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Abstract: Introduction: Diabetes is a major contributor to cardiovascular disease. There is a growing body of evidence pointing towards intra-myocellular lipid accumulation as an integral etiological factor. Here we aimed to determine the effect of two common fatty acids on lipid accumulation and cellular stress in primary cardiomyocytes. Methods: We evaluated lipid accumulation biochemically (by triacylglyceride assay and radiolabeled fatty acid uptake assay) as well as histologically (by BODIPY 493/503 staining) in mouse and rat neonatal cardiomyocytes treated with saturated (palmitate) or mono-unsaturated (oleate) fatty acids. Endoplasmic reticulum (ER) stress was evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting. Cell viability was assessed by propidium iodide staining. Results: We found that both oleate and palmitate led to significant increases in intracellular lipid in cardiomyocytes; however there were distinct differences in the qualitative nature of BODIPY staining between oleate and palmitate treated cardiomyocytes. We also show that palmitate caused significant apoptotic cell death and this was associated with ER stress. Interestingly, co-administration of oleate with palmitate abolished cell death, and ER stress. Finally, palmitate treatment caused a significant increase in ubiquitination of Grp78, a key compensatory ER chaperone. Conclusion: Palmitate causes ER stress and apoptotic cell death in primary cardiomyocytes and this is associated with apparent differences in BODIPY staining compared to oleate treated cardiomyocytes. Importantly, the lipotoxic effects of palmitate are abolished with the co-administration of oleate.
The main focus of this research was to apply Metabolic Control Analysis to quantitative investigation of the regulation of respiration by components of the Mitochondrial Interactosome (MI, a supercomplex consisting of ATP Synthasome, mitochondrial creatine kinase (MtCK), voltage dependent anion channel (VDAC), and tubulin) in permeabilized cardiomyocytes. Flux control coefficients (FCC) were measured using two protocols: 1) with direct ADP activation, and 2) with MtCK activation by creatine (Cr) in the presence of ATP and pyruvate kinase-phosphoenolpyruvate system. The results show that the metabolic control is much stronger in the latter case: the sum of the measured FCC is 2.7 versus 0.74 (ADP activation). This is consistent with previous data showing recycling of ADP and ATP inside the MI due to the functional coupling between MtCK and ANT and limited permeability of VDAC for these compounds, PCr being the major energy carrier between the mitochondria and ATPases. In physiological conditions, when the MI is activated, the key sites of regulation of respiration in mitochondria are MtCK (FCC = 0.93), adenine nucleotide translocase ANT (FCC = 0.95) and CoQ cytochrome c oxidoreductase (FCC = 0.4). These results show clearly that under the physiological conditions the energy transfer from mitochondria to the cytoplasm is regulated by the MI supercomplex and is very sensitive to metabolic signals.
Content available remote Elevation of the adenylate pool in rat cardiomyocytes by S-adenosyl-L-methionine.
Rapid resynthesis of the adenylate pool in cardiac myocytes is important for recovery of contractility and normal function of regulatory mechanisms in the heart. Adenosine and adenine are thought to be the most effective substrates for nucleotide synthesis, but the possibility of using other compounds has been studied very little in cardiomyocytes. In the present study, the effect of S-adenosyl-L-methionine (SAM) on the adenylate pool of isolated cardiomyocytes was investigated and compared to the effect of adenine and adenosine. Adult rat cardiomyocytes were isolated using the collagenase perfusion technique. The cells were incubated in the presence of adenine derivatives for 90 min followed by nucleotide determination by HPLC. The concentrations of adenine nucleotides expressed in nmol/mg of cell protein were initially 22.1 ± 1.4, 4.0 ± 0.3 and 0.70 ± 0.08 for ATP, ADP and AMP, respectively (n = 10, ±S.E.M.), and the total adenylate pool was 26.8 ± 1.6. In the presence of 1.25 mM SAM in the medium, the adenylate pool increased by 5.2 ± 0.4 nmol/mg of cell protein, but only if 1 mM ribose was additionally present in the medium. No changes were observed with SAM alone. A similar increase (by 4.9 ± 0.6 nmol/mg protein) was observed after incubation with 1.25 mM adenine plus 1 mM ribose, but no increase was observed if ribose was omitted. Adenosine at 0.1 or 1.25 mM concentrations also caused an increase in the adenylate pool (by 5.2 ± 1.0 and 5.2 ± 0.9 nmol/mg protein, respectively), which in contrast to the SAM or adenine was independent of the additional presence of ribose. Thus, S-adenosyl-L-methionine could be used as a precursor of the adenylate pool in cardiomyocytes, which is as efficient in increasing the adenylate pool after 90 min of incubation as adenosine or adenine. Nucleotide synthesis from SAM involves the formation of adenine as an intermediate with its subsequent incorporation by adenine phosphoribosyltransferase.
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