiomyocytes are mononucleated and proliferative) and adolescent sheep (at 6 months of age when all cardiomyocytes contribute to heart growth by hypertrophy). A microarray was utilized to determine the expression of known miRNAs within the damaged and undamaged tissue regions in fetal and adolescent hearts after MI. Results:73 miRNAs were up-regulated and 58 miRNAs were down-regulated significantly within the fetal infarct compared to remote cardiac samples. From adolescent hearts 69 non-redundant miRNAs were up-regulated and 63 miRNAs were down-regulated significantly in the infarct area compared to remote samples. Opposite differential expression profiles of 10 miRNAs within tissue regions (Infarct area, Border zone and Remote area of the left ventricle) occurred between the fetuses and adolescent sheep. These included miR-558 and miR-1538, which when suppressed using LNA anti-miRNAs in cell culture, increased cardiomyoblast proliferation. Conclusion:There were significant differences in miRNA responses in fetal and adolescent sheep hearts following a MI, suggesting that the modulation of novel miRNA expression may have therapeutic potential, by promoting proliferation or repair in a damaged heart.Mitchell C. Lock, Ross L. Tellam, Jack R. T. Darby, Jia Yin Soo, Doug A. Brooks, Mike Seed ... et al

Fine-mapping of prostate cancer susceptibility loci in a large meta-analysis identifies candidate causal variants

Prostate cancer is a polygenic disease with a large heritable component. A number of common, low-penetrance prostate cancer risk loci have been identified through GWAS. Here we apply the Bayesian multivariate variable selection algorithm JAM to fine-map 84 prostate cancer susceptibility loci, using summary data from a large European ancestry meta-analysis. We observe evidence for multiple independent signals at 12 regions and 99 risk signals overall. Only 15 original GWAS tag SNPs remain among the catalogue of candidate variants identified; the remainder are replaced by more likely candidates. Biological annotation of our credible set of variants indicates significant enrichment within promoter and enhancer elements, and transcription factor-binding sites, including AR, ERG and FOXA1. In 40 regions at least one variant is colocalised with an eQTL in prostate cancer tissue. The refined set of candidate variants substantially increase the proportion of familial relative risk explained by these known susceptibility regions, which highlights the importance of fine-mapping studies and has implications for clinical risk profiling. © 2018 The Author(s).Prostate cancer is a polygenic disease with a large heritable component. A number of common, low-penetrance prostate cancer risk loci have been identified through GWAS. Here we apply the Bayesian multivariate variable selection algorithm JAM to fine-map 84 prostate cancer susceptibility loci, using summary data from a large European ancestry meta-analysis. We observe evidence for multiple independent signals at 12 regions and 99 risk signals overall. Only 15 original GWAS tag SNPs remain among the catalogue of candidate variants identified; the remainder are replaced by more likely candidates. Biological annotation of our credible set of variants indicates significant enrichment within promoter and enhancer elements, and transcription factor-binding sites, including AR, ERG and FOXA1. In 40 regions at least one variant is colocalised with an eQTL in prostate cancer tissue. The refined set of candidate variants substantially increase the proportion of familial relative risk explained by these known susceptibility regions, which highlights the importance of fine-mapping studies and has implications for clinical risk profiling. © 2018 The Author(s).Peer reviewe

Development of a method to determine cytochrome P450 1A2, 2C9, 2D6 and 3A4 activity sheep hepatic microsomes

INTRODUCTION:Ex vivo studies of human fetal hepatic drug metabolism are uncommon as it requires access to functional liver tissue and therefore raises practical and ethical concerns. Large animal models provide an alternative opportunity to study changes in cytochrome P450 (CYP) activity in the mother and fetus during pregnancy. We aimed to develop methods to determine the activity of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in sheep hepatic microsomes. METHODS:We identified optimal conditions to determine the activity of CYP1A2 (using the probe drug phenacetin), CYP2C9 (diclofenac), CYP2D6 (dextromethorphan) and CYP3A4 (midazolam) by varying techniques for microsome extraction, probe drug concentration, incubation time and microsome concentration. The specificity of each probe drug was assessed by determining the rate of metabolism when specific CYP enzyme inhibitors were included in the reaction. RESULTS:The optimum incubation time and probe drug concentration was six hours with 5 μM phenacetin (CYP1A2), four hours with 10 μM diclofenac (CYP2C9), 30 min with 1 μM of midazolam (CYP3A4) and 10 min with 1 μM dextromethorphan (CYP2D6). For both CYP2D6 and CYP3A4 reactions required 20 μg of microsomal protein, whereas for CYP1A2 and CYP2C9, reactions required 40 μg of microsomal protein. Metabolism of phenacetin, dextromethorphan and midazolam was reduced by specific enzyme inhibitors, but the specific CYP2C9 inhibitor sulfaphenazole did not substantially inhibit diclofenac metabolism. DISCUSSION:This study identifies the optimal conditions for determining CYP activity in maternal sheep hepatic microsomes. In doing so, we have developed a standardised protocol for assessment of microsomal activity of CYP3A4, CYP1A2 and CYP2D6, but we were unable to optimise conditions for assessment of CYP2C9. This approach can be applied to investigate the impact of pregnancy complications on maternal and fetal hepatic drug metabolism.Grace M. McBride, Jia Yin Soo, Tamara Varco, Janna L. Morrison, Michael D. Wiese ... et al

The impact of intrauterine growth restriction on cytochrome P450 enzyme expression and activity

With the increased prevalence of non-communicable disease and availability of medications to treat these and other conditions, a pregnancy free from prescribed medication exposure is rare. Up to 99% of women take at least one medication during pregnancy. These medications can be divided into those used to improve maternal health and wellbeing (e.g., analgesics, antidepressants, antidiabetics, antiasthmatics), and those used to promote the baby's wellbeing in either fetal (e.g., anti-arrhythmics) or postnatal life (e.g., antenatal glucocorticoids). These medications are needed for pre-existing or coincidental illnesses in the mother, maternal conditions induced by the pregnancy itself through to conditions that arise in the fetus or that will be encountered by the newborn. Thus, medications administered to the mother may be used to treat the mother, the fetus or both. Metabolism of medications is regulated by a range of physiological processes that change during pregnancy. Other pathological processes such as placental insufficiency can in turn have both immediate and lifelong adverse health consequences for babies. Individuals born growth restricted are more likely to require medications but may also have an altered ability to metabolise these medications in fetal and postnatal life. This review aims to determine the effect of suboptimal fetal growth on the fetal expression of the drug metabolising enzymes (DMEs) that convert medications into active or inactive metabolites, and the transporters that remove both these medications and their metabolites from the fetal compartment.Grace M. McBride, Michael D. Wiese, Jia Yin Soo, Jack R.T. Darby, Mary J. Berry, Tamara J. Varcoe, Janna L. Morriso

Imaging inflammation using an activated macrophage probe with Slc18b1 as the activation-selective gating target

Activated macrophages have the potential to be ideal targets for imaging inflammation. However, probe selectivity over non-activated macrophages and probe delivery to target tissue have been challenging. Here, we report a small molecule probe specific for activated macrophages, called CDg16, and demonstrate its application to visualizing inflammatory atherosclerotic plaques in vivo. Through a systematic transporter screen using a CRISPR activation library, we identify the orphan transporter Slc18b1/SLC18B1 as the gating target of CDg16. © 2019, The Author(s

Differential response to injury in fetal and adolescent sheep hearts in the immediate post in the immediate post-myocardial myocardial myocardial infarction period

Aim: Characterizing the response to myocardial infarction (MI) in the regenerative sheep fetus heart compared to the post-natal non-regenerative adolescent heart may reveal key morphological and molecular differences that equate to the response to MI in humans. We hypothesized that the immediate response to injury in (a) infarct compared with sham, and (b) infarct, border, and remote tissue, in the fetal sheep heart would be fundamentally different to the adolescent, allowing for repair after damage. Methods: We used a sheep model of MI induced by ligating the left anterior descending coronary artery. Surgery was performed on fetuses (105 days) and adolescent sheep (6 months). Sheep were randomly separated into MI (n = 5) or Sham (n = 5) surgery groups at both ages. We used magnetic resonance imaging (MRI), histological/immunohistochemical staining, and qRT-PCR to assess the morphological and molecular differences between the different age groups in response to infarction. Results: Magnetic resonance imaging showed no difference in fetuses for key functional parameters; however there was a significant decrease in left ventricular ejection fraction and cardiac output in the adolescent sheep heart at 3 days post-infarction. There was no significant difference in functional parameters between MRI sessions at Day 0 and Day 3 after surgery. Expression of genes involved in glucose transport and fatty acid metabolism, inflammatory cytokines as well as growth factors and cell cycle regulators remained largely unchanged in the infarcted compared to sham ventricular tissue in the fetus, but were significantly dysregulated in the adolescent sheep. Different cardiac tissue region-specific gene expression profiles were observed between the fetal and adolescent sheep. Conclusion: Fetuses demonstrated a resistance to cardiac damage not observed in the adolescent animals. The manipulation of specific gene expression profiles to a fetal-like state may provide a therapeutic strategy to treat patients following an infarction.Mitchell C. Lock, Jack R. T. Darby, Jia Yin Soo, Doug A. Brooks, Sunthara Rajan Perumal, Joseph B. Selvanayagam, Mike Seed, Christopher K. Macgowan, Enzo R. Porrello, Ross L. Tellam and Janna L. Morriso

Detecting metabolic differences in fetal and adult sheep adipose and skeletal muscle tissues

The primary metabolic pathway required to produce ATP differs as a result of tissue type, developmental stage and substrate availability. We utilised molecular and histological techniques to define the metabolic status in fetal and adult, adipose and skeletal muscle tissues. Redox ratios of these tissues were also determined optically by two-photon microscopy. Adult perirenal adipose tissue had a higher optical redox ratio than fetal perirenal adipose tissue, which aligned with glycolysis being used for ATP production; whereas adult skeletal muscle had a lower optical redox ratio than fetal skeletal muscle, which aligned with OXPHOS activity being utilised for ATP production. We have compared traditional molecular and microscopy techniques of metabolic tissue characterisation with optical redox ratios to provide a more comprehensive report on the dynamics of tissue metabolism.Jack R. T. Darby, Alexandra Sorvina, Christie A. Bader, Mitchell C. Lock, Jia Yin Soo ... Douglas A. Brooks ... et al