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The incidence of type 2 diabetes (T2D) is increasing globally, with long-term implications for human health and longevity. Heart disease is the leading cause of death in T2D patients, who display an elevated risk of an acute cardiovascular event and worse outcomes following such an insult. The underlying mechanisms that predispose the diabetic heart to this poor prognosis remain to be defined. This study developed a pre-clinical model (Rattus norvegicus) that complemented caloric excess from a high-fat diet (HFD) and pancreatic ß-cell dysfunction from streptozotocin (STZ) to produce hyperglycaemia, peripheral insulin resistance, hyperlipidaemia and elevated fat mass to mimic the clinical features of T2D. Ex vivo cardiac function was assessed using Langendorff perfusion with systolic and diastolic contractile depression observed in T2D hearts. Cohorts representing untreated, individual HFD- or STZ-treatments and the combined HFD + STZ approach were used to generate ventricular samples (n = 9 per cohort) for sequential and integrated analysis of the proteome, lipidome and metabolome by liquid chromatography-tandem mass spectrometry. This study found that in T2D hearts, HFD treatment primed the metabolome, while STZ treatment was the major driver for changes in the proteome. Both treatments equally impacted the lipidome. Our data suggest that increases in ß-oxidation and early TCA cycle intermediates promoted rerouting via 2-oxaloacetate to glutamate, γ-aminobutyric acid and glutathione. Furthermore, we suggest that the T2D heart activates networks to redistribute excess acetyl-CoA towards ketogenesis and incomplete ß-oxidation through the formation of short-chain acylcarnitine species. Multi-omics provided a global and comprehensive molecular view of the diabetic heart, which distributes substrates and products from excess ß-oxidation, reduces metabolic flexibility and impairs capacity to restore high energy reservoirs needed to respond to and prevent subsequent acute cardiovascular events.
Assuntos
Diabetes Mellitus Tipo 2 , Cardiomiopatias Diabéticas , Animais , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Ácidos Graxos/metabolismo , Humanos , Insulina , Proteoma , RatosRESUMO
Heart failure (HF) with left ventricular diastolic dysfunction is a growing global concern. This study evaluated myocardial oxidized nicotinamide adenine dinucleotide (NAD+) levels in human systolic and diastolic HF and in a murine model of HF with preserved ejection fraction, exploring NAD+ repletion as therapy. We quantified myocardial NAD+ and nicotinamide phosphoribosyltransferase levels, assessing restoration with nicotinamide riboside (NR). Findings show significant NAD+ and nicotinamide phosphoribosyltransferase depletion in human diastolic HF myocardium, but NR successfully restored NAD+ levels. In murine HF with preserved ejection fraction, NR as preventive and therapeutic intervention improved metabolic and antioxidant profiles. This study underscores NAD+ repletion's potential in diastolic HF management.
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Success in gene therapy in treating human disease makes this technology attractive to enhance athletic performance, creating the need for gene doping detection. In 2021, World Anti-Doping Agency (WADA) approved the first gene doping test. Here, we describe a new method to detect doping with four additional genes, follistatin, growth hormone 1, growth hormone-releasing hormone and insulin-like growth factor 1, that may improve performance by increasing muscle size and strength. The method utilises four hydrolysis probe-based polymerase chain reaction (PCR) assays that target the transgenes based on the coding sequence of the four endogenous genes. The assays are specific, reproducible and capable to detect five copies of transgene in the presence of very similar endogenous gene in 25,000 times excess. To underpin reliable and comparable routine method performance by doping testing laboratories, a synthetic reference material for the method was designed and generated following the ISO Guide 35. The complete method was validated in blood samples using plasma as extraction matrix and QIAamp DNA blood midi DNA extraction kit. All blood samples from different donors (n = 8) simulated to be negative or positive (1500 transgene copies spiked per millilitre of blood) for the transgenes were reported correctly. The new method that targets four additional genes will extend the capabilities of laboratories involved in doping control to protect athletes' health, fairness and equality.
Assuntos
Atletas , Dopagem Esportivo , Humanos , Transgenes , Reação em Cadeia da Polimerase/métodos , Terapia Genética , DNARESUMO
There is an urgent need for models that faithfully replicate heart failure with preserved ejection fraction (HFpEF), now recognized as the most common form of heart failure in the world. In vitro approaches have several shortcomings, most notably the immature nature of stem cell-derived human cardiomyocytes [induced pluripotent stem cells (iPSC)] and the relatively short lifespan of primary cardiomyocytes. Three-dimensional 'organoids' incorporating mature iPSCs with other cell types such as endothelial cells and fibroblasts are a significant advance, but lack the complexity of true myocardium. Animal models can replicate many features of human HFpEF, and rodent models are the most common, and recent attempts to incorporate haemodynamic, metabolic, and ageing contributions are encouraging. Differences relating to species, physiology, heart rate, and heart size are major limitations for rodent models. Porcine models mitigate many of these shortcomings and approximate human physiology more closely, but cost and time considerations limit their potential for widespread use. Ex vivo analysis of failing hearts from animal models offer intriguing possibilities regarding cardiac substrate utilisation, but are ultimately subject to the same constrains as the animal models from which the hearts are obtained. Ex vivo approaches using human myocardial biopsies can uncover new insights into pathobiology leveraging myocardial energetics, substrate turnover, molecular changes, and systolic/diastolic function. In collaboration with a skilled cardiothoracic surgeon, left ventricular endomyocardial biopsies can be obtained at the time of valvular surgery in HFpEF patients. Critically, these tissues maintain their disease phenotype, preserving inter-relationship of myocardial cells and extracellular matrix. This review highlights a novel approach, where ultra-thin myocardial tissue slices from human HFpEF hearts can be used to assess changes in myocardial structure and function. We discuss current approaches to modelling HFpEF, describe in detail the novel tissue slice model, expand on exciting opportunities this model provides, and outline ways to improve this model further.
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Insuficiência Cardíaca , Animais , Células Endoteliais , Insuficiência Cardíaca/terapia , Humanos , Miocárdio , Miócitos Cardíacos , Volume Sistólico , SuínosRESUMO
Aims: Cysteine (Cys) is a major target for redox post-translational modifications (PTMs) that occur in response to changes in the cellular redox environment. We describe multiplexed, peptide-based enrichment and quantitative mass spectrometry (MS) applied to globally profile reversible redox Cys PTM in rat hearts during ischemia/reperfusion (I/R) in the presence or absence of an aminothiol antioxidant, N-2-mercaptopropionylglycine (MPG). Parallel fractionation also allowed identification of irreversibly oxidized Cys peptides (Cys-SO2H/SO3H). Results: We identified 4505 reversibly oxidized Cys peptides of which 1372 were significantly regulated by ischemia and/or I/R. An additional 219 peptides (247 sites) contained Cys-SO2H/Cys-SO3H modifications, and these were predominantly identified from hearts subjected to I/R (n = 168 peptides). Parallel reaction monitoring MS (PRM-MS) enabled relative quantitation of 34 irreversibly oxidized Cys peptides. MPG attenuated a large cluster of I/R-associated reversibly oxidized Cys peptides and irreversible Cys oxidation to less than nonischemic controls (n = 24 and 34 peptides, respectively). PRM-MS showed that Cys sites oxidized during ischemia and/or I/R and "protected" by MPG were largely mitochondrial, and were associated with antioxidant functions (peroxiredoxins 5 and 6) and metabolic processes, including glycolysis. Metabolomics revealed I/R induced changes in glycolytic intermediates that were reversed in the presence of MPG, which were consistent with irreversible PTM of triose phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), altered GAPDH enzyme activity, and reduced I/R glycolytic payoff as evidenced by adenosine triphosphate and NADH levels. Innovation: Novel enrichment and PRM-MS approaches developed here enabled large-scale relative quantitation of Cys redox sites modified by reversible and irreversible PTM during I/R and antioxidant remediation. Conclusions: Cys sites identified here are targets of reactive oxygen species that can contribute to protein dysfunction and the pathogenesis of I/R.
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Antioxidantes/farmacologia , Cisteína/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Oxirredução , Processamento de Proteína Pós-Traducional , Animais , Modelos Animais de Doenças , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/metabolismo , Peptídeos/metabolismo , Proteoma , Proteômica/métodos , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
AIMS: Sleep apnoea and congestive heart failure (CHF) commonly co-exist, but their interaction is unclear. Metabolomics may clarify their interaction and relationships to outcome. METHODS AND RESULTS: We assayed 372 circulating metabolites and lipids in 1919 and 1524 participants of the Framingham Heart Study (FHS) (mean age 54 ± 10 years, 53% women) and Women's Health Initiative (WHI) (mean age 67 ± 7 years), respectively. We used linear and Cox regression to relate plasma concentrations of metabolites and lipids to echocardiographic parameters; CHF and its subtypes heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF); and sleep indices. Adenine dinucleotide phosphate (ADP) associated with left ventricular (LV) fractional shortening; phosphocreatine with LV wall thickness; lysosomal storage molecule sphingomyelin 18:2 with LV mass; and nicotine metabolite cotinine with time spent with an oxygen saturation less than 90% (ß = 2.3 min, P = 2.3 × 10-5 ). Pro-hypertrophic metabolite hydroxyglutarate partly mediated the association between LV wall thickness and HFpEF. Central sleep apnoea was significantly associated with HFpEF (P = 0.03) but not HFrEF (P = 0.5). There were three significant metabolite canonical variates, one of which conferred protection from cardiovascular death [hazard ratio = 0.3 (0.11, 0.81), P = 0.02]. CONCLUSIONS: Energetic metabolites were associated with cardiac function; energy- and lipid-storage metabolites with LV wall thickness and mass; plasma levels of nicotine metabolite cotinine were associated with increased time spent with a sleep oxygen saturation less than 90%, a clinically significant marker of outcome, indicating a significant hazard for smokers who have sleep apnoea.
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Insuficiência Cardíaca , Síndromes da Apneia do Sono , Adulto , Idoso , Ecocardiografia , Feminino , Ventrículos do Coração/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes da Apneia do Sono/complicações , Volume SistólicoRESUMO
Men are significantly more susceptible to non-melanoma skin cancers than women, and the androgen receptor (AR) is widely distributed in the skin, suggesting a ro\le for androgens acting via AR. Therefore, we explored the role of androgen action via AR in susceptibility to experimental 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin carcinogenesis and in skin structural development of male and female mice. We demonstrate that both the male gender and androgen action via AR modify the susceptibility to carcinogen-induced skin cancer, but the effect depends on the carcinogenesis model used. Following systemic DMBA exposure, males were significantly (p < 0.05) more susceptible to DMBA-induced experimental skin cancer than females and AR inactivation significantly delayed cancer detection in both male (median time to palpable tumours 19 vs. >35 weeks (wild-type [WT] vs. AR knockout [ARKO], p < 0.001) and female (27 vs. >35 weeks, p = 0.008)) mice. In contrast, following DMBA/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced multistage local skin carcinogenesis, AR inactivation protected against formation of DMBA-induced skin cancers in both male and female mice. The skin structure was also affected by gender effect as well as the AR inactivation and could at least partly explain the different responses between the carcinogenesis models (systemic vs. topical). In addition, AR inactivation modified Cox-1 and Cox-2 expression in the skin, suggesting possible molecular mechanism for the AR effect on skin. Finally, some gender differences are observed also in ARKO mice insensitive to androgens, suggesting that factors other than androgens also play a role in gender-dependent skin carcinogenesis.