Subsarcolemmal and intermyofibrillar mitochondria proteome differences disclose functional specializations in skeletal muscle

Skeletal muscle is a highly specialized tissue that contains two distinct mitochondria subpopulations, the subsarcolemmal (SS) and the intermyofibrillar (IMF) mitochondria. Although it is established that these mitochondrial subpopulations differ functionally in several ways, limited information exists about the proteomic differences underlying these functional differences. Therefore, the objective of this study was to biochemically characterize the SS and IMF mitochondria isolated from rat red gastrocnemius skeletal muscle. We separated the two mitochondrial subpopulations from skeletal muscle using a refined method that provides an excellent division of these unique mitochondrial subpopulations. Using proteomics of mitochondria and its subfractions (intermembrane space, matrix and inner membrane), a total of 325 distinct proteins were identified, most of which belong to the functional clusters of oxidative phosphorylation, metabolism and signal transduction. Although more gel spots were observed in SS mitochondria, 38 of the identified proteins were differentially expressed between the SS and IMF subpopulations. Compared to the SS mitochondrial, IMF mitochondria expressed a higher level of proteins associated with oxidative phosphorylation. This observation, coupled with the finding of a higher respiratory chain complex activity in IMF mitochondria, suggests a specialization of IMF mitochondria toward energy production for contractile activity.     

Populations of rat skeletal muscle mitochondria after exercise and immobilization

We slightly modified an existing procedure (Palmer et al., J. Biol. Chem. 252: 8731-8739, 1977) to isolate two distinct populations of mitochondria from rat skeletal muscle; initial brief Polytron homogenization released the subsarcolemmal mitochondria, and brief exposure of the resultant intact myofibrils to the proteolytic enzyme, Nagarse, extracted the intermyofibrillar mitochondria. The intermyofibrillar mitochondria differed from the subsarcolemmal mitochondr. ia by higher state III respiration measurements and enzymatic activities. These two populations of mitochondria were then isolated from the gastrocnemius muscle that had been induced to perform different amounts of contractile activity. The endurance training program of daily running significantly increased state III respiration and respiratory control index in the subsarcolemmal mitochondria, but the program did not increase these measurements in the intermyofibrillar mitochondria. In addition, 2 days of hindlimb immobilization resulted in a significant decrease in state II respiration and the respiratory control index of the subsarcolemmal mitochondria; however, immobilization did not affect the intermyofibrillar mitochondria. These measurements suggest that the subsarcolemmal mitochondria adapt in response to chronic changes in the level of contractile activity.     

Increase in the adenine nucleotide translocase content of duckling subsarcolemmal mitochondria during cold acclimation

Intermyofibrillar and subsarcolemmal mitochondria were isolated from duckling gastrocnemius muscle. The adenine nucleotide translocase (ANT) content of subsarcolemmal mitochondria was found to be half of that present in intermyofibrillar mitochondria. In addition, cold acclimation resulted in a 1.7-fold increase in subsarcolemmal mitochondrial ANT content, with intermyofibrillar mitochondrial ANT remaining constant. This change in mitochondrial ANT content correlates with the previously reported cold-induced change in the sensitivity of mitochondria to palmitate-inhibited ATP synthesis [Roussel et al. (1998) FEBS Lett. 439, 258-262]. It is suggested that the mitochondrial ANT content enhances or reduces the fatty acid uncoupling activity in tissue, depending on the energetic state of mitochondria.     

Mitochondrial subpopulations and heterogeneity revealed by confocal imaging: possible physiological role?

Heterogeneity of mitochondria has been reported for a number of various cell types. Distinct mitochondrial subpopulations may be present in the cell and may be differently involved in physiological and pathological processes. However, the origin and physiological roles of mitochondrial heterogeneity are still unknown. In mice skeletal muscle, a much higher oxidized state of subsarcolemmal mitochondria as compared with intermyofibrillar mitochondria has been demonstrated. Using confocal imaging technique, we present similar phenomenon for rat soleus and gastrocnemius muscles, where higher oxidative state of mitochondrial flavoproteins correlates also with elevated mitochondrial calcium. Moreover, subsarcolemmal mitochondria demonstrate distinct arrangement and organization. In HL-1 cardiomyocytes, long thread mitochondria and small grain mitochondria are observed irrespective of a particular cellular region, showing also heterogeneous membrane potential and ROS production. Possible physiological roles of intracellular mitochondrial heterogeneity and specializations are discussed.     

Physical and Functional Association of Lactate Dehydrogenase (LDH) with Skeletal Muscle Mitochondria

The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. To examine whether skeletal muscle mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO₂) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO₂. However, subsequent addition of NAD⁺ significantly increased JO₂, and was abolished by the inhibitor of mitochondrial pyruvate transport, α-cyano-4-hydroxycinnamate. In experiments with isolated subsarcolemmal and intermyofibrillar mitochondrial subpopulations, only subsarcolemmal exhibited NAD⁺-dependent lactate oxidation. To further investigate the details of the physical association of LDH with mitochondria in muscle, immunofluorescence/confocal microscopy and immunoblotting approaches were used. LDH clearly colocalized with mitochondria in intact, as well as permeabilized fibers. LDH is likely localized inside the outer mitochondrial membrane, but not in the mitochondrial matrix. Collectively, these results suggest that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.     

Proteomic analysis of succinate dehydrogenase and ubiquinol-cytochrome c reductase (Complex II and III) isolated by immunoprecipitation from bovine and mouse heart mitochondria

The oxidative phosphorylation system (OXPHOS) consists of five multi-enzyme complexes, Complexes I-V, and is a key component of mitochondrial function relating to energy production, oxidative stress, cell signaling and apoptosis. Defects or a reduction in activity in various components that make up the OXPHOS enzymes can cause serious diseases, including neurodegenerative disease and various metabolic disorders. Our goal is to develop techniques that are capable of rapid and in-depth analysis of all five OXPHOS complexes. Here, we describe a mild, micro-scale immunoisolation and mass spectrometric/proteomic method for the characterization of Complex II (succinate dehydrogenase) and Complex III (ubiquinol-cytochrome c reductase) from bovine and rodent heart mitochondria. Extensive protein sequence coverage was obtained after immunocapture, 1D SDS PAGE separation and mass spectrometric analysis for a majority of the 4 and 11 subunits, respectively, that make up Complexes II and III. The identification of several posttranslational modifications, including the covalent FAD modification of flavoprotein subunit 1 from Complex II, was possible due to high mass spectrometric sequence coverage.     

Dexamethasone treatment specifically increases the basal proton conductance of rat liver mitochondria

We investigated the role that mitochondrial proton leak may play in the glucocorticoid-induced hypermetabolic state. Sprague-Dawley rats were injected with dexamethasone over a period of 5 days. Liver mitochondria and gastrocnemius subsarcolemmal and intermyofibrillar mitochondria were isolated from dexamethasone-treated, pair-fed and control rats. Respiration and membrane potential were measured simultaneously using electrodes sensitive to oxygen and to the potential-dependent probe triphenylmethylphosphonium, respectively. Five days of dexamethasone injection resulted in a marked increase in the basal proton conductance of liver mitochondria, but not in the muscle mitochondrial populations. This effect would have a modest impact on energy expenditure in rats.     

Substrate-specific changes in mitochondrial respiration in skeletal and cardiac muscle of hibernating thirteen-lined ground squirrels

During torpor, the metabolic rate (MR) of thirteen-lined ground squirrels (Ictidomys tridecemlineatus) is considerably lower relative to euthermia, resulting in part from temperature-independent mitochondrial metabolic suppression in liver and skeletal muscle, which together account for ~40 % of basal MR. Although heart accounts for very little (<0.5 %) of basal MR, in the present study, we showed that respiration rates were decreased up to 60 % during torpor in both subsarcolemmal (SS) and intermyofibrillar (IM) mitochondria from cardiac muscle. We further demonstrated pronounced seasonal (summer vs. winter [i.e., interbout] euthermia) changes in respiration rates in both mitochondrial subpopulations in this tissue, consistent with a shift in fuel use away from carbohydrates and proteins and towards fatty acids and ketones. By contrast, these seasonal changes in respiration rates were not observed in either SS or IM mitochondria isolated from hind limb skeletal muscle. Both populations of skeletal muscle mitochondria, however, did exhibit metabolic suppression during torpor, and this suppression was 2- to 3-fold greater in IM mitochondria, which provide ATP for Ca²⁺- and myosin ATPases, the activities of which are likely quite low in skeletal muscle during torpor because animals are immobile. Finally, these changes in mitochondrial respiration rates were still evident when standardized to citrate synthase activity rather than to total mitochondrial protein.     

Monocarboxylate transporters in subsarcolemmal and intermyofibrillar mitochondria

Whether subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria contain monocarboxylate transporters (MCTs) is controversial. We have examined the presence of MCT1, 2, and 4 in highly purified SS and IMF mitochondria. These mitochondria were not contaminated with plasma membrane, sarcoplasmic reticulum or endosomal compartments, as the marker proteins for these sub-cellular compartments (Na+-K+-ATPase, Ca2+-ATPase, and the transferrin receptor) were not present in SS or IMF mitochondria. MCT1, MCT2, and MCT4 were all present at the plasma membrane. However, MCT1 and MCT4 were associated with SS mitochondria. In contrast, the IMF mitochondria were completely devoid of MCT1 and MCT4. However, MCT2 was associated with both SS and IMF mitochondria. These observations suggest that SS and IMF mitochondria have different capacities for metabolizing monocarboxylates. Thus, the controversy as to whether mitochondria can take up and oxidize lactate will need to take account of the different distribution of MCTs between SS and IMF mitochondria.     

Distinct Functional Roles of Cardiac Mitochondrial Subpopulations Revealed by a 3D Simulation Model

Experimental characterization of two cardiac mitochondrial subpopulations, namely, subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), has been hampered by technical difficulties, and an alternative approach is eagerly awaited. We previously developed a three-dimensional computational cardiomyocyte model that integrates electrophysiology, metabolism, and mechanics with subcellular structure. In this study, we further developed our model to include intracellular oxygen diffusion, and determined whether mitochondrial localization or intrinsic properties cause functional variations. For this purpose, we created two models: one with equal SSM and IFM properties and one with IFM having higher activity levels. Using these two models to compare the SSM and IFM responses of [Ca²⁺], tricarboxylic acid cycle activity, [NADH], and mitochondrial inner membrane potential to abrupt changes in pacing frequency (0.25–2 Hz), we found that the reported functional differences between these subpopulations appear to be mostly related to local [Ca²⁺] heterogeneity, and variations in intrinsic properties only serve to augment these differences. We also examined the effect of hypoxia on mitochondrial function. Under normoxic conditions, intracellular oxygen is much higher throughout the cell than the half-saturation concentration for oxidative phosphorylation. However, under limited oxygen supply, oxygen is mostly exhausted in SSM, leaving the core region in an anoxic condition. Reflecting this heterogeneous oxygen environment, the inner membrane potential continues to decrease in IFM, whereas it is maintained to nearly normal levels in SSM, thereby ensuring ATP supply to this region. Our simulation results provide clues to understanding the origin of functional variations in two cardiac mitochondrial subpopulations and their differential roles in maintaining cardiomyocyte function as a whole.     

Differential effects of thyroid status on regional H2O2 production in slow- and fast-twitch muscle of ducklings

Birds seem to employ powerful physiological strategies to curb the harmful effects of reactive oxygen species (ROS) because they generally live longer than predicted by the free radical theory of aging. However, little is known about the physiological mechanisms that confer protection to birds against excessive ROS generation. Hence, we investigated the ability of birds to control mitochondrial ROS generation during physiologically stressful periods. In our study, we analyzed the relationship between the thyroid status and the function of intermyofibrillar and subsarcolemmal mitochondria located in glycolytic and oxidative muscles of ducklings. We found that the intermyofibrillar mitochondria of both glycolytic and oxidative muscles down regulate ROS production when plasma T₃ levels rise. The intermyofibrillar mitochondria of the gastrocnemius muscle (an oxidative muscle) produced less ROS and were more sensitive than the pectoralis muscle (a glycolytic muscle) to changes in plasma T₃. Such differences in the ROS production by glycolytic and oxidative muscles were associated with differences in the membrane proton permeability and in the rate of free radical leakage within the respiratory chain. This is the first evidence which shows that in birds, the amount of ROS that the mitochondria release is dependent on: (1) their location within the muscle; (2) the type of muscle (glycolytic or oxidative) and (3) on the thyroid status. Reducing muscle mitochondrial ROS generation might be an important mechanism in birds to limit oxidative damage during periods of physiological stress.     

CRIF1 Is Essential for the Synthesis and Insertion of Oxidative Phosphorylation Polypeptides in the Mammalian Mitochondrial Membrane

Although substantial progress has been made in understanding the mechanisms underlying the expression of mtDNA-encoded polypeptides, the regulatory factors involved in mitoribosome-mediated synthesis and simultaneous insertion of mitochondrial oxidative phosphorylation (OXPHOS) polypeptides into the inner membrane of mitochondria are still unclear. In the present study, disruption of the mouse Crif1 gene, which encodes a mitochondrial protein, resulted in a profound deficiency in OXPHOS caused by the disappearance of OXPHOS subunits and complexes in?vivo. CRIF1 was associated with large mitoribosomal subunits that were located close to the polypeptide exit tunnel, and the elimination of CRIF1 led to both aberrant synthesis and defective insertion of mtDNA-encoded nascent OXPHOS polypeptides into the inner membrane. CRIF1 interacted with nascent OXPHOS polypeptides and molecular chaperones, e.g., Tid1. Taken together, these results suggest that CRIF1 plays a critical role in the integration of OXPHOS polypeptides into the mitochondrial membrane in mammals.     

Influence of intensity of food restriction on skeletal muscle mitochondrial energy metabolism in rats

Variable durations of food restriction (FR; lasting weeks to years) and variable FR intensities are applied to animals in life span-prolonging studies. A reduction in mitochondrial proton leak is suggested as a putative mechanism linking such diet interventions and aging retardation. Early mechanisms of mitochondrial metabolic adaptation induced by FR remain unclear. We investigated the influence of different degrees of FR over 3 days on mitochondrial proton leak and mitochondrial energy metabolism in rat hindlimb skeletal muscle. Animals underwent 25, 50, and 75% and total FR compared with control rats. Proton leak kinetics and mitochondrial functions were investigated in two mitochondrial subpopulations, intermyofibrillar (IMF) and subsarcolemmal (SSM) mitochondria. Regardless of the degree of restriction, skeletal muscle mass was not affected by 3 days of FR. Mitochondrial basal proton conductance was significantly decreased in 50% restricted rats in both mitochondrial subpopulations (46 and 40% for IMF and SSM, respectively) but was unaffected in other groups compared with controls. State 3 and uncoupled state 3 respiration rates were decreased in SSM mitochondria only for 50% restricted rats when pyruvate + malate was used as substrate (-34.5 and -38.9% compared with controls, P < 0.05). IMF mitochondria respiratory rates remained unchanged. Three days of FR, particularly at 50% FR, were sufficient to lower mitochondria energetic metabolism in both mitochondrial populations. Our study highlights an early step in mitochondrial adaptation to FR and the influence of the severity of restriction on this adaptation. This step may be involved in an aging-retardation process.     

The effect of strength training on estimates of mitochondrial density and distribution throughout muscle fibres

The purpose of this study was to investigate the effect of strength training (12 weeks, 3 days/week, four lower-body exercises) of young individuals (mean age 23.6 years) on estimates of mitochondrial distribution throughout muscle fibres. A control group (mean age 21. 7 years) was followed simultaneously. Skeletal muscle biopsy samples were obtained from the vastus lateralis, pre- and post-training. The regional distribution of subsarcolemmal and intermyofibrillar mitochondrial populations was determined using quantitative histochemical staining of succinate dehydrogenase (SDH) in type I and II muscle fibres. Strength training resulted in significant increases of 26% and 28% in the cross-sectional area of type I and II fibres, respectively (P < 0.05). Overall SDH activity decreased by 13% with strength training (P < 0.05). The decrease in SDH activity with strength training between fibre types and between subsarcolemmal and intermyofibrillar regions of muscle fibres was not different. Fibre area and SDH activity was unchanged in the control group. We conclude that the muscle hypertrophy associated with strength training results in reduced density of regionally distributed mitochondria, as indicated by the reduction in the activity of SDH.     

Differences in proton leak kinetics, but not in UCP3 protein content, in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria from fed and fasted rats

We have investigated the effect of 24-h fasting on basal proton leak and uncoupling protein (UCP) 3 expression at the protein level in subsarcolemmal and intermyofibrillar skeletal muscle mitochondria. In fed rats, the two mitochondrial populations displayed different proton leak, but the same protein content of UCP3. In addition, 24-h fasting, both at 24 and 29 degrees C, induced an increase in proton leak only in subsarcolemmal mitochondria, while UCP3 content increased in both the populations. From the present data, it appears that UCP3 does not control the basal proton leak of skeletal muscle mitochondria.     

Characterisation of oxidative phosphorylation in skeletal muscle mitochondria subpopulations in pig: a study using top-down elasticity analysis

In skeletal muscle, two mitochondrial populations are present which, on the basis of their localisation, are termed intermyofibrillar and subsarcolemmal mitochondria (IMF and SS, respectively). These two populations have different biochemical characteristics and show different responses to physiological stimuli. In this paper, we characterise the oxidative phosphorylation of SS and IMF using 'top-down' elasticity analysis. We excluded the possibility that their different characteristics can be attributed to a different degree of breakage of the two types of mitochondria due to the different isolation procedures used in their preparation. The higher respiration rate and higher respiratory control ratio shown by IMF compared with those shown by SS are principally due to the higher activities of the reactions involved in substrate oxidation as confirmed by the measurement of cytochrome oxidase activity. There is no difference in the leak of protons across the inner mitochondrial membrane between IMF and SS; a faster rate of ATP synthesis and turnover is driven by the lower membrane potential in SS compared with in IMF.     

Localization of mRNAs encoding human mitochondrial oxidative phosphorylation proteins

The mitochondrial oxidative phosphorylation (OXPHOS) proteins are encoded by both nuclear and mitochondrial DNA. The nuclear-encoded OXPHOS mRNAs have specific subcellular localizations, but little is known about which localize near mitochondria. Here, we compared mRNAs in mitochondria-bound polysome fractions with those in cytosolic, free polysome fractions. mRNAs encoding hydrophobic OXPHOS proteins, which insert into the inner membrane, were localized near mitochondria. Conversely, OXPHOS gene which mRNAs were predominantly localized in cytosol had less than one transmembrane domain. The RNA-binding protein Y-box binding protein-1 is localized at the mitochondrial outer membrane and bound to the OXPHOS mRNAs. Our findings offer new insight into mitochondrial co-translational import in human cells.     

Morphometry of muscle fibre types in the carp (Cyprinus carpio L.)

Summary Ultrastructural parameters of muscle fibre types of the carp (Cyprinus carpio L.) were measured and compared with their contractile properties. In red fibres, which are slower than pink fibres, the relative length of the junction between the T system and the sarcoplasmic reticulum (T-SR junction) is smaller and the Z lines are thicker than in pink fibres. Pink fibres have a smaller relative length of T-SR junction than white fibres from the axial muscles. The two types of red fibres present in carp muscle also differ in their relative lengths of T-SR junction. Significant differences in the relative areas of the SR were not found.The relative volume of myofibrils in red fibres is two-thirds that in pink fibres, a difference that is not reflected in the maximal isometric tetanic tensions of these types. Red fibres, which are less easily fatigued than pink fibres, have larger relative volumes of subsarcolemmal and intermyofibrillar mitochondria. Small pink fibres have a larger relative volume of subsarcolemmal mitochondria than large pink fibres, but have a similar relative volume of intermyofibrillar mitochondria. Small and large pink fibres differ in the relative volumes of their membrane systems, but have similar relative lengths of T-SR junction.     

Mitochondria in cardiomyocyte Ca signaling

Ca²⁺ signaling is of vital importance to cardiac cell function and plays an important role in heart failure. It is based on sarcolemmal, sarcoplasmic reticulum and mitochondrial Ca²⁺ cycling. While the first two are well characterized, the latter remains unclear, controversial and technically challenging.In mammalian cardiac myocytes, Ca²⁺ influx through L-type calcium channels in the sarcolemmal membrane triggers Ca²⁺ release from the nearby junctional sarcoplasmic reticulum to produce Ca²⁺ sparks. When this triggering is synchronized by the cardiac action potential, a global [Ca²⁺]_i transient arises from coordinated Ca²⁺ release events. The ends of intermyofibrillar mitochondria are located within 20 nm of the junctional sarcoplasmic reticulum and thereby experience a high local [Ca²⁺] during the Ca²⁺ release process. Both local and global Ca²⁺ signals may thus influence calcium signaling in mitochondria and, reciprocally, mitochondria may contribute to the local control of calcium signaling. In addition to the intermyofibrillar mitochondria, morphologically distinct mitochondria are also located in the perinuclear and subsarcolemmal regions of the cardiomyocyte and thus experience a different local [Ca²⁺].Here we review the literature in regard to several issues of broad interest: (1) the ultrastructural basis for mitochondrion – sarcoplasmic reticulum cross-signaling; (2) mechanisms of sarcoplasmic reticulum signaling; (3) mitochondrial calcium signaling; and (4) the possible interplay of calcium signaling between the sarcoplasmic reticulum and adjacent mitochondria.Finally, this review discusses experimental findings and mathematical models of cardiac calcium signaling between the sarcoplasmic reticulum and mitochondria, identifies weaknesses in these models, and suggests strategies and approaches for future investigations.     

Calcium uptake by two preparations of mitochondria from heart

Ca²⁺ transport and respiratory characteristics of two preparations of cardiac mitochondria (Palmer, J.W., Tandler, B. and Hoppel, C.L. (1977) J. Biol. Chem. 252, 8731–8739) isolated using polytron homogenization (subsarcolemmal mitochondria) and limited Nagarse exposure (intermyofibrillar mitochondria) are described.The Nagarse procedure yields mitochondria with 50% higher rates of oxidative phosphorylation than the polytron-prepared mitochondria in both rat and dog. Rat hear intermyofibrillar mitochondria contain 50% more cytochrome aa₃ than the polytron preparation, whereas in the dog, cytochrome aa₃ content is not significantly different. Cytochrome oxidase activities and cytochrome c, c₁ and b contents were comparable in both populations of rat and dog heart mitochondria.The V of succinate-supported Ca²⁺ accumulation for Nagarse-prepared mitochondria from rat heart was 1.8-fold higher than the polytron-prepared mitochondria. In dog heart, the Nagarse preparation showed a 3.0-fold higher V for Ca²⁺ uptake compared to the polytron preparation. A lower apparent affinity for Ca²⁺ was demonstrated in the intermyofibrillar mitochondria for both species (K_m is 2–2.5-fold higher). The Hill coefficient was 1 both mitochondrial types. Subsarcolemmal mitochondria from both species were treated with Nagarse to determine the role of this treatment on the observed differences. Nagarse did not alter any kinetic parameter of Ca²⁺ uptake.The properties of these mitochondria with reference to their presumed intracellular location may pertain to the role of mitochondria as an intracellular Ca²⁺ buffering mechanism in contractile tissue.