MuRF1 activity is present in cardiac mitochondria and regulates reactive oxygen species production in vivo

Erratum: https://link.springer.com/article/10.1007/s10863-014-9597-1MuRF1 is a previously reported ubiquitin-ligase found in striated muscle that targets troponin I and myosin heavy chain for degradation. While MuRF1 has been reported to interact with mitochondrial substrates in yeast two-hybrid studies, no studies have identified MuRF1’s role in regulating mitochondrial function to date. In the present study, we measured cardiac mitochondrial function from isolated permeabilized muscle fibers in previously phenotyped MuRF1 transgenic and MuRF1−/− mouse models to determine the role of MuRF1 in intermediate energy metabolism and ROS production. We identified a significant decrease in reactive oxygen species production in cardiac muscle fibers from MuRF1 transgenic mice with increased α-MHC driven MuRF1 expression. Increased MuRF1 expression in ex vivo and in vitro experiments revealed no alterations in the respiratory chain complex I and II function. Working perfusion experiments on MuRF1 transgenic hearts demonstrated significant changes in glucose oxidation. This is an factual error as written; however, total oxygen consumption was decreased. This data provides evidence for MuRF1 as a novel regulator of cardiac ROS, offering another mechanism by which increased MuRF1 expression may be cardioprotective in ischemia reperfusion injury, in addition to its inhibition of apoptosis via proteasome-mediate degradation of c-Jun. The lack of mitochondrial function phenotype identified in MuRF1−/− hearts may be due to the overlapping interactions of MuRF1 and MuRF2 with energy regulating proteins found by yeast two-hybrid studies reported here, implying a duplicity in MuRF1 and MuRF2’s regulation of mitochondrial function.Funding support from Medical Research Council, United Kingdom; National Institutes of Health, United States; British Heart Foundation, United Kingdo

LC-quadrupole/Orbitrap high-resolution mass spectrometry enables stable isotope-resolved simultaneous quantification and 13C-isotopic labeling of acyl-coenzyme A thioesters

Acyl-coenyzme A thioesters (acyl-CoAs) are evolutionarily conserved, compartmentalized, and energetically activated substrates for biochemical reactions. The ubiquitous involvement of acyl-CoAs in metabolism, including the tricarboxylic acid cycle, fatty acid metabolism, amino acid degradation, and cholesterol metabolism highlights the broad applicability of applied measurements of acyl-CoAs. However, quantitation of acyl-CoA levels provides only one dimension of metabolic information and a more complete description of metabolism requires the relative contribution of different precursors to individual substrates and pathways. Using two distinct stable isotope labeling approaches, acyl-CoAs can be labeled with either a fixed [(13)C(3)(15)N(1)] label derived from pantothenate into the CoA moiety, or via variable [(13)C] labeling into the acyl-chain from metabolic precursors. Liquid chromatography-hybrid quadrupole/Orbitrap high resolution mass spectrometry using parallel reaction monitoring, but not single ion monitoring, allowed the simultaneous quantitation of acyl-CoAs by stable isotope dilution using the [(13)C(3)(15)N(1)] label and measurement of the incorporation of labeled carbon atoms derived from [(13)C(6)]-glucose, [(13)C(5)(15)N(2)]-glutamine and [(13)C(3)]-propionate. As a proof-of-principle we applied this method to human B-cell lymphoma (WSU-DLCL2) cells in culture, to precisely describe the relative pool size and enrichment of isotopic tracers into acetyl-, succinyl-, and propionyl-CoA. This method will allow highly precise, multiplexed, and stable isotope resolved determination of metabolism to refine metabolic models, characterize novel metabolism, and test modulators of metabolic pathways involving acyl-CoAs

Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs)

The obligate-intracellular pathogen Chlamydia trachomatis (Ct) has undergone considerable genome reduction with consequent dependence on host biosynthetic pathways, metabolites and enzymes. Long-chain acyl-CoA synthetases (ACSLs) are key host-cell enzymes that convert fatty acids (FA) into acyl-CoA for use in metabolic pathways. Here, we show that the complete host ACSL family [ACSL1 and ACSL3–6] translocates into the Ct membrane-bound vacuole, termed inclusion, and remains associated with membranes of metabolically active forms of Ct throughout development. We discovered that three different pharmacologic inhibitors of ACSL activity independently impede Ct growth in a dose-dependent fashion. Using an FA competition assay, host ACSLs were found to activate Ct branched-chain FAs, suggesting that one function of the ACSLs is to activate Ct FAs and host FAs (recruited from the cytoplasm) within the inclusion. Because the ACSL inhibitors can deplete lipid droplets (LD), we used a cell line where LD synthesis was switched off to evaluate whether LD deficiency affects Ct growth. In these cells, we found no effect on growth or on translocation of ACSLs into the inclusion. Our findings support an essential role for ACSL activation of host-cell and bacterial FAs within the inclusion to promote Ct growth and development, independent of LDs

Role of AMPK signalling pathway during compensatory growth in pigs

Abstract Background The molecular basis of compensatory growth in monogastric animals has not yet been fully explored. Herewith, in this study we aim to determine changes in the pig skeletal muscle transcriptome profile during compensatory growth following a feed restriction period. A RNA-Seq experiment was performed with a total of 24 females belonging to a Duroc commercial line. Half of the animals received either a restricted (RE) or ad libitum (AL) diet during the first fattening period (60–125 d of age). After that, all gilts were fed ad libitum for a further ~30 d until the age of ~155 d, when animals were slaughtered and samples of gluteus medius muscle were harvested to perform RNA-Seq analyses and intramuscular fat content determination. Results During the period following food restriction, RE animals re-fed ad libitum displayed compensatory growth, showed better feed conversion rate and tended to deposit more subcutaneous fat than AL fed animals. Animals were slaughtered in the phase of accelerated growth, when RE animals had not completely compensated the performance of AL group, showing lower live and carcass weights. At intramuscular level, RE gilts showed a higher content of polyunsaturated fatty acids during the compensatory growth phase. The comparison of RE and AL expression profiles allowed the identification of 86 (ǀlog2Fold-Changeǀ > 1, padj < 0.05) differentially expressed (DE) genes. A functional categorization of these DE genes identified AMPK Signaling as the most significantly enriched canonical pathway. This kinase plays a key role in the maintenance of energy homeostasis as well as in the activation of autophagy. Among the DE genes identified as components of AMPK Signaling pathway, five out of six genes were downregulated in RE pigs. Conclusions Animals re-fed after a restriction period exhibited a less oxidative metabolic profile and catabolic processes in muscle than animals fed ad libitum. The downregulation of autophagy observed in the skeletal muscle of pigs undergoing compensatory growth may constitute a mechanism to increase muscle mass thus ensuring an accelerated growth rate. These results reveal that the downregulation of AMPK Signaling plays an important role in compensatory growth in pigs