Glucose fluctuation promotes mitochondrial dysfunctions in the cardiomyocyte cell line HL-1.

AIMS: Glycemic variability has been suggested as a risk factor for diabetes complications but the precise deleterious mechanisms remain poorly understood. Since mitochondria are the main source of energy in heart and cardiovascular diseases remain the first cause of death in patients with diabetes, the aim of the study was to evaluate the impact of glucose swings on mitochondrial functions in the cardiomyocyte cell line HL-1. METHODS: HL-1 cells were exposed to low (LG, 2.8 mmol/l), normal (NG, 5.5 mmol/l), high (HG, 25 mmol/l) or intermittent high glucose (IHG, swing between low and high) every 2h during 12h (short-time treatment) or every 12h during 72h (long-time treatment). Anaerobic catabolism of glucose was evaluated by measuring glucose consumption and lactate production, oxidative phosphorylation was evaluated by polarography and ATP measurement, mitochondrial superoxide anions and the mitochondrial membrane potential (MMP) were analysed using fluorescent probes, and the protein oxidation was measured by oxyblot. RESULTS: IHG and HG increased glucose consumption and lactate production compared to LG and NG but without any difference between short- and long-time treatments. After 72h and unlike to LG, NG and HG, we didn't observe any increase of the mitochondrial respiration in the presence of succinate upon IHG treatment. IHG, and to a lesser extent HG, promoted a time-dependent decrease of the mitochondrial membrane potential compared to LG and NG treatments. HG and IHG also increased superoxide anion production compared to LG and NG both at 12 and 72h but with a higher increase for IHG at 72h. At last, both HG and IHG stimulated protein oxidation at 72h compared to LG and NG treatments. CONCLUSIONS: Our results demonstrated that exposure of HL-1 cells to glucose swings promoted time-dependent mitochondrial dysfunctions suggesting a deleterious effect of such condition in patients with diabetes that could contribute to diabetic cardiomyopathy.

Silicon Supply Improves Nodulation and Dinitrogen Fixation and Promotes Growth in Trifolium incarnatum Subjected to a Long-Term Sulfur Deprivation.

In many crops species, sulfur (S) deprivation negatively affects growth, seed yield quality and plant health. Furthermore, silicon (Si) is known to alleviate many nutritional stresses but the effects of Si supply on plants subjected to S deficiency remain unclear and poorly documented. The objective of this study was to evaluate whether Si supply would alleviate the negative effects of S deprivation on root nodulation and atmospheric dinitrogen (N2) fixation capacity in Trifolium incarnatum subjected (or not) to long-term S deficiency. For this, plants were grown for 63 days in hydroponic conditions with (500 µM) or without S and supplied (1.7 mM) or not with Si. The effects of Si on growth, root nodulation and N2 fixation and nitrogenase abundance in nodules have been measured. The most important beneficial effect of Si was observed after 63 days. Indeed, at this harvest time, a Si supply increased growth, the nitrogenase abundance in nodules and N2 fixation in S-fed and S-deprived plants while a beneficial effect on the number and total biomass of nodules was only observed in S-deprived plants. This study shows clearly for the first time that a Si supply alleviates negative effects of S deprivation in Trifolium incarnatum.

TRPM4 non-selective cation channel in human atrial fibroblast growth.

Cardiac fibroblasts play an important role in cardiac matrix turnover and are involved in cardiac fibrosis development. Ca2+ is a driving belt in this phenomenon. This study evaluates the functional expression and contribution of the Ca2+-activated channel TRPM4 in atrial fibroblast phenotype. Molecular and electrophysiological investigations were conducted in human atrial fibroblasts in primary culture and in atrial fibroblasts obtained from wild-type and transgenic mice with disrupted Trpm4 gene (Trpm4-/-). A typical TRPM4 current was recorded on human cells (equal selectivity for Na+ and K+, activation by internal Ca2+, voltage sensitivity, conductance of 23.2 pS, inhibition by 9-phenanthrol (IC50 = 6.1 × 10-6 mol L-1)). Its detection rate was 13% on patches at days 2-4 in culture but raised to 100% on patches at day 28. By the same time, a cell growth was observed. This growth was smaller when cells were maintained in the presence of 9-phenanthrol. Similar cell growth was measured on wild-type mice atrial fibroblasts during culture. However, this growth was minimized on Trpm4-/- mice fibroblasts compared to control animals. In addition, the expression of alpha smooth muscle actin increased during culture of atrial fibroblasts from wild-type mice. This was not observed in Trpm4-/- mice fibroblasts. It is concluded that TRPM4 participates in fibroblast growth and could thus be involved in cardiac fibrosis.

Impact of T161, Y318 and S363 alanine mutations on regulation of the human delta-opioid receptor (hDOPr) induced by peptidic and alkaloid agonists.

Previously, we showed a differential regulation of the human delta-opioid receptor (hDOPr) by etorphine and [D-Pen2, D-Pen5] enkephalin (DPDPE). To understand the molecular basis of such differences, we introduced 3 alanine mutations at the residues T161. Y318 and S363. Both wild type (WT) and hDOPr mutants were expressed in HEK cells containing endogenous arrestins or CFP-tagged arrestin 3, then desensitization, internalization, recycling and phosphorylation were studied. In a context of endogenous arrestin expression, a major difference in DOPr desensitization was observed between agonists that was modified with the T161A mutation upon etorphine and with the S363A substitution upon DPDPE exposure. While both agonists induced a major receptor internalization, T161A and S363A impaired DOPr sequestration only for etorphine. However, similar level of S363 phosphorylation was measured between agonists. When CFP-tagged arrestin 3 was over-expressed, a similar profile of desensitization was measured for both agonists. In this context, all the 3 alanine mutations decreased etorphine-induced receptor desensitization. Using FRET, we showed similar interactions between WT hDOPr and arrestin 3 under DPDPE and etorphine stimulation which were delayed by both the Y318A and the S363A substitutions for etorphine. Finally, hDOPr recycling was qualitatively evaluated by microscopy and showed neither arrestin 3/hDOPr colocalization nor major impact of alanine mutations except for the S363A which impaired internalization and recycling for etorphine. The T161, Y318 and S363 residues of hDOPr could underlie the differential regulation promoted by DPDPE and etorphine.

Molecular imaging of endothelial activation and mineralization in a mouse model of accelerated atherosclerosis.

PURPOSE: Preclinical imaging of endothelial activation and mineralization using both positron emission tomography (PET) and magnetic resonance (MR) remains scarce. PROCEDURES: A group of uremic ApoE-/- (Ur), non-uremic ApoE-/- (NUr), and control C57Bl/6 J mice (Ctl) were investigated. Mineralization process was assessed using sodium fluoride ([18F]NaF) PET, and MR imaging combined with intravenous injection of MPIO-αVCAM-1 was used to evaluate endothelial activation. Micro- and macrocalcifications were evaluated by flame atomic absorption spectroscopy and von Kossa staining, respectively. RESULTS: Ur mice showed an active and sustained mineralization process compared to Ctl mice (p = 0.002) using [18F]NaF PET imaging. Calcium plasma level was increased in Ur (2.54 ± 0.09 mM, n = 17) compared to NUr and Ctl mice (2.24 ± 0.01, n = 22, and 2.14 ± 0.02, n = 27, respectively; p < 0.0001). Likewise, vascular calcium content was increased in Ur (0.51 ± 0.06 µg Ca2+ per milligram of dry weight aorta, n = 11) compared to NUr (0.27 ± 0.05, n = 9, p = 0.013) and Ctl (0.28 ± 0.05, n = 11, p = 0.014). Ur mice also had a higher inflammatory state using MPIO-αVCAM-1 MR (p global = 0.01, post hoc analysis Ur vs. Ctl p = 0.003) associated with increased VCAM-1 expression (p global = 0.02). Aortic remodeling at the level of the brachiocephalic trunk, brachiocephalic trunk itself, and aortic arch in Ur mice was also demonstrated using MR. CONCLUSIONS: Preclinical molecular imaging allowed in vivo characterization of the early phase of atherosclerosis. [18F]NaF PET showed early and sustained vascular mineralization in uremic ApoE-/- mice. MPIO-αVCAM-1 MR imaging demonstrated aortic endothelial activation, predominantly in segments with vascular remodeling.

Identification of a novel splice site mutation in the SERAC1 gene responsible for the MEGDHEL syndrome.

BACKGROUND: MEGDHEL is an autosomal recessive syndrome defined as 3-MEthylGlutaconic aciduria (3-MGA) with Deafness, Hepatopathy, Encephalopathy, and Leigh-like syndrome on magnetic resonance imaging, due to mutations in the SERAC1 (Serine Active Site Containing 1) gene, which plays a role in the mitochondrial cardiolipin metabolism. METHODS: We report the case of a young patient who presented with a convulsive encephalopathy, 3-methylglutaconic aciduria, deafness, and bilateral T2 hypersignals of the putamen and the thalami, who passed away at 8 years of age. RESULTS: Analysis of nuclear genes using an ampliSeq™ targeted custom panel disclosed two compound heterozygous variants in the SERAC1 gene: a nonsense substitution in exon 4, c.202C>T, resulting in a premature stop codon (p.Arg68*), and a novel variant at a canonical splicing site upstream exon 4 (c.129-1G>C). mRNAs sequencing from the fibroblasts of the patient showed that the splice site variant resulted in exon 3 skipping without frameshift while Western blot experiments showed the absence of SERAC1 expression compared to controls and abnormal filipin staining. CONCLUSION: We showed that the loss of the putative transmembrane domain of SERAC1, due to a novel splice site variant, impairs the protein expression and is responsible for the MEGDHEL syndrome.

A 2 bp deletion in the mitochondrial ATP 6 gene responsible for the NARP (neuropathy, ataxia, and retinitis pigmentosa) syndrome.

Mitochondrial (mt) DNA-associated NARP (neurogenic muscle weakness, ataxia, and retinitis pigmentosa) syndrome is due to mutation in the MT-ATP6 gene. We report the case of a 18-year-old man who presented with deafness, a myoclonic epilepsy, muscle weakness since the age of 10 and further developed a retinitis pigmentosa and ataxia. The whole mtDNA analysis by next-generation sequencing revealed the presence of the 2 bp microdeletion m.9127-9128 del AT in the ATP6 gene at 82% heteroplasmy in muscle and to a lower load in blood (10-20%) and fibroblasts (50%). Using the patient's fibroblasts, we demonstrated a 60% reduction of the oligomycin-sensitive ATPase hydrolytic activity, a 40% decrease in the ATP synthesis and determination of the mitochondrial membrane potential using the fluorescent probe tetramethylrhodamine, ethyl ester indicated a significant reduction in oligomycin sensitivity. In conclusion, we demonstrated that this novel AT deletion in the ATP6 gene is pathogenic and responsible for the NARP syndrome.