Amniotic fluid metabolomics identifies impairment of glycerophospholipid and amino acid metabolism during congenital Zika syndrome development.

PURPOSE: Our main goal is to identify the alterations in the amniotic fluid (AF) metabolome in Zika virus (ZIKV)-infected patients and their relation to congenital Zika syndrome (CZS) progression. EXPERIMENTAL DESIGN: We applied an untargeted metabolomics strategy to analyze seven AF of pregnant women: healthy women and ZIKV-infected women bearing non-microcephalic and microcephalic fetuses. RESULTS: Infected patients were characterized by glycerophospholipid metabolism impairment, which is accentuated in microcephalic phenotypes. Glycerophospholipid decreased concentration in AF can be a consequence of intracellular transport of lipids to the placental or fetal tissues under development. The increased intracellular concentration of lipids can lead to mitochondrial dysfunction and neurodegeneration caused by lipid droplet accumulation. Furthermore, the dysregulation of amino acid metabolism was a molecular fingerprint of microcephalic phenotypes, specifically serine, and proline metabolisms. Both amino acid deficiencies were related to neurodegenerative disorders, intrauterine growth retardation, and placental abnormalities. CONCLUSIONS AND CLINICAL RELEVANCE: This study enhances our understanding of the development of CZS pathology and sheds light on dysregulated pathways that could be relevant for future studies.

Replicative Study in Performance-Related Genes of Brazilian Elite Soccer Players Highlights Genetic Differences from African Ancestry and Similarities between Professional and U20 Youth Athletes.

Classically, genetic association studies have attempted to assess genetic polymorphisms related to human physiology and physical performance. However, the heterogeneity of some findings drives the research to replicate, validate, and confirmation as essential aspects for ensuring their applicability in sports sciences. Genetic distance matrix and molecular variance analyses may offer an alternative approach to comparing athletes' genomes with those from public databases. Thus, we performed a complete sequencing of 44 genomes from male Brazilian first-division soccer players under 20 years of age (U20_BFDSC). The performance-related SNP genotypes were obtained from players and from the "1000 Genomes" database (European, African, American, East Asian, and South Asian). Surprisingly, U20_BFDSC performance-related genotypes had significantly larger FST levels (p < 0.00001) than African populations, although studies using ancestry markers have shown an important similarity between Brazilian and African populations (12-24%). U20_BFDSC were genetically similar to professional athletes, showing the intense genetic selection pressure likely to occur before this maturation stage. Our study highlighted that performance-related genes might undergo selective pressure due to physical performance and environmental, cognitive, and sociocultural factors. This replicative study suggests that molecular variance and Wright's statistics can yield novel conclusions in exercise science.

Aging Triggers Mitochondrial Dysfunction in Mice.

Direct analysis of isolated mitochondria from old mice enables a better understanding of heart senescence dysfunction. Despite a well-defined senescent phenotype in cardiomyocytes, the mitochondrial state in aged cardiomyocytes is still unclear. Here, we report data about mitochondrial function in old mice. Isolated cardiomyocytes' mitochondria were obtained by differential centrifugation from old and young mice hearts to perform functional analyses of mitochondrial O2 consumption, transmembrane potential, ROS formation, ATP production, and swelling. Our results show that mitochondria from old mouse hearts have reduced oxygen consumption during the phosphorylative states of complexes I and II. Additionally, these mitochondria produced more ROS and less ATP than those of young hearts. Mitochondria from old hearts also showed a depolarized membrane potential than mitochondria from young hearts and, as expected, a greater electron leak. Our results indicate that mitochondria from senescent cardiomyocytes are less efficient in O2 consumption, generating more ROS and producing less ATP. Furthermore, the phosphorylative state of complexes I and II presents a functional defect, contributing to greater leakage of protons and ROS production that can be harmful to the cell.

Decellularized Extracellular Matrix Powder Accelerates Metabolic Maturation at Early Stages of Cardiac Differentiation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

During fetal development, cardiomyocytes switch from glycolysis to oxidative metabolism to sustain the energy requirements of functional cells. State-of-the-art cardiac differentiation protocols yield phenotypically immature cardiomyocytes, and common methods to improve metabolic maturation require multistep protocols to induce maturation only after cardiac specification is completed. Here, we describe a maturation method using ventricle-derived decellularized extracellular matrix (dECM) that promoted early-stage metabolic maturation of cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs). Chemically and architecturally preserved particles (45-500 µm) of pig ventricular dECM were added to hiPSCs at the start of differentiation. At the end of our maturation protocol (day 15 of cardiac differentiation), we observed an intimate interaction between cardiomyocytes and dECM particles without impairment of cardiac differentiation efficiency (approx. 70% of cTNT+). Compared with control cells (those cultured without pig dECM), 15-day-old dECM-treated cardiomyocytes demonstrated increased expression of markers related to cardiac metabolic maturation, MAPK1, FOXO1, and FOXO3, and a switch from ITGA6 (the immature integrin isoform) to ITGA3 and ITGA7 (those present in adult cardiomyocytes). Electrical parameters and responsiveness to dobutamine also improved in pig ventricular dECM-treated cells. Extending the culture time to 30 days, we observed a switch from glucose to fatty acid metabolism, indicated by decreased glucose uptake and increased fatty acid consumption in cells cultured with dECM. Together, these data suggest that dECM contains endogenous cues that enable metabolic maturation of hiPSC-CMs at early stages of cardiac differentiation.

Modelling premature cardiac aging with induced pluripotent stem cells from a hutchinson-gilford Progeria Syndrome patient.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic disorder that causes accelerated aging and a high risk of cardiovascular complications. However, the underlying mechanisms of cardiac complications of this syndrome are not fully understood. This study modeled HGPS using cardiomyocytes (CM) derived from induced pluripotent stem cells (iPSC) derived from a patient with HGPS and characterized the biophysical, morphological, and molecular changes found in these CM compared to CM derived from a healthy donor. Electrophysiological recordings suggest that the HGPS-CM was functional and had normal electrophysiological properties. Electron tomography showed nuclear morphology alteration, and the 3D reconstruction of electron tomography images suggests structural abnormalities in HGPS-CM mitochondria, however, there was no difference in mitochondrial content as measured by Mitotracker. Immunofluorescence indicates nuclear morphological alteration and confirms the presence of Troponin T. Telomere length was measured using qRT-PCR, and no difference was found in the CM from HGPS when compared to the control. Proteomic analysis was carried out in a high-resolution system using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). The proteomics data show distinct group separations and protein expression differences between HGPS and control-CM, highlighting changes in ribosomal, TCA cycle, and amino acid biosynthesis, among other modifications. Our findings show that iPSC-derived cardiomyocytes from a Progeria Syndrome patient have significant changes in mitochondrial morphology and protein expression, implying novel mechanisms underlying premature cardiac aging.

Plasma metabolome study reveals metabolic changes induced by pharmacological castration and testosterone supplementation in healthy young men.

Testosterone is a hormone that plays a key role in carbohydrate, fat, and protein metabolism. Testosterone deficiency is associated with multiple comorbidities, e.g., metabolic syndrome and type 2 diabetes. Despite its importance in many metabolic pathways, the mechanisms by which it controls metabolism are not fully understood. The present study investigated the short-term metabolic changes of pharmacologically induced castration and, subsequently, testosterone supplementation in healthy young males. Thirty subjects were submitted to testosterone depletion (TD) followed by testosterone supplementation (TS). Plasma samples were collected three times corresponding to basal, low, and restored testosterone levels. An untargeted metabolomics study was performed by liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) to monitor the metabolic changes induced by the altered hormone levels. Our results demonstrated that TD was associated with major metabolic changes partially restored by TS. Carnitine and amino acid metabolism were the metabolic pathways most impacted by variations in testosterone. Furthermore, our results also indicated that LH and FSH might strongly alter the plasma levels of indoles and lipids, especially glycerophospholipids and sphingolipids. Our results demonstrated major metabolic changes induced by low testosterone that may be important for understanding the mechanisms behind the association of testosterone deficiency and its comorbidities.

Sarcopenic metabolomic profile reflected a sarcopenic phenotype associated with amino acid and essential fatty acid changes.

INTRODUCTION: Although sarcopenia greatly affects health and quality of life in older people, its pathophysiological causes are not fully elucidated. To face this challenge, omics technologies can be used. The metabolome gives a vision of the interaction between the genome and the environment through metabolic networks, thus contributing in clarifying the pathophysiology of the sarcopenic phenotype. OBJECTIVES: The main goal of this study was to compare the plasma metabolome of sarcopenic and non-sarcopenic older people. METHODS: Cross-sectional study of 20 sarcopenic and 21 non-sarcopenic older subjects with available frozen plasma samples. Non-targeted metabolomic study by ultra-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) analysis with later bioinformatics data analysis. Once the significantly different metabolites were identified, the KEGG database was used on them to establish which were the metabolic pathways mainly involved. RESULTS: From 657 features identified, 210 showed significant differences between the study groups, and 30 had a FoldChangeLog2 > 2. The most interesting metabolic pathways found with the KEGG database were the biosynthesis of amino acids, arginine and proline metabolism, the biosynthesis of alkaloids derived from ornithine, linoleic acid metabolism, and the biosynthesis of unsaturated fatty acids. CONCLUSIONS: The study results allowed us to confirm that the concept of "sarcopenic phenotype" is also witnessed at the plasma metabolite levels. The non-targeted metabolomics study can open a wide view of the sarcopenic features changes at the plasma level, which would be linked to the sarcopenic physiopathological alterations.

Cues from human atrial extracellular matrix enrich the atrial differentiation of human induced pluripotent stem cell-derived cardiomyocytes.

New robust and reproducible differentiation approaches are needed to generate induced pluripotent stem cell (iPSC)-derived cardiomyocytes of specific subtypes in predictable quantities for tissue-specific disease modeling, tissue engineering, and eventual clinical translation. Here, we assessed whether powdered decellularized extracellular matrix (dECM) particles contained chamber-specific cues that could direct the cardiac differentiation of human iPSCs toward an atrial phenotype. Human hearts were dissected and the left ventricle (LV) and left atria (LA) were isolated, minced, and decellularized using an adapted submersion decellularization technique to generate chamber-specific powdered dECM. Comparative proteomic analyses showed chamber-specific dECM segregation, with atrial- and ventricle-specific proteins uniquely present in powdered dECM-hA and dECM-hV, respectively. Cell populations differentiated in the presence of dECM-hA showed upregulated atrial molecular markers and a two-fold increase in the number of atrial-like cells as compared with cells differentiated with dECM-hV or no dECM (control). Finally, electrophysiological data showed an increase in action potentials characteristic of atrial-like cells in the dECM-hA group. These findings support the hypothesis that dECM powder derived from human atria retained endogenous cues to drive cardiac differentiation toward an atrial fate.

Tissue-engineered human embryonic stem cell-containing cardiac patches: evaluating recellularization of decellularized matrix.

Decellularized cardiac extracellular matrix scaffolds with preserved composition and architecture can be used in tissue engineering to reproduce the complex cardiac extracellular matrix. However, evaluating the extent of cardiomyocyte repopulation of decellularized cardiac extracellular matrix scaffolds after recellularization attempts is challenging. Here, we describe a unique combination of biochemical, biomechanical, histological, and physiological parameters for quantifying recellularization efficiency of tissue-engineered cardiac patches compared with native cardiac tissue. Human embryonic stem cell-derived cardiomyocytes were seeded into rat heart atrial and ventricular decellularized cardiac extracellular matrix patches. Confocal and atomic force microscopy showed cell integration within the extracellular matrix basement membrane that was accompanied by restoration of native cardiac tissue passive mechanical properties. Multi-electrode array and immunostaining (connexin 43) were used to determine synchronous field potentials with electrical coupling. Myoglobin content (~60%) and sarcomere length measurement (>45% vs 2D culture) were used to evaluate cardiomyocyte maturation of integrated cells. The combination of these techniques allowed us to demonstrate that as cellularization efficiency improves, cardiomyocytes mature and synchronize electrical activity, and tissue mechanical/biochemical properties improve toward those of native tissue.

Exogenous 10 kDa-Heat Shock Protein Preserves Mitochondrial Function After Hypoxia/Reoxygenation.

Humoral factors released during ischemic preconditioning (IPC) protect the myocardium against ischemia/reperfusion (I/R) injury. We have recently identified 10 kDa-heat shock protein (HSP10) and a fraction of small 5-10 kDa peptides (5-10-sP) in the coronary effluent of IPC-treated hearts and demonstrated their cardioprotective potential. We here used our isolated mitochondria model to characterize the impact of exogenous HSP10 and 5-10-sP on mitochondria function from myocardium subjected to I/R injury. Isolated perfused rat hearts were submitted to 30-min global ischemia and 10-min reperfusion. Before ischemia, isolated hearts were infused with saline or 5-10-sP, with or without a mitochondrial ATP-sensitive-K+-channel blocker (5HD 10 µmol·L-1) or PKC inhibitor (chelerythrine 10 µmol·L-1), before I/R. HSP10 (1 µmol·L-1) was infused into isolated hearts before I/R without blockers. At 10-min reperfusion, the mitochondria were isolated and mitochondrial function was assessed. In a subset of experiments, freshly isolated mitochondria were directly incubated with HSP10 or 5-10-sP with or without 5HD or chelerythrine before in vitro hypoxia/reoxygenation. Infusion of 5-10-sP (n = 5) and HSP10 (n = 5) into isolated hearts before I/R improved mitochondrial ADP-stimulated respiration, ATP production and prevented mitochondrial ROS formation compared to the I/R group (n = 5); this effect was abrogated by 5HD and chelerythrine. In freshly isolated mitochondria with in vitro hypoxia/reoxygenation, HSP10 (n = 16) and 5-10-sP (n = 16) incubation prevented reductions of mitochondrial ADP-stimulated respiration (91.5 ± 5.1 nmol O2/min/mg PTN), ATP production (250.1 ± 9.3 µmol ATP/200µg PTN), and prevented mitochondrial ROS production (219.7 ± 9.0 nmol H2O2/200µg PTN) induced by hypoxia/reoxygenation (n = 12, 51.5 ± 5.0 nmol O2/min/mg PTN; 187 ± 21.7 µmol ATP/200 µg PTN; 339.0 ± 14.3 nmol H2O2/200 µg PTN, p < 0.001, respectively). 5HD reduced the ADP-stimulated respiration in the HSP10 group (65.84 ± 3.3 nmol O2/min/mg PTN), ATP production (193.7 ± 12.1 µmol ATP/200µg PTN) and increased ROS in the 5-10-sP group (274.4 ± 21.7 nmol H2O2/200 µg PTN). Mitochondria are a target of the cardioprotection induced by 5-10-sP and HSP10. This protection is dependent of PKC and mKATP activation. HSP10 can act directly on mitochondria and protects against hypoxia/reoxygenation injury by mKATP activation.

Different Signatures of High Cardiorespiratory Capacity Revealed With Metabolomic Profiling in Elite Athletes.

PURPOSE: High cardiorespiratory capacity is a key determinant of human performance and life expectancy; however, the underlying mechanisms are not fully understood. The objective of this pilot study was to investigate biochemical signatures of endurance-performance athletes using high-resolution nontargeted metabolomics. METHODS: Elite long-distance runners with similar training and anthropometrical records were studied. After athletes' maximal oxygen consumption (V˙O2max) was measured, they were divided into 2 groups: low V˙O2max (<65 mL·kg-1·min-1, n = 7) and high V˙O2max (>75 mL·kg-1·min-1, n = 7). Plasma was collected under basal conditions after 12 hours of fasting and after a maximal exercise test (nonfasted) and analyzed by high-resolution LC-MS. Multivariate and univariate statistics were applied. RESULTS: A total of 167 compounds were putatively identified with an LC-MS-based metabolomics pipeline. Partial least-squares discriminant analysis showed a clear separation between groups. Significant variations in metabolites highlighted group differences in diverse metabolic pathways, including lipids, vitamins, amino acids, purine, histidine, xenobiotics, and others, either under basal condition or after the maximal exercise test. CONCLUSIONS: Taken together, the metabolic alterations revealed in the study affect cellular energy use and availability, oxidative stress management, muscle damage, central nervous system signaling metabolites, nutrients, and compound bioavailability, providing new insights into metabolic alterations associated with exercise and cardiorespiratory fitness levels in trained athletes.

R534C mutation in hERG causes a trafficking defect in iPSC-derived cardiomyocytes from patients with type 2 long QT syndrome.

Patient-specific cardiomyocytes obtained from induced pluripotent stem cells (CM-iPSC) offer unprecedented mechanistic insights in the study of inherited cardiac diseases. The objective of this work was to study a type 2 long QT syndrome (LQTS2)-associated mutation (c.1600C > T in KCNH2, p.R534C in hERG) in CM-iPSC. Peripheral blood mononuclear cells were isolated from two patients with the R534C mutation and iPSCs were generated. In addition, the same mutation was inserted in a control iPSC line by genome editing using CRISPR/Cas9. Cells expressed pluripotency markers and showed spontaneous differentiation into the three embryonic germ layers. Electrophysiology demonstrated that action potential duration (APD) of LQTS2 CM-iPSC was significantly longer than that of the control line, as well as the triangulation of the action potentials (AP), implying a longer duration of phase 3. Treatment with the IKr inhibitor E4031 only caused APD prolongation in the control line. Patch clamp showed a reduction of IKr on LQTS2 CM-iPSC compared to control, but channel activation was not significantly affected. Immunofluorescence for hERG demonstrated perinuclear staining in LQTS2 CM-iPSC. In conclusion, CM-iPSC recapitulated the LQTS2 phenotype and our findings suggest that the R534C mutation in KCNH2 leads to a channel trafficking defect to the plasma membrane.

Metabolomic profiling suggests systemic signatures of premature aging induced by Hutchinson-Gilford progeria syndrome.

INTRODUCTION: Hutchinson-Gilford Progeria Syndrome (HGPS) is an extremely rare genetic disorder. HGPS children present a high incidence of cardiovascular complications along with altered metabolic processes and an accelerated aging process. No metabolic biomarker is known and the mechanisms underlying premature aging are not fully understood. OBJECTIVES: The present work aims to evaluate the metabolic alterations in HGPS using high resolution mass spectrometry. METHODS: The present study analyzed plasma from six HGPS patients of both sexes (7.7 ± 1.4 years old; mean ± SD) and eight controls (8.6 ± 2.3 years old) by LC-MS/MS in high-resolution non-targeted metabolomics (Q-Exactive Plus). Targeted metabolomics was used to validate some of the metabolites identified by the non-targeted method in a triple quadrupole (TSQ-Quantiva). RESULTS: We found several endogenous metabolites with statistical differences between control and HGPS children. Multivariate statistical analysis showed a clear separation between groups. Potential novel metabolic biomarkers were identified using the multivariate area under ROC curve (AUROC) based analysis, showing an AUC value higher than 0.80 using only two metabolites, and tending to 1.00 when increasing the number of metabolites in the AUROC model. Taken together, changed metabolic pathways involve sphingolipids, amino acids, and oxidation of fatty acids, among others. CONCLUSION: Our data show significant alterations in cellular energy use and availability, in signal transduction, and lipid metabolites, adding new insights on metabolic alterations associated with premature aging and suggesting novel putative biomarkers.

Paradoxical effect of testosterone supplementation therapy on cardiac ischemia/reperfusion injury in aged rats.

Aging is followed by numerous physiological limitations that reduce health span, particularly cardiovascular and metabolic disorders. Testosterone supplementation therapy (TST) has been widely used in the treatment of aging dysfunctions in either adult or aged patients, although recent evidence have suggested that the incidence of myocardial infarction might be increased in elderly patients. So far, though, the effects of TST in the progression of cardiac ischemia/reperfusion (IR) injury in aged hearts remain unclear. Male aged (23-24 months old) and adult (6 months old) Wistar rats were treated with placebo (Old + Placebo n = 5 / Adult + Placebo n = 5) or TST (Old + TST n = 7 / Adult + TST n = 5) for 30 days. After euthanasia, artificially-perfused isolated rat hearts were submitted to IR. Cardiac expression levels of genes encoding α and ß myosin heavy chain (MHC), ryanodine receptor (RyR), brain-natriuretic peptide (BNP), sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a), glucose-regulated protein 78 kDa (GRP78), eukaryotic initiation factor 2α (eIF2α), C/EBP-homologous protein (CHOP), caspase 3 and B cell lymphoma 2 (Bcl-2) were accessed by qRT-PCR. Protein levels of CHOP, p-Akt, and p-glycogen synthase kinase 3ß (p-GSK-3ß) were measured by Western Blot. Compared to placebo-treated aged rats, Old + TST group exhibited increased heart weight and up-regulation of αMHC mRNA expression levels, whereas ßMHC mRNA expression (p < 0.05). During reperfusion, left ventricular developed pressure, dP/dt+, dP/dt-, and cardiac contractile function index were increased in Old + TST rat hearts (p < 0.05), whereas infarct size was increased (p < 0.05) in comparison with Old + Placebo group. p-Akt levels of Old + TST rat hearts were decreased when compared to Old + Placebo group. Conversely, TST did not promote significant effects in adult rat hearts. Taken together, these findings suggest that myocardial stunning and infarct size of aged hearts were distinctly affected by TST.

Novel strategies for clinical investigation and biomarker discovery: a guide to applied metabolomics.

Metabolomics is an emerging technology that is increasing both in basic science and in human applications, providing a physiological snapshot. It has been highlighted as one of the most wide ranging and reliable tools for the investigation of physiological status, the discovery of new biomarkers and the analysis of metabolic pathways. Metabolomics uses innovative mass spectrometry (MS) allied to chromatography or nuclear magnetic resonance (NMR). The recent advances in bioinformatics, databases and statistics, have provided a unique perception of metabolites interaction and the dynamics of metabolic pathways at a system level. In this context, several studies have applied metabolomics in physiology- and disease-related works. The application of metabolomics includes, physiological and metabolic evaluation/monitoring, individual response to different exercise, nutritional interventions, pathological processes, responses to pharmacological interventions, biomarker discovery and monitoring for distinct aspects, such as: physiological capacity, fatigue/recovery and aging among other applications. For metabolomic analyses, despite huge improvements in the field, several complex methodological steps must be taken into consideration. In this regard, the present article aims to summarize the novel aspects of metabolomics and provide a guide for metabolomics for professionals related to physiologist and medical applications.

Single-Nucleotide-Polymorphism-Panel Population-Genetics Approach Based on the 1000 Genomes Database and Elite Soccer Players.

Purpose: Soccer is one of the most popular sports worldwide, a physical activity of great physiological demand and complexity. Currently, numerous trials involving physiological responses such as hypertrophy, energy expenditure, vasodilation, cardiac output, VO2max, and recovery have supported the possibility of genomic predictors' affecting performance. In a complementary way to association studies with single nucleotide polymorphisms (SNPs), the objective was to evaluate if the use of population genetics data from human-genomics databases can provide information for a better understanding of the relationship between heritability and sport performance. Methods: The study included 25 healthy male professional soccer players (25.5 [4.3] y, 177.4 [6.4] cm, 76.4 [6.4] kg, body fat 10.5% [4.3%]) from the Brazilian first-division soccer club. Anthropometric measurements and field and isokinetic tests were performed to evaluate performance and physiologic parameters of subjects. Moreover, 10 genetic polymorphisms previously related to performance were genotyped. The genotypes of the same polymorphisms were obtained for 2504 individuals from the populations deposited in the 1000 Genomes database. A principal-component analysis and matrix genetic-distances approach (Fst) were evaluated. Results: As expected, the admixture Brazilian population has numerous genetic similarities with the European and American populations from genomic databases. Although the African component is absolutely recognized in genomes from the Brazilian population, using the specific performance-related SNPs, surprisingly the African population was one of the most genetically distant of the players (P < .00001). Conclusions: The early results suggest a selective pressure on genes of elite soccer players, possibly related simultaneously to physical-performance, environmental, cognitive, and sociocultural aspects.

Aging-related compensated hypogonadism: Role of metabolomic analysis in physiopathological and therapeutic evaluation.

Aging is a complex process that increases the risk of chronic disease development. Hormonal and metabolic alterations occur with aging, such as androgen activity decrease. Studies aim to understand the role of testosterone replacement therapy (TRT) in males, however biomarkers and the metabolic responses to TRT are not well characterized. Therefore, the present study investigated TRT effect in young adult and aged rats by metabolomics. Male Wistar rats were divided into four groups: adult and adult + testo (6months), old and old + testo (25-27months). TRT animals received daily testosterone propionate (1 mg/kg/subcutaneous). TRT changed the testicular weight index decrease induced by aging but did not change the body weight and liver weight index. Sera were analyzed by liquid chromatograph high resolution mass spectrometry (LCMS/MS). Testosterone was quantified by target LCMS/MS. A total of 126 metabolites were detected with known identification altered by TRT by non-target metabolomics analysis. Multivariate statistics shows that all groups segregated individually after principal component analysis. The treatment with testosterone induced several metabolic alterations in adult and old rats that were summarized by variable importance on projection score, metabolite interaction and pathway analysis. Aging-related hypogonadism induces a pattern of systemic metabolic alterations that can be partially reversed by TRT, however, this treatment in aged rats induces novel alterations in some metabolites that are possible new targets for monitoring in patients submitted to TRT.

Bone-Marrow-Derived Mesenchymal Stromal Cells (MSC) from Diabetic and Nondiabetic Rats Have Similar Therapeutic Potentials / Células Estromais Mesenquimais (CEM) Derivadas de Medula Óssea de Ratos com e sem Diabetes têm Potencial Terapêutico Similar

Abstract Background: Diabetes mellitus is a severe chronic disease leading to systemic complications, including cardiovascular dysfunction. Previous cell therapy studies have obtained promising results with the use bone marrow mesenchymal stromal cells derived from healthy animals (MSCc) in diabetes animal models. However, the ability of MSC derived from diabetic rats to improve functional cardiac parameters is still unknown. Objectives: To investigate whether bone-marrow-derived MSC from diabetic rats (MSCd) would contribute to recover metabolic and cardiac electrical properties in other diabetic rats. Methods: Diabetes was induced in Wistar rats with streptozotocin. MSCs were characterized by flow cytometry, morphological analysis, and immunohistochemistry. Cardiac electrical function was analyzed using recordings of ventricular action potential. Differences between variables were considered significant when p < 0.05. Results: In vitro properties of MSCc and MSCd were evaluated. Both cell types presented similar morphology, growth kinetics, and mesenchymal profile, and could differentiate into adipogenic and osteogenic lineages. However, in an assay for fibroblast colony-forming units (CFU-F), MSCd formed more colonies than MSCc when cultured in expansion medium with or without hydrocortisone (1 µM). In order to compare the therapeutic potential of the cells, the animals were divided into four experimental groups: nondiabetic (CTRL), diabetic (DM), diabetic treated with MSCc (DM + MSCc), and diabetic treated with MSCd (DM + MSCd). The treated groups received a single injection of MSC 4 weeks after the development of diabetes. MSCc and MSCd controlled hyperglycemia and body weight loss and improved cardiac electrical remodeling in diabetic rats. Conclusions: MSCd and MSCc have similar in vitro properties and therapeutic potential in a rat model of diabetes induced with streptozotocin.

Resumo Fundamentos: O diabetes mellitus é uma doença crônica grave que leva a complicações sistêmicas, como a disfunção cardiovascular. Estudos anteriores de terapia celular obtiveram resultados promissores com utilização de células estromais mesenquimais (CEM) derivadas de medula óssea de animais saudáveis (CEMc) em modelos de animais diabéticos. No entanto, a capacidade das CEM derivadas de ratos diabéticos em melhorar parâmetros cardíacos funcionais é ainda desconhecida. Objetivos: Investigar se CEM derivadas de medula óssea de ratos diabéticos (CEMd) poderiam contribuir para a recuperação metabólica e de propriedades elétricas cardíacas em outros ratos também com diabetes. Métodos: O diabetes foi induzido em ratos Wistar com estreptozotocina. As CEM foram caracterizadas por citometria de fluxo, análise morfológica e imunohistoquímica. A função elétrica cardíaca foi analisada através de registro do potencial de ação ventricular. As diferenças entre as variáveis foram consideradas significativas quando p < 0,05. Resultados: As propriedades in vitro das CEMc e CEMd foram avaliadas. Ambos os tipos celulares apresentaram morfologia, cinética de crescimento e perfil mesenquimal semelhante, e puderam ser diferenciadas em linhagens adipogênica e osteogênica. No entanto, em ensaios para unidades formadoras de colônias de fibroblastos (UFC-F), as CEMd formaram mais colônias em comparação às CEMc quando cultivadas em meio com ou sem hidrocortisona (1 µM). Para comparar o potencial terapêutico das células, os animais foram divididos em quatro grupos experimentais: não diabéticos (CTRL), diabéticos (DM), diabéticos tratados com CEMc (DM + CEMc) e diabéticos tratados com CEMd (DM + CEMd). Os grupos tratados receberam uma única injeção de CEM 4 semanas após o estabelecimento do diabetes. Ambas CEMc e CEMd controlaram a hiperglicemia e a perda de peso corporal e melhoraram o remodelamento elétrico cardíaco em ratos com diabetes. Conclusão: As CEMd e CEMc possuem propriedades in vitro e potencial terapêutico semelhante em um modelo de rato com diabetes induzido por estreptozotocina. (Arq Bras Cardiol. 2017; 109(6):579-589)