Browsing by Author "Adam, Tasneem"

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    Investigating the possible cytoprotective effects of melatonin isomer against simulated ischemic injury
    (2017) Victor, Laikyn; Lecour, Sandrine; Adam, Tasneem
    Introduction: The presence of melatonin in wine contributes to the cardioprotective effect of regular and moderate consumption of wine against lethal ischemia/reperfusion injury. Recently, the presence of melatonin isomers has been identified in red wine, but whether or not these isomers confer any physiological properties is unknown. Aim: The aim of our study was to establish a cell culture model of simulated ischemia to study and compare the possible cytoprotective effects of dietary melatonin and a melatonin isomer against an ischemic insult and to explore the possible role of melatonin receptors in this effect. Methods: H9C2 cardiac fibroblast cells were subjected to simulated ischemia by exposure to 1mM H₂O₂ following a 30min pre-treatment with 75ng/L (dietary concentration), 1μM (pharmacological concentration, 0.232mg/L) melatonin or/and 75mg/L (dietary concentration) melatonin isomer. To determine the role of melatonin receptors, cells were pre-treated with the melatonin receptor inhibitor, luzindole (10 μM) for 1h prior to H₂O₂ treatment. At the end of the simulated ischemic insult, cell viability was assessed using trypan blue staining. Mitochondrial respiration in permeabilized H9C2 cells was measured using the Oroboros Instrument, at two different time points: at the end of a 30min pre-treatment with either 75ng/L melatonin or 75mg/L melatonin isomer, or the afore mentioned pre-treatments prior to a 15min treatment of 1mM H₂O₂. Results: A simulated ischemic insult with 1mM H₂O₂ reduced cell viability from 92.9±1.5% to 28.4±1.4% (p<0.001 vs control). Pre-treatment with the dietary concentrations of melatonin or the melatonin isomer improved the cell viability to a similar extent as a pre-treatment with the pharmacological concentration of melatonin (74.4±3.1%, 73.9±2.7% and 69.0±1.2%, p<0.001 vs H₂O₂ and p<0.01 vs H₂O₂ respectively). A combined pre-treatment of melatonin and the melatonin isomer did not add further cytoprotective benefit. Addition of luzindole fully abolished the cytoprotective effect of dietary melatonin (29.7±2.4%, p<0.001 vs H₂O₂ + Mel), but only partially abolished the cytoprotective effect of the melatonin isomer (41.4±3.6%). Both dietary concentrations of melatonin and the melatonin isomer did not affect mitochondrial respiration in permeabilized H9C2 cells. Conclusion: Our findings suggest that both dietary melatonin and the melatonin isomer confer cytoprotection against a simulated ischemic insult, an effect which is mediated, at least in part, via the activation of melatonin receptors. Both melatonin and melatonin isomers present the advantage to be potentially safe and inexpensive therapies against ischemic heart disease.
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    Open Access
    Nuclear respiratory factor 1: a novel inhibitor of the human gene promoter of the cardiac isoform of acetyl-coenzyme a carboxylase
    (2008) Adam, Tasneem; Hatter Cardiovascular, Essop, M F
    Introduction: Nuclear respiratory factor-1 (NRF-1) is a pivotal transcriptional modulator controlling the expression of nuclear genes encoding mitochondrial proteins. Activation of 5'-AMP-activated protein kinase (AMPK) has been associated with enhanced NRF-1 gene promoter binding activity. Furthermore, NRF-1 has been linked with increased gene expression of carnitine palmitoyltransferase-1 (CPT-1), the rate-limiting mitochondrial fatty acid (FA) transfer enzyme. Upstream, the cardiac-enriched isoform of acetyl-CoA carboxylase (ACCl3) synthesises malonyl-CoA, a potent inhibitor of CPT-1 and FA 13-oxidation. Since ACCl3 induction elevates malonyl-CoA levels, we hypothesised that AMPK activates NRF-1 thereby inhibiting ACCl3 expression in the heart to ultimately increase FA !3-oxidation. Methods: A 1,317 bp human ACCl3 gene promoter-luciferase construct (pPll13-1317/+65-Luc) was transiently transfected into rat neonatal cardiomyocytes ± the following expression constructs: NRF-1 (pSG5-NRF-1) and dominant negative NRF-1 (pCMV-HA-dnNRF-1). To elucidate whether AMPK is involved in the transcriptional regulation of ACCl3 gene, the transfected cardiomyocytes were treated with AMPK activators, i.e. AICAR or metformin for 24 hours. Transfections were also performed with upstream stimulatory factor 1 (USF1), a known transactivator of the human ACCl3 gene promoter. In addition, the effect of NRF-1 on endogenous USF1 transcriptional activity was evaluated with a luciferase construct containing multiple copies of USF1-specific enhancer elements (pUSF1-Luc). Results: NRF-1 overexpression reduced ACCl3 gene promoter activity by 56 ± 8.8% (p<0.001 vs. pPlll3-1317/+65-Luc). Cotransfection with the dnNRF-1 construct abrogated this effect. Addition of AICAR dose-dependently decreased ACCl3 gene promoter activity (p<0.05 vs. untreated control). In contrast, NRF-1 attenuation of ACCl3 gene promoter activity was not altered by AICAR treatment. Exposure to metformin did not affect the ACCl3 gene promoter activity at baseline, and in response to NRF-1. NRF-1 inhibited the USF1-dependent upregulation of ACCl3 gene promoter activity by 58 ± 7.5% (p<0.001 vs. pPlll3-1317/+65-Luc + USF1), an effect reversed with the dnNRF-1 construct. In addition, NRF-1 reduced endogenous USF1 transcriptional activity by 55 ± 6.2% (p<0.001 vs. pUSF1-Luc). This effect was abolished with the dnNRF-1 construct. The NRF-1-mediated decrease in endogenous USF1 transcriptional activity was independent of AMPK activation with AICAR. The ACC~ mRNA expression level was unaffected at baseline, and by NRF-1 overexpression. However, AICAR (2 mM) treatment markedly reduced the ACC~ mRNA expression level (p<0.05 vs. vehicle) at baseline, but had no effect in response to NRF-1 overexpression. Conclusion: Our data reveal a unique, inhibitory role for NRF-1 in the transcriptional regulation of human ACC~ gene in the mammalian heart, independent of AMPK activation.