Protein composition and function of red and white skeletal muscle mitochondria

Abstract

Glancy B, Balaban RS. Protein composition and function of red and white skeletal muscle mitochondria. Am J Physiol Cell Physiol 300: C1280 -C1290, 2011. First published February 2, 2011 doi:10.1152/ajpcell.00496.2010.-Red and white muscles are faced with very different energetic demands. However, it is unclear whether relative mitochondrial protein expression is different between muscle types. Mitochondria from red and white porcine skeletal muscle were isolated with a Percoll gradient. Differences in protein composition were determined using blue native (BN)-PAGE, two-dimensional differential in gel electrophoresis (2D DIGE), optical spectroscopy, and isobaric tag for relative and absolute quantitation (iTRAQ). Complex IV and V activities were compared using BN-PAGE in-gel activity assays, and maximal mitochondrial respiration rates were assessed using pyruvate (P) ϩ malate (M), glutamate (G) ϩ M, and palmitoyl-carnitine (PC) ϩ M. Without the Percoll step, major cytosolic protein contamination was noted for white mitochondria. Upon removal of contamination, very few protein differences were observed between red and white mitochondria. BN-PAGE showed no differences in the subunit composition of Complexes I-V or the activities of Complexes IV and V. iTRAQ analysis detected 358 mitochondrial proteins, 69 statistically different. Physiological significance may be lower: at a 25% difference, 48 proteins were detected; at 50%, 14 proteins were detected; and 3 proteins were detected at a 100%. Thus any changes could be argued to be physiologically modest. One area of difference was fat metabolism where four ␤-oxidation enzymes were ϳ25% higher in red mitochondria. This was correlated with a 40% higher rate of PCϩM oxidation in red mitochondria compared with white mitochondria with no differences in PϩM and GϩM oxidation. These data suggest that metabolic demand differences between red and white muscle fibers are primarily matched by the number of mitochondria and not by significant alterations in the mitochondria themselves.proteomics; fast and slow-twitch muscle; iTRAQ; energetics; oxidative phosphorylation SKELETAL MUSCLE-specific oxygen consumption can increase over 100-fold from rest to maximal oxygen uptake (V O 2max ) (52). An additional threefold increase in power output can be achieved during maximal anaerobic exercise (43). Thus skeletal muscle is faced with a wide range of energetic demands dependent on both exercise intensity and duration. Slow and sustained activities such as maintaining posture or low-intensity exercise result in the recruitment of red, oxidative muscle fibers. Conversely, shorter bouts of high-intensity exercise are accomplished by the activation of white, glycolytic myocytes. Accordingly, protein expression in red and white fibers is tuned to meet their respective energetic demands. White muscle is characterized by a predominance of glycolytic enzymes and the fast isoforms of contractile proteins, whereas red muscle has greater abundance of contractile protein slow isoforms and higher ...

Mitochondrial Diagnostics: A Multiplexed Assay Platform for Comprehensive Assessment of Mitochondrial Energy Fluxes

“…Mitochondrial Percoll purification was carried out as previously described (Glancy and Balaban, 2011; Graham, 2001), with some modifications. Mitochondrial pellets (~5mgs) were resuspended in 1ml of sucrose wash buffer (250mM sucrose, 10mM HEPES (pH = 7.2), 0.1% BSA).…”

“…Efforts to optimize the platform for new applications must consider several critical factors, including: a) tissue and cell type-specific differences in matrix dehydrogenase profiles, b) prioritization of substrate conditions when mitochondrial yield is limiting, and c) purity of tissue-specific mitochondrial isolations and potential need for sucrose- or Percoll-based purification protocols (Glancy and Balaban, 2011; Graham, 2001). To this point, it is noteworthy that the simple isolation protocol (without a sucrose-based buffer) used in the current work was sufficient to produce fractions with similar amounts of mitochondrial protein between tissues (Figures S4A and S4B).…”

“…However, additional mitochondrial purification may be required for tissue types in which cytosolic, ER and/or lipid contamination within the mitochondrial fraction is more problematic. These considerations could become more important in studies where proteomics analyses are desired (Glancy and Balaban, 2011). Finally, the substrate conditions employed herein were selected to cover the primary metabolic pathways operating in muscle mitochondria, as evidenced by the dehydrogenase profiles of the tissues.…”

Metabolite Profile Differences between Beef Longissimus and Psoas Muscles during Display

“…Muscles with more red muscle fibers (PM) will have more mitochondria and capillary density than muscles with more white fibers (LL). Hence TCA and glycolytic substrates will be utilized at different rates in red muscles than in white muscles (Kushmerick et al, 1992;Glancy and Balaban, 2011). Recently metabolomics was used to study color stability in ovine meat (Subbaraj et al, 2016).…”

Identification of differences in microRNA transcriptomes between porcine oxidative and glycolytic skeletal muscles

“…used two-dimensional gel electrophoresis and mass spectrometry to build a reference map of proteins and identified hundreds of distinct gene products including metabolic, transport, and contractile proteins [8]. Moreover, it was reported that the different energetic demands of white and red muscles were matched primarily by the different numbers of mitochondrial proteins in the two tissues [9]. Recently, a number of studies have focused on small non-coding RNAs, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), which are involved in the transcriptional and post-transcriptional regulation of gene expression.…”